**Ameliorative E**ff**ect of** *Bacillus subtilis* **on Growth Performance and Intestinal Architecture in Broiler Infected with Salmonella**

#### **Alaeldein M. Abudabos 1,\*, Muttahar H. Ali 1, Mohammed A. Nassan <sup>2</sup> and Ahmad A. Saleh <sup>3</sup>**


Received: 6 March 2019; Accepted: 12 April 2019; Published: 23 April 2019

**Simple Summary:** Salmonellosis is a dangerous disease in broilers that causes huge economic losses. We assumed that instead of antibiotics, a Bacillus-based probiotic may serve as an alternative to alleviate the negative effects of *Salmonella* infection. A control group with no feed additive, a positive control supplemented with a standard antibiotic and two groups that were supplemented with different strains and levels of *Bacillus subtilis* were the experimental animals of the present study. It was revealed that supplementation of probiotic bacteria induced similar results in terms of feed intake, body weight gain and feed efficiency in comparison with the group treated with antibiotics. In addition, the dimensions of intestinal villi were also improved in the probiotic-treated birds. As concluded from the results of the present study, probiotic bacteria could be used as an alternative to antibiotics against *Salmonella* in broilers.

**Abstract:** A total of 600 day-old broiler chicks (Ross 308) confirmed for the absence of *Salmonella* were randomly allocated to five treatments each with 10 replicates: negative control (basal diet only); positive control (basal diet) + infected with *Salmonella*; T1, *Salmonella* infected + avilamycin; T2, *Salmonella* infected + *Bacillus subtilis* (ATCC PTA-6737; 2 <sup>×</sup> 107 CFU/g) and T3, *Salmonella* infected + *B. subtilis* (DSM 172999; 1.2 <sup>×</sup> 106 CFU/g). The results revealed that feed intake (FI) and body weight (BW) were significantly (*p* < 0.01) lower in T1 compared to T2. The feed conversion ratio (FCR) was significantly (*p* < 0.01) lower in T2 and T3 compared to other treatments. Similarly, the performance efficiency factor (PEF) was also significantly (*p* < 0.01) higher in T2 and T3 compared to positive control. Villus height was significantly (*p* < 0.01) higher in T2 compared to all other treatments. However, villus width and surface area were significantly (*p* < 0.01) higher in T1. In conclusion, dietary supplementation with *B. subtilis* improved growth and intestinal health by reversing the negative effects of Salmonellosis.

**Keywords:** broiler; growth; intestinal villi; *Salmonella*

#### **1. Introduction**

In the modern broiler industry, antimicrobials used as growth promoters are among the most popular synthetic agents used in poultry production for the improvement of feed efficiency and the reduction of microbial pathogenesis [1,2]. Antimicrobials as an additive have produced promising results; however, their regular use has caused drug resistance and residues in eggs and meat [3]. Under such circumstances, many countries are considering a strict ban or have already banned (European Union) the use of Antimicrobial growth promoters (AGPs) [4–6]. Therefore, there is a necessity to find suitable alternatives that could replace AGPs. Recently natural products have gained special interest, since they improve growth performance and reduce mortality rates as an effect of infectious agents [7–9].

*Salmonella* is one of the most important poultry diseases causing heavy economic losses through stunted growth and increased mortality rates [6,8]. Incidences of *Salmonella* are most frequent during the starter phase, since the immune system of the chick is not well developed [9]. Chickens are frequently exposed to *Salmonella* during their life and micro-organisms can be readily transferred to humans through the consumption of poultry meat, causing severe gastrointestinal symptoms [10]. A number of practices are used to reverse the symptoms of salmonellosis in broilers including the use of probiotics [9,11]. Probiotics are used in poultry production due to the wide range of their positive effects [11]. Probiotics are now considered an alternative to antibiotics and added in the animals' diet as a microbial supplement [12]. It has been reported that probiotics enhance growth, provide protection against a wide range of pathogens and improve immunity [11–13]. *Bacillus subtilis* is naturally isolated from the gut of chickens and it is known to produce antimicrobial substances such as surfactants [11]. Recently, it was reported that *B. subtilis* improved the growth and antioxidant status in broilers exposed to *Salmonella* [8]. In the literature, positive effects of the two strains of *B. subtilis* have been reported; however, their comparative effects have not been described. The aim of the present study was to evaluate the effects of two different strains of *B. subtilis* on the performance and intestinal health of *Salmonella*-infected broilers during the starter phase.

#### **2. Methods**

#### *2.1. Animals and Feeding Practices and Randomization*

A total of 600 day-old broiler chicks (Ross 308) were randomly divided into five treatments (10 replicates and 12 birds per replicate). On arrival, all chicks were confirmed for the absence of *Salmonella*. The experiment was carried out in an environmentally controlled closed poultry unit. Straw was used as bedding material on the concrete floor. Initially the temperature was set to 31 ◦C and gradually decreased to a thermoneutral temperature of 22–24 ◦C and a relative humidity of 70%. An automated exhaust fan drew outside air in at 45.8 m3/min. The photoperiod was maintained at 23:1 L:D at the intensity rate of 20 lux using cool light fluorescent tubes. The stocking density was maintained at 50 kg/m2.

Broiler chicks were raised according to the recommendations of the Ross guide. A standard starter (0–15) diet with isocaloric and isonitrogenous contents was offered in a mash form based on corn-SBM and was formulated to meet the requirements of the broilers (Table 1). On day 1, chicks received one of five treatments randomly as follows: negative control (basal diet); positive control (basal diet) + infected with *Salmonella enterica*, subspecies *typhimurium*; T1, infected with *Salmonella* + avilamycin (0.2 g/kg); T2, infected + probiotics that have viable spores of *B. subtilis* (ATCC PTA-6737; (2 <sup>×</sup> 107 CFU/g) and T3, infected <sup>+</sup> *B. subtilis* (DSM 17299; 1.2 <sup>×</sup> 106 CFU/g).


**Table 1.** Dietary composition of broiler chicken starter diets.


**Table 1.** *Cont.*

<sup>1</sup> Vitamin-mineral premix contains the following per kg: vitamin A, 2,400,000 IU; vitamin D, 1,000,000 IU; vitamin E, 16,000 IU; vitamin K, 800 mg; vitamin B1, 600 mg; vitamin B2, 1600 mg; vitamin B6, 1000 mg; vitamin B12, 6 mg; niacin, 8000 mg; folic acid, 400 mg; pantothenic acid, 3000 mg; biotin 40 mg; antioxidant, 3000 mg; cobalt, 80 mg; copper, 2000 mg; iodine, 400; iron, 1200 mg; manganese, 18,000 mg; selenium, 60 mg; zinc, 14,000 mg.

#### *2.2. Challenge Inoculum*

On the second day, all groups apart from the negative control were orally administered with a <sup>3</sup> <sup>×</sup> <sup>10</sup><sup>9</sup> live culture of *Salmonella enterica* subspecies *typhimurium* as described by Abudabos et al. [9].

#### *2.3. Growth Performance*

Growth performance parameters such as body weight (BW), feed intake (FI) and feed conversion rate (FCR) were recorded at five-day intervals. The BW was measured on an electronic digital balance with a sensitivity of 0.1 g (Berkley, Columbia, SC, USA). The production efficiency factor (PEF) was calculated as described by Abudabos et al. [6].

#### *2.4. Sitophological Measurements*

Histological study of intestinal tissue was conducted as described by Rahman et al. [14]. On day 15, 10 birds per treatment were randomly selected. For histological studies, about 2 <sup>×</sup> 2 cm2 long samples from the proximal portion of the ileum were collected, fixed and then embedded in paraffin. The tissues were sectioned to roughly 5 mm small pieces and stained (hematoxylin and eosin). Ten well-oriented villi per sample were selected and measured using a simple microscope (Olympus) connected to an image analysis system.

#### *2.5. Statistical Analysis*

All statistical analyses were performed using the Statistical Analysis System [15]. Means were compared by the method described by Steel and Torrie [16] and a significant level was obtained by the Duncan multiple-range test [17].

#### *2.6. Ethical Approval*

The study was approved by the Committee on Care and Use of Animals, King Saud University, Saudi Arabia (1127/18/DAP).

#### **3. Results**

#### *3.1. Growth Performance*

The results of growth performance for the control and experimental groups are provided in Table 2. Feed intake was significantly (*p* < 0.01) higher in T2 compared to T1. Similarly, BW was significantly (*p* < 0.01) higher in T2 compared to T1 and the positive control. As a result, FCR was significantly (*p* < 0.01) lower in T2 and T3 compared to the positive control. Similarly, PEF was also significantly (*p* < 0.01) higher in T2 and T3 compared to the positive control. Although PEF in T1 was not significantly different from T2 and T3, values were slightly better in T2 and T3 compared to T1.

**Table 2.** Means ± SE of feed intake (FI), body weight (BW), feed conversion ratio (FCR), body weight (BW) and performance efficiency factor (PEF) for the cumulative starter period (0 to 15 days of age).


a,b,c Means within a column differ significantly (*p* < 0.01). T1, infected + avilamycin at a rate of 0.2 g/kg; T2, infected + probiotics that have viable spores (2 × 107 CFU/g) of *Bacillus subtilis* (ATCC PTA-6737); T3: infected + *B. subtilis* (DSM 17299;1.2 × 10<sup>6</sup> CFU/g).

#### *3.2. Intestinal Histology*

The effects of *B. subtilis* on histological structures of broiler chickens are presented in Table 3. Villus height was significantly (*p* < 0.01) higher in T2 compared to all other treatments. However, villus width and surface area were significantly (*p* < 0.01) higher in T1 compared to the positive control group. Villus width was not statistically significant between the negative control and T1 groups.

**Table 3.** Means ± SE of villi height (L), width (W) and villi total area (TA) of ileum in broiler chickens at 15 days.


a,b,c Means within a column differ significantly (*p* < 0.01). T1, infected + avilamycin at the rate of 0.2 g/kg; T2, infected + probiotics that have viable spores (2 × 107 CFU/g) of *B. subtilis* (ATCC PTA-6737); T3: T3, infected + *B. subtilis* (DSM 17299 1.2 × 10<sup>6</sup> CFU/g).

#### **4. Discussion**

In the current study, growth performance and intestinal histological parameters were improved in the probiotic-treated birds infected with *Salmonella*. The results were similar to those of the antibiotic-treated birds. Probiotics are considered one of the viable alternatives to antibiotics, particularly in view of the recent ban of regular use of AGPs in poultry diet [6]. In the present study, the growth performance and intestinal architecture were significantly deteriorated in the *Salmonella*-infected birds. Reduced growth and lesion in the intestinal villi are some of the most prominent signs of salmonellosis, leading to heavy economic losses [8].

Interestingly, the results of the probiotic-treated birds were comparable to those of the birds fed antibiotic supplements. The improved growth performance of broilers infected with different kinds of pathogens such as *Clostridium* and *Salmonella* in response to supplementation of *B. subtilis* or phytogentics has been published recently [8,9]. The positive effects of probiotics are well documented, e.g., improved performance (body weight gain and feed conversion rate); enhanced immune response and healthy intestine [11]. The effects of the two types of probiotics on the performance of the birds were not significantly different. The efficacy of probiotic use is linked to genetics, nutritional status, frequency, dose, specificity of the strain, survival and stability [11]. A number of mechanisms through which probiotics produce positive effects are involved, such as the reduction of intestinal pH, production of volatile fatty acids and suppression of pathogenetic bacteria through competitive exclusion [18].

As indicated in the current study, villus dimensions were restored as an effect of probiotic supplementation. Dietary probiotics have been shown to enhance the intestinal microbiome in a positive direction [8,9]. The intestinal villi secrete different kinds of defensive mucins such as MUC2 and MUC3 from the goblet cells [11]. In addition, probiotic bacteria improve the humoral and cellular immunity through increased production of delayed hypersensitivity, respiratory burst of macrophages, antibody production, natural killer cells, interleukins, cytokines, antibody-secreting cells and T lymphocytes [11,18].

#### **5. Conclusions**

Dietary supplementation with *B. subtilis* improved the growth performance and gut health of *Salmonella*-infected broiler chickens.

**Author Contributions:** Formal analysis, M.H.A.; Investigation, A.A.S.; Methodology, M.A.N.; Project administration, A.M.A.

**Funding:** This research was funded by the Deanship of Scientific Research at King Saud University, grant number RGP-273.

**Conflicts of Interest:** The authors have no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

### **E**ff**ect of Soybean Isoflavones on Growth Performance, Immune Function, and Viral Protein 5 mRNA Expression in Broiler Chickens Challenged with Infectious Bursal Disease Virus**

**Mahmoud Mostafa Azzam 1,2,3,**†**, Shou-qun JIANG 1,\*, Jia-li CHEN 1, Xia-jing LIN 1,**†**, Zhong-yong GOU 1, Qiu-li FAN 1, Yi-bing WANG 1, Long LI <sup>1</sup> and Zong-yong JIANG 1,4,\***


Received: 27 February 2019; Accepted: 17 April 2019; Published: 16 May 2019

**Simple Summary:** Infectious bursal disease virus (IBDV) is characterized by inflammation and subsequent atrophy of the bursa of Fabricius and immune suppression. However, nutritional strategies are able to ameliorate the negative effects of viral infections. Therefore, the aim of the present study was to determine the effect of different levels of soybean isoflavones (SI) on broiler chickens challenged with IBDV. Based on the findings, supplemental 10~20 mg/kg SI may have a positive effect on broiler chickens infected with IBDV, probably because SI decrease the severity of bursa lesions and viral protein 5 mRNA expression, and have strong antioxidant activity.

**Abstract:** A total of 200 one-day-old male broilers were assigned to five groups, and each group consisted of four replicates with 10 birds per replicate. Chicks were fed the basal diet with 0 (non-infected control), 0 (infected control), 10, 20, and 40 mg/kg soybean isoflavones (SI) for 42 days. At 21 days of age, chickens were inoculated with an infectious bursal dose (causing 50% morbidity) of the infectious bursal disease virus (IBDV) BC 6/85 strain by the eye-drop and nasal route (except for the non-infected group). Average daily gain (ADG) and average daily feed intake (ADFI) decreased (*p* < 0.05) in broilers infected with infectious bursal disease virus (IBDV) from 22 to 42 days. However, infected broilers fed 10 and 20 mg SI/kg had the maximum (*p* <0.05) ADG and ADFI from 1 to 42 days. Body weight (BW) increased (*p* < 0.05) in infected broilers fed the 10 and 20 mg SI /kg diet. The bursa weight at 7 days post-infection (dpi) was increased (*p* < 0.05) by the supplemental 10 mg SI/kg diet. Infected broilers showed the highest (*p* < 0.05) bursa lesions, with an average score of 4.0 ± 0.0, while the severity of bursa lesions was decreased (*p* < 0.05) at 3 dpi and 7 dpi by the supplemental 20 mg SI/kg diet. Supplemental SI at 20 mg/kg decreased (*p* < 0.05) the viral protein 5 (VP5) mRNA expression at 3 dpi and 7 dpi. The level of interferon gamma (IFNγ) was elevated (*p* < 0.05) in the infected group at 3 dpi and 7 dpi as compared with the control group, while its level was decreased *(p* < 0.05) by supplemental 10 mg/kg SI at 3 dpi. The level of nuclear factor κB in the bursal tissue showed the lowest value (*p* < 0.05) with supplemental 10 and 20 mg SI/kg diet at 7 dpi. Supplemental 10, 20, 40 mg/kg SI improved (*p* < 0.05) the serum total antioxidant

activity (T-AOC) in infected broilers at 3 dpi. In addition, the serum level of malondialdehyde (MDA) decreased (*p* < 0.05) in the group fed 20 mg/kg SI at 7 dpi. In conclusion, supplemental 10~20 mg/kg SI may have a positive effect on broiler chickens infected with IBDV, probably because SI decrease the severity of bursa lesions and viral protein 5 mRNA expression, and have strong antioxidant activity.

**Keywords:** broilers; IBDV; soybean isoflavones; immune function; viral protein 5 mRNA expression

#### **1. Introduction**

Infectious bursal disease (IBD), or Gumboro disease, is an acute, highly contagious disease of young chickens caused by the infectious bursal disease virus (IBDV), characterized by inflammation and subsequent atrophy of the bursa of Fabricius, and immune suppression and mortality, generally at 3 to 6 weeks of age [1–4]. It has been shown that IBDV induces suboptimal feed conversion and weight gain [5]. In addition, immune dysfunction decreases the growth performance and increases carcass condemnation rates, but increases the rate of mortality and morbidity due to secondary viral and bacterial infections [6].

Recently, bioactive compounds in feedstuffs or feed additives are considered as an important strategy to boost immunity in modern poultry production [7–10]. Isoflavones are natural molecules available in edible plants, particularly in soybeans, red clover, and kudzu root [11,12]. Isoflavones, as phenolic compounds, are the main phytoestrogens of soybeans [13]. Isoflavones, including genistein, daidzein, and glycitein, are similar in structure to 17-β-estradiol. Soy isoflavones are used as a supplement to improve growth performance, antioxidant activity, and immune function [14–19]. These reasons caused us to hypothesize that supplemental soy isoflavones may improve the performance and immune function of broilers chickens infected with IBDV.

This study was conducted to investigate the ability of isoflavones in the amelioration of oxidative stress and immune function of broilers chickens challenged with IBDV.

#### **2. Materials and Methods**

The experimental protocol was reviewed and approved by the Institute of Animal Science, Guangdong Academy of Agricultural Sciences, China (GAASISA-2015-03).

#### *2.1. Birds, Virus, and Diets*

A total of 200 one-day-old Lingnan yellow-feathered male broilers were obtained from a commercial hatchery Guangdong Wiz Agricultural Science and Technology Co., Guangzhou, China) and raised under standard conditions with free access to water and feed. The strain of IBDV, BC 6/85, is a classic strain of virulent IBDV used as a standard challenge strain in China and was purchased from the China Institute of Veterinary Drug Control (Haidian District, Beijing, China). Nutrient levels of the diets were based on the National Research Council [20] recommended nutrient requirements for broiler chickens (Table 1)


**Table 1.** Ingredient and composition of the basal diets for Chinese yellow-feathered broilers at 1–21 and 22–42 days of age (as fed-basis).

<sup>1</sup> Supplied per kilogram of diet: vitamin A, 14,700 IU; vitamin D3, 3300 IU; vitamin E, 20 IU; vitamin K3, 3.9 mg; vitamin B1, 3 mg; vitamin B2, 9.6 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; nicotinic acid, 60 mg; pantothenic acid, 18 mg; folic acid, 1.5 mg; biotin, 0.36 mg; FeSO4·7H2O, 80 mg; CuSO4·5H2O, 8 mg; MnO, 80 mg; KI, 0.38 mg; and NaSeO3, 0.44 mg. The carrier was zeolite. <sup>2</sup> Values were calculated from data provided by Feed Database in China (2016) except that crude protein was analysed.

#### *2.2. Experimental Design*

On the first day of the experiment, 200 one-day-old yellow-feathered male broiler chickens were weighed and allotted randomly to five treatment groups, each of which included four replicates of 10 birds. Broilers were placed in floor pens (1 × 2 m). The litter thickness was 5 cm of sawdust. All birds were fed the same basal diet, supplemented with 0 (non-infected control group), 0 (infected control), 10, 20, or 40 mg/kg soybean isoflavones (SI) (supplied by Newland Feed Science and Technology Co., Guangdong, China).These treatments are described as non-infected control, IBDV (0 SI), IBDV (10 SI), IBDV (20 SI), and IBDV (40 SI), respectively (Table 2).

**Table 2.** The design of the experimental study.


SI: synthetic soybean isoflavones; IBDV: infectious bursal disease virus.

At 21 days of age, chickens were inoculated with the bursal infectious dose causing 50% morbidity of the IBDV BC 6/85 strain by the eye-drop and nasal route, except for the non-infected control group.

A pre-experiment had been conducted to titrate the optimal dose of the inoculation. By administering the chosen dose, visible pathological changes were visible on the bursa of Fabricius at 5 days post-infection (dpi) without evident mortality. During the experiment, which lasted 42 days, the infected and non-infected groups of chickens were housed in equivalent but separate places.

#### *2.3. Growth Performance*

Broiler chickens were weighed at 1, 21, and 42 days of age. Average daily feed intake was determined on a per pen basis. The feed conversion ratio (FCR) was calculated. Mortality and health status were recorded daily.

#### *2.4. Blood Sampling and Laboratory Analyses*

Eight broilers per treatment group (two birds per replicate) were selected randomly and bled into tubes (5 mL per bird) from a wing vein at 3 and 7 dpi to collect the serum, and then the broilers were slaughtered and bursa of Fabricius was collected and weighed from each broiler. The half of bursa was fixed in 4% buffered formaldehyde. Another half was snap-frozen with liquid nitrogen and stored at −80◦C to analyze viral protein 5 (VP5) mRNA expression.

#### *2.5. Histology of Bursa of Fabricius*

The collected tissues of bursa of Fabricius were fixed with 4% formaldehyde solution for 24 h. Serial sections were cut at 5 μm were dehydrated, cleared, embedded in paraffin, deparaffinized in xylene, rehydrated, and stained with hematoxylin and eosin. Three sections were made for each sample to observe the bursal lesion and to measure the degree of damage of the bursal follicle using light microscope. To observe the bursal lesion and to measure the degree of damage of the bursal follicle, the histopathological changes of the bursa of Fabricius were scored according to the methods of Sharma et al. [21] and Kim et al. [22]: 0 = normal bursa of Fabricius; 1 = 1–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 76–100% of follicles showing cellular depletion.

#### *2.6. Viral Protein 5 (VP5) mRNA Expression*

Total RNAs was extracted from the Bursal homogenates using QiAmp Viral RNA Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Gene-specific primers for viral protein 5 (VP5) and the endogenous reference gene (β-actin) are shown in Table 3. Briefly, the 50-μL reaction mixture contained 10 μL of extracted RNA, 10 μL of 5· RT-PCR buffer, 2 μL primer F, 2 μL primer R, 2 μL dNTP mix containing 400 μM each of dATP, dGTP, dCTP, and dTTP, and 2 μL of Qiagen One Step Enzyme Mix. A fragment of 94 bp of the 5 noncoding region was amplified using a PCR reaction with the SYBR Premix PCR kit (Takara, Dalian, China). The PCR program was 95◦C for 10 min followed by 40 cycles of 95◦C for 15 s and 60 ◦C for 60 s. The standard curve was generated using pooled samples and efficiency was calculated from standard curves. Each sample was run in duplicate and a no-template control was included. Specificity of the amplification was verified via melting curve analysis and the specificity of the product was confirmed by electrophoresis on a 1.2% agarose gel, with purification using a DNA purification kit (Takara), and sequencing (Shanghai Sangon Biotech Co. Ltd., Shanghai, China). The difference of the cycle threshold (Ct) value for the 18s rRNA was less than 0.5 across all treatments, and therefore was considered to be an appropriate endogenous control. Average gene expression relative to the endogenous control for each sample was calculated using the 2−ΔΔ*C*<sup>t</sup> method [23]. The calibrator for each studied gene was the average <sup>Δ</sup>*C*t value of the control group.


**Table 3.** Sequences of primers for the real-time PCR.

<sup>1</sup> viral protein 5(encodes a 17-kDa non structural polypeptide).

#### *2.7. Determination of Antioxidant Capacity in the Serum*

Serum total antioxidant activity (T-AOC) and malonaldehyde (MDA) were measured spectrophotometrically. A total antioxidant capacity assay kit (A015–1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used according manufacturer's instructions and was expressed in U/mL. The MDA levels were assayed using the thiobarbituric acid method [24], reading the absorbance at 532 nm with the spectrometer.

#### *2.8. Determination of Bursal Immunologic Indices*

Bursa of Fabricius samples were thawed at 4◦C and homogenized in 10 volumes of cold normal saline. The homogenates were then centrifuged at 20,000× *g* for 20 min at 4 ◦C and the supernatant was collected for analyses. Diluent solution and standard samples were added at 100 μL per well in duplicate wells. The plate was incubated for 2 h at 37 ◦C, followed by three washings with wash solution. The immunological indicators of interleukin-2 (IL-2), interleukin-6 (IL-6), interferon gamma) IFNγ), and nuclear factor κB were (NF-κB) determined by ELISA kits. The kits were purchased from Shanghai Jianglai Biotechnology Co., Ltd. (Shanghai, China), and the specific operation was carried out according to the instructions.

#### *2.9. Statistical Analyses*

The replicate was the experimental unit. The effects of SI supplementation levels were examined by one-way ANOVA using the general linear model GLM procedures of SAS software (v9.2, SAS Institute, Cary, NC, USA). In the absence of SI, IBDV-infected and non-infected controls were compared by *t*-tests. Significance was declared at *p* < 0.05. All data are expressed as means ± SE.

#### **3. Results and Discussions**

Some of the main strategies during stress periods such as viral infections are to boost the immune function, maximize antioxidant ability, and minimize lipid peroxidation. Therefore, this study was conducted to investigate the ability of isoflavones in the amelioration of oxidative stress and in the immune function of broilers chickens challenged with IBDV.

#### *3.1. Bursa of Fabricius*

The effect of SI supplementation on bursa of Fabricius weight and index of broilers challenged with infectious bursal disease virus is shown in Table 4. In addition, the bursa lesion score is shown in Table 5.


**Table 4.** Effect of adding soy isoflavones on bursa development of IBDV-challenged broilers 1.

<sup>1</sup> Data are means of eight broilers chickens per treatment (two broilers/replicate). Capital letters indicate statistically significant (*p* < 0.05) differences between control group and IBDV group by Student's *t*-test; small letters indicate statistically significant (*p* < 0.05) differences between IBDV (0 SI), IBDV (10 SI), IBDV (20 SI) and IBDV (40 SI).


**Table 5.** Effect of adding soy isoflavones on bursa lesion score of IBDV-challenged broilers 1.

<sup>1</sup> Data are means of eight broilers chickens per treatment (two broilers/replicate). Capital letters indicate statistically significant (*p* < 0.05) differences between the control group and IBDV group by Student's *t*-test; small letters indicate statistically significant (*p* < 0.05) differences between IBDV (0 SI), IBDV (10 SI), IBDV (20 SI) and IBDV (40 SI).Note: Histopathological score of bursa of Fabricius: 0 = normal bursa of Fabricius; 1 = 1–25%; 2 = 26–50%;3 = 51–75%; and 4 = 76–100% of follicles showing cellular depletion.

The weight (g) and the index of bursa of Fabricius (%) were reduced significantly *(p* < 0.05) in broiler chicks infected with IBDV as compared with those of the control group (non-infected) at 3 dpi and 7 dpi. However, supplemental 10 mg of SI increased the bursa weight significantly *(p* < 0.05) at 7 dpi. Our finding is in agreement with the findings of Li et al. [25]. They reported that a significant decrease in the bursa to body weight ratios (B/BW) had appeared at 7 dpi. Bursa lesions in infected broiler had an average score of 4.0 ± 0.0 compared with non-infected control (*p* < 0.05). However, the severity of bursa lesions was decreased (*p* < 0.05) at 3 dpi and 7 dpi by the supplemental 20 mg SI/kg diet. This is in agreement with the result of Li et al. [25]. They found that bursa lesion score was 4.0 ± 0.0 at 3 dpi in IBDV-infected group.

In terms of bursal damage, the control group (non-infected) had no signs of bursal damage (Figure 1), while all infected broilers had bursal damage at 3 dpi and 7 dpi (Figures 2 and 3). This finding is in agreement with that of Li et al. [25], who demonstrated a depletion of lymphoid cells in bursal follicles was observed microscopically. In the present study, infected broilers fed a 20 mg SI/kg diet had the lowest amount of bursal damage. As shown in Figure 3, the architecture is almost clear between the follicles, the lining epithelium was less corrugated, there was less necrosis and heterophil invasion, and fewer fibrous tissues were observed. It has been shown that IBDV infection induced a temporary or permanent destruction of the bursa of Fabricius and other lymphoid organs [26,27]. Destruction of B cells contributes to IBDV-induced immune suppression [28].

**Figure 1.** Normal bursal tissue section stained with hematoxylin and eosin (H&E) in the non-infected group.

**Figure 2.** Bursal tissue section stained with hematoxylin and eosin (H&E) of each group at 3 dpi (200×). (**A**) is from the IBDV (0 SI); (**B**) is from the IBDV (10 SI); (**C**) is from the IBDV (20 SI); and (**D**) is from the IBDV (40 SI).

**Figure 3.** Bursal tissue section stained with hematoxylin and eosin (H&E) of each infected group at 7 dpi (200×). (**A**) is from the IBDV (0 SI); (**B**) is from the IBDV (10 SI); (**C**) is from the IBDV (20 SI); and (**D**) is from the IBDV (40 SI). (1) Bursa microscopically revealed complete loss of architecture. (2) Bursa with infectious bursitis presenting necrosis and heterophil invasion. (3) The lining epithelium was highly corrugated. (4) There was no intact lymphoid follicle. (5) The entire area was filled up by fibrous tissue. The IBDV+20 SI group was the best among the infected groups (the architecture is almost clear between follicles, the lining epithelium was less corrugated, and there was less necrosis, heterophil invasion, and fibrous tissue).

#### *3.2. Viral Protein 5 (VP5) mRNA Expression*

Viral protein 5 (VP5) expression was higher (*p* < 0.05) in broilers infected with IBDV as compared with those of the control group at 3 dpi and 7 dpi (Table 6). However, supplemental 20 mg/kg of SI reduced VP5expression at 3 dpi and 7 dpi. It has been indicated that RT-PCR was a sensitive test to detect the IBDV [2,29,30]. According to our knowledge, no previous study investigated the effects of SI on VP5. VP5 is one protein employed by IBDV to induce the programmed cell death process [31]. It has been suggested that VP5 might play an important role as anti-apoptotic protein at an early stage of IBDV infection [32]. In addition, Qin and Zheng [33] suggested that VP5 as an anti-apoptotic protein is an important factor to support viral replication at the early stage of IBDV infection. The anti-apoptotic activity of VP5 was noticed at 8 or 12 h post-infection [34], while VP5 induced apoptosis were found after 24 h post-infection [35].

**Table 6.** Effect of adding soy isoflavones on the IBDV mRNA expression of bursal tissue in IBDV—challenged broilers 1.


<sup>1</sup> Data are means of eight broilers chickens per treatment (two broilers/replicate). Capital letters indicate statistically significant (*p* < 0.05) differences between control group and IBDV group by Student's *t*-test; small letters indicate statistically significant (*p* < 0.05) differences between IBDV (0 SI), IBDV (10 SI), IBDV (20 SI), and IBDV (40 SI).

#### *3.3. Immune Function*

Effect of adding soy isoflavones on immunity of IBDV-challenged broilers is presented in Table 7.


**Table 7.** Effect of adding soy isoflavones on bursal immune response of IBDV-challenged broilers 1.

<sup>1</sup> Data are the means of eight broilers chickens per treatment (two broilers/replicate). IL-2; interleukin 2; IL-6: interleukin 6; IFNγ: interferon gamma; NF-κB: nuclear factorκB. Capital letters indicate statistically significant (*p* < 0.05) differences between the control group and IBDV group by Student's *t*-test; small letters indicate statistically significant (*p* < 0.05) differences between IBDV (0 SI), IBDV (10 SI), IBDV (20 SI) and IBDV (40 SI).

The bursal concentration of interleukin 2 (IL-2) was lower *(p* < 0.05) in challenged broilers, while supplemental 20 mg of SI increased its level *(p* < 0.05) at 3 dpi, which is considered an acute stage of IBDV infection. IL-2 is a cytokine secreted by activated T lymphocytes, which has an important role in regulation of host response to pathogenic challenge [36]. In addition, the bursal concentration of interleukin 6(IL-6) was lower (*p* < 0.05) in the infected group, while supplemental 10 mg of SI increased its level (*p* < 0.05) at 3 dpi.

Long et al. [37] reported that IBDV infection increased IFN-γ mRNA relative expression in the bursa of Fabricius. Interferon-γ is one of the proinflammatory Th1 cytokines [38]. The level of interferon gamma (IFNγ) in the bursa of Fabricius was elevated (*p* < 0.05) in infected broilers with IBDV at 3 dpi and 7 dpi as compared with the control group, while its level was decreased (*p* < 0.05) by supplemental 10 mg/kg SI at 3 dpi. In the current study, the effect of dietary SI in immune function was significant during early stages of infection, and it was obviously significant at 10 and 20 mg SI/kg diet. It is well known that infectious bursal disease (IBD) disease peaks between 2 to 5 day post infection and is practically cleared by day 7 [39].

The bursal level of nuclear factorκB (NF-κB) was higher in infected broilers with IBDV at 7 dpi, while its level was decreased (*p* < 0.05) by supplemental 10 and 20 mg SI at 3 dpi and 7 dpi (Table 8). It has been shown that SI are associated with cell survival, cell cycle, inflammation, and apoptosis, and they suppress nuclear factor (NF)-κB and other signaling pathways [40]. In addition, it has been reported that NF-κB activity was blunted more efficiently by genistein, probably due to its additional antioxidant effect [41]. In the present study, supplemental 10, 20, and 40 mg/kg SI significantly (*p* < 0.05) improved the serum T-AOC in infected broilers at 3 dpi (Table 8). In addition, the serum level of MDA was decreased (*p* < 0.05) in the group fed 20 mg/kg SI at 7 dpi.


**Table 8.** Effect of adding soy isoflavones on serum antioxidant index of IBDV-challenged broilers 1.

<sup>1</sup> Data are means of eight broilers chickens per treatment (two broilers/replicate). <sup>2</sup> Total antioxidant activity (T-AOC), malonaldehyde (MDA). Capital letters indicate statistically significant (*p* < 0.05) differences between control group and IBDV group by Student's *t*-test; small letters indicate statistically significant (*p* < 0.05) differences between IBDV (0 SI), IBDV (10 SI), IBDV (20 SI), and IBDV (40 SI).

Huang et al. [19] reported that SI improved the immune function in young piglets fed oxidized fish oil. Moreover, Lv et al. [42] reported that genistein (GEN boosted the anti-viral capacity of broilers chickens. They reported that the Newcastle disease (ND) and IBD antibody titers in the GEN group were higher (*p* < 0.05) than broilers in the control group.

#### *3.4. Antioxidant Capacity and Oxidative Stress*

In the present experiment, supplemental 10, 20, and 40 mg/kg SI significantly (*p* < 0.05) improved the serum T-AOC in infected broilers at 3 dpi (Table 8). It has been reported that SI have antioxidant properties via detoxifying free radical species and up-regulating antioxidant genes [40].The serum level of MDA was decreased(*p* < 0.05) in the group fed 20 mg/kg SI at 7 dpi. The level of MDA can be a marker of the level of lipid peroxidation endogenously, which is the result of diminished antioxidant protection as levels of reactive oxygen species and antioxidants ROS increase or there is weak antioxidant activity.

#### *3.5. Growth Performance*

Infected broilers with IBDV had decreased (*p* < 0.05) average daily gain (ADG) and average daily feed intake (ADFI) from 22 to 42 days. However, infected broilers fed 10 and 20 mg/kg had the maximum *(p* < 0.05) ADG and ADFI from 1 to 42 days. In addition, body weight (BW) was increased (*p* < 0.05) in infected broilers fed 10 and 20 mg/kg (Table 9).


**Table 9.** Effect of adding soy isoflavones on growth performance of IBDV-challenged broilers 1.

<sup>1</sup> Data are means of four replications per treatments, with 10 broilers per replicate. Capital letters indicate statistically significant (*p* < 0.05) differences between the control group and IBDV group by Student's *t*-test; small letters indicate statistically significant (*p* < 0.05) differences between IBDV (0 SI), IBDV (10 SI), IBDV (20 SI), and IBDV (40 SI). ADFI: average daily feed intake; FCR: feed conversion ratio.

It has been reported that IBDV decreased weight gain and feed efficiency [5]. Recently, Wang and Wu [43] reported that SI alleviated the growth suppression induced by dextran sulfate sodium in mice. In addition, Greiner et al. [44,45] reported that soybean genistein (200 mg/kg) and daidzein (200 or 400 mg/kg) could improve growth in virally challenged pigs. There is a positive effect of SI on infected broiler chickens with IBDV, probably because SI decrease the severity of bursa lesions and viral protein 5 mRNA expression, and have strong antioxidant activity.

In the present study, no broilers died due to IBDV infection. These findings are in agreement with other studies [46,47].

#### **4. Conclusions**

Supplemental 10~20 mg/kg SI may have a positive effect on broiler chickens infected with IBDV, probably because SI decrease the severity of bursa lesions andviral protein 5 mRNA expression, and have strong antioxidant activity.

**Author Contributions:** Data curation, M.M.A., J.-l.C., X.-j.L., Z.-y.G., Q.-l.F., and L.L.; Formal analysis, J.-l.C. and X.-j.L.; Investigation, M.M.A., J.-l.C., Q.-l.F., and Y.-b.W.; Methodology, X.-j.L., Y.-b.W., and L.L.; Project administration, S.-q.J. and Z.-y.G.; Supervision, S.-q.J.; Writing—original draft, M.M.A.; Writing—review and editing, S.-q.J. and Z.-y.J.

**Funding:** This study was supported by National Key R&D Project (2018 YFD0500600), National Natural Science Foundation of China (31802104), the Natural Science of Guangdong Province (2017A030310096), the "Twelve-Five" National Science and Technology Support Program (2014BAD13B02), Earmarked Fund for Modern Agro-industry Technology Research System (CARS-41) from the Ministry of Agriculture, Scientific and Technological Project (2017B020202003) from the Department of Science and Technology of Guangdong Province, and Grant No. 201804020091 from Guangzhou Science Technology and Innovation Commission, Presidential Foundation of the Guangdong Academy of Agricultural Sciences (201620, 201805, 201807B, 201809B and 201908), PR China.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **E**ff**ects of Phytase Supplementation to Diets with or without Zinc Addition on Growth Performance and Zinc Utilization of White Pekin Ducks**

#### **Youssef A. Attia 1,3, Nicola F. Addeo 2, Abd Al-Hamid E. Abd Al-Hamid <sup>3</sup> and Fulvia Bovera 2,\***


Received: 28 March 2019; Accepted: 21 May 2019; Published: 25 May 2019

**Simple Summary:** The environment sustainability of farms is extremely important for the future of the world. In this context, the lowering of the pollution from intensive poultry farms is necessary. Due to its low levels and low digestibility in feeds, Zn is often overdosed in poultry feed, and its excess in the excreta can accumulate in the soil, inhibiting the growth of soil microorganism as well as altering their morphology and metabolism, thus reducing the crop yield and quality. Enzymes, such as phytase, can breakdown the linkage of Zn with phytic acid in vegetable feeds, thus increase the Zn availability for animal digestion. In this way, very low supplementation of Zn to the diets can meet the requirement of poultry.

**Abstract:** The effect of phytase and inorganic Zn supplementation was studied in 180 male White Pekin ducks (WPD) from 1 to 56 days of age. The birds were divided into four groups fed the same basal diet (containing 26 ppm of Zn from raw materials): the control group did not receive Zn supplementation; the second group was supplemented with 30 ppm of Zn oxide; and the third and fourth groups were fed the control and the 30 ppm diets, respectively, both supplemented with 500 U of *E. coli* phytase. Each group contained five replicates of nine ducks. The body weight and feed intake were recorded at 1, 28 and 56 days of age. At 56 days of age, five birds/group were used to measure feed digestibility and five other birds/group were slaughtered. Zn at 30 ppm increased the body weight gain (BWG, *p* < 0.01) and feed intake (*p* < 0.05) and improved the feed conversion (FCR, *p* < 0.05) of the growing ducks. The Zn retention and Zn level in the excreta increased (*p* < 0.01) due to Zn supplementation. The addition of phytase improved BWG (*p* < 0.01) and FCR (*p* < 0.05) of growing ducks. The use of phytase reduced (*p* < 0.01) the level of Zn in duck excreta. Phytase supplementation to the basal diet at 30 ppm seems to be adequate to meet Zn requirements for ducks without further Zn additions.

**Keywords:** ducks; zinc oxide; phytase; growth performance; zinc utilization

#### **1. Introduction**

Zinc (Zn) is an essential trace mineral with several roles in animal metabolism, acting as a functional component of more than 200 enzymes [1,2]. In the NRC guidelines [3], Zn requirements for ducks are not provided; therefore, the dietary requirement of Zn for ducks is based on those for other bird species.

In poultry nutrition, Zn is required for eggshell deposition [4]; inadequate amount of Zn negatively affects the feed intake, growth rate and feed conversion ratio of broilers [5]. In addition, abnormalities in the immune responses, as well as reproduction, skeletal and skin disorders can be tied to the deficiency of Zn in poultry diets [6].

In general, the level of Zn in feedstuffs is low [3] and in vegetable products this element is also poorly available for digestion because its chelation to the phytic acid [7]. Thus, the addition of Zn to poultry diets is a common practice. A comparison between NRC [3] recommendation and modern commercial strains of broilers suggests that industries often use a big safety margin of Zn in feed formulation [5], also considering that amount of Zn up to 2000 mg/kg in poultry diets does not negatively affect the bird performance [8]. However, high amount of Zn in the diets is responsible of the high excretion of this trace element into the environment [9] as fecal Zn content linearly increases with Zn dietary levels [10]. Thus, the European Commission has recently established a maximum limit for the total Zn content, including the supplemental premix, of poultry diets at 100 ppm [11]. Therefore, the knowledge of the specific Zn requirements can reduce its supplementation in poultry diets, without affecting animal health, welfare and productivity [2].

A possible solution could be the use of enzymes associated with vegetables. Phytase is a useful additive that improves the nutritive value of feedstuffs rich in phytic acid and also reduces environmental pollution related to nitrogen, and several metals (Cu, Zn, Fe, and Mg) by improving their availability to the animal and decreasing their excretion into the environment [12–16]. Yu et al. [17] indicated that phytate reduces the Zn absorption in the broiler intestinal tract; therefore, it can be hypothesized that adding the phytase to the diets, the amount of Zn available for poultry digestion can be increased.

The objective of this study was to investigate the effects of phytase supplementation to diets with or without Zn addition on productive performance and physiological traits of growing ducks. The addition of phytase to the diet with Zn oxide aimed to verify if only the supplementation of inorganic Zn is enough to sustain animal performance or if more Zn can provide further improvements.

#### **2. Materials and Methods**

#### *2.1. Experimental Design, Birds, Diets, and Husbandry*

All procedures were approved by the Animal and Poultry Production Department, Faculty of Agriculture, Damanhour University (Egypt) that recommends animal rights and welfare.

One hundred eighty one-day-old male White Pekin ducks (*Anas platyrhynchos* domestica, WPD) were homogeneously distributed into four groups fed the same starter and finisher diets (basal diets). The groups were subjected to four dietary treatments as follow: the first group (control) was fed basal diets unsupplemented with Zn oxide or phytase; the second group was fed the basal diets supplemented with 30 ppm of Zn oxide (72% Zn); the third group was fed the basal diets supplemented with 500 U of *E. coli* phytase (*E. coli* 6-phytase, 500 U/kg diet; Danisco Animal Nutrition, England); and the fourth group was fed the basal diets supplemented with 30 ppm of Zn oxide and 500 U of *E. coli* phytase. The basal (starter and finisher) diets were obtained by using a Zn-free trace mineral mixture and contained 26 ppm of Zn from raw materials without Zn supplementation, as measured by Atomic Absorption Spectrometry analysis. The starter diet (fed from 1 to 35 days of age) and the finisher diet (36–56 days of age) were formulated according to NRC [3] recommendations and their ingredients and chemical-nutritional characteristics [18] are reported in Table 1.


**Table 1.** Ingredients and chemical-nutritional characteristics of the basal diets fed to White Pekin ducks during the starter (1–35 days of age) and the finisher (36–56 days) periods.

\* Vit + Min Premix provides the following (per kg of diet): Vitamin A, 1800 mg retinol; Vitamin E, 6.67 mg d-alpha-tocopherol; menadione, 2.5 mg; Vit D3, 50 mcg cholecalciferol; riboflavin, 2.5 mg; Ca pantothenate, 10 mg; nicotinic acid, 12 mg; choline chloride, 300 mg; vitamin B12, 4 mcg; vitamin B6, 5 mg; thiamine, 3 mg; folic acid, 0.50 mg; biotin 0.2 mg; Mn, 80 mg; Fe, 40 mg; Cu, 4 mg; Se, 0.10 mg.

Each diet was fed to five replicates consisting of nine male WPD each. Each replicate was housed in floor pens (1.0 m × 2 m) furnished with wood shavings. The brooding temperature was 34, 32, 30 and 28 ◦C during Weeks 1, 2, 3 and 4, respectively, and thereafter the temperature inside the house was about 27 ◦C. The light program provided 24 h of light on the first day; and then the lighting was gradually reduced to 10 h/day at 21 days of age. The light was supplied continuously. Water and mash form of feed were offered ad libitum.

#### *2.2. Data Collection*

The ducks were individually weighed at 1, 28, and 56 days of age in the morning, before offering feed. The remaining, scattered and consumed feed were measured during the periods 1–28, 28–56 and 1–56 days for each replicate; thus, the average feed intake per bird was calculated as the ratio between feed intake and the number of ducks per replicate. The feed conversion ratio (FCR) was calculated as units of feed intake required to produce one unit of gain in live body weight in the periods 1–28, 28–56 and 1–56 days. The mortality rate was recorded along the entire experimental period. At the end of the trial (56 days of age), five birds per treatment were randomly chosen, weighed after being fasted overnight, and slaughtered according to the Islamic guidelines. Feathers were plucked, the inedible parts (head, feet, and inedible viscera) were removed and the remaining (dressed) carcass was weighed. The feathers, liver, spleen, gizzard, heart, pancreas, and abdominal fat were separated and individually weighed. The percentage carcass yield and the percentages of internal organ weights relative to live body weight were calculated. A 50/50 (*w*/*w*) sample of skinless breast and thigh meat

was weighed and kept in an electric drying oven at 70 ◦C until a constant weight was reached. The dried flesh was finely ground through a suitable mixer, passed through a sieve (1 mm2), and then carefully mixed and stored in tightly sealed glass containers for subsequent analysis. The physical characteristics of a sample mixture of breast and thigh meats were evaluated. The ability of meat to hold water (WHC) and meat tenderness were measured according to the methods of Volvoinskaia and Kelman [19]. The pH was measured as described by Aitken et al. [20]. The color intensity (optical density) of meat was determined according to the method of Husani et al. [21].

At 56 days of age, five ducks per group were housed in individual cages and used to evaluate the nutrient digestibility of the experimental diets. The birds were housed in individual cages. The methodology involved a four-day adaptation period followed by a three-days excreta collection period. After each day of collection, the excreta samples were dried to come to equilibrium with the atmosphere, weighed, ground and, finally, mixed together and stored in screw-top glass jars until analysis. The proximate chemical composition of the feed and excreta was according to the official methods of Association of Official Analytical Chemists (AOAC) [18].

The Zinc content was determined after ashing of the samples with 10 mL of concentrated sulfuric acid. Three drops of bichloric acid were added and the samples were incubated at room temperature for 2 h. Zinc concentration in the diets, liver, bones, excreta, and plasma were determined by atomic absorption spectroscopy (GBC Avanta Z, GBC Scientific Equipment, Braeside, Australia) using a standard curve. The apparent retention of Zn was calculated by dividing the difference between the amount consumed and that excreted by the amount consumed.

Blood samples were collected from wing vein from five ducks per treatment and placed into heparinized tubes. The plasma was separated by centrifugation at 1500× *g* for 15 min and stored at −18 ◦C until analysis. The plasma levels of Zn and Cu were determined by atomic absorption spectrometry after processing the samples as previously described.

#### *2.3. Statistical Analysis*

The data were analyzed using a two-way ANOVA of the General Linear Model (GLM) procedure of SAS [22] in which Zn and phytase supplementations were the main effects. The potential interactions between the effects were also evaluated. A probability of less or equal to 0.05 was considered significant, based on the Student Newman–Keuls Test of mean differences among treatments [22]. The data are reported based on the main effects and significant interactions. The differences among mortality rate were analyzed by chi-square test.

#### **3. Results**

The grower and the finisher basal diets used in the trial contained 26 ppm of Zn from raw materials (Table 1) as determined by atomic absorption spectrometry. The data on in vivo performance are reported in Table 2.

The mortality rate was not statistically different among the experimental groups. The addition of 30 ppm of Zn to the basal diets increased the body weight gain (*p* < 0.01) and feed intake (*p* < 0.05), and improved the FCR (*p* < 0.05) of ducks considering the entire period of the trial. The supplementation of phytase also improved (*p* < 0.01) BWG and FCR from 1 to 56 days, but the feed intake was not different from the control group. Except for the feed intake, the interaction between the two tested factors was significant: when no Zn was included in the diet, the addition of phytase improved both FCR and BWG; however, when 30 ppm of Zn were added to the basal diet, the addition of phytase did not improve the duck performance.

The addition of Zn to the diets reduced (*p* < 0.01) Zn retention (Table 3) and increased the level of Zn in tibia (*p* <0.01), liver (*p* < 0.05) and excreta (*p* < 0.01).



\**n* = 180 bird as 45 bird per treatment group for body weight gain and *n* = 20 replicates as five replicates per each treatment for feed intake and feed conversion ratio. RMSE, Root mean square error. a–d means with different superscripts in the same column in similar treatment group are significantly different; NS, not significant.

 \*.


**Table3.**Effectofzincsupplementation,withandwithoutphytaseaddition,onZnretentionandtissueandexcrementconcentrations,andplasmaZnand

48

different; NS, not significant.

In addition, the level of Zn and Cu in the plasma increased (*p* < 0.01) due to Zn inclusion in the basal diets. The addition of phytase increased the level of Zn in tibia (*p* < 0.05) and liver (*p* < 0.01) as well as the concentration of Zn and Cu in plasma (*p* < 0.01) but decreased the Zn content in the excreta (*p* < 0.01). The interaction between Zn level and phytase was significant for tibia ash, plasma Zn and plasma Cu. The use of phytase significantly decreased the tibia ash when 30 ppm of Zn oxide were added to the diets, but it did not happen for the Zn-free diet. The addition of phytase to the basal diet increased Zn and Cu concentration by 13.4% and 34.4%, respectively, while the addition of phytase to 30 ppm Zn diets increased the Zn and Cu plasma levels by 9.4% and 29.7%, respectively.

The addition of Zn to the basal diet decreased (*p* < 0.01) the percentage of gizzard but the other carcass traits were unaffected (Table 4). The use of phytase decreased the percentage of liver (*p* < 0.01) and abdominal fat (*p* < 0.01). There was a significant interaction between Zn level and phytase supplementation on gizzard percentage. Results indicate that phytase increased gizzard percentage of ducks fed 30 ppm Zn diet but had no effect when added to the basal diets.


\* *n* = 20 samples as five sample per each treatment; RMSE, Root mean square error. means with different superscripts in the same column in similar treatment groups aredifferent; NS, not significant.

#### **4. Discussion**

The natural presence of Zn in the diets from the raw materials is not enough alone to adequately sustain the duck growth. In our trial, the addition of 30 ppm of Zn oxide to the basal diets improved the animal performance: the increase of feed intake was responsible for the increased body weight gain, giving a more favorable FCR. Cufadar and Bahtiyarca [23] indicated that 30 ppm of Zn was adequate for growth performance of male WPD. The live weight of the ducks at the end of the trial (56 days) was lower than the data recorded in the literature. However, as reported by Dodu [24], the imported breed of ducks, along the years, was mixed with local populations giving genetic lines differing for some growing characteristics from the original breed. In particular, Dodu [24] indicated that the body weight at 56 days of some Pekin ducks bred in Romania was around 2 kg. In a previous study, Attia et al. [25] found similar body weight for 56-day-old Pekin ducks breed in Egypt.

The supplementation of Zn to the basal diet strongly increased its amount in the excreta, also due to the lowering of the retention rate. In fact, the primary mechanism of trace minerals homeostasis is the modification of the trace minerals absorption and excretion in the gut [25–27]. Cao et al. [28] observed that bone and fecal Zn contents were significantly increased when the diets of chickens were supplemented with organic and inorganic sources of Zn. The significant increase of gizzard percentage due to the addition of Zn in the present trial could be justified by the increased feed intake which could play a physical effect on gizzard expansion.

The improved BWG and FCR in ducks supplemented with phytase diets were not due to an increase of feed intake: in fact, the percentage of gizzard was also unchanged between the groups. The positive effect of phytase on growth performance of WPD could be attributed to the increase in the availability of others inorganic and organic nutrients [14,29–31]. The positive effect of Zn on BWG was probably due to an improved activity of the copper-requiring metalloenzymes, such as ceruloplasmin, cupro-zinc superoxide dismutase and cytochrome c oxidase, which have a very important role as anti-oxidants in the metabolism [32]. In addition, looking at the interaction effect, the use of the basal diet without Zn supplementation induced lower growth rate than that with an addition of 500 U of phytase or 30 ppm of Zn. The increased growth rate due to phytase or Zn supplementation resulted in an improved FCR. The phytase improved Zn utilization, as evidenced by the increase of Zn in plasma and its decrease in the excreta, but the effect on Zn retention was weak. In the literature, the effect of phytase on Zn availability is contradictory: phytase is reported to increase the availability of dietary Zn [4,23] as well as the bone Zn content in pigs and chicks [33], but to have no significant effect on Zn digestibility and apparent absorption percentage of Zn, Fe, or Cu in chicks [34]. These differences could be ascribed to the differences in the metabolism among the species, the different dietary composition or Zn level in the basal diet. Dietary Zn at 800 ppm negatively affected phytate breakdown by phytase [35] as a result of a conformational change in the phytate moiety, thereby making it less accessible to phytase.

The effect of phytase on plasma Zn content was stronger in WPD fed the basal diet than in those fed diets supplemented with 30 ppm of Zn oxide (13.3% vs. 9.4%). These results are consistent with those reported by Mohana and Nys [36]. In addition, the value for Zn retention found herein agrees with those reported by other authors [10,36]. Similar to the present findings, Jondreville et al. [15] found that 100 FTU of phytase were equal to 1 ppm of Zn, and that the Zn excretion could be reduced by 10% if a corn–soybean diet were supplemented with 500 FTU phytase/kg diet.

In the present study, the phytase significantly increased plasma content of Cu, according to Attia et al. [37,38]. Revy et al. [30] reported a positive effect of phytase on Cu availability due to the effect of phytase on phytate-mineral complex formation. However, Jondreville et al. [15] reported that microbial phytase had a negative effect on the liver Cu content in chicks and pigs, likely because of the negative effect of Zn on Cu availability due to release of Zn by phytase [39].

The lower percentage of the abdominal fat and liver in WPD fed diets with phytase may be attributed to the reapportioning of nutrients towards growth rather than fat accumulation. Similar results were reported by Attia et al. [12,29]. Furthermore, Cufadar and Bahtiyarca [23] reported that increasing dietary phytase at three dietary Zn levels increased the results for all carcass parameters, although the effects were not proportional to the level of dietary phytase; rather, phytase prevented the deleterious effects of dietary Zn on carcass traits. This might explain the positive effect of phytase on the growth and the decrease of the abdominal fat of WPD in the present study. Orban et al. [40], Attia et al. [12,29] and Cufadar and Bahtiyarca [23] found that phytase significantly increased the carcass weight, neck, thigh, back + breast and wings of broilers.

#### **5. Conclusions**

The natural presence of Zn in raw materials is not enough alone to satisfy the Zn requirements of the growing ducks. The addition of 30 ppm of Zn or 500 U of phytase to the basal diet increased the growth rate and improved the FCR of the ducks. However, the addition of 30 ppm of Zn oxide also increased the level of Zn in the excreta, while the addition of 500 U of phytase had an opposite effect and is more appropriate to reduce the potential risks for environmental pollution.

**Author Contributions:** Conceptualization, Y.A.A.; Data curation, A.A.-H.E.A.A.-H. and F.B.; Investigation, Y.A.A.; Supervision, Y.A.A.; Writing—original draft, A.A.-H.E.A.A.-H.; Writing—review & editing, N.F.A. and F.B.

**Funding:** This article was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah. The authors, therefore, acknowledge with thanks the Deanship of Scientific Research for technical and financial support.

**Acknowledgments:** The authors thank the late Hassan Saber Zeweil, Department of Animal and Fish Production, Faculty of Agriculture (Saba Basha), Alexandria University, and late Mohamed Drawish Sahledom, Secondary School of Agriculture, Damanhour, Ministry of Education, for their help and support during data collection and Lab analyses.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Assessment of Residual Feed Intake and Its Relevant Measurements in Two Varieties of Japanese Quails (***Coturnixcoturnix japonica***) under High Environmental Temperature**

**Moataz M. Fathi 1,2,\*, Ibrahim Al-Homidan 1, Tarek A. Ebeid 1,3, Ahmed Galal <sup>2</sup> and Osama K. Abou-Emera 1,4**


Received: 21 April 2019; Accepted: 22 May 2019; Published: 30 May 2019

**Simple Summary:** Residual feed intake (RFI) is an important factor in improving poultry production and laying performance, particularly for poultry raised under heat stress. An experiment was conducted to assess RFI and its related measurements in Japanese quails (*Coturnixcoturnix japonica*) of two varieties (gray and white) reared under high environmental temperatures.The current results confirmed that raising gray quails for egg production under high environmental temperature is recommended. Multiple regression analysis clearly identified a significant effect of metabolic body weight and egg mass for the computation of expected feed intake rather than body weight gain in both varieties of Japanese quails.

**Abstract:** Three hundred and ten 12-week-old laying Japanese quails (*Coturnixcoturnix japonica*) from gray and white varieties (155 each) were randomly selected from the initial population and kept in individual battery cages. The measurements of growth and egg production were determined to derive residual feed intake (RFI). The relationship between RFI and egg quality, blood parameters, and carcass characteristics was also determined. The results indicated that the gray quails had significantly higher egg mass and lower broken eggs compared to the white quails. A significant increase of eggshell strength and shell percentage was found in eggs produced from gray quails compared to their white counterparts, although their shell thickness means weresimilar. The results of multiple regression analysis clearly identified a significant effect of metabolic body weight and egg mass for the computation of expected feed intake, rather than body weight gain, in both varieties of Japanese quails. A strong positive correlation between RFI and feed intake in both gray and white quail varieties was found. The same trend was also observed for feed conversion ratio (FCR). Therefore, including RFI in the selection criteria of Japanese quails in order to improve FCR under high environmental temperature is highly recommended.

**Keywords:** residual feed consumption; quail; high environmental temperature

#### **1. Introduction**

Feed expenses represent almost 70% of the gross cost of poultry production. Lowering costs of maintenance processes would leave more energy remaining for higher output. Minimizing residual feed intake (RFI) and, in turn, improving feed efficiency would be beneficial for more efficient quail hens, particularly under high environmental temperatures. However, a bird's ability to convert consumed feed to produce eggs and/or meat is greatly influenced by genotype and environmental factors. Birds that require less feed than expected for maintenance and production requirements have a negative RFI and are desirable in poultry breeding programs to reduce feed costs. RFI has increasingly become a critical factor for measuring feed efficiency and is commonly considered one of the target traits in animal breeding programs [1]. However, many selection programs take RFI into consideration to improve economic productive traits of synthetic or commercial strains. It has already been reported that RFI could be used in selection programs in laying hens and quails. Altan et al. [2] indicated that the selection for RFI in Japanese quails (*Coturnixcoturnix japonica*) might provide a tool to improve the efficiency of feed utilization without significant negative changes in egg production and egg quality traits and with a decreased susceptibility of the laying hens to stress. Most researchers concluded that a four-week recording period provides sufficient information for the genetic evaluation of residual feed intake in many species of poultry [3–7].

Direct selection for more efficient birds is becoming one of the primary goals in breeding programs of laying hens [7]. Improving feed efficiency is of great economic concern for commercial egg producers to maximize project outcomes. Traditionally, feed efficiency has been improved by selection for increased egg mass and decreased body weight and getting a correlated response in feed efficiency [2]. Identifying birds that require less feed for body maintenance could improve feed efficiency. However, selection for feed conversion ratio can lead to unfavorable changes in the component traits. Additionally, direct selection for feed efficiency requires measurement of individual feed intake, which is time consuming and very expensive. Well-designed feeders are also required to prevent feed wastage. On the other hand, additional criteria for feed utilization should be involved. Residual feed intake may be used as selection criteria to attain these goals [2]. To our knowledge, there are no previous reports on the residual feed intake of different lines or varieties of Japanese quails raised under hot ambient temperature [4]. Due to the need to adjust patterns of feed consumption according to ambient temperature, the present study was carried out to estimate RFI, as well as its relationship with productive traits, in two varieties of Japanese quails under high environmental temperature.

#### **2. Materials and Methods**

#### *2.1. Birds, Housing, and Management*

A total of 310 laying Japanese quails of two varieties (gray and white) (155 each) were randomly selected from an initial population of 800-day-old hens that were reared to point of lay and transferred to individual battery cages. Each hen was individually housed in a wire cage (20 × 20 × 20 cm) supplied with an individual feed trough in the front and a nipple drinker. The quail received a laying ration containing 18% crude protein (CP) and 2850 kcal/kg metabolizable energy (ME) during the experimental period. Throughout the experiment, feed and water were available ad libitum. Birds were exposed to a lighting period of 16 h per day.All quails received uniform care and management practices throughout the whole experimental period. The average high and low ambient temperatures recorded during the experimental period were 38.9 ◦C and 24.3 ◦C, respectively. No vaccination or medication was performed. The use and handling of quails were approved by the Ethical Committee of Qassim University.

#### *2.2. Egg Production Parameters*

Starting from 12weeks of age, egg production (weight and number), feed intake (FI), and body weight (initial and final) were determined for each hen over a four-week experimental period. Egg production (%) was calculated as total laid eggs divided by the total number of days (28 days). Feed intake was measured on a cage basis and combined with egg production data to calculate feed conversion ratio (FCR).

#### *2.3. Egg Quality Measurements*

A total of 460 eggs (230 eggs from each variety) were collected at 16 weeks of age. External and internal egg quality measurements were assessed according to Fathi et al. [8]. Each egg was weighed to the nearest 0.1 g. Egg width (equatorial axis) and egg length (longitudinal axis) were measured using Vernier caliper to 0.1 mm. Egg shape was calculated according to the following formula:

$$\text{Egg shape} = \text{(egg width)} \text{(egg length)} \times 100 \tag{1}$$

The breaking strength for intact eggs was determined in Kg/cm<sup>2</sup> using Egg Force ReaderTM, Orka Food Technology Ltd. (Wanchai, Hong Kong). The liquid contents were put aside and the shell plus membranes were washed under running water to remove the adherent albumen. The wet eggshell was left for 24 h at room temperature for drying and then weighed to the nearest 0.01 g. The relative weight of dry eggshell was calculated on the basis of egg weight. To measure shell thickness, pieces from three different regions (two poles and equator) of each eggshell with intact membranes were measured with a dial gauge micrometer to the nearest 0.01 mm. The height of thick albumen and egg yolk was measured by placing the liquid content on a balanced surface using a tripod micrometer. Then, the yolk was separated and rolled on tissue papers to remove the residual albumen. Albumen weight was calculated by subtracting the yolk and shell weight from egg weight. The weight of eggshell, yolk, and albumen were expressed as a percentage of egg weight. Haugh units (HU) were calculated according to the following formula:

$$\text{HUU} = 100 \log \left( \text{H} - 1.7 \text{W}^{0.37} + 7.57 \right) \tag{2}$$

where H is the albumen height (mm) and W is the egg weight (g). Yolk color was measured by comparing yolk color to the DSM yolk color fan.

#### *2.4. Carcass and Internal Organs*

At the end of the experiment, 50 quails (25 from each variety) were randomly assigned for carcass yield assessment. After a pre-slaughter fasting period of 4 h, the quails were weighedand slaughtered by cutting their jugular veins. Following a 2-min bleeding time, each quail was dipped in a hot water bath at 60 ◦C for 60 s. and manually defeathered. Head and feet were removed. The carcass was eviscerated manually and weighed. Upon evisceration, the weight of eviscerated carcass, liver, heart, and gizzard was recorded and expressed as a percentage of live body weight according to Fathi et al. [9]. To minimize variations in the carcass procedure, all dissections were carried out by the same person.

#### *2.5. Blood Hematology and Plasma Biochemistry*

During slaughter, 25 blood samples were collected from each variety in heparinized tubes. The hematological parameters were determined by using Automatic Fully Digital Hematology Analyzer (BC-3000 Plus, Shenzhen Mindray, Bio-Medical Electronics Co., LTD, Shenzhen, China). These parameters were total count of red blood cells (RBC), hemoglobin (HGB), hematocrit (HT), and thrombocytes. The collected blood samples were centrifuged at 4000× rpm for 15 min. The resulting plasma samples were frozen at −20 ◦C for further analysis. The plasma concentrations of total protein, albumen, total cholesterol, and triglycerides were spectrophotometrically determined according to Fathi et al. [10] using commercial reagent kits (Stanbio Laboratory, Boerne, TX, USA). The globulin was calculated as the difference between the total protein and albumen.

#### *2.6. Calculation of Residual Feed Intake and Statistical Analysis*

Expected feed intake was computed using mid-metabolic body weight (BW0.75), body weight gain (ΔBW), and total egg mass (EM) for a given time considered by multiple regression analysis. Residual feed intake (RFI) was calculated as the difference between observed feed intake (OFI) and expected feed intake (EFI) for each experimental hen using the PROC REG procedure of JMP Ver. 11 (SAS, Cary, NC, USA) [11]. Each variety had its own partial regression coefficients according to the following multiple regression equation:

$$\text{EFI} = \text{aBW}\_{\text{i}}^{0.75} + \text{bEM}\_{\text{i}} + \text{c\Delta BW}\_{\text{i}} + \text{d} \tag{3}$$

where EFI = expected feed intake of hen i (g); BWi 0.75 = mean metabolic body weight of hen i (g0.75); EMi = egg mass production of hen i (g); ΔBWi = body weight gain (g); a, b, and c = partial regression coefficients; d = intercept.

Student *t* test analysis was applied to separate between means. All data were presented as means and the pooled SEM. Correlation coefficient was computed between RFI and some studied traits within each variety using PROC CORR procedure.

#### **3. Results and Discussion**

Productive performance of two varieties of Japanese quails is shown in Table 1. No significant difference for body weight (initial and final), weight gain, FI, or FCR was identified between the varieties. Gray quails had significantly higher (*p* < 0.02) egg mass and egg production percentage than that of white quails. A superiority of egg production in brown variety Japanese quails compared to both gray and white ones was previously detected [12]. Broken eggs were significantly (*p* < 0.05) affected by the quail variety. The gray quails recorded the lower value (1.13%) compared to the white quails (2.06%). Mortality levels fell within the normal range and there was no significant difference between quail varieties.


**Table 1.** Productive performance of two varieties of Japanese quails (*Coturnixcoturnix japonica*).

*N* = 155 quails/ variety; FI: feed intake; FCR: feed conversion ratio; a,b mean values in a raw without a common superscript are significantly different.

Internal and external egg quality characteristics are presented in Table 2. Shape index was significantly (*p* < 0.01) higher in eggs produced from white quails compared to their gray counterparts. Consistent with our results, Yilmaz et al. [13] and Sari et al. [14] reported that the egg shape index depended on the plumage color of the quails. They found that the mean shape index obtained from the gray plumage line was significantly lower than that of the white plumage line. In contrast to our results, Bagh et al. [10] did not find a significant difference between gray and white lines for all physical properties of egg quality. A numerical increase (*p* = 0.08) in HU was found in gray quails when compared with white quails. However, Bagh et al. [12] reported that there was no significance difference among the quail varieties for HU. In terms of yolk properties, there were no significant

differences between quail varieties for yolk color, yolk index, and yolk percentage. Significantly (*p* < 0.04) higher albumen percentage was foundin eggs produced from white variety compared to the gray variety. In regard to eggshell quality, a significant (*p* < 0.01) increase in eggshell breaking strength was found in eggs produced from gray quails (1.43 kg/cm2) compared to the white quails (1.34 kg/cm2). Also, gray quails had a significantly (*p* < 0.01) higher relative weight of eggshell (9.4%) compared to that of their white counterparts (9.0%). However, shell thickness did not exhibit a significant difference due to the effect of variety. This advantage in eggshell strength associated with gray quails may be attributed to better ultrastructural featuresin comparison to eggshells of the white variety. Changes in external and internal quality characteristics of eggs obtained from quails with different plumage colors have previously been reported [12–14]. However, literature on the external and internal quality characteristics of eggs obtained from quail varieties with different plumage color under high environmental conditions is very limited.


**Table 2.** Internal and external egg quality of two varieties of Japanese quails.

*N* = 230 intact eggs/variety; HU: Haugh units; <sup>1</sup> DSM yolk color fan. a,b mean values in a raw without a common superscript are significantly different.

Plasma biochemical and hematological parameters of gray and white feathered Japanese quails are summarized in Table 3. No significant effect on the blood biochemical variables was detected due to variety, except for cholesterol level. White quails had a significant (*p* < 0.01) increase in cholesterol level (198.5 mg/dL) compared to the gray variety (152.5 mg/dL). In terms of blood hematology, the white feathered quails had significantly higher levels of RBC, HGB, and HT compared to the gray quails. Generally, hematological parameters fell within the normal range for quails [15]. These results indicate that different genotypes in the present study were of normal physiological status.



*N* = 25 quails/variety; RBC: red blood cells; HGB: hemoglobin; HT: hematocrit; a,b mean values in a raw without a common superscript are significantly different.

Results of carcass traits studied as affected by variety of quails are shown in Table 4. It was found that neither carcass percentage nor giblets (liver, heart, and gizzard) significantly differed due to variety effect. However, an insignificant increase (*p* = 0.15) in carcass % was found in white quails (63.3%) compared to their gray counterparts (62.4%). Similar to the present study, Charati and Esmailizadeh [16] found that genotype had no significant effect on carcass percentage in white and wild (gray) Japanese quails. In contrast, several previous studies reported that feather color had a significant effect on live weight and carcass characteristics in Japanese quails. The white feathered quails had less body weight than that of the wild-type [17–19]. Similarly, Vali et al. [20] found significant differences in two quail strains for carcass weight, carcass percentage, and the relative weight of breast and femur.


**Table 4.** Carcass traits of two varieties of Japanese quails.

*N* = 25 quails/variety.

The results of the multiple regression analysis are listed in Table 5. Below the table, the prediction equations for the expected feed consumption for each variety have been provided. RFI is defined as the difference between the realized feed consumption and the expected feed consumption, which was estimated based on metabolic BW, body weight gain, and EM [3,21]. As shown, the partial regression coefficients for metabolic body weight and egg mass had significant effects in computing expected feed intake in both quail varieties. The intercept value also had a significant effect. On the other hand, body weight gain (Δ BWT) did not significantly affect the computation of RFI in either the gray variety (*p* = 0.08) or the white variety (*p* = 0.63). Estimates of regression coefficients in the models of gray and white quails were close. Similar to our findings, Badawe et al. [3] found thatthe prediction of feed intake and residual feed intakederived from multiple regression analysis was significantly affected by metabolic body weight and egg mass in laying chickens (*gallus gallus*).

**Table 5.** Partial regression coefficients for factors affecting expected feed intake of two varieties of Japanese quails.


*N* = 155 individual records/variety, \**p* < 0.05, \*\**p* < 0.01. Prediction equations: Y = 914.6 − 0.96 Δ BWT + 5.21 (BWT)0.75 − 1.64 EM (Gray variety), Y = 396.2 − 0.36 Δ BWT + 11.84 (BWT)0.75 − 1.11 EM (White variety); where Y stands for expected feed consumption, Δ BWT = body weight gain, (BWT)0.75 = metabolic body weight, EM = total egg mass.

Phenotypic correlations between RFI and some studied traits are presented in Table 6. A strong positive correlation between RFI and FI in gray and white quail varieties (0.89 and 0.91, respectively) was found. Notably, the correlation between RFI and FI in our study was much higher than those estimated in previous works on laying chickens [7,22,23]. The selection for low RFI could reduce FI without significant changes in EM [7]. FCR is widely used but not a suitable selection trait because of its complex correlations with growth and production traits [6,7]. As shown in Table 6, a significantly high

correlation was recorded between RFI and FCR (0.55 and 0.49, respectively). These strong relationships have indicated that selection for negative RFI would genetically improve feed efficiency and reduce feed intake. These results are consistent with those of Zhang et al. [1], who found a high phenotypic correlation between RFI and FCR (0.55). Moreover, RFI was strongly correlated with FI (0.82) in a random population of Pekin duck (*anas paltyrhynchos*). Similarly, RFI was positively correlated with FI in laying hens of chickens [22–24] and Japanese quails [2]. It is worthy to note that there was a low or neglected correlation between RFI and both egg weight and egg production% for the quail varieties. A low correlation between RFI with body weight gain was found (close to zero) in both quail varieties. Our results are in accordance with the findings of Luiting and Urff [25] and Altan et al. [2], who also described that the phenotypic correlation of RFI with both egg mass and body weight was almost zero. Likewise, these results are in agreement with the findings of Varkoohi et al. [5], reflecting the fact that RFI is phenotypically independent of weight gain. On the other hand, phenotypic correlations between RFI and both blood parameters and carcass traits were found to be rather low and insignificant. No significant relationship was observed between RFI with live body weight and eviscerated carcass weight [26].


**Table 6.** Phenotypic correlations between RFI and some studied traits in Japanese quails.

\* *p* < 0.05, \*\* *p* < 0.01.

#### **4. Conclusions**

The current results indicate that the egg mass significantly increased in the gray variety of Japanese quails compared to the white variety. Additionally, gray quails had a significantly lower percentage of broken eggs. Color variations in Japanese quails should be considered when selecting for type of production. We recommended raising gray quails for egg production under high environmental temperature, while the white variety may be more suitable for meat type. Results derived from multiple regression analysis clearly identified a significant effect of metabolic body weight and egg mass in computing expected feed intake rather than body weight gain in both varieties of Japanese quails. Including residual feed intake in the selection criteria of Japanese quailsin order to improve feed conversion ratio under high environmental temperature is highly recommended.

**Author Contributions:** Experimental design: M.M.F.; Experiment execution and data collection: M.M.F., I.A.-H., T.A.E., and O.K.A.-E.; Data processing and statistical analysis: M.M.F. and A.G.; Final revisions: M.M.F. All authors have been involved in developing, writing, and commenting on the manuscript. All authors read and approved the final manuscript.

**Funding:** This research was funded by Qassim University.

**Acknowledgments:** This work was supported by Qassim University, Al-Qassim, KSA. Also, A. Al-Moshawah must be thanked for his technical assistance during the course of the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Intestinal Morphology in Broiler Chickens Supplemented with Propolis and Bee Pollen**

### **Ivana Prakatur 1, Maja Miskulin 2, Mirela Pavic 3, Ksenija Marjanovic 2, Valerija Blazicevic 4, Ivan Miskulin 2,\* and Matija Domacinovic <sup>1</sup>**


Received: 22 May 2019; Accepted: 27 May 2019; Published: 31 May 2019

**Simple Summary:** Consumers are becoming more aware of the nutritional value of foods, and they want to consume food that provides health benefits beyond the provision of essential nutrients. Chicken meat could fulfil the above requirements due to its high nutrient content and relatively low caloric value, and it serves as an interesting basis for functional foods. In this study, we evaluated the effects of propolis and bee pollen, as potential additives, on the intestinal morphology and absorptive surface areas of broiler chickens. The results of this study showed that supplementation of broilers with propolis and/or bee pollen has a profoundly beneficial effect on intestinal morphology and absorptive surface areas. Thus, these natural additives could be used as alternative additives in modern broiler production, while chicken meat can be even more beneficial for human health.

**Abstract:** The aim of this study was to determine the influence of dietary supplementation with propolis and bee pollen on the intestinal morphology and absorptive surface areas of chickens. Two hundred day-old Ross 308 chickens (100 male and 100 female) were equally allocated into five groups. Throughout the whole study, the control group of chickens was fed with a basal diet, while the experimental groups of chickens were fed with the same diet supplemented with propolis and bee pollen: P1 = 0.25 g of propolis/kg + 20 g of bee pollen/kg; P2 = 0.5 g of propolis/kg; P3 = 1.0 g of propolis/kg; P4 = 20 g of bee pollen/kg. The duodenal villi of chickens from all experimental groups were significantly higher and wider (*p* < 0.001), while their duodenal villi crypts were significantly deeper (*p* < 0.001) in comparison with these parameters in chickens from the control group. The villus height to crypt depth ratio, as well as the absorptive surface areas of broiler chickens, were significantly increased (*p* < 0.001) in experimental groups of chickens in comparison with the control group. These findings suggest that dietary supplementation with propolis and bee pollen has a beneficial effect on broilers chickens' intestinal morphophysiology.

**Keywords:** intestinal morphology; duodenum; intestinal villi; intestinal absorption; broilers feeding; propolis; bee pollen

#### **1. Introduction**

Propolis and bee pollen belong to a group of natural substances of animal and vegetable origin with intense antioxidant and antimicrobial properties [1]. The bioactive components of propolis and bee pollen include flavonoids, phenolic acids and their derivatives, which are also responsible for the bactericidal, antiviral, antifungal, analgesic, anti-inflammatory, antioxidant, immunostimulating and immunomodulating effects of these compounds in humans and animals [1–3].

A large number of previous studies have suggested an increase in the production performance of chickens fed with propolis and/or bee pollen [4–9]. These effects could be related to the effect of propolis extract on gastrointestinal microbiota, which increases levels of beneficial bacteria and decreases pathogenic types [10]. This modulation of microbiota could promote intestinal health, since the beneficial bacteria could provide improved feed digestibility and protection against pathogens via competitive exclusion through a variety of mechanisms [11,12].

With consideration of the above, and also the fact that the European Commission banned the use of antibiotics as growth agents in 2006 [7,13], the use of natural feeding additives such as propolis or bee pollen is very important in terms of improvement of performance, health status and immune systems in broiler chickens [1,14].

The small intestine is an important organ responsible for the digestion and absorption of nutrients from the diet. Any changes in its function affect the function of other organs and systems in the organism [15]. There are only a few studies that have previously evaluated the effect of propolis and bee pollen on the intestinal morphology of broiler chickens, and their results are contradictory [12,16–19]. However, some of them have proven that these natural additives improved intestinal morphophysiology [16–19].

The aim of this study was to determine the influence of dietary supplementation with propolis and bee pollen on the intestinal morphology and absorptive surface areas of broiler chickens.

#### **2. Materials and Methods**

#### *2.1. Animals and Diets*

A total of 200 (100 male and 100 female) day-old Ross 308 broiler chickens were evenly distributed by gender for use in the present study. The feeding trial of the broilers was carried out on a farm in Eastern Croatia under the supervision of the Division for Animal Production and Biotechnology, Faculty of Agrobiotechnical Sciences Osijek, Josip Juraj Strossmayer University of Osijek. The experimental protocol was approved by the Committee for Animal Welfare of the Faculty of Agrobiotechnical Sciences Osijek, Josip Juraj Strossmayer University of Osijek (Approval code: 602-04/18-01/01; 2158-94-02-18-01).

The experiment was a completely randomized design, and broilers were allocated into five dietary treatments with two replicate groups of 20 birds per pen (5 diets × 2 replicates). The groups of broilers were housed under the same conditions during the whole experimental period. Temperature, humidity, and lighting in the facility were maintained within the optimum limits according to the manufacturer's recommendations for the Ross 308 hybrid [20]. Breeding was conducted on wooden sawdust (10 cm depth) and lasted for six weeks (42 days). During the study, feed and water were offered to broilers ad libitum. For ensuring effective monitoring of all the investigated indicators, all the broilers were marked with a leg ring on the seventh day of the feeding trial.

During days 1–21 of the study, broilers were fed a mixture of broiler starter. During days 22–42 of the study, broilers were fed a mixture of broiler finisher. The composition and calculated analyses of feed mixtures used in the feeding of the broilers are shown in Table 1. Throughout the study, the control group (K) of the broilers was fed a standard diet without additives, while the experimental groups of broilers (P1, P2, P3 and P4) were fed the same diet supplemented with propolis and/or bee pollen: the P1 group was offered a diet supplemented with 0.25 g of propolis and 20 g of bee pollen per kg of diet; the P2 group was offered a diet supplemented with 0.5 g of propolis per kg of diet; the P3 group was offered a diet supplemented with 1.0 g of propolis per kg of diet; the P4 group was offered a diet supplemented with 20 g of bee pollen per kg of diet. The doses of bee pollen and propolis were selected on the basis of known broiler chicken gastrointestinal tract physiology and through series of pilot studies on a small number of animals. The inclusion of propolis and bee pollen into the feed mixture was performed using a vertical mixer (Briketstroj Ltd., Valpovo, Croatia).


**Table 1.** The composition and calculated analysis of feed mixtures used in the feeding of the broilers.

\* Each 1 kg of premix contained: vitamin A 1200,000 IU; vitamin D3 200,000 IU; vitamin E 3000 mg; vitamin K3 250 mg; vitamin B1 150 mg; vitamin B2 600 mg; vitamin B6 200 mg; vitamin B12 1 mg; folic acid 50 mg; niacin 4400 mg; Ca pantothenate 1500 mg; biotin 10mg; choline chloride 50,000 mg; iron 5000 mg; copper 700 mg; manganese 8000 mg; zinc 5000 mg; iodine 75 mg; cobalt 20 mg; magnesium 750 mg; selenium 15 mg; antioxidant butylated hydroxytoluene (BHT) 10,000 mg; methionine 100,000 mg; herbal carrier 1000 g.

Samples of raw propolis and bee pollen used in this study were obtained from apiaries located in naturally preserved areas of continental Croatia (around the city of Osijek, Eastern Croatia). Propolis and bee pollen were crushed mixed, in powder form, with dry feed mixture using a vertical mixer. Bearing in mind that the biological activity of propolis and bee pollen depends on the components of polyphenolic fraction, mainly flavonoids, in the propolis and bee pollen samples used in this study, the amount of total flavonoids (expressed as equivalents of quercetin) was determined by a colorimetric method according to Chang et al. [21]. The results of this analysis are shown in Table 2. The analysis was performed at the Department of Health Ecology within the Croatian Institute of Public Health in Zagreb, Croatia accredited according to HRN EN ISO/IEC 17025:2000.

**Table 2.** The amount of total flavonoids (mg/g) in propolis and bee pollen, expressed as equivalents of quercetin.


#### *2.2. Sample Collection, Measurements and Analysis*

At the end of the feeding trial (i.e., day 42), 10 birds from each group were randomly selected and slaughtered for a necropsy examination. Fifty duodenal samples (10 from each group) were collected from the birds directly after slaughter and fixed in 10% neutralized formalin. The duodenal samples were 2 cm long and dissected at the midpoint of the duodenum. The fixed tissue samples were transported to the Department of Pathology and Forensic Medicine, Faculty of Medicine Osijek, where they were further processed. The tissues were then dehydrated with increasing concentrations of ethyl alcohol (70%, 90%, 96% and 100%), cleared in xylene and embedded in paraffin. The paraffin blocks were then cut using microtome, into four 5-μm-thick discontinuous paraffin-embedded sections per broiler duodenal sample that were stained with hematoxylin and eosin (H&E) and examined under a light microscope (Olympus CX40), while representative fields were photographed and digital images were captured for morphometric analysis. A computer morphometric program, Quick Photo Micro 3.0, was used for morphometric measuring the duodenal villi height and base width of the villi. The same computer program was used for measuring the duodenal villi crypt depth. For the measurement of duodenal villi height, cross-sections of 10 villi were randomly selected. The criterion for villus selection was based on the presence of intact lamina propria. Villus height and width, as well as crypt depth, were measured at 40× the objective magnification. The villus height was measured as the distance from the apex of the villus to the junction of the villus and crypt [22]. The villus width was measured as the distance from the junction to the basement membrane of the epithelial cell at the bottom of the crypt at the bottom third of the length of the villus (base width of the duodenal villi) [23]. All the measurements taken from 10 villi per one sample were counted from four different preparations from each duodenal segment for each bird, and were expressed as the average duodenal villi height and average base width of the duodenal villi for each bird. Finally, 10 average heights of duodenal villi, as well as 10 average base widths of the duodenal villi from 10 birds were expressed as the average height of the villi for a group and the average base width of the villi for a group [22]. The duodenal villi crypt depth was measured from the base of the villus to the mucosa [23]. All the measurements from 10 crypts were counted from four different preparations from each duodenal segment for each bird. Averaged depth measurements of 10 crypts were expressed as the average duodenal villi crypt depth for each bird. Finally, 10 average depths of duodenal villi crypts of 10 birds were expressed as the average depth of duodenal villi crypts of the group [22]. The ratio of villus to crypt was estimated by dividing the villus height by the crypt depth [23]. The absorptive surface area of the duodenal villus was estimated by considering a villus as a cylindrical structure [23]. Villus absorptive surface area was calculated using the formula: Villus absorptive surface area = 2π × (average villus width/2) × villus height [23,24].

#### *2.3. Statistical Analysis*

The normality of the data distribution was tested by a Kolmogorov–Smirnov test; all data were processed by methods of descriptive statistics. The numerical variables were described as the median and interquartile ranges. A Kruskal–Wallis test was used for the comparison of numerical variables among the groups. The level of statistical significance was set at *p* < 0.05. Statistical analysis was performed using the statistical package Statistica for Windows 2010 (version 10.0, StatSoft Inc., Tulsa, OK, USA).

#### **3. Results**

Morphometric analysis of the duodenal villi of broiler chickens revealed differences between the control and experimental groups of chickens at the tissue structure level on the 42nd day of the feeding trial, as shown in Table 3. The duodenal villi of chickens from all the experimental groups were significantly higher (*p* < 0.001), while their base was significantly wider (*p* < 0.001) in comparison to those in chickens from the control group. There was a statistically significant difference in duodenal villi crypt depth between the groups of chicken (*p* < 0.001).


**Table 3.** The values of evaluated parameters of duodenal villi of broiler chickens on the 42nd day of the feeding trial.

\* Kruskal–Wallis test. abcde: Medians within a row with different superscripts are different; K = control group; P1 = feed mixture + 0.25 g of propolis/kg of feed mixture + 20 g of bee pollen/kg of feed mixture; P2 = feed mixture + 0.5 g of propolis/kg of feed mixture; P3 = feed mixture + 1.0 g of propolis/kg of feed mixture; P4 = feed mixture + 20 g of bee pollen/kg of feed mixture.

The histological representations of the duodenal villi of broiler chickens from all the groups are shown in Figures 1–5.

**Figure 1.** Histological representation of the duodenal villi of broiler chickens from the control group of chickens (K) (H&E; ×100).

**Figure 2.** Histological representation of the duodenal villi of broiler chickens from the P1 experimental group of chickens (H&E; ×100).

**Figure 3.** Histological representation of the duodenal villi of broiler chickens from the P2 experimental group of chickens (H&E; ×100).

**Figure 4.** Histological representation of the duodenal villi of broiler chickens from the P3 experimental group of chickens (H&E; ×100).

**Figure 5.** Histological representation of the duodenal villi of broiler chickens from the P4 experimental group of chickens (H&E; ×100).

The study also revealed that there was a statistically significant difference in the villus height-to-crypt depth ratio on the 42nd day of the feeding trial between the control and experimental group of chickens (*p* < 0.001), as shown in Table 4.


**Table 4.** The villus height-to-crypt depth ratio of broiler chickens on the 42nd day of the feeding trial.

\* Kruskal–Wallis test. (Q1–Q3) = interquartile range; K = control group; P1 = feed mixture + 0.25 g of propolis/kg of feed mixture + 20 g of bee pollen/kg of feed mixture; P2 = feed mixture + 0.5 g of propolis/kg of feed mixture; P3 = feed mixture + 1.0 g of propolis/kg of feed mixture; P4 = feed mixture + 20 g of bee pollen/kg of feed mixture.

The study further showed that there was a statistically significant difference between the average values of the absorptive surface areas of the duodenal villi of broiler chickens on the 42nd day of the feeding trial between the control and experimental group of chickens (*p* < 0.001) (see Figure 6).

**Figure 6.** The average absorptive surface area of duodenal villi according to the group of broiler chickens on the 42nd day of the feeding trial (Kruskal–Wallis test, *p* < 0.001).

#### **4. Discussion**

Morphometric results of the duodenal villi in chickens on day 42nd of feeding trial revealed that it was significantly higher, while its base was significantly wider in the experimental groups compared to the controls. These results are consistent with the results of the study by Wang et al. [16], who demonstrated that chickens fed a diet supplemented with a mixture of bee pollen had significantly higher and wider intestinal villi of the duodenum, jejunum and ileum in comparison to the chickens fed a control diet. The same authors further determined that the observed differences were greater during the early stages of development of the gastrointestinal system [16]. The results of the present study are also consistent with the results of a study by Tekeli et al. [25], who showed that the addition

of ginger and propolis extract both separately and in combination in the diet resulted in a significant increase in the length of the intestinal villi of the jejunum in chickens from the experimental groups when compared to chickens of the control group. On the other hand, Eyng et al. [17] showed that the intestinal villi of the duodenum of chickens fed a diet supplemented with various amounts of propolis were shorter or lower when compared to the intestinal villi of the chickens in the control group.

Considering the morphometric results of the duodenal villi crypt depths in chickens on the 42nd day of fattening, this study showed that there were significant differences in the depths of duodenal villi crypts between the chickens from the experimental groups compared to the chickens from the control group. This result is consistent with that of Eyng et al. [17], who showed that the crypt of the intestinal villi of the duodenum of chickens fed a diet supplemented with various amounts of propolis were deeper compared to crypt of the intestinal villi of the duodenum of chickens from the control group.

All the previously mentioned results of this study can be attributed to the beneficial effect of the biologically active components of propolis and/or bee pollen. These components participate in controlling the proliferation of pathogenic bacteria and the consequent avoidance of possible damage to the intestinal mucosa, which also leads to the reduction of morphometric measures of the intestinal villi [17,26].

Within the explanation of the identified influence of propolis and/or bee pollen on the histological features of chickens' intestines, it is important to keep in mind that diet composition is in fact the main factor that can modify the histological appearance or morphology of the intestine and, consequently, its absorptive capacity, which ultimately defines the growth performance of fattening chickens [27]. It is further known that the intestinal villi are quickly and continuously adjusted as a response to conditions in the lumen of the intestine (that are strongly influenced by diet composition) reflecting the dynamic environment inside the intestines of animals. Accordingly, longer intestinal villi are associated with an increase in the absorptive surface of the intestines and also with an increase of the absorption capacity of the intestine [28]. This finding was also demonstrated in the present study, since the absorptive surface area of duodenal villi in all experimental groups were increased in comparison to that of the control broilers.

Previous studies have already confirmed that longer intestinal villi indicate an improved ability to absorb nutrients in the intestine [29,30]. In addition, it has been proven that longer villi are associated with active cell mitosis, which provides a greater absorptive potential of villi for various nutrients [31,32]. Deeper intestinal villi crypts indicate a rapid metabolism of tissue in order to allow the renewal of the intestinal villi, if there is a need for its regeneration [27]. Lowering the height of the villi or reducing crypt depths of intestinal villi may lead to a reduction in the absorption of nutrients [33].

This study further showed that there was a statistically significant difference in the villus height to crypt depth ratio on the 42nd day of the feeding trial between the control and experimental groups of chickens. This result is highly important, bearing in mind that a higher ratio of villous height and crypt depth refers to a greater capacity of nutrient digestibility and absorption in chickens [34]. Namely, it has been proven that shorter intestinal villi relative to crypt depth are related to a smaller number of absorptive cells and a larger number of secretory cells. Secretory cells are responsible for the secretion of mucins that form a mucinous lining of the intestinal epithelium, thus increasing the number of secretory cells and leading to an increased secretion of mucin. Changes in the quantity or composition of mucin of the intestinal mucosal surface can reduce the absorption of nutrients and/or increase the amount of energy required to maintain function of the intestines [27,35].

In present study, all the experimental groups of chickens had deeper crypts of the intestinal villi of the duodenum in relation to chickens from the control group, which is a clear indicator of higher proliferative activity in the mucosa of these intestinal villi. Higher proliferative activity in the mucosa of the intestinal villi indicates better digestibility and absorption of consumed feed mixtures in the experimental groups of chickens that were fed a mixture with the addition of propolis and/or bee pollen. The latter has also been shown in studies of other substances of pronounced antimicrobial and antioxidant properties, such as, for example, garlic and some herbal extracts [36–38].

The clarification of antimicrobial and antioxidant effects of all the previously mentioned substances, including propolis and bee pollen, re-emphasizes the role of their phenolic components such as various flavonoids, phenolic acids and their derivatives that they have the ability to protect the intestinal villi and increase the absorption of nutrients [38]. It is believed that these biologically active components exert their antioxidant activity both at the cellular and at the tissue level [39]. Apart from their antioxidants, their antimicrobial activity should also be significant as these bioactive agents can modulate the gut ecosystem. Due to the synergism of antioxidant and antimicrobial activity of biologically active phenolic compounds from propolis and bee pollen, a further positive effect on the utilization of nutrients has been achieved [40,41].

The present study revealed some original solutions regarding the applied dosage of investigated natural supplements and their specific combinations in broilers feeding, but was not without limitations. Due to the commonly accepted '3Rs', the authors had the justifiable wish to minimize the number of animals used in this study that had already been used in similar studies [3]. However, considering the tested natural feeding additives and main objective of this study, the authors believe that the described design of the study did not affect the results.

In conclusion, the present study showed that the addition of propolis and/or bee pollen to feed mixtures has a significant protective effect on the gut tissue of chickens, which is reflected through better morphometric measures of the duodenal villi and duodenal villi crypts of chickens from all the experimental groups in relation to chickens from the control group. Following the results of this study, the addition of 0.5 g of propolis per kg of feed mixture showed the strongest positive effect on chicken guts. The promising and encouraging results of this study emphasize the importance of the further evaluation of the administration level of investigated supplements in order to maximize their positive effects on the gut tissue of chickens and, consequently, the overall health of broiler chickens.

**Author Contributions:** Conceptualization, I.P., M.M., I.M. and M.D.; Methodology, I.P., M.P., K.M., V.B. and M.D.; Investigation, I.P., M.M., I.M. and M.D.; Formal analysis, I.P., M.M., I.M. and V.B.; Data curation, I.P., M.M. and I.M.; Writing—original draft preparation, I.P., M.M. and I.M.; Writing—review & editing, M.P., K.M., V.B. and M.D.; Visualization, I.M., K.M. and V.B.; Supervision, M.D.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Whole-Life or Fattening Period Only Broiler Feeding Strategies Achieve Similar Levels of Omega-3 Fatty Acid Enrichment Using the DHA-Rich Protist,** *Aurantiochytrium limacinum*

### **Jason D. Keegan 1, Giorgio Fusconi 2, Mauro Morlacchini <sup>2</sup> and Colm A. Moran 3,\***


Received: 20 May 2019; Accepted: 5 June 2019; Published: 6 June 2019

**Simple Summary:** In many parts of the world, the human population does not consume sufficient quantities of omega-3 fatty acids. Humans can potentially reduce the risk or severity of a variety of illnesses by simply increasing their dietary intake of omega-3 fatty acids, with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) being particularly beneficial. Fish are a rich source of these important fatty acids, but many individuals do not consume fish and so the enrichment of more commonly consumed foods has been explored as a method to increase the consumption of omega-3 fatty acids. The fatty acid content of chicken meat reflects the fatty acid composition of their diet and so poultry meat can be easily enriched by introducing omega-3 rich ingredients into poultry diets. In this study we fed broilers diets supplemented with a DHA-rich protist, *Aurantiochytrium limacinum* for their whole life (42 days) or for the final 21-day fattening period to investigate which strategy represented a more efficient use of the DHA-rich ingredient. As similar levels of enrichment were found from both feeding durations tested, the study indicates that feeding for the 21-day fattening period is the more efficientperiod of dietary inclusion for *Aurantiochytrium limacinum*.

**Abstract:** The fatty acid composition of broiler chicken tissues can be increased by adding omega-3 rich ingredients to their diets. The purpose of this study was to compare the levels of tissue enrichment observed following the supplementation of broilers with the docosahexaenoic acid (DHA)-rich protist, *Aurantiochytrium limacinum* (AURA) for their whole life (42 days) or for the final 21-day fattening period. Day-old chicks (n = 350) were distributed among 35 pens (10 birds per pen) with each pen randomly assigned to one of five treatments: Control; 0.5% AURA from day 0–42; 1% AURA from day 0–42; 0.5% AURA from day 21–42; 1% AURA from day 21–42. Production parameters were recorded over the course of the study and the fatty acid profile of the breast, thigh, liver, kidney and skin with adhering fat was quantified at the end of the feeding period. The level of supplementation had a significant impact on the degree of omega-3 tissue enrichment, however, no differences were observed when the same dose was provided for 21 or 42 days. These results indicate that supplementation with AURA for a period of 21 days does not negatively affect broiler productivity and is the most efficient strategy to increase the nutritional value of broiler products.

**Keywords:** broilers; DHA; omega-3; fatty acids; enrichment

#### **1. Introduction**

Increasing consumer awareness of the health benefits associated with the consumption of omega-3 polyunsaturated fatty acids (n-3 PUFA) has resulted in a greater demand for n-3 PUFA rich produce [1]. As a result, the industry has been developing strategies to increase the n-3 PUFA content of commonly consumed foods [2]. By feeding chickens diets rich in n-3 PUFA, the fatty acid content of chicken meat can be altered to contain much higher quantities of n-3 PUFA, which can lead to health benefits for the consumer without making significant changes to their diet [2]. There are a variety of n-3 PUFA rich ingredients that have been used to supplement poultry diets in order to increase the n-3 PUFA content of their meet and eggs. Linseed, in various forms, can be a good source of alpha-linoleic acid (ALA) and can significantly increase tissue n-3 FA content [3]. However, some antinutritional features of linseed limit its inclusion in poultry diets [3]. In addition, most of the health benefits associated with omega-3 fatty acids have been attributed to increasing the intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [4]. While the human body can convert ALA to EPA and DHA, this process is inefficient and as such, the direct consumption of at least 250 mg of EPA+DHA per day is recommended [5]. Fish are a rich source of EPA + DHA but are only consumed regularly by small proportions of the population [6]. For those who do not consume fish, chicken has been found to contribute up to 28% of the dietary intake of EPA+DHA in the UK [7] and 24% of EPA + Docosapentaenoic acid (DPA)+DHA intake in Australia [8], despite the fact that chicken is not a naturally rich source of n-3 PUFA [9].

Fish meal and oil can also be used as a feed ingredient for poultry [10], but while this does increase the EPA + DHA content of the meat, it can also lead to a deterioration in consumer acceptability [11,12]. In addition, using fish meal/oil to supplement animal diets is not considered sustainable and could not supply the n-3 PUFA requirements of the world's growing population [13].

*Schizochytrium* spp. and *Aurantiochytrium limacinum* are both members of the Thraustochytrid family of heterotrophic protists, commonly classified as microalgae, but with fungus-like characteristics. The Thraustochytrids are primary producers of n-3 PUFA in the marine food chain and can be grown in a sustainable manner on a large scale using heterotrophic fermentation. Various authors have used *Schizochytrium* as a dietary supplement for broilers, and have detected increased tissue n-3 PUFA concentrations without any major impact on the organoleptic properties of the meat or on broiler productivity [14,15]. *A. limacinum* (AURA) is a closely related species that can be provided as a dietary supplement and has been shown to increase the DHA concentrations of cows milk, pork, chicken meat and eggs [16–21].

Previous work indicated that AURA is well tolerated by broilers with no negative impact on broiler health or productivity observed when broiler diets were supplemented at a level of 5% for their whole life [16]. Significant increases of tissue DHA concentrations were also observed when feeding broilers for the final 21-day fattening period only [17]. When comparing these similar studies, the results suggested that feeding for the entire life of the broiler may represent a more cost efficient use of dietary AURA, reaching higher levels of enrichment potentially due to the accumulation of DHA in the tissues over the whole life of the bird [17]. These comparisons were drawn from studies of similar design, carried out in the same facility, but at different times. As the studies did not occur concurrently, differences between the levels of enrichment could be attributed to factors other than the duration of supplementation. Therefore, in the current study we aimed to investigate the most cost-effective duration of supplementation by comparing the levels of enrichment achieved when birds were provided with supplementary AURA for their whole lives or for the final 21-day fattening period only.

#### **2. Materials and Methods**

The research protocol and animal care were conducted in accordance with European Union Directive 2010/63/EU covering the protection of animals used for experimental or other scientific purposes, and according to the recommendation of the Commission of 2007/526/CE covering the accommodation and care of animals used for experimental and other scientific purposes. The live animal portion of the study was carried out at the CERZOO research center which is authorized by the Italian Ministry of Health to employ animals for experimental or other scientific purposes. The study was conducted to investigate the effect of supplementing broiler diets with *A. limacinum* (AURA; ALL-G-RICH®; CCAP 4087/2; Alltech Inc., Nicholasville, KY, USA) over their whole life (WL; Day 0–42) or for the 21-day fattening period only (FP; Day 21–42) on production parameters and the fatty acid content of breast, thigh, liver, kidney and skin samples. The analytical composition of AURA was determined prior to the start of the study using the following methods: crude protein (AOAC 990.03), crude fat (AOAC 954.02), fatty acid composition (AOAC 996.06, with matrix validation [22]), moisture (AOAC 930.15) and ash (AOAC 942.05).

The study was carried out using male Ross 308 broilers housed in 1.4 × 1.6 m pens. Food and water were provided ad libitum using one feeder per pen and using an internal water system network. The study site was equipped with a dynamic ventilation system. Heating was provided from positive water aerotherms and positive pressure ventilation was achieved by single, variable-speed fans linked to temperature sensors. The temperature program was recommended by the breeder [23] and was automatically controlled and programmed to gradually reduce the temperature as follows: 25–30 ◦C (D1); 22–27 ◦C (D1–D7); 19–25 ◦C (D7–D14); and 18–25 ◦C (D14–D42). The higher temperature at the beginning of the study was guaranteed by specific 150-watt lamps mounted in each pen. The relative humidity ranged from 60–80% (D1–D7) and 40–80% (D7–D42). The lighting program was 18 hours light, and 6 hours dark.

Day-old chicks (n = 350) were randomly distributed among 35 pens (10 birds per pen) with each pen randomly assigned to one of the five following treatments: 0% AURA day 0–42 (Control); 0.5% AURA from day 0–42 (0.5%WL); 1% AURA from day 0–42 (1%WL); 0.5% AURA from day 21–42 (0.5%FP); 1% AURA from day 21–42 (1%FP). The ingredients of the diets are shown in Table 1 and were designed to meet the nutrient levels recommended for Ross 308 broilers [24]. The experimental diets were produced at the CERZOO feed mill and provided as a crumble from day 0–10, or as pellets from day 10–42. The nutrient content of the experimental diets was determined at the Institute of Food Science and Nutrition (Faculty of Agricultural Sciences, Food and Environment, Catholic University of Sacred Heart, Piacenza, Italy) using the following methods: dry matter (ISO 6496); crude protein (Gazzetta Ufficiale Serie Generale n.92 of 21.04.96); crude fat (Directive EEC 84/4/EEC); crude fibre (Directive EEC n.92/89); crude ash (NEN 3329, ISO 5984-2002 (E)); starch by polarimetric method (Directive CEE n. 72/199 and ISO 10520:1997 (E)); sugars ( Gazzetta Ufficiale CEE n. L155 of 12.07.71); energy content was calculated according to the equation proposed in G.U. CE n. L54 22.02.09. Individual fatty acids were quantified using AOAC Met. 996.06.2001, recently validated for chicken feed [25]. In brief, the fatty acids in each sample were trans-esterified in situ with 1.5 N HCl in methanol, in the presence of toluene. The toluene contained methyl tricosanoate which acted as the internal standard. The resultant fatty acid methyl esters (FAMEs) and toluene were then extracted. The FAMEs were then separated, identified, and quantified by gas chromatography equipped with a flame ionization detector (GC-FID).

The mg of FAME per 100 g of sample was then calculated using the following formula:

$$\text{FAME, mg FA/100 g wet sample} = \frac{A\_X \times CF\_X \times W\_{IS} \times 100}{A\_{IS} \times W\_S \times 1.04 \times 1000}$$

where AX = area counts for the FA; AIS = area counts for internal standard (C23:0); CFX = instrumental response factor for FA (EPA = 0.98, DHA = 0.99, GLA = 0.98); WIS = weight of IS added to sample in mg; WS = weight of sample in mg; 1.04 = conversion factor from methyl ester to fatty acid.

The AURA used in the study contained 71.7 g of crude fat/100 g biomass and 16.0 g DHA/100 g biomass with a significant amount of palmitic acid (34.0 g/100 g biomass). Additionally, the AURA contained 13% crude protein, 2.63% ash and 1.97% moisture. The analytical composition of the experimental diets is shown in Table 2, while the fatty acid composition of the diets is shown in Table 3. Daily bird health, mortality and culling records were maintained. Pen live weight on days 0, 21 and

42 were recorded in addition to the feed intake and feed refusals. One bird per replicate was killed (according to annex I of the Reg. CE n. 1099/2009 of the council of the 24/09/2009 concerning the protection of animals during slaughter) on day 43 and both kidneys, the whole liver, breast, thigh and skin with adhering fat samples were taken post-mortem. The left breast and thigh were used for the determination of dry matter (UNI ISO 1442), crude fat (AOAC 991.36), protein (UNI ISO 937) and ash (AOAC 991.36). The fatty acid content of both kidneys, the liver and the right (skinless) breast, (skinless) thigh and skin with adhering fat were quantified using the method recently validated for chicken tissues [26]. FAME calculations were as described above for chicken feed.

**Table 1.** Ingredient composition of the five experimental diets supplemented with 0, 0.5 or 1% *Aurantiochytrium limacinum* (AURA) for their whole life (WL; day 0–42) and for the fattening period only (FP; day 21–42).


<sup>1</sup> The content of vitamins and trace minerals per kg of premix (Rovimix B extra /1), produced by DSM Nutritional Product—Istituto delle Vitamine, Segrate (MI), Italy, is as follows: vit. A: 2,000,000 IU/kg; vit. D3: 600,000 IU/kg; vit. E: 15,000 mg/kg; vit. K3: 612 mg/kg; vit. B1: 400 mg/kg; vit. B2: 1200 mg/kg; D-pantothenic acid: 2778 mg/kg; vit. B6: 1200 mg/kg; vit. B12: 6 mg/kg; vit. C: 20,000 mg/kg; Niacin: 8000 mg/kg; Folic acid: 250 mg/kg; Biotine: 30 mg/kg; choline chloride: 100,000 mg/kg; Mn: 26,000 mg/kg; Fe: 10,000 mg/kg; Cu: 1,000 mg/kg; Zn: 15,000 mg/kg; I: 200 mg/kg; Se: 40 mg/kg. Excipient: calcium carbonate: 42.00%; spent grapes: 25.06%.

**Table 2.** The analytical composition of the five experimental diets supplemented with 0, 0.5 or 1% *Aurantiochytrium limacinum* for their whole life (WL; day 0–42) and for the fattening period only (FP; day 21–42).


<sup>1</sup> The crumbled feed provided from day 0–10 and pelleted feed provided from day 10–21 were analysed separately and so the values presented here represent the mean of these two analyses <sup>2</sup> Metabolisable energy calculated according to Gazzetta Ufficiale CE n. L54, February 22, 2009.

**Table 3.** The fatty acid composition of the five experimental diets supplemented with 0, 0.5 or 1% *Aurantiochytrium limacinum* for their whole life (WL; day 0–42) and for the fattening period only (FP; day 21–42).


<sup>1</sup> The crumbled feed provided from day 0–10 and pelleted feed provided from day 10–21 were analysed separately; the values presented here represent the mean of these two analyses <sup>2</sup> Eicosapentaenoic acid <sup>3</sup> Docosahexaenoic acid.

Differences between the treatment groups were determined using the general linear model procedure of Minitab (Minitab, v18, State College, PA, USA) with Tukey's post hoc analysis used to determine the differences between the treatment groups. Regression analysis was used to determine whether the estimated DHA intake per bird could predict the DHA content of breast and thigh meat. DHA intake per bird was calculated by dividing the intake per pen by the number of birds present and then multiplying by the DHA content detected for each experimental diet.

#### **3. Results**

#### *3.1. Bird Health and Performance*

Mortality was below 5% for each treatment group with an overall mortality of 3.14% observed. The performance of the birds in terms of their weight, weight gain, feed intake and feed conversion ratio (FCR) are summarised in Table 4. There was no significant difference between the groups in terms of weight at the beginning of the study. The 1%WL and 0.5%WL groups differed significantly in terms of weight by day 21 and in terms of their average daily gain during the first period (D 0–21) with the latter gaining significantly more weight. By day 42, the 1%WL group differed significantly to the control in terms of weight and average daily gain, with lower values again observed for the 1%WL group. Average daily feed intake differed between the 1%FP group and the 0.5%WL groups between day 0 and 21 and between the control and 1%WL groups between day 21 and 42, and overall. Thigh weight differed significantly between the control and 1%WL group with the latter weighing significantly less than the control at the end of the trial.


**Table 4.** The effect of dietary supplementation with 0, 0.5 or 1% Aurantiochytrium limacinum for their whole life (WL; day 0–42) and for the fattening period only (FP; day 21–42).

<sup>1</sup> Average Daily Gain; <sup>2</sup> Average Daily Feed Intake; <sup>3</sup> Feed Conversion Ratio; <sup>4</sup> European Production Efficiency Factor calculated as ((Average grams gained/day X % survival rate)/Feed Conversion X 10); a,b Means within a row that do not share a superscript differ significantly.

#### *3.2. Meat Fatty Acid Content*

The effects of supplementation with AURA on the concentration of selected fatty acids (i.e., those fatty acids that differed significantly between treatments or were of interest overall) in chicken breast, thigh, kidney, liver or skin with adhering fat are shown in Table 5. The full fatty acid profile determined for each tissue/organ is provided in Supplementary Table S1. The highest DHA concentrations were detected in the liver followed by the skin with adhering fat, thigh and kidney, with the breast meat having the lowest DHA content of all the sampled tissues. In all cases, the DHA content of the tissues/organs of supplemented birds were significantly higher than the unsupplemented control group. For breast and thigh samples both 1% groups had significantly more DHA than all other treatments with similar levels of enrichment observed for the 1%WL and 1%FP groups. The groups supplemented with 0.5% AURA were enriched to a similar degree with no difference observed between the 0.5%WL and 0.5%FP treatments. In the liver, both 1% treatments were again the most enriched with the duration of supplementation not effecting the level of enrichment. In the kidney, the 1%WL and both FP treatments were enriched to a similar degree, with the 0.5%WL group having significantly less DHA than the 1%WL group. In the skin the 1%WL treatment had significantly more DHA than each of the other treatments, with all remaining supplemented groups enriched to a similar degree. A quadratic relationship between DHA intake and tissue/organ DHA content was observed for the thigh, kidney and liver samples, while a linear relationship was observed for breast and skin samples (Figure 1). The efficiency of DHA transfer from the feed to the breast and thigh was estimated and indicated that feeding for the final 21 days resulted in a more efficient transfer of DHA from the feed to the tissues for both breast and thigh (Figure 2).



Means that do not share a superscript differ significantly. <sup>1</sup> Eicosapentaenoic acid; <sup>2</sup> Docosapentaenoic acid; <sup>3</sup> Docosahexaenoic acid; <sup>4</sup> Total Omega 3 = {C18:3n3+C20:3n3+C20:5n3+C22:5n3+C22:6n3}; <sup>5</sup> Total Omega 6 = {C18:2n6cis+C18:3n6+C20:2n6+C20:3n6+C20:4n6+C22:4n6}. a,b,c Means within a row that do not share a superscript differ significantly.

**Figure 1.** Scatterplots with regression lines for docosahexaenoic acid (DHA) intake (mg/day) against DHA content (mg/100g) detected in breast (**A**) thigh (**B**), kidney (**C**), liver (**D**) and skin with adhering fat (**E**). The r2 value, significance of the relationship (*p*) and model equation are shown for each graph in panel **F**.

The concentration of DPA did not differ significantly between any of the treatment groups for any of the tissues/organs sampled. The highest levels of EPA enrichment were observed in the skin with adhering fat, followed by the kidney, liver, thigh and breast. For each tissue/organ sampled the highest EPA concentration was detected in the 1%WL group, with this group having more EPA than the control group in each case. The 1%FP group also had significantly more EPA than the control group in each case with the exception of the breast tissue samples. The 1%FP group was enriched to a similar degree as the 1%WL group in each tissue with the exception of the skin, in which the 1%WL group had significantly more EPA (Table 5). These differences contributed to significant differences between the groups in terms of their total n-3 PUFA concentration, with groups receiving the 1% AURA treatments generally enriched to a greater degree. For each tissue the C22:4n-6 concentration detected in the supplemented groups was generally significantly lower than the control, however, the same trend was not observed for the total n-6 concentrations, which did not differ between the treatment and control groups. The n-6/n-3 ratio for both 1% treatments were significantly lower than the control group in every tissue/organ sample. Both 0.5% AURA treatments also had significantly lower n-6/n-3 ratios in every tissue/organ samples with the exception of the skin.

**Figure 2.** Mean efficiency of DHA transfer (± 95% C.I.) from feed to the breast (**A**) or thigh (**B**) tissues. Transfer efficiency was estimated for one bird per pen (n = 7 per treatment) as follows; (mg DHA in feed × average daily feed intake x 42 days) ÷ (mg DHA/g breast or thigh × breast or thigh weight) × 100. a,b Columns that do not share a letter differ significantly (*p* < 0.001).

#### **4. Discussion**

In our previous studies, the addition of AURA was found to have no effect on any of the broiler productivity parameters measured [16,17]. In contrast, the 1%WL group in the current study was found to have a lower average weight than the control group by day 42, likely due to a significantly lower average daily feed intake between day 21 and 42. For the whole 42 day period, feed intake did not differ between the groups with no differences in feed conversion ratio, European Production Efficiency Factor, dressing % or breast weight observed. In our previous study, investigating supplementation at up to 5% of the diet, no differences in productivity were observed between the groups [16] which may suggest that the differences observed in the current study were not as a result of the inclusion of AURA. Some other n-3 PUFA rich ingredients have been shown to negatively impact broiler productivity, with the anti-nutritional properties of linseed limiting its inclusion in broiler diets [3]. In contrast, some authors have shown improvements in weight gain, feed intake and FCR when providing omega-3 rich *Schizochytrium* [10,27] or AURA [28]. The effects on the 1%WL group could be considered minor and considering there was no differences between the two groups supplemented for the fattening period only, supplementation with AURA is unlikely to reduce the productivity of broilers in practice.

It has previously been demonstrated that supplementing the diets of broilers with AURA is an effective method that can increase the concentration of DHA in broiler tissues [16,17]. When comparing the results of our previous studies, feeding 0.5% AURA for 42 days resulted in a similar level of tissue enrichment as feeding 1% AURA for 21 days [16,17]. Interestingly, the cost of supplementation (with an approximate AURA cost of €7 per kg) for 42 days at a level of 0.5% of the diet (€0.18 per bird) was lower than the cost of providing AURA at a level of 1% of the diet for 21 days (€0.26 per bird). These studies were carried out in the same research facility, over a two-year period using similar diets, however, differences in the level of enrichment observed between the studies could have been caused by factors other than the duration of feeding. As such, it was important to test the effect of AURA feeding duration on tissue DHA enrichment in a single experiment. Here we found that feeding a lower amount of AURA over a longer period did not lead to similar levels of enrichment as feeding higher levels over a shorter period. Furthermore, we found no effect of duration of feeding (i.e., 21 or 42 days) on the level of enrichment observed at each dose. The cost of supplementing the birds for 21 days at 1% of the diet in the current study would be an estimated €0.23 per bird. Feeding at a rate of 0.5% for the whole life of the bird would be less expensive (€0.16 per bird), but the degree of enrichment achieved remained significantly below that of birds supplemented at a rate of 1% for a shorter duration. Feeding for the final 21-day fattening period only is further supported by the significantly higher transfer efficiencies observed for the FP treatments when compared to the WL treatments. These results indicate that the most efficient feeding strategy is to feed for the final 21-day fattening period only.

The level of enrichment observed in the breast samples of birds supplemented with AURA at a rate of 0.5% fell within the range of values reported from our previous studies (23–35 mg DHA/100 g breast) when broilers were supplemented at the same rate. The DHA content of the thigh samples from the current study (47 and 54 mg DHA/100 g thigh following 21 and 42 days, respectively) exceeded the range of 24–43 mg DHA/100 g thigh reported from our previous studies. A similar trend was observed for the 1% AURA treatments, with the DHA concentration of breast samples from the current study (50 and 55 mg DHA/100 g breast following 21 or 42 days, respectively) and those from the thigh samples (80 and 86 mg/100 g thigh after 21 or 42 days respectively) exceeding the range reported from the breast (36–48 mg DHA/100 g breast) and thigh (42–50 mg DHA/100 g thigh) from our previous trials [16,17]. The fat content of the tissues in the current study was generally higher than those of the tissues from previous studies, increasing their capacity for DHA enrichment. However, the degree of fat deposition has been shown to be unaffected by increasing levels of n-3 PUFA with other nutritional and genetic factors likely responsible for the differences observed [29], between the overall levels of fat detected in the different studies.

Zuidhof et al. [30] investigated the effect of the duration of dietary supplementation of broilers using ground full-fat flaxseed as a source of n-3 PUFA and found that feeding for 24.1 days was the most cost effective duration that achieved an adequate level of tissue enrichment. Kanakari et al. [31] also used flaxseed to supplement broiler diets and showed that feeding for 2–4 weeks prior to slaughter would be sufficient for broilers to accumulate the same amount of n-3 LCPUFA as being fed for six weeks, which is similar to the findings of the current study. When supplementing the diets of broilers with fish oil accompanied by either flaxseed, lard or rapeseed, Konieczka et al., [32] found that feeding for a duration of three weeks before slaughter resulted in significantly higher EPA + DHA concentrations than feeding for one or two weeks only. Our previous work in laying hens indicated that supplementation at a level of 1% of the diet increased the DHA content of eggs for the first 24 days, with no significant increase in the concentration of egg DHA after that point [18]. Overall, these results, using a variety of different n-3 PUFA sources, indicate that feeding for periods longer than 2–4 weeks is not a cost-effective use of the supplemental ingredient.

For a food to be labelled "a source of omega-3" in the European Union (EU) it must contain at least 40 mg EPA+DHA/100 g, while to be labelled "high in omega-3" it must contain 80 mg EPA + DHA/100 g [33]. For the breast meat, both the 0.5%WL and the 0.5%FP groups fell just short of this target with 35 and 33 mg EPA + DHA/100g detected respectively. The thigh samples however, did meet these criteria with 59 mg and 51 mg EPA + DHA/100 g detected in the 0.5%WL and 0.5%FP groups respectively. The breast samples of the 1%WL and 1%FP groups met the criteria to be considered a source of omega-3, with 60 and 54 mg EPA+DHA/100 g detected respectively, while the thigh samples could be considered high in omega-3 with 94 and 87 mg EPA+DHA/100 g detected respectively. Based on these results, the broilers would need to be supplemented at a rate of 1% of the diet so that both the breast and thigh meat could be labelled as "a source of omega-3" in the EU.

No EPA was detected in any of the diets provided, but despite this, an increased inclusion of AURA generally led to increased tissue concentrations of EPA. Similar results have been obtained in our previous trials [16,17], with the increase likely due to the retro-conversion of DHA to EPA [34]. In contrast, no differences were observed between the treatments in terms of the tissue n-3 DPA concentration. This is in keeping with the results of our previous study, when supplementing broilers with 0, 0.25, 0.5 and 1 % AURA, resulted in no differences in n-3 DPA concentrations between treatments in breast tissue samples, while in the thigh samples, the 0.5% treatment had significantly less n-3 DPA than the control group with no differences observed between the other groups [17]. Small but significant increases in breast n-3 DPA were observed when birds were supplemented with AURA at a rate of 2.5% and 5% of the diet with 10.19 and 10.52 mg n-3 DPA/100 g detected respectively, while at 0 and 0.5% of the diet 9.02 and 9.02 mg n-3 DPA/100 g were detected respectively. It does not appear that this increase in tissue n-3 DPA is due to some form of retro-conversion from DHA. It could more likely be a result of the elongation of EPA, which was present in higher concentrations in the 2.5 and 5% treatments. The elongation of EPA to n-3 DPA is more efficient than the elongation of EPA to DHA [35]. In humans, supplementary DHA from an algal source (containing no EPA) was found to significantly increase blood EPA concentrations while reducing the concentration of n-3 DPA indicating that DHA supplementation could negatively influence the concentration of n-3 DPA, possibly as result of competition at the level of fatty acid esterification [36]. These findings indicate that supplementary DHA-rich AURA, when provided at a level of 0.5–1% of the diet can also increase the tissue concentrations of the beneficial fatty acid EPA, despite it being absent from the experimental diets.

The significant increases in both EPA and DHA contributed to the higher total n-3 PUFA concentrations detected in many of the tissue/organ samples. The concentration of C20:4n6 was significantly lower in most of the tissue/organ samples from supplemented birds, however, this did not translate into lower total n-6 PUFA concentrations in any of the tissues/organs. Importantly, supplementation led to lower n-6/n-3 ratios, in every treatment and in every tissue/organ with the exception of the WL skin with adhering fat treatments. Reductions in a person's overall n-6/n-3 ratio have been shown to improve the health outcomes of patients suffering from a variety of illnesses, including cardio-vascular diseases and some forms of cancer, with the effective ratio based on the type of illness in question [37]. Consuming this AURA enriched broiler meat could help to reduce the overall n-6/n-3 ratio of a person's diet.

No attempt was made to assess the effect of supplementation on the consumer acceptability of the meat of supplemented birds. However, supplementing broiler diets with marine protists at a level of 2.8% of the diet was previously found to have no impact on consumer acceptability [14]. As the inclusion rates observed in this trial were below this, it is unlikely that the consumer acceptability would be affected. However, it would be beneficial to determine the consumer acceptability of these enriched meats when fresh and following a period of storage. Recent work investigating the frozen storage stability of DHA found significant reductions in the tissue DHA content after a period of 12 or 24 weeks [26]. Chicken meat for human consumption is unlikely to be stored for this extended period of time and over this duration the DHA concentration of breast meat decreased by 35%, 35%, 29.4% and 27.8% in broilers supplemented with AURA at a rate of 0%, 0.5%, 1.5% and 2.5% of the diet. As such, it would be beneficial to establish the effects of storage on the stability of DHA and the consumer acceptability of the AURA enriched broiler meat over a shorter time period.

#### **5. Conclusions**

These results indicate that providing AURA as a dietary supplement for broilers for the final 21-day fattening period can improve the nutritional quality of broiler meat by significantly increasing the DHA and EPA concentrations of tissues and organs, as well as lowering the n-6/n-3 ratio without significantly impacting productivity.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-2615/9/6/327/s1. Table S1: The full fatty acid profile of breast, thigh, liver, kidney and skin (with adhering fat), taken from birds fed diets supplemented with A. limacinum at a rate of 0, 0.5 or 1% for their whole life (WL; day 0–42) or for the fattening period only (FP; day 21–42).

**Author Contributions:** Conceptualization and methodology, C.A.M., M.M. and G.F.; supervision, M.M and G.F.; project administration, C.A.M. and M.M.; data curation, M.M. and J.D.K.; formal analysis, M.M., J.D.K. and G.F.; writing—original draft preparation, M.M. and J.D.K.; writing—review and editing, C.A.M., J.D.K., M.M., and G.F.

**Funding:** The research was funded by Alltech SARL (France).

**Acknowledgments:** The authors would like to express their gratitude to the following: Rebecca Timmons (Alltech Inc., USA) for her technical input on *Aurantiochytrium limacinum* and Tuoying Ao (Alltech Inc., USA) for his knowledge of the application of *A. limacinum* in broiler nutrition.

**Conflicts of Interest:** The authors C.A.M. and J.D.K. are employees of Alltech which produces and markets ALL-G-RICH®, the commercial product used in this study

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

### **E**ff**ect of Dietary Modulation of Selenium Form and Level on Performance, Tissue Retention, Quality of Frozen Stored Meat and Gene Expression of Antioxidant Status in Ross Broiler Chickens**

**Doaa Ibrahim 1,\*, Asmaa T.Y. Kishawy 1,\*, Safaa I. Khater 2, Ahmed Hamed Arisha 3, Haiam A. Mohammed 3, Ahmed Shaban Abdelaziz 4, Ghada I. Abd El-Rahman <sup>5</sup> and Mohamed Tharwat Elabbasy 6,7**


Received: 23 April 2019; Accepted: 5 June 2019; Published: 11 June 2019

**Simple Summary:** Although the importance of usage of selenium as essential trace element in poultry production has been proven, the best source and level has not been fully addressed yet. Three different dietary selenium forms with three different levels were chosen to be added in broiler diet. Met-Se or nano-Se up to 0.6 mg/kg increased their performance and was more efficiently retained in the body than SeS. Frozen stored meat quality was improved in a dose-dependent manner especially with both Met-Se and nano-Se. Nano-Se was more potent than Met-Se, which in turn was more potent than inorganic Se against oxidative stress, which improved the quality of meat under frozen conditions.

**Abstract:** This study compares between different selenium forms (sodium selenite; SeS, selenomethionine; Met-Se or nano-Se) and levels on growth performance, Se retention, antioxidative potential of fresh and frozen meat, and genes related to oxidative stress in Ross broilers. Birds (n = 450) were randomly divided into nine experimental groups with five replicates in each and were fed diets supplemented with 0.3, 0.45, and 0.6 mg Se/kg as (SeS, Met-Se), or nano-Se. For overall growth performance, dietary inclusion of Met-Se or nano-Se significantly increased (*p* < 0.05) body weight gain and improved the feed conversion ratio of Ross broiler chicks at the level of 0.45 and 0.6 mg/kg when compared with the group fed the same level of SeS. Se sources and levels significantly affected (*p* < 0.05) its concentrations in breast muscle, liver, and serum. Moreover, Se retention in muscle was higher (*p* < 0.05) after feeding of broiler chicks on a diet supplemented with Met-Se or nano-Se compared to the SeS group, especially at 0.6 mg/kg. Additionally, higher dietary levels from Met-Se or nano-Se significantly reduced oxidative changes in breast and thigh meat in the fresh state and after a four-week storage period and increased muscular pH after 24 h of slaughter. Also, broiler's meat in the Met-Se and nano-Se groups showed cooking loss and lower drip compared to the SeS group (*p* < 0.05). In the liver, the mRNA expression levels of glutathione peroxidase, superoxide dismutase, and catalase were elevated by increasing dietary Se levels from Met-Se and nano-Se groups up to

0.6 mg/kg when compared with SeS. Therefore, dietary supplementation with 0.6 mg/kg Met-Se and nano-Se improved growth performance and were more efficiently retained than with SeS. Both sources of selenium (Met-Se and nano-Se) downregulated the oxidation processes of meat during the first four weeks of frozen storage, especially in thigh meat, compared with an inorganic source. Finally, dietary supplementation of Met-Se and nano-Se produced acceptable Se levels in chicken meat offered for consumers.

**Keywords:** broilers; selenium sources-levels; selenium retention; antioxidant capacity; frozen meat

#### **1. Introduction**

Selenium (Se) is an important trace nutrient for the maintenance, growth, and animals and humans health [1]. It improves the nutritive value and of meat quality [2]. As feed additives, Se can enhance growth productivity in broiler chickens [3]. Selenium is an important constituent of at least 25 selenoproteins involved in various physiological processes, including immune function, reproduction, and the maintenance of antioxidant defenses to avoid tissue damage. Selenium deficiency results in a number of disorders and injuries in poultry, such as skeletal myodegeneration, exudative diathesis (ED), muscular hemorrhages, atrophy of pancreas, decreased production of eggs, liver injury, reduced hatchability, and inhibited growth of bursal and thymic [4], and increase susceptibility of humans to certain degenerative diseases, such as cancer [5]. The fortification of poultry meat with Se represents a viable strategy for increasing human intake of Se. The national research council (NRC) [6] recommendations established a low selenium level (0.15 mg/kg) for the supplementation of broilers. This level is not adequate to avoid production losses resulted from selenium deficiency disorders [7]; consequently, there is a need to increase dietary selenium levels. Moreover, Se bioavailability not only depends on its physical form but also on dietary concentration and the levels of other trace elements. Excess levels of Se can be toxic when provided above the biological requirement. Thus, meeting Se requirements and optimizing performance is an important step in modern poultry production. Practically, selenium can be added for poultry's diet in the form of inorganic Se, organic Se, and most recently, nano-Se. The inorganic form of selenium (Se selenite) is primarily and commonly used for dietary supplementation, and exhibits a very narrow border between its dietary requirement and its toxicity [8]. Recently, it has been recognized that organic Se has a higher rate of tissue retention and bioavailability thus lower toxicity than inorganic Se, so it is preferable to inorganic Se in broilers [9]. In addition, organic Se is deposited more efficiently in breast muscle than inorganic forms [10]. With the development of nanotechnology, nano-Se has attracted widespread research interest due to its high catalytic efficiency and higher adsorbing capacity, and has exhibited strong absorption efficiencies and lower toxicity than inorganic Se [11]. Moreover, recent studies found that nano-Se has a higher effectiveness in controlling selenoenzymes and displays less toxicity than selenium-selenite [12]. Moreover, supplementation of dietary Se could also enhance oxide dismutase (SOD), glutathione peroxidase, (GPx) and catalase (CAT) activities, and reduce oxidative stress and lipid peroxidation biomarkers, consequently reducing oxidative stress in broilers [13]. Moreover, Se plays a key role in the signaling of redox via removal of hydrogen peroxide and lipid hydroperoxides using glutathione as an ultimate electron donor [14]. These antioxidant properties of Se have also been shown to continue in postmortem muscle tissue and prevent lipid oxidation [15]. For this reason, many dietary regimes in animal nutrition have been established to produce Se-enriched meat in order to increase human Se consumption [16]. The type and level of available Se is important to meet broilers' dietary requirements and optimize their production without producing any hazardous effects on broilers or human health. Definitive comparative studies to fully exploit the benefits of dietary supplementation with different available Se sources and levels in Ross broilers remains poorly investigated. Thus, the aim of the present study was to compare the bioavailability of different levels and sources of Se on performance,

Se retention, lipid oxidative stability of meat, meat quality, and mRNA expression of some selected genes related to antioxidant capacity in Ross broiler chickens.

#### **2. Materials and Methods**

#### *2.1. Selenium Sources*

Sodium selenite (SS) and selenomethionine (Met-Se) were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO, USA and Sel-Plex; Alltech Inc., Nicholasville, KY, USA, respectively. nano-Se powder was prepared according to [17] where 4 mL of 25 mM GSH containing 15 mg of bovine serum albumin were mixed with one ml of 25 mM sodium selenite. The resulting red suspension was dialyzed against double-distilled water for 96 h. Every 24 h, the water was replaced to isolate the oxidized GSH from the nano-Se. Then, nano-Se and bovine serum albumin were lyophilized. The phase characterization and morphology of nanoparticles were analyzed by means of X-ray diffraction (XRD) using EMPYREAN diffractometer and JEM-200CX transmission electron microscopy (TEM) working at 30 kV as shown in (Figure 1). XRD patterns corresponding to the (100), (101), (110), (102), (111), (201), (003), (202), (210), and (211) planes of the formed nano-Se were observed at 20 angles of 23.6◦, 29.9◦, 41.4◦, 43.8◦, 45.4◦, 51.8◦, 55.9◦, 61.8◦, 65.3◦, and 68.3◦, respectively [18]. The nano-Se was spherical in shape with average size 42 ± 1.4 nm (total count of 100 NPs).

**Figure 1.** Characterization of nano-Se (**A**,**B**): (**A**) Morphology of the formed nano-Se pictured by transmission electron microscopy (TEM) and; (**B**) X-ray diffraction (XRD) pattern of the nano-Se.

#### *2.2. Birds and Experimental Procedures*

Four hundred and fifty, one-day-old, Ross broiler chicks (Ross 308) were individually weighed and divided to nine dietary groups, each group consisting of five replicates of ten chicks each per floor pen. Broiler chicks were fed diets containing inorganic Se (sodium selenite; SeS), organic Se, (selenomethionine, Met-Se), or nano-Se, each at three levels 0.3, 0.45, or 0.6 mg/kg Se (as fed). The basal diet was formulated to meet nutrient requirements of Ross broilers according to [19] except Se (Table 1). Diets were fed from 1 to 38 d including starter (1–11 d), grower (12–22 d), and finisher (23–38 d) diets. All chicks were given ad libitum access to feed and water. The environmental temperature was kept at 32 ◦C for the 1st week and then gradually decreased until reached 23 ◦C. All the experimental procedures were performed at the Institute of Nutrition and Clinical Nutrition and Poultry Farm following the Faculty of Veterinary Medicine guidelines and in accordance with the protocols approved by Institutional Animal Care and Use committee at Zagazig University (Approval no: ZU-IACUC/2/F/123/2018).

The proximate analysis of feed ingredients was done according to the standard method of [20]. For Se analysis in feed, one gram of feed was heated for 5 h in a furnace at 550 ◦C for ashing. Mixture from 3 N HCl (10 mL) and an ashed sample was heated until the solution became clear. After cooling, the sample was filtered and diluted to 50 mL with 0.1 N HCl. For analysis of selenium, lanthanum 185.4 L 50 gm/kg was added to 6 mL of the sample solution. Then, analysis was achieved by a spectrometer at a wavelength of 400 nm [20].


**Table 1.** The ingredients and nutrient levels of the basal diet (on dry matter basis).

\* Provided for each kilogram of diet: Vitamin A, 10,000 IU; vitamin E, 7200 IU; vitamin D3, 3000 IU; vitamin K, 2 mg; vitamin B1, 2640 mg; vitamin B6, 1200 mg; calcium pantothenate, 10 mg; nicotinamide, 50 mg; biotin, 40 mg; choline chloride, 500 mg; folic acid, 0.5 mg; cobalamin, 0.01 mg; calcium, 9000 mg; manganese, 120 mg phosphorus, 2100 mg; sodium, 3700 mg; iron, 110 mg; copper, 10 mg; zinc, 100 mg; iodine 1.1 mg. <sup>b</sup> Calculated values except selenium.

#### *2.3. Growth Parameter Measurement*

Live body weight (LBW) and feed intake of broiler chicks/pen were estimated individually at 21 and 38 d of age to calculate live body weight, body weight gain (BWG), total feed intake, feed conversion ratio (FCR) and relative growth rate (RGR).

#### *2.4. Sampling and Analytical Procedures*

At the end of the feeding trail, tissues samples (liver and breast meat) were collected from five birds/replicates that were slaughtered (slaughtering house under supervision of Institutional Animal Care and Use Committee at Zagazig University Faculty of Veterinary Medicine) and handled and kept at −20 ◦C until analysis of selenium content and meat quality tests. Blood samples were collected with or without anticoagulant, then plasma and serum were kept at −20 ◦C until the analysis of selenium content and chemical analysis was performed.

#### 2.4.1. Tissue Retention of Selenium

Briefly, liver and breast muscle were weighed (0.1 g) and mixed with of HNO3 (8 mL) then digested by microwave. After that, deionized water was added to produce a 10 mL volume. The selenium content was determined following the procedure of [21] by atomic absorption spectrophotometer (Shimadzu Ltd., Shimane Shimadzu, Japan).

#### 2.4.2. Selenium Content in Serum Constituents

Selenium content was measured in serum by atomic absorption spectrophotometer (AA6501, Shimadzu Ltd., Japan). Plasma samples were used for measuring of aspartate amino transferase (AST), alanine glutamyl transferase (ALT), and creatinine calorimetrically by diagnostic kits (MAK055, MAK052, and C4255, respectively) manufactured by Sigma-Aldrich.

#### 2.4.3. Laboratory Analysis for Meat Quality

#### Meat pH and Drip and Cooking Loss in Meat Samples

Breast meat was used to determine postmortem pH (t = 0.5 and 24 h) by pH meter. Drip loss was estimated according to [22] (percent; proportional weight loss of a sample suspended for 72 h in a closed plastic bag under refrigerated conditions at 4 ◦C). After storage at −20 ◦C, cooking loss was determined (percent; weight loss proportionate of a sample after cooking for 40 min in a water bath at 70 ◦C followed by cooling).

#### Preparation of Samples for Total Antioxidant Capacity

Six hours after slaughter and handling, breast meat was cut into cubes of approximately 3 cm square); visible connective tissues and fat were removed. These muscle cubes mixed with distilled water then homogenized and centrifuged and used for measuring total antioxidative markers as free radical scavenging assay using 2,2 -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) thiobarbituric acid reactive substances (TBARS) assay, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and ferric reducing/ antioxidant (FRAP) assay.

Thiobarbituric acid-reactive assay: Oxidation was evaluated on the first day and after one week from storage by the thiobarbituric acid assay described by [23]. Perchloric acid (27 mL, 3.83% v/v) was added to of meat sample (5 g) then homogenized for 1 min and filtered by filter paper, then 2 mL thiobarbituric acid was added to supernatants and incubated in a water bath (100 ◦C) for 20 min. Subsequently, immediate cooling to room temperature and centrifugation for 15 min was performed, then the absorbance was read by the spectrophotometer at 532 nm. The results were then calculated according to the standard curve and values were expressed as mg malondialdehyde (MDA)/kg meat.

ABTS assay: The total antioxidant capacity of chicken breast and thigh meat was analyzed by Trolox-equivalent antioxidant capacity (TAC) assay [24]. Briefly, the reaction between 14 mM ABTS [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] with an equal volume of 4.9 mM potassium persulfate was catalyzed to stimulate the formation ABTS+ radical cation formation, then incubated in the dark at room temperature for 12–16 h. After that, 10 μL of meat homogenate was added to the ABTS+ solution (1.0 Ml) and mixed thoroughly and after 60 s absorbance was read at 734 nm.

DPPH assay: The scavenging activity of the muscle samples was analyzed by 1,1-diphenyl-2 picrylhydrazyl radical (DPPH) [25]. Briefly, the meat samples were homogenized in distilled water and then centrifuged. The supernatant was mixed with ethanol and DPPH radical solution and incubated in a dark room for 10 min. Next, the absorbance measurement was read at 517 nm. The ability to scavenge the DPPH radical was expressed as μM per g of wet muscle tissue.

FRAP assay: Ferric reducing antioxidant power (FRAP) assay [26] was carried out on meat homogenates. The meat samples were homogenized in potassium phosphate buffer, centrifuged, and the supernatant was collected. Then, supernatant (1 mL) was collected and added to FRAP buffer (3 mL) containing 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine) in 40 mM HCl, and 20 mM Fe2Cl3 was added to 300 mM acetate buffer. Immediately after mixing, the absorbance was measured at 593 nm. A standard curve was prepared with FeCl2. The antioxidant power of the samples was expressed as μM of Fe2<sup>+</sup> per 1 g wet muscle tissue.

#### *2.5. RNA Extraction, Reverse Transcription, and Quantitative Real-Time PCR.*

At the end of the feeding trial (day 38), three birds per group were randomly selected, marked and injected with tert-butyl hydroperoxide, 0.2 mmol/kg body weight, intraperitoneally purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA, CAS Number 75- 91-2) to induce the oxidative stress. Birds were slaughtered, and liver samples were collected 48 h post-injection. From liver tissue, the total RNA was extracted by RNeasy Mini Kit; Qiagen, Cat. No. 74104. according to the manufacturer regulation. The extracted RNA was quantified using the NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA). The first strand cDNA was synthesized using kits of RevertAidTM H Minus (Fermentas Life Science, Pittsburgh, PA, USA). One μL of this cDNA was blended with 12.5 <sup>μ</sup>L of 2<sup>×</sup> SYBR® Green PCR mix with ROX from BioRad, 5.5 <sup>μ</sup>L of RNase free water, and 0.5 μL (10 pmol/μL) of each forward and reverse primer for the selected genes were added. The primers' sequences of catalase, glutathione peroxidase, and superoxide dismutase genes involved in antioxidant function were designed as previously described in [27]. The real-time PCR amplification was carried out with Rotor-Gene Q2 plex (Qiagen Inc., Valencia, CA, USA) with the following conditions; initial denaturation at 95 ◦C for 10 min and 40 cycles at 95 ◦C for 15 s and 60 ◦C for 1 min. Relative fold changes in the expression of target genes measured in triplicate were estimated by the comparative 2−ΔΔCt method with the GAPDH gene as an internal control to normalize target gene expression levels [28].

#### *2.6. Statistical Analysis*

Data were submitted to a 2-way ANOVA, using PASW statistics 18 (SPSS Inc., Chicago, IL, USA) to clarify the effects of dietary Se sources, its levels, and their interaction. Gene expression data were statistically analyzed using one-way ANOVA and relevant figures were generated by Graphpad Prism 7 (GraphPad Software Inc., San Diego, CA, USA). Tukey's test was used to separate the means when the treatment difference was significant (*p* < 0.05). All data were expressed as the mean ± SEM. Statistical significance was considered at *p* ≤ 0.05.

#### **3. Results and Discussion**

#### *3.1. Growth Performance*

The effects of different dietary treatments on overall growth performance parameters (1–38 days) are presented in Table 2. The present study showed that the interaction between different dietary sources and levels of Se had significant (*p* < 0.05) effect on the body weight and gain of broilers at 38 days. The groups supplemented with selenomethionine (Se-Met) and nano-Se showed a significant increase (*p* < 0.05) in body weight and gain of Ross broiler chicks when compared with selenite selenium (SeS). Moreover, variety of levels and sources of Se played an important role in our study as when Se- Met or nano-Se were added to diets, body weight and gain increased as dietary Se levels increased, while higher levels in the SeS group at Se concentration of 0.3–0.45 mg/kg diet caused declines as dietary SeS levels increased. Different sources and levels of Se had no effect on feed intake (*p* < 0.05). Feed conversion ratio (FCR) of broilers was affected by the Se sources and levels as they play an important role in improving FCR. The FCR was improved by dietary supplementation of Se-Met or nano-Se, while the best FCR was established with a level of 0.6 mg Se/kg diet from Se-Met

or nano-Se, followed by groups supplemented with 0.3–0.45 mg/kg from Se-Met or nano-Se. Our data demonstrated that the application of dietary Se-Met or nano-Se up to 0.6 mg/kg resulted in the maximum growth rate of broiler chicks, while the same dose from SeS tended to reduce the growth performance of broilers chicks. These results proved that such selected dietary Se-Met or nano-Se levels had higher bioavailability than inorganic forms of Se. These results are in agreement with [29] who found a decline in body weight gain and feed utilization as supplemental inorganic Se increased, while for nano-Se, average daily gain, FCR, and survival ratio reached their highest levels at an Se concentration of 0.15–1.20 mg/kg. [13] Showed that feeding of broilers on 0.3 and 0.5 mg/kg from nano-Se significantly improved FCR and increased tissue selenium content. Our findings were also in agreement with those of [30], who described no differences in feed intake among broilers fed diets supplemented with either organic or inorganic forms of Se. Broilers chicks groups fed on 0.2 mg/kg diet from organic selenium or nano-Se had a similar growth rate as compared to the group supplemented with the same level of Se-selenite [31]. Our findings were also in agreement with those of [32] who reported that increased selenium levels had improved average daily gain in the same time there was no differences on average daily gain between nano-selenium and organic selenium in broiler chickens. The function of Se on growth rate may relate to its role in the selenoprotein P and selenoenzymes type I iodothyronine deiodinase expression, which have critical roles in the synthesis of thyroid hormones and Se transport [33]. Moreover, our results of increased growth performance with selenium methionine and nano-Se could possibly due to an increased thyroid hormone regulating the body's energy metabolism and increased digestibility of protein [34]. The results of this study suggest that different Se sources and levels may be necessary to optimize the performance of broilers, and that the form of organic Se may be of importance.

**Table 2.** Effects of dietary sources and levels of Se (mg/kg) on growth performance of broilers over 38 days.


SeS = sodium selenite; Met-Se = selenomethionine; nano-Se = nano-selenium; BW = body weight; BWG = body weight gain; FI = feed intake; FCR = feed conversion ratio, RGR = relative growth rate. a,b,c Means within a row carrying different superscript letters denote significant differences (*p* < 0.05).

#### *3.2. Selenium Retention in Serum, Muscle, and Liver*

In the present study, the different Se sources and levels had significantly affected (*p* < 0.05) Se concentrations in serum, liver, and breast muscle of broilers (Table 3). The groups fed on a diet supplemented with nano-Se and Met-Se showed higher (*p* < 0.05) serum, liver, and breast muscle Se concentrations when compared with those fed diets supplemented with SeS, indicating that nano-Se and Se-Met were better retained in the body than SeS, although the effect of Se-Met was more prominent for tissue Se retention than nano-Se at the same lowered level. Accumulation of minerals in tissues is considered an indicator for mineral utilization [35]. The concept of increasing Se content in human foods by altering dietary Se sources and level given to livestock is now of interest to nutritionists [36]. Wang et al. [37] stated that transport and uptake of selenium by broiler intestinal cells were higher in nano-Se than that of SeS. The difference in retention of Se between Se yeast and SeS or nano-Se may be clarified by the probable metabolic pathways and absorption process for Se from different Se sources [29]. The safe limit of Se in human food has been established at 2.0 mg/kg for the United States [38]. This level agreed with our results that up to 0.6 mg/kg of Se in broiler diets precipitates less than 1 mg/kg in meat with all sources of Se. Selenium uptake from Se-selenite occurs by passive diffusion contributing the poor availability of Se-selenite [39], and up to 50–75% of consumed Se-selenite

is lost through urine. Another limitation of adding selenite to feed is the short period storage of Se in the animal's body [40]. Our results of Se retention in tissue in accordance with those of [41], who demonstrated that broiler chicks fed on dietary organic Se had higher (*p* < 0.05) Se content in breast muscle and liver than those fed diets fortified by SeS. [36] also proved that the contents of Se in liver and muscles were affected by dietary Se supplementation, and retention of Se was increased when organic Se was supplemented as compared with inorganic Se. Cai et al. [13] stated that increasing dietary nano-Se increased the concentration of selenium in liver and muscle tissue (*p* < 0.01). An explanation for increased tissue content from nano-Se may be attributable to improved intestinal absorption of nano-Se due to smaller particle size and larger surface area [42]. SeS and nano-Se, on the other hand, are changed to the transitional selenide and then employed for synthesis of selenoprotein or methylated and after that excreted. However, Met-Se contains a large amount of selenomethionine. When recognized as a Se species, it can be altered to selenocysteine through the trans-selenation pathway and then lysed to selenide. So Met-Se might be simply utilized in the tissue than SeS or nano-Se [43]. Another property of Met-Se involves the chemical similarity between Met-Se and Met, which permits the body to use them interchangeably in protein synthesis as Met-tRNA cannot distinguish between Met and Met-Se, which makes it possible to build Se reserves in the body [2].

#### *3.3. The E*ff*ect of Di*ff*erent Levels and Sources of Se on Selected Serum Parameters*

The activity of liver enzymes including ALT and AST were not significantly affected by the interaction between different levels and sources of Se. The same trend was recorded for serum creatinine values (Table 3). Selim et al. [44] stated that activity of liver enzymes including ALT and AST were not significantly affected by addition of Zn-Se-Meth, P-Nano-Se, or L-nano-Se in broiler diets. Moreover, increasing the supplemental Se level from 0.3 to 0.45 ppm in broiler diets could not cause any significant difference in plasma creatinine level. In previous studies, [44] found that liver enzymes were not affected by adding different forms of Se (inorganic, organic, or nano) at levels up to 0.3 mg Se/kg diet.



means ± standard error.

#### *3.4. Antioxidant Potential of Di*ff*erent Sources and Levels of Se*

These data showed that the expression pattern of selected antioxidant-related genes (glutathione peroxidase, GPx, super oxide dismutase, SOD and catalase, CAT) in relation to different Se levels and sources was addressed in (Figure 2). The expression of GPx mRNA significantly increased in groups fed nano-Se at 0.6 mg/kg diet followed by groups supplemented by 0.3 and 0.45 mg/kg diet from Met-Se and nano-Se when compared with SeS with the same levels. The highest expression of SOD was observed in groups supplemented with 0.45 and 0.6 mg/kg diet from nano-Se followed by the group supplemented by a 0.3 mg/kg diet from nano-Se and groups supplemented by 0.45 and 0.6 mg/kg diet from Met-Se, when compared with SeS supplemented group. The mRNA expression of catalase significantly increased with an increasing level of nano-Se and Met-Se when compared with SeS. The antioxidant enzymes such as SOD, CAT, and GPx [45], and non-enzymatic constituents such as glutathione (GSH) [46], play an important role for keeping the animal health, and physiological antioxidant systems. Selenium is a cofactor in several selenoproteins and the antioxidant selenoenzymes as glutathione peroxidase (GPx), thus its functional role is associated with the Se concentration in tissues [47]. Xiao et al. [48] demonstrated that the supplementation of Se in the maternal diet significantly (*p* < 0.05) enhanced the activity of GPx, T-SOD, and CAT in heat stress treated chick embryos when compared with the basal diet, as the levels of GPx1 mRNA were significantly (*p* < 0.05) elevated by adding Se. This may be clarified by higher Se retention in maternal Met-Se treatment [49], which aids in the production of more selenoproteins to preserve chick embryos with a higher antioxidant level. Under heat stress, [35] reported that the addition of organic Se significantly improved GPx activities as compared with broilers fed with inorganic Se. [50] established that the highest GPx activity and lowest MDA content in blood and testis were attained in the treatment of 0.5 mg/kg, as the GPx enzymes were involved in scavenging toxic H2O2 [51]. In animal research, the activity of GPx enzymes and their expression genes in tissues were correlated with the concentration of Se added to feed [47]. This finding is also in accordance with [52], who described that Se deficiency caused the reduction of GPx mRNA levels in four GPx genes found in chicken livers. The superoxide dismutase (SOD) and CAT are important antioxidant enzymes for poultry. The superoxide anion is transformed to H2O2 by SOD [53], and CAT changes H2O2 into water [54], although Se is not a component of SOD and CAT. Our results also indicated that nano-Se and Met-Se increased the mRNA expression of these genes. Yuan. [49] showed that in broiler breeding experiments, hepatic GPx1 and TrxR1 mRNA levels in Met-Se groups were higher (*p* < 0.05) than that in the SeS group.

**Figure 2.** Effects of dietary Se source and level on the relative antioxidant enzymes expression (**A**–**C**). (**A**) Glutathione peroxidase (GPx); (**B**) super oxide dismutase (SOD); and (**C**) catalase (CAT) in the liver of broiler chickens at 40 days. SeS = sodium selenite, Met-Se = selenomethionine, nano-Se = nano-selenium. a,b,c,d Different superscript letters denote significant difference (*p* < 0.05). Values are means ± standard error.

#### *3.5. E*ff*ect of Di*ff*erent Se Sources and Levels on Meat Quality*

The role of diets supplemented with different Se sources and levels on breast meat quality in broiler chickens are shown in Table 4. Compared with SeS, dietary Met-Se and nano-Se inclusion in broiler diet improved meat quality, especially as Se levels increased from 0.45 to 0.6 mg/kg.

#### *3.6. Post-Mortem pH of Meat, Cooking Loss and Drip Loss*

Breast meat from groups that received an increased level of Met-Se and nano-Se exhibited increased (*p* < 0.05) pH 0.5 and 24 h later when compared with the SeS groups. In addition, birds in the Met-Se and nano-Se groups, specially at high levels, had lesser drip and cooking loss group (*p* < 0.05) compared to those in the SeS groups. The presence of Se in animal diets are a key influence on meat water retention, with the form and level regulating the variation in meat drip loss [55]. The results of our study agreed with [56], who found that the drip loss was lower and water-holding capacity was higher in pigs fed with organic selenium. It has been reported by some authors that the mechanism by which antioxidants modify the water-holding capacity and drip loss of meat can be attributed to their ability to maintain muscle membranes' integrity post-mortem [57], while others have suggested that proteolysis and protein oxidation acts as an essential factor for determining the moisture retention of meat [58]. Lambert et al. [59] reported that the accumulation of a large amount lactic acid in the muscles, combined with a cessation of blood circulation which induces cellular hypoxia and results in a decreased pH after slaughter, changed the permeability of cell membrane and decreased the water-holding capacity. But our study demonstrated increased water-holding capacity of breast meat in broilers fed on Met-Se and nano-Se. This may be explained by the metabolic conversion of glucose to lactic acid in post-mortem muscle being delayed with organic Se or nano-Se supplementation, thus improving the water-holding capacity of meat and decreasing drip loss [60]. It has been reported elsewhere that 0.3 mg/kg Met-Se supplementation resulted in an increase in the pH of the breast meat of broilers [61] and in geese [62] as compared with 0.3 mg/kg SS supplementation. Other studies demonstrated that water-holding capacity is affected by organic Se supplementation [63] and nano-Se [64]. Cai et al. [13] proved that application of nano-Se increases the ability of broiler muscle proteins to attract water, thus reducing drip loss percentage. The present study further indicates that the role of Met-Se and nano-Se on the biochemistry of muscle tissue is more prominent than with SeS.

#### *3.7. Thiobarbituric acid Reactive Substances (TBRAS) Content of Meat as a Marker for Lipid Oxidation*

Frozen storage of all analyzed meat significantly increased (*p* < 0.05) the TBRAS values in breast and thigh meat, with the lowest values for TBRAS recorded in breast meat, which could be related to the total lipid content. With increasing dietary level of organic Se and nano-Se, the TBRAS values decreased in breast and thigh meat when compared with SeS supplementation (Table 4). Exposure to different physiochemical or pathological conditions has recently been shown to be one of the main predisposing agents controlling free radical formation in the body [65]. On the other hand, chicken meat enriched with polyunsaturated fatty acids (PUFA) augmenting the meat susceptibility to oxidation progressions [66]. Bakhshalinejad [32] reported that oxidation resistance of broiler meat was higher in case of supplementation of organic of Se and the higher concentration of Se the higher glutathione peroxidase activity, total antioxidant capacity and malondialdehyde formation. Oxidation of lipids produces free radicals, leading to mutagenesis, carcinogenesis, and aging of the cell [67]. The antioxidant role of Se has also been shown to continue post-mortem in muscle tissue, where it is reported Se reduced oxidization of lipids in meat and had an effect on its quality [15]. Providing Se-enriched meat for human consumption by manipulating animal feed therefore also protects the quality of meat [68]. In this respect, [69] showed that inclusion of Se in poultry diets provides Se-enriched meat and protects the meat from oxidation after slaughter, increasing the stability of the meat against various storage conditions which accelerate the oxidation processes that destroy membrane lipids, consequently reducing the meat's nutritional value [70]. Similarly, higher protection of muscle samples against lipid oxidation have been demonstrated by Se yeast with broilers [61] and turkey meat [71]. In addition, the breeders' diet supplemented with Se also provides antioxidant protection of lipid rich tissues, which was detected by lower TBARS values after slaughter [72]. Calvo et al. [73] found that birds supplemented with organic Se had lower malondialdehyde (MDA) concentrations in muscle samples than the SeS group with the same storage time. In agreement with our results on muscle pH, it has been reported that the pH reduction could accelerate lipid oxidation due to the enhanced autoxidation of hemoglobin at reduced pH [74]. With decreasing muscle pH, higher TBARS values have been reported [75].

#### *3.8. Total Antioxidant Capacity of Meat*

The presence of antioxidants in poultry meat is a powerful factor influencing its quality. Once antioxidant defense systems are debilitated, dysfunction of all body cells and tissues may occur. Thus to keep body functions optimal, antioxidant levels are important [76]. As Se plays major role in protecting cells against oxidative stress, measuring the antioxidant biomarkers is a beneficial tool for evaluating the Se antioxidative role. In the present study ABTS, DPPH, and FRAP assays were used to estimate antioxidant capacities, as theses assay reflect the antioxidant properties of meat [77].

DPPH Assay: Thigh meat was characterized by significantly higher DPPH free radical scavenging ability than breast meat. The supplementation of nano-Se and organic Se at higher levels (0.6 mg/kg) into the Ross broiler diet increased the ability of meat to scavenge free radical DPPH and this capacity increased with the storage period (Table 4). Using specific sources from selenium in poultry diet increases the meat's ability to scavenge the free radical DPPH, due to Se antioxidative functions. During frozen storage, the removal ability of DPPH augmented in all examined samples of chicken meat, demonstrating that Se is stable in the meat [78].

ABTS Assay: The ability of breast and thigh meat to scavenge free radical ABTS were affected by dietary inclusion of Met-Se and nano-Se up to 0.6 mg/kg. During frozen storage, the ability of the meat parts to remove the free radical ABTS tended to increase, reaching the highest values after four weeks of storage (Table 4). These results agree with [78], who stated a higher antioxidative potential of chickens breast to remove free radicals tended to increase during frozen storage, reaching the highest values after storage period of 90 days. This can be accompanied by moisture loss as a result of evaporation, besides alterations in proteins structure and lipids due to oxidation progressions. Also, implementation of Met-Se and nano-Se to chicken diets significantly rises the breast's ability and thigh tissues to scavenge the synthetic free radical ABTS when compared with SeS.

FRAP Assay: In general, the capacity of the thigh myofibrillar protein to reduce Fe3<sup>+</sup> to Fe2<sup>+</sup> was higher than in breast myofibrillar protein. In the first three hours, dietary inclusion of 0.45 and 0.6 mg/kg diet of Met-Se and nano-Se had the same reducing capacity of Fe3<sup>+</sup> to Fe2<sup>+</sup>, while after four weeks the reducing capacity of Fe3<sup>+</sup> to Fe2<sup>+</sup> was more prominent in breast meat and thigh meat for groups supplemented with nano-Se. It is well understood that Se is vital for the intra- and extra-cellular antioxidant systems of the body [79]. Selenium is also effective in delaying post-mortem oxidation responses [15]. The association between meat quality and oxidation resistance of muscle is well recognized. Huff-Lonergan et al. [58] described that changes in the antioxidant defense system of animals and muscles would affect water-holding capacity, meat proteolysis and calpain activity, thus quality characteristics of meat. In former studies, the water-holding capacity and chicken muscles color were enhanced by dietary Se addition [80]. Se application to chicken diets causes a significant increase in the iron reduction ability for both sets of the leg and back muscles, which can be associated with the higher Se retention in the lipids-rich parts [78]. Li et al. [81] described how total protein solubility, pH at 45 min, and myofibrillar protein solubility decreased while cooking loss was improved after feeding broiler chickens 0.3 mg/kg of either Met-Se or nano-Se as compared with SeS. Muscle proteins comprise connective tissue, sarcoplasmic and myofibrillar, [82]. Protein solubility resulted from protein denaturation during muscle ageing. In addition, denaturation of muscle protein is associated with antioxidant capacity [83]. When muscle amino acids as cysteine, tryptophan are oxidized, disulfide bonds and carbonyl are produced. At that time, the protein structure is destroyed, which would decrease the solubility of protein [84]. Current study, revealed significant increases in the iron reduction capacity which can be related to higher deposition of Met-Se and nano-Se in breast and thigh of chickens specially when supplemented with higher dose (0.6 mg /kg diet) compared with SeS supplementation, which could be a consequence of improved antioxidant capacity.



SeS = Sodium selenite; Met-Se =

selenomethionine;

 nano-Se =

nano-selenium;

 means within a row carrying different superscript letters denote significant difference (*<sup>p</sup>* < 0.05).

#### **4. Conclusions**

Our results suggested that in Ross broiler chickens, dietary supplementation of either Met-Se or nano-Se up to 0.6 mg/kg increased their performance and was more efficiently retained in the body than SeS. In addition, under stress the antioxidant resistance of broilers fed selected higher levels of Met-Se or nano-Se was enhanced. Moreover, frozen stored meat quality was improved in a dose-dependent manner with both Met-Se and nano-Se. Nano-Se was more potent than Met-Se, which in turn was more potent than inorganic Se against oxidative stress, which improved the quality of meat under frozen conditions.

**Author Contributions:** Data curation, A.T.Y.K., and A.H.A.; Formal analysis, D.I.; Funding acquisition, A.S.A., G.I., A.E.-R. and M.T.E.; Investigation, H.A.M.; Methodology, D.I., A.T.Y.K., and S.I.K.; Project administration, S.I.K.; Resources, A.T.Y.K.; Software, A.T.Y.K., and A.H.A.; Writing—original draft, D.I.; Writing—review & editing, A.T.Y.K.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **E**ff**ect of In Ovo Injection of L-Arginine in Di**ff**erent Chicken Embryonic Development Stages on Post-Hatchability, Immune Response, and Myo-D and Myogenin Proteins**

**Sivakumar Allur Subramaniyan 1, Da Rae Kang 1, Jin Ryong Park 1, Sharif Hasan Siddiqui 1, Palanisamy Ravichandiran 2,3, Dong Jin Yoo 2,3, Chong Sam Na 1,\* and Kwan Seob Shim 1,\***


Received: 8 May 2019; Accepted: 5 June 2019; Published: 14 June 2019

**Simple Summary:** In the current study, we hypothesized that the in ovo injection of L-arginine (L-Arg) at different stages of embryonic development, which would have positive effects on the survival rate, hatching rate, immunoglobulin M (IgM) levels, heat shock proteins (HSPs) such as HSP-47, HSP-60, and HSP-70, and muscle development markers as well: Mainly, myoblast determination protein (myoD) and myogenin in pectoral muscles. As indicated, the in ovo injection of L-Arg resulted in an increased hatch rate and weight, survival rate, higher levels of IgM, and myogenin and MyoD expression in the muscles. At the same time, a decrease in the level of HSP-47, HSP-60, and HSP-70 expressions in the tissues was observed on the 14th day of injection compared to the eighth and 18th day of the injection period. In addition, the in ovo injection of L-Arg decreased the serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) concentration in serum as well micronuclei and nuclear abnormality in the blood on the 14th day of the incubation period. Hence, the 14th day would be a suitable day for the injection of L-Arg to promote the hatching rate and muscle growth of broiler chickens.

**Abstract:** The aim of this study was to evaluate the effect of in ovo injection with different ratios of L-arginine (L-Arg) into Ross broiler eggs at three different embryonic developmental stages (eighth day (d), 14th day, and 18th day) on the survival, hatchability, and body weight (BW) of one-day-old hatched chicks. Additionally, we have analyzed the levels of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT), the protein expression of heat shock proteins (HSPs), and we have also determined micronuclei (MN) and nuclear abnormality (NA). In addition, the genotoxic effect was observed in peripheral blood cells such as the presence of micronuclei and nuclear abnormalities in the experimental groups. The results showed that survival and hatching rates as well as body weight were increased on the 14th day of incubation compared to the eighth and 18th day of incubation at lower concentrations of L-Arg. Moreover, the levels of SGOT and SGPT were also significantly (*p* < 0.05) increased on the 14th day of incubation at the same concentration (100 μg/μL/egg) of injection. In addition, immunoglobulin (IgM) levels were increased on the 14th day of incubation compared to other days. The protein expressions of HSP-47, HSP-60, and HSP-70 in the liver were significantly down-regulated, whereas the expression of myogenin and myoblast determination protein (MyoD) were significantly up-regulated on the 14th day after

incubation when treated with all different doses such as 100 μg, 1000 μg, and 2500 μg/μL/egg, namely 3T1, 3T2, and 3T3, respectively. However, the treatment with low doses of L-Arg down-regulated the expression levels of those proteins on the 14th day of incubation. Histopathology of the liver by hematoxylin and eosin (H&E) staining showed that the majority of liver damage, specifically intracytoplasmic vacuoles, were observed in the 3T1, 3T2, and 3T3 groups. The minimum dose of 100 μg/mL/egg on the 14th day of incubation significantly prevented intracytoplasmic vacuole damages. These results demonstrate that in ovo administration of L-Arg at (100 μg/μL/egg) may be an effective method to increase chick BW, hatch rate, muscle growth-related proteins, and promote the immune response through increasing IgM on the 14th day of the incubation period.

**Keywords:** embryonic development; heat shock proteins; immunoglobulin; intracytoplasmic vacuoles; L-arginine

#### **1. Introduction**

The selection of chickens (*Gallus gallus*) for meat production has led to the generation of inbred strains that show accelerated growth performance, particularly enhanced muscle growth that mostly occurs during embryogenesis [1,2]. During embryogenesis, nutrients and energy are mainly acquired from yolk, which mainly contains lipids and low levels of carbohydrates [3]. Subsequently, the health of the embryo and post-hatch chicken depends on gluconeogenesis from essential amino acids [4,5]. In recent years, researchers have found that the administration of amino acids into fertilized broiler eggs, which is called in ovo feeding, may provide poultry companies with an alternative method to increase the hatchability and muscle growth weight of newly hatched chicks [6,7]. The supplementation of nutrients into fertilized broiler eggs influences embryo development and growth during incubation and the post-hatch growth performance of chicks [7]. The nourishment and supplementation with bioactive substances such as bioactive amino acids, polyphenols, and prebiotics can enhance the immune system, decrease osteoporosis, and decrease the risk of heart diseases [8]. Similarly, previous reports have indicated that the amino acids, carbohydrates, and vitamins that are applied to eggs through in ovo feeding can improve the hatching rate, body weight, survival rate, growth performance, and marketing size [9]. Moreover, an earlier study demonstrated that the in ovo feeding site and time can affect hatchability [10].

During embryonic development, the chorioallantoic membrane develops, which can vascularize on the 12th day of the incubation period. Moreover, the embryo is surrounded by the amniotic fluid, which remains in contact with the embryonic gastrointestinal tract and enables the transport of substances from the air chamber into the intestine [11]. Several genes are associated with cellular interactions and differentiation during the organogenesis of the eye, ear, brain, skin, and tissues such as bones and cartilages; the expression of those genes is either transient or initiated during later stages of embryogenesis [12]. Some authors have indicated that the injection of amino acids into the egg on the first day is sufficient to fully support embryonic development [13,14], leading to increased hatching and breast weight [15]. It has been demonstrated that the injection of sucrose and dextrin into chicken embryos can result in a greater percentage of pectoral muscle weight than the control [5,16]. Recently, it has been reported that chicken embryos injected with L-glutamine on the first day of incubation can increase the fiber area, pectoral muscle mass, and endothelial cell proliferation while stimulating vasculogenesis and angiogenesis [17]. The in ovo administration of amino acids or peptides increases the expression levels of MyoD1 and paired box protein 7 (Pax7), which are necessary for muscle growth during embryogenesis [18].

Standardization of the injection site, needle length, and embryonic age using amino acid (Lys + Met + Cys, Thr + Gly + Ser or Ile + leu + Val) with 11-mm and 24 mm-needles on the seventh and 14th day of incubation has resulted in poor hatchability and poor muscle growth markers [7]. An in ovo injection

of glutamine in conjugation with (silver nanoparticles) Ag NPs on the first day of chicken embryos increased the muscle mass [19], and L-arginine (L-Arg) in one-day-old quail embryos increased the hatchability and growth performance [20]. In our study, we checked different time intervals (eighth day, 14th day, and 18th day) and different doses of L-Ar (100, 1000, and 2500 μg/100 μL/egg) for responses related to the survival rate, hatchability, body weight, and muscle growth-related proteins such as myogenin and MyoD and immunoglobulin M (IgM) levels.

#### **2. Materials and Methods**

#### *2.1. Ethics Statement*

The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Chonbuk National University, with the project number 2017R1D1A1B03032217. Animal care and handling are in compliance with the regulations of the IAEC Guidelines for the Euthanasia of Animals: 2015 Edition. The sampling procedures complied with the "Guidelines on Ethical Treatment of Experimental Animals" (2015) No. CBNU 2015048 set by the Ministry of Science and Technology, Korea.

#### *2.2. Chemicals*

L-arginine, hematoxylin and eosin (H&E), and periodic acid-Schiff's (PAS) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Chemiluminescent for band detection was purchased from Thermo Scientific (Rockford, IL, USA). Antibodies were purchased from ENZO Life Science (Farmingdale, NY, USA). All the laboratory glassware was acquired from Falcon Lab ware (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

#### *2.3. Experimental Design and Incubation*

Ross 1040 broiler chicken eggs were obtained from Samhwa-Won Jong, South Korea. On the first day of incubation, eggs were weighed (60 ± 1.36 g) and separated into different groups. Eggs were randomly divided into 13 groups (4 × 20 × 3 = replication × eggs × injection) as described in Table 1. L-Arg was injected at three concentrations (100 μg, 1000 μg, and 2500 μg/100μL/egg) on the eighth, 14th, and 18th day of the incubation period, respectively. A 0.100-mL of L-Arg (1% PBS) was injected. Immediately after the injection, the hole was sealed with liquid paraffin. Then, eggs were placed in an incubator for 20 days under standard conditions (temperature, 37.8 ◦C; humidity, 60%). On the 18th day, eggs were transferred to hatching boxes and promptly placed in a hatcher incubator with humidity maintained at 60% and temperature set at 37 ◦C to hatch chicks.


**Table 1.** Experimental design for dose (L-Arg) and embronic stage (eighth day, 14th day, and 18th day) fixation.

Note: In Ovo Injection and Treatment Groups.

#### *2.4. Survival Rate Measurement*

Embryos' survival rates during the incubation period were measured on the eighth day. Treated eggs were checked to determine the number of live and dead eggs as well as fertilized and non-fertilized ones among the total number of eggs. At 18th day of incubation, after injection, the eggs live eggs were moved to another hatching incubator with their respective experimental group. The survival rate was calculated with the following Equation (1):

$$\text{Survival rate } \%= \frac{\text{No. of live eggs}}{\text{No. of fertilized eggs}} \times 100\tag{1}$$

#### *2.5. Hatching Rate and Body Weight Measurements*

On the 21st day, hatched chicks were moved from the hatcher incubator to hatching boxes to determine hatching rates. These hatched chicks were kept without food and water at 32 ◦C. Then, they were weighed to record their live body weights. The hatching rate was calculated with the following Equation (2):

$$\text{Hatching rate } \%= \frac{\text{No. of chicks matched on 21st day}}{\text{No. of fertilized eggs that were in } ovo \text{ fed}} \times 100\tag{2}$$

#### *2.6. Biochemical Indices*

At the end of the experimental period, the hatched chicks were sacrificed under anesthesia (diethyl ether). Blood was collected from the jugular vein in tubes for serum separation. A small amount of collected blood was immediately smeared on clean grease-free microscope slides and air-dried for micronuclei (MN) and nuclear abnormality (NA). The breast muscle and liver were collected and washed in ice-cold saline for further study. The body was cut opened; muscle and liver samples were excised, washed with ice-cold saline, and then homogenized with 0.1 M of cold phosphate buffer, pH 7.4. Concentrations of serum glutamate pyruvate transaminase (SGPT) and erum glutamate oxaloacetate transaminase (SGOT) in serum were measured using commercial kits (Asan Pharamaceuticals Co., Ltd., Seoul, Korea).

#### *2.7. Micronuclei (MN) and Nuclear Abnormality (NA) Tests Using Periodic Acid Schi*ff*'s (PAS) Staining*

MN and NA were assayed in the liver by standard methods presented elsewhere [21,22]. Blood samples collected from the first day of the hatching period were immediately smeared on clean grease-free microscope slides and air dried. Afterwards, slides were fixed with methanol for 5 min at room temperature, gently rinsed with running tap water for 1 min, and immersed in a periodic acid solution for 5 min at room temperature. Then, these slides were rinsed using DH2O, immersed in PAS Schiff's reagent for 15 min at room temperature, and gently washed with running tap water for 5 min. Finally, counter-staining was performed with a hematoxylin solution for 90 s. Then, slides were rinsed in running tap water for 30 s, air dried, and examined with a light microscope (100×) using immersion oil.

#### *2.8. Measurement of IgM Concentration in Serum*

Serum samples were collected from individual experimental animals to determine serum immunoglobulin (Ig) M levels using chicken IgM ELISA kit (Abcam, Suite B2304, Cambridge, MA 02139-1517, USA) following the manufacture's specification. IgM levels were analyzed based on absorbance values measured at 450 nm.

#### *2.9. Analysis of Heat-Shock Proteins (HSPs) by Western Blot*

Proteins were extracted from 100 mg of muscle samples using radioimmunoprecipitation assay (RIPA) buffer to determine the protein expression levels of HSP-47, HSP-60, HSP-70, myoD, and myogenin in experimental groups. Protein concentrations were determined using a BIO-RAD protein assay kit (BIO-RAD). Extract samples containing 50 μg of protein were solubilized in *Laemmli bu*ff*er,* separated by 12% acrylamide gel, and then transferred to Hybond-P PVDF membranes (GE Healthcare Inc., Amersham, UK) for 60 min at 200 mA. Then, these PVDF membranes were blocked with 5% skimmed milk powder in 0.5 M of Tris-buffered saline (pH 7.4) with 0.05% Tween 20 (TBST) at room temperature for 2 h. Western immunoblotting with HSP-47, HSP-60, HSP-70, Myo-D, and myogenin are primary antibodies (1:2500 dilution) took place overnight. After washing three times with TBST, these membranes were probed with HRP-conjugated secondary antibodies (1:5000 dilutions) for 60 min at room temperature, and then washed three times with TBST (10 min each wash). Protein bands were visualized using a Chemiluminescent assay kit from Thermo Scientific for 1–5 min. Bands were imaged with an iBright™ CL1000 Imaging System (Invitrogen in Thermo Fisher Scientific, Life Technologies Korea LLC, Jeonju-si, Jeollabuk-do, Korea) and quantified using Image J Software. The relative density of the band was normalized to that of β-actin as an internal control.

#### *2.10. In Silico Molecular Docking Studies*

To understand the mechanism of interaction of L-Arg with heat shock protein, crystal structures of GroEL mutant A109C (PDB ID: 5OPW) [23] and human HSP70 substrate binding domain L542Y mutant (PDB ID: 5XIR) [24] were downloaded from the Protein Data Bank. Molecular docking studies were performed using the GLIDE program [25] (Version 8.5, Schrodinger LLC, New York, NY, USA). To analyze docking results and execute the protocol, the Maestro user interface (Version 8.5, Schrodinger LLC, New York, NY, USA) was employed. Validation of the protocol was performed by redocking. The structure of L-Arg was sketched using ACD/chemsketch (Freeware version). The GLIDE grid generation wizard was used to define the docking space. Docking was performed using XP (Extra Precision mode) docking protocol.

#### *2.11. Histopathological Study of the Liver*

Livers were collected after chickens were sacrificed, immediately fixed with 10% neutral buffered formalin (NBF), and processed in an auto processor (Excelsior ES, Thermo Scientific, Waltham, MA, USA). After embedding in paraffin, 5-μm sections were made and subjected to H&E staining. Digital images were obtained using a Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany) at fixed 100× (200×) magnification.

#### *2.12. Statistical Analysis*

All the values are presented as mean ± SD from 12 determinations from each group and statistically analyzed using Duncan test following ANOVAs with SAS® software, version 9.4, (Institute of INC, North Carolina, USA).

#### **3. Results and Discussion**

#### *3.1. Survival Rate and Hatchability*

Survival rate was significantly (*p* < 0.05) increased in the 2T1 and 3T1 groups than that in other groups. The lowest survival rate was observed in 1T3, 3T2, and 3T3 groups (Figure 1). These results showed that the survival rates differed depending upon the injection period and the concentration of L-Arg. Embryos may utilize in ovo administered amino acids to improve energy status and save muscle protein to improve their enteric development, hatching, and survival rate [26]. In our study, the same mechanism might have occurred; the administration of L-Arg could improve the survival

rate at the minimal concentration (3T1) on the 14th day of the injection period (Figure 1). However, during incubation, an excess of amino acids such as glycine and proline failed to improve embryo development [27]. The same attributes could have been observed in our current study: that a maximum concentration of L-Arg affects the embryonic growth.

**Figure 1.** Effects of in ovo injections at different concentrations of L-arginine (L-Arg) with different developmental embryonic stages on survival rate. Small characters indicate significant differences among experimental groups at *p* < 0.01. Values are presented as mean ± SD from 12 determinations. Data were analyzed using Duncan test following ANOVAs with SAS® software, version 9.4, (Institute of INC, North Carolina, USA).

Different concentrations of L-Arg injected in embryos can influence biological molecules and toxicity during embryogenesis. However, several studies have reported that higher doses of L-Arg become toxic, which can cause significantly increased mortality rates and impaired weight gain, whereas chicks injected with lower concentrations of L-Arg (1.0%) showed better growth performance than those injected with a higher concentration (1.5%) of L-Arg [28]. In a parallel effect revealed in our current study to a lower concentration of L-Arg (2T1) on the 14th day of injection increasing the hatching rate (96.29%), body weight (64.25 g) also increased the survival rate (98.03%) compared to other groups (Figures 1–3). The in ovo injection of L-Arg to late-term embryos can increase the body weight (5% to 6%) compared to controls [5]. In addition, in ovo administration of all 20 different amino acids can increase chick weight by 3.6% and 2.1%, respectively [10]. The in ovo administration of amino acids might have stimulated the utilization of amino acids with a concomitant decrease in the degradation of amino acids by the embryo [29]. In ovo feeding of L-Arg resulted in higher embryo weight due to an increase in muscle mass [30]. The in ovo injection of L-Arg could be utilized by the embryo, resulting in increased muscle mass and a heavy embryo, which can increase the hatching rate [31]. L-Arg may attenuate adverse effects of rearing chickens under cold ambient temperatures or at high altitudes [32]. Furthermore, feeding broiler chickens with a diet that is deficient in L-Arg under cold stress at high altitudes can depress nitric oxide synthesis, decrease feed intake, reduce body weight gain, and increase the right ventricle to total ventricle weight ratio, mortality rate, and ascites mortality [31]. A previous study reported that a lower percentage (1.36%) of L-Arg supplemented to broiler eggs was more easily digestible than a higher percentage of arginine, and it could obtain the highest egg weight [33]. Albeit, a low dose of L-Arg stimulates the secretion of the growth hormone, which could increase the body weight [21]. The same mechanism might occur in our present study; L-Arg appeared to improve the body weight of chicks in group 2T1 (Figure 3). However, the body weights did not significantly vary among the other groups. Hatchability, gut microflora population, immune-related gene expression, and muscle fiber increased as a result of the 12–14-days in ovo injection of various substances such as Raffinose, *Lactococcus lactis*, and *Silybum marianum* extract [34–36]. Hence, a low dose (2T1) of L-Arg injected on the 14th day of the incubation period could improve body weight;

the reason behind this might be that the lower concentration of L-Arg may stimulate the growth hormones in the middle stages of embryonic development when compared to the early (eighth day) and late (18th day) embryonic stages.

**Figure 2.** Effects of in ovo injections at different concentrations of L-Arg with different developmental embryonic stages on hatching rates. Small characters indicate significant differences among experimental groups at *p* < 0.01. Values are presented as mean ± SD from 12 determinations. Data were analyzed using Duncan test following ANOVAs with SAS® software, version 9.4, (Institute of INC, North Carolina, USA).

**Figure 3.** Effects of in ovo injections at different concentrations of L-Arg with different developmental embryonic stages on Chicks weight. Small characters indicate significant differences among experimental groups at *p* < 0.01. Values are presented as mean ± SD from 12 determinations. Data were analyzed using Duncan test following ANOVAs with SAS® software, version 9.4, (Institute of INC, North Carolina, USA).

#### *3.2. Biochemical Indices (SGOT and SGPT)*

Elevated SGOT and SGPT levels indicate improper liver function due to damages of the cell integrity and cell membrane in the liver. Our results revealed that the injection of L-Arg at all doses except the lower dose affected SGOT and SGPT levels on the eighth and 18th-day embryonic stages (Figure 6A). SGOT and SGPT levels were significantly decreased in the 2T1 and 2T2 groups of embryos compared to 2T3, 3C1, 3T1, 3T2 and 3T3. Increased levels of SGOT and SGPT in the blood are conducive to liver function damage [37–39]. In fact, free radicals can attack hepatocytes and release stored SGPT to re-enter the blood serum [40]. A lower concentration of L-Arg supplementation caused a greater percentage reduction in SGOT and SGPT levels in sickle cell anemia subjects [39]. The supplementation of L-Arg to mice in higher concentrations showed that increased SGOT and SGPT levels had been linked to damage to hepatic cells and hemolysis [41]. The cause of liver damage is unclear. Hence, confirming that a higher concentration of L-Arg might have damaged the hepatic cell through the elevation of SGOT and SGPT in the 1T1, 1T2, 1T3, 3T1, 3T2 and 3T3 groups. On the other hand, the 2T1 group of injected chicken embryos could re-back the SGOT and SGPT levels compared to the other groups. Stimulating the action of nitric oxide (NO) production by L-Arg results showed that it improved the degree of the hepatocellular structure by blocking of B-cell lymphoma-2 (Bcl-2) and tumor necrosis factor-α (TNF-α) [42]. In addition, L-Arg at 1 g/day decreased the liver enzymes such as SGOT and SGPT through increasing the nitric oxide (NO) synthesis. NO synthase plays an important role in liver injury through inducible nitric oxide synthase (iNOS) pathways [43]. The same mechanism could be involved in our current study, too. This same mechanism that might have occurred in our study could be that the production of NO reduces necrosis and apoptosis by attenuation of the inflammatory pathway, which in turned prevented the hepatotoxicity. Moreover, it also improved the hepatobiliary function, and the ultrastructure of liver results reduced the SGOT and SGPT levels in L-Arg treatment in the lower dose (2T1) on the 14th day injection of embryos (Figure 4).

#### *3.3. Micronuclei (MN) and Nuclear Abnormality (NA) Tests Using Periodic Acid-Schi*ff*'s (PAS) Staining*

The wide use of different doses of L-Arg at three different incubation periods requires examining the genotoxic activity in peripheral blood by the method of [44]. MN and NA tests were conducted to examine peripheral blood cells in all the groups of experimental chicks (Figure 5). The MN test can measure subcellular processes of chromosomal breaks (clastogenesis) or cell spindle malfunctions (aneugenesis) as well as the formation of mitochondrial disruption and nuclear DNA, which can lead to mitochondria-dependent apoptosis in chicken embryos as an indicator of chromosomal damage [45]. Similar results were obtained in our current experiment: the MN and NA in peripheral blood erythrocytes were observed, which clearly demonstrates the higher genotoxicity of a high dose of L-Arg on the eighth, 14th, and 18th day of the incubation period. Moreover, the 2T1 and 2T2 groups showed a normal architecture of nuclei in peripheral blood cells, which was similar to the control group. Figure 5 shows marked inflammation around the periportal region with microvesicular and macrovesicular fatty infiltration (yellow arrows).

**Figure 5.** Photomicrographs of erythrocytes with normal nuclei in the peripheral blood cells of experimental groups. Micronuclei and nuclear abnormalities such as blebbed nuclei or lobed nuclei are indicated by an arrow ("→").

#### *3.4. Protein Analysis by Western Blot*

Western blot was performed in muscles to determine whether the different doses of L-Arg supplemented at various days of the incubation period may alter the protein levels of the HSP family such as HSP-47, HSP-60, and HSP-70. As shown in Figure 6, the protein expressions of HSP-47, HSP-60, and HSP-70 were significantly (*p* < 0.01) down-regulated in the 2T1 group compared to other groups. Moreover, their levels in the 3T2 and 3T3 groups were significantly (0.01) up-regulated compared to the 2T1 and 2T2 groups, although the protein expressions of HSP-60 and HSP-70 showed no significant difference (*p* < 0.01) among the 1T1, 1T2, 1T3, and 2T1 groups. Moreover, HSP-46, HSP-60, and HSP-70 were down-regulated in the 2T1 group compared to those in the other groups, whereas there was no significant difference in their levels between 1C and 2T1. HSP-70 is a reliable index of stress in chickens, while "3-hydroxyl-3-methyl-glutaryl coenzyme A reductase" has been used as an indicator of stress [46]. Pretreatment with L-Arg markedly reduced the dramatic down-regulation of HSP-60 and HSP-70 in hypoxic rat model. The increased expression of HSP-60 and HSP-70 might be related to their leakages from tissue, which can cause tissue injury due to free radical production [47,48]. Tissue injury might be caused by nitric oxide, a free radical, through the stimulation of endothelial cells and neutrophils that is generated from a higher dose of L-Arg [49]. Hence, the present results may suggest that the increased levels of HSP-47, HSP-60, and HSP70 in high doses of L-Arg may have a major role in tissue injury. The results of the study show that the increase of HSP-60 and 70 may be involved in tissue injury in the 3T1, 3T2, and 3T3 groups due to free radical production. The 2T1 group can prevent tissue injury via the down-regulation of HSP-46, HSP-60, and HSP-70. Moreover, the protein expressions of myogenin and MyoD were significantly up-regulated in the 2T1 group, whereas they were down-regulated in the 3T1, 3T2, and 3T3 groups compared to the other experimental groups.

**Figure 6.** (**A**) Effects of expression levels of L-Arg, heat shock protein (HSP)-47, HSP-60, and HSP-70 as well as myogenin and myoblast determination (MyoD) protein expressions in different stages of chicken embryos at different doses. Small characters indicate significant differences among experimental groups at *p* < 0.01. (**B**) The bar graph represents the quantitative expression of different proteins in all the groups. Data are expressed as the ratio of relative intensity with β-actin. Values are presented as mean ± SD from 12 determinations.

Oxidative stress can cause muscle atrophy by reducing myogenic differentiation markers such as myogenin and MyoD in skeletal muscles [50]. Some growth factors, namely cytokines and oncogenes, suppress the activity of myogenin and MyoD, thus resulting in decreased in the mass of muscle, which is defined as muscle atrophy [51]. A previous study reported that myogenic regulatory factors—mainly MyoD and MRF4—are only expressed later in different embryonic muscle groups as a result of increased muscle mass [52]. L-Arg increased the muscle cell as well as myogenin and MyoD under oxidative stress. Moreover, results from a previous experiment demonstrated that lower doses of

L-Arg could promote HSP70 expression in pig intestine [53]. Nevertheless, our present study has proved that increasing the concentration of L-Arg on the eighth and 18th day of the injection period could up-regulate the expression of HSP-60 and HSP-70; this effect might be through whey protein hydrolysate, which indicates the improper use of a functional food ingredient. Moreover, the L-Arg on the 14th day with (100 μg/100 μL/egg) promoted the muscle mass through the up-regulation of MyoD and myogenin due to their free radical scavenging activity.

#### *3.5. Measurement of IgM Concentration in Serum*

Concentrations of immune response markers such as IgM in all the experimental groups were analyzed. The duration and amount of L-Arg supplementation may influence immune status. Short-term supplementary L-Arg can influence the immunity power, because L-Arg has antioxidant and anti-inflammatory effects [54,55]. It can attenuate inflammatory reactions by suppressing the generation of inflammatory mediators such as inflammatory cytokines and C-reactive protein, which play major roles in the progression of tissue damage and organ dysfunction [56]. The treatment of L-Arg shows improved renal function through improved immune function [57]. Levels of IgM could provide an overall picture of immune function. It has been recently demonstrated that L-Arg can increase the specific immune response against infectious bursal disease (IBD) in chickens [58]. L-Arg provided by treatment has been reported to be the sole precursor of nitric oxide with lots of immune functions and growth performance [59]. These same biological attributes might be present after a low dose of L-Arg injection on the 14th day of the incubation period. It may improve immunity via the generation of IgM and the suppression of inflammatory cytokines and C-reactive protein (Figure 7).

**Figure 7.** L-Arg induces immunoglobulin M (IgM) levels in different stages of chicken embryos at different doses. Small characters indicate significant differences among experimental groups at *p* < 0.01. Values are presented as mean ± SD from 12 determinations. Data were analyzed using Duncan test following ANOVAs with SAS® software, Version 9.4, (Institute of INC, North Carolina, USA).

#### *3.6. Histopathology (H&E) Staining*

Figure 8 shows the histology of liver of all the experimental groups. Sections from the control group exhibited a complete structure and regular shape of liver cells. Sections from the 1T1 and 2T1 groups showed normal hepatocyte gap compared to the 1C and 1C1 groups. Sections from the 1T3, 2T3, 3T1, 3T2, and 3T3 groups appeared with intracytoplasmic vacuoles in hepatocytes around the centrilobular regions. Moreover, hepatocyte tubes were surrounded by inflammatory cells and showed necrosis with nuclear fragmentation in the 3T2 and 3T3 groups. The hepatocyte gap was increased in the 2T3, 3T1, 3T2, and 3T3 groups. The hepatocyte gap appeared in normal architecture in the 1C, 1C1, 2C1, 3C1, 1T1, and 2T2 groups. The degeneration of livers was observed for birds when treated with 167 and 334 mg/L of L-Arg, which had an adverse effects on organs [60]. The liver after treatment with L-Arg (334 mg/L) had congested vascular spaces and periportal mononuclear inflammatory infiltration [61]. The addition of L-Arg to poultry diets is required to avoid harmful influences of excessive free radicals produced during normal metabolism [62]. Dietary L-Arg supplementation plays a key role in enhancing meat quality. Increased L-Arg and betaine supplementation alleviates total body fat deposition and fatty liver [63,64]. Additionally, supplementation with high doses (50% and 100%) of L-Arg has negative effects on the structure of the liver of Sasso birds proved by H&E staining. However, our current results showed that in ovo injection with low doses (2T1) of L-Arg on the 14th day of egg embryo development did not have any negative effects compared to higher doses of L-Arg on the eighth or 18th day of the incubation period.

**Figure 8.** Histopathology of liver using hematoxylin and eosin straining. Sections from control chicks' hepatic lobule indicate complete structures. The liver cell has a regular shape that is within normal limits. Intracytoplasmic vacuoles are shown in hepatocytes around the centrilobular region in the 1T3, 2T3, 3T1, 3T2, and 3T3 groups. Hepatocyte marked inflammation around the periportal region, with microvesicular and macrovesicular fatty infiltration (yellow arrows) liver cells appearing near necrosis with nuclear fragmentation in the 3T2 and 3T3 groups. The hepatocyte gap was also increased in the 2T3 3T1, 3T2, and 3T3 groups. There was no difference between the control and the 2T1 group.

#### *3.7. In Silico Molecular Docking Studies*

The in silico molecular docking of L-Arg was studied. The entire glide, E model scores, and hydrogen bond interactions are presented in Figure 9. The main aim of the molecular modeling study is to understand the interactions of functional groups present in L-Arg with residues of targeted proteins theoretically. Heat shock protein 70 (HSP70) is one of the main nonglobin proteins, which has a similar structure in almost all living organisms. Between organisms as varied as yeast, chicken, Drosophila, and human, HSP70 is highly conserved. A previous study has described that HSPs are antigenically linked to the chicken HSPs by means of rabbit polyclonal antibodies [25]. In the same way, the similarity of HSPs extends to a DNA sequence. The chicken and human HSP70 genes are 64–72% similar (homologous) in the obtained amino acid sequences [65]. In addition, the HSP70 gene(s) of chicken, Drosophila, mouse, and human are very much conserved with the comparison of the DNA sequence [66,67]. Therefore, in the present study, solution structure for the human HSP70 substrate binding domain L542Y mutant (PDB id: 5XIR) was selected for the in silico molecular modeling study with L-arginine [68].

**Figure 9.** (**A**) 2D docking interaction of L-arginine with the active site of 5XIR. (**B**) 3D docking of L-arginine in the active site of 5XIR. (**C**) Docking packing representation of L-arginine with suitable binding pockets of 5XIR.

As presented in Figure 9, the main aim of the molecular modelling study is to understand interactions of functional groups present in L-Arg with residues of targeted proteins theoretically. In L-Arg, primary and secondary amines have strong hydrogen bonding interactions with protein residues. In brief, the molecular interaction of L-Arg with 5XIR protein and primary and secondary amines established a strong affinity with protein resides such as ASP A 328, ASP A 83, and HIS A 401. In addition, the carboxyl group present in the molecule also exhibits good hydrogen bonding interactions with LYS A 498. In a similar way, L-arginine with 5XIR protein showed good molecular interactions with ASP A 54, PHE A 49, GLU A 25, THR A 26 and GLY A 29 (Figure 9A).

The molecular modelling study revealed that L-Arg was bound onto similar active site cavity in the protein molecule. Superposition of interactions of an active site of L-Arg and amino acid residues of 5XIR protein is evidently portrayed in Figure 9B,C. The molecular docking of L-Arg with 5XIR protein is exposed to seven hydrogen bonding interactions, respectively with corresponding active site of the protein molecule. Besides, L-Arg showed strong hydrogen bonding interactions with good surface molecular interactions due to the presence of primary, secondary amines, and carboxylic acid groups. These relationships among 5XIR protein, and L-Arg might explain the experimental activity of them. Further research is going on in a due course to explore their possible modes of action. It is possible that L-Arg might block the activity of HSPs, and activation of Myogenin and Myo-D as well improved the immunoglobulin levels, thereby regulating the muscle growth (Figure 10).

**Figure 10.** Possible mechanism of high doses and low doses of L-Arg on toxicity and muscle growth in chicken embryo.

#### **4. Conclusions**

In this study, we described a suitable embryonic developmental stage for the accessibility of in ovo injection using L-Arg at different concentrations for the first time. The injection of L-Arg on the 14th day at 100 μg/μL/egg enhanced both hatching and survival rates. It also increased body weight and immune response (IgM). In addition to oxidative stress, a sign of genotoxic effect was also observed in peripheral blood cells in which a presence of micronuclei and nuclear abnormalities such as blebbed nuclei, lobed nuclei, and notched nuclei were observed in the 2T3, 3T1, 3T2, and 3T3 groups. Histology from the control and 2T1 groups showed normal architecture, while injection on the 18th day of incubation and first day of chicks showed liver tissue damage. Overall results demonstrate that the optimum dose is 100 μg/μL/egg, and the optimum injection stage is on the 14th day to improve the immunity, hatching, and survival rate, which can be used for the poultry industry. In ovo injection in early and late embryonic stages could not offer good benefits for survival, hatching rate, or muscle development. If we choose the middle stage of embryonic development for in ovo injection, L-Arg might be able to promote muscle growth and improve the immune power without inducing adverse effects on the liver.

**Author Contributions:** Conceived and designed the experiments: S.A.S., K.S.S. Performed the experiments and measurements of serum biochemical parameters: S.A.S., D.R.K., J.R.P. and S.H.S. Protein analysis by western blot: S.A.S. Histopathology (H&E) staining: S.A.S. In silico molecular docking studies: P.R., D.J.Y and C.S.N.

**Funding:** This research was funded by National Research Foundation of Korea: Project No. NRF-2017R1D1A1B03032217 and 2017R1D1A3B03028490.

**Acknowledgments:** This research was supported by the Basic Science Research Program, National Research Foundation of Korea (NRF), Ministry of Education (Project No. NRF-2017R1D1A1B03032217 and 2017R1D1A3B03028490) and the "Research Funds of Chonbuk National University in 2018". We thank to Choi, Eun-Jin, Center for University Research Facility (CURF) at Chonbuk National University.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**

1. Berri, C.; Wacrenier, N.; Millet, N.; Le Bihan-Duval, E. Effect of selection for improved body composition on muscle and meat characteristics of broilers from experimental and commercial lines. *Poult. Sci.* **2001**, *80*, 833–838. [CrossRef] [PubMed]


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **E**ff**ects of Dietary Supplementation of L-Carnitine and Excess Lysine-Methionine on Growth Performance, Carcass Characteristics, and Immunity Markers of Broiler Chicken**

**Seyed Mohammad Ghoreyshi 1, Besma Omri 2, Raja Chalghoumi 2, Mehrdad Bouyeh 1, Alireza Seidavi 1,\*, Mohammad Dadashbeiki 3, Massimo Lucarini 4, Alessandra Durazzo 4, Rene van den Hoven <sup>5</sup> and Antonello Santini 6,\***


Received: 18 March 2019; Accepted: 12 June 2019; Published: 16 June 2019

**Simple Summary:** L-carnitine, lysine, and methionine are amino acids of important nutritional and nutraceutical interest and are used as dietary supplements to improve feed quality characteristics in broiler chicken. This study investigated the effect of different levels of L-carnitine and extra levels of lysine-methionine on growth performance, carcass characteristics, and some immune system markers. The findings of this study showed that the diet with around 30% of lysine-methionine content increased the back thoracic vertebrae and the proventriculus weights. A combination of lysine-methionine (level equal to NRC recommendations) with L-carnitine (15% and 75%) improved the immune response of broiler chickens against Newcastle and Gumboro diseases by stimulating the antibody production.

**Abstract:** L-carnitine as well as lysine and methionine are amino acids of important nutritional and nutraceutical interest and are used in nutritional strategies as dietary supplements to improve feed quality characteristics in animals and broiler chicken in particular. This study investigated the effect of different levels of L-carnitine and extra levels of lysine-methionine on growth performance, carcass characteristics, and some immune system markers. Two hundred seventy male Ross 308 broilers were a fed control diet (C) and eight different diets supplemented with an excess of amino acids. In the experimental diets, identified as D1, D2, D3, D4, D5, D6, D7, and D8, extra L-carnitine, lysine, and methionine were added in excess with respect to the American National Research Council (NRC) recommendations: L-carnitine equal to NRC (D1), control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine equal to NRC (D2), control diet supplemented with lysine equal to NRC, methionine equal to NRC, and L-carnitine at 15% in excess of NRC (D3), control diet supplemented control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 15% in excess of NRC (D4), control diet supplemented lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at

15% in excess of NRC (D5), control diet supplemented with lysine equal to NRC recommendations, methionine equal to NRC recommendations, and L-carnitine at 75% in excess of NRC (D6), control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 75% in excess of NRC (D7); and control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC (D8). During the starter and growth phases, feed intake was not affected by dietary treatment (*p* > 0.05). By contrast, body weight and FCR were both affected (*p* < 0.01) during the starter period. During the finisher phase, feed consumption was affected (*p* < 0.05) by dietary treatment. Feed intake of broilers fed on C, D3, D6, and D7 were statistically similar (*p* > 0.05) (1851.90, 1862.00, 1945.10, and 1872.80 g/pen/day, respectively) and were higher (*p* < 0.05) than 1564.40 g/pen/day (D5). With the exception of drumsticks, neck, back thoracic vertebrae, and proventriculus weights, the economical carcass segments were not affected (*p* > 0.05) by the dietary supplementation of amino acids. Duodenum and ileum weights and lengths decreased with amino acid supplementation (*p* < 0.05). IgT and IgG titers against Sheep Red Blood Cells (SRBC) for both primary and secondary responses were not affected by dietary treatments (*p* > 0.05). Dietary amino acids supplementation did not affect IgM titer after the secondary challenge (*p* > 0.05) and had a significant effect (*p* < 0.05) on serum antibody titers in broilers vaccinated against Newcastle disease (NCD) and Gumboro 's disease at the 27th and 30th days, respectively.

**Keywords:** amino acids; dietary supplementation; broiler; growth performance; humoral immunity

#### **1. Introduction**

Nowadays, the growing demand for poultry meat has resulted in pressure on breeders to increase the growth rate of birds, the feed efficiency, the size of breast muscles, and the reduction in abdominal fatness [1]. Therefore, research is being oriented toward improving the techniques of poultry meat production. The improvement in carcass compositions with additives has become a focus on nutrition research. As an example, the addition of amino acids and metabolic intermediates to diets may lower the abdominal fat deposition in poultry. One example is L-carnitine, the biologically active form of carnitine, which is synthesized in the liver, kidney, and brain [2] from the essential amino acids' lysine and methionine, that can be considered as L-carnitine precursors [3,4]. L-carnitine (δ-trimethylamino-β-hydroxybutyrate) is a quaternary hydrosoluble amine with a small molecular weight that occurs naturally in microorganisms, plants, and animals [5]. Its concentration in animals varies according to species [6], tissue type [4,7], nutritional status of the animal [8,9], and the feed quality [10]. Dietary effects of L-carnitine, lysine, and methionine supplementations on the growth performance and body composition of broiler chickens are still poorly understood. Many studies have suggested that the dietary addition of lysine and methionine in excess with respect to the American National Research Council (NRC) [11] recommendations may result in enhanced performances, especially with regard to breast meat yield, body weight gain, and feed conversion ratio [12–21]. Corzo et al. [22] reported how a high dietary density of amino acids can lead to increased breast meat related to an increase in lean muscle tissue. Moreover, Mukhtar et al. [10] reported that a significant improvement in amino acids feed intake improves the average body weight gain and feed conversion ratio. On the other hand, Si et al. [15] concluded that the level of methionine should not be increased if lysine is in excess of its minimum needs.

It has been also reported that the dietary supplementation of lysine and methionine can improve the immunity of broiler chickens against different diseases [23–26]. Moreover, it has been reported that methionine constructively affects the immune system by improving both cellular and humoral responses [27–29]. The mechanisms proposed to explain methionine interference in the immune system is the T cells proliferation, which are sensitive to intracellular glutathione and cysteine levels, compounds that participate in the methionine metabolism [30].

L-carnitine supplementation is used to improve broiler productivity [31], and its bioavailability depends on the composition of the diet. Theoretically, supplementing broiler diets with adequate content of L-carnitine would facilitate the fatty acids β-oxidation and decrease the esterification reactions and triacylglycerols storage in the adipose tissue [3,32,33]. However, the impact of extra supplied L-carnitine may depend on the magnitude of its endogenic biosynthesis from lysine and methionine in the presence of Fe2<sup>+</sup> and a number of vitamins (e.g., ascorbate, niacin, and pyridoxine), which are required as cofactors for the enzymes involved in the metabolic pathway of L-carnitine [4,34–36]. Some authors reported that abdominal fat deposition in broilers is reduced by L-carnitine supplementation without a significant effect on daily gain or feed conversion [37], while others observed no impact of dietary L-carnitine supplementation on abdominal fat composition [38]. Nonetheless, Bouyeh and Gevorgyan [39] and Celik et al. [40] showed that the growth performance of broilers was improved by L-carnitine supplementation. A study by Hosseintabar et al. [33] evaluated the effects of different levels of L-carnitine, lysine, and methionine on the blood concentrations of energy, protein, and lipid metabolites of male broiler chickens and concluded that, compared to a standard diet, the addition of 150 mg/kg of L-carnitine plus 15% lysine and methionine sustained a low plasmatic total cholesterol concentration compared to a standard diet. El-Wahab et al. [41] reported the broilers fed high levels of lysine and methionine with a surplus amount of L-carnitine (350 mg kg−<sup>1</sup> to the diet) led to significantly lower cholesterol levels vs. a low L-carnitine intake. To the knowledge of the authors, the effect of simultaneous feed supplementation with L-carnitine and excess lysine-methionine on growth performance, carcass characteristics, and immunity markers of broiler chicken has not been studied before.

There is a need to standardize the dose of lysine-methionine and L-carnitine supplementation in the diet of broiler chickens not only to enhance their growth performance and carcass characteristics but also to improve their immune response. Hence, the main objective of this study has been to evaluate the effect of dietary supplementation of different levels of L-carnitine with or without an excess of lysine-methionine compared to dietary nutrient requirements on broiler chickens' growth performance, humoral immunity markers, and carcass characteristics during a 6-week rearing trial.

#### **2. Materials and Methods**

#### *2.1. Animal Welfare and Ethics*

All procedures related to animals' care and sampling were conducted under the approval of the Institution's Ethic Committee at the Department of Animal Science, Rasht Branch, Islamic Azad University, Rasht (Iran) (protocol N◦ 105/19) before the beginning of the experimental trial.

#### *2.2. Experimental Diets Preparation*

A control diet (C) for broiler chicken based on corn and soybean-meal was prepared according to the dietary nutrient requirements of broilers [1]. The ingredients and chemical composition of the control diet are given in Table 1. Thereafter, eight amino acids-supplemented diets (indicated as D1, D2, D3, D4, D5, D6, D7, and D8) were prepared by mixing the control diet thoroughly with the designated supplements at the required incorporation levels as shown in Table 2.

As stated above, the L-carnitine, lysine, and methionine levels in starter, grower, and finisher feeds for the control diet were determined according to the NRC [11] recommendations. In the experimental diets, D1, D2, D3, D4, D5, D6, D7, and D8, extra L-carnitine, lysine, and methionine (Carniking1, Lonza Ltd., Basel, Switzerland) were added in excess to the NRC [11] recommendations, as shown in Table 2, since NRC [11] recommended diets are suggested for feeding Ross 308 broiler chickens because of fewer phases of feeding periods and lower workloads [42].


**Table 1.** Ingredients and chemical compositions of the control diet.

\* Calcium Pantothenate: 4 mg/g; Niacin: 15 mg/g; Vitamin B6: 13 mg/g; Cu: 3 mg/g; Zn: 15 mg/g; Mn: 20 mg/g; Fe: 10 mg/g; K: 0.3 mg/g; \*\* Vitamin A: 5000 IU/g; Vitamin D3: 500 IU/g; Vitamin E: 3 mg/g; Vitamin K3: 1.5 mg/g; Vitamin B2: 1 mg/g.



\* L-carnitine (American National Research Council (NRC)): Starter period: 17.8 mg/kg; Grower period: 18.1 mg/kg; Finisher period: 22.9 mg/kg; \*\* lysine (NRC): Starter period: 1.41%; Grower period: 1.26%; Finisher period: 1.22%; \*\*\* methionine (NRC): Starter period: 0.61%; Grower period: 0.57%; Finisher period: 0.48%.

#### *2.3. Animals and Experimental Design*

Two hundred seventy 1-day-old, male Ross 308 broiler chicks obtained from a local commercial hatchery were used in this experiment. Chicks were randomly distributed into 27 pens (9 groups × 3 replications, each replication included 10 chicks). Each group was allocated to one of the nine dietary treatments indicated above. Birds were given starter feed from 1 to 21 days, a grower feed from 22 to 35 days, and a finisher feed from 36 to 42 days of age. Feed and water were provided ad libitum throughout the experimental assay. For the growth performance traits, the experimental unit was the pen. For the carcass and immunity traits, the experimental unit was the chicken.

#### *2.4. Growth Performance Monitoring*

Tens of birds per pen were weighed together on the 1st, 21st, and the 35th days of age to determine the live body weight and the weight gain. The feed consumption and feed conversion ratio (FCR) were also calculated for each growing phase as follows: from the 1st to the 21st day, from the 22nd day to the 35th day, and from the 36th to the 42nd day of the 42 days experimental study as described by Bouyeh and Gevorgyan [39].

#### *2.5. Carcass Characteristics Determination*

As shown by Panda et al. [43–45], at the end of the experiment (day 42), three broilers per same treatment (e.g., one broiler per same diet per pens) (*n* = 3) were randomly selected, weighed without prior fasting, and scarified between 9:00 am and 10:00 am by cervical dislocation to evaluate the characteristics of the carcass. After skin removal and total evisceration, the feet were separated from the carcass in the tibio–tarsal joint. Economic carcass and gastrointestinal segments were removed, weighed, and the ratios of each segment to body weight were calculated.

#### *2.6. Humoral Immune Response Measurements*

Non-pathogenic antigens of Sheep Red Blood Cells (SRBC) were used to monitor the humoral immune response of broilers. The SRBC were purchased from a local Iranian supplier. A suspension was prepared by mixing 1 mL of phosphate-buffered saline (PBS) with 10 mL of SRBC. Six birds per same treatment (e.g., two broilers per same diet per pens) (*n* = 6) were subcutaneously injected in the breast with 0.5 mL of SRBC suspension on the 22nd and the 36th days of the experimental trial.

Then, seven days after each sensitization (28 and 42 days, respectively), antibody titers against SRBC were measured by a hemagglutination inhibition (HI) test according to Cunningham [46]. All antibody titers were recorded according to previous studies [47,48].

Birds were also vaccinated against infectious bronchitis (IB) on the 1st and 16th days of age, against Newcastle disease (NCD) on the 8th and 20th days of age, and against Gumboro's disease on the 14th and 23rd days of age. The humoral immune responses of chickens to the IB virus at the 23rd day of age, to the NCD virus at 27th day of age, and to the Gumboro virus at the 30th day of age were measured using the HI and ELISA methods as described by references [47,48]. Blood samples were collected from the brachial vein. Serum was separated by centrifugation (3000× *g* rpm for 15 min), and antibody titers against IB, NCD, and Gumboro virus were measured using commercially available ELISA kits (Bio-check BV, Gouda, Holland) according to the manufacturer's instructions. The absorbance of controls and samples were read at a wavelength of 405 nm using an ELISA reader (Bio-Tek Instruments Inc. ELX 800, Winooski, VT, USA).

#### *2.7. Statistical Analysis*

All data were subjected to an ANOVA statistical analysis with the General Linear Model (GLM) procedure of SAS [49]. The GLM was used according to the following model:

$$\mathcal{Y}\_{\rm ijk} = \mu + \alpha\_{\rm j} + \beta\_{\rm k} + (\alpha\beta)\_{\rm jk} + \varepsilon\_{\rm ijk} \tag{1}$$

where Yijk = the jth observation on the ith treatment, μ = overall mean, α<sup>j</sup> = the main effect of the L-carnitine level, β<sup>K</sup> = the main effect of the methionine-lysine level, αβjk = the effect of the interaction of L-carnitine and of methionine-lysine treatments, and εijk = The random error.

#### **3. Results**

#### *3.1. Growth Performance*

Table 3 summarizes the results of the growth performance using the different diets. During the starter and growth phases, feed intake was not affected by dietary treatment (*p* > 0.05). By contrast, body weight and FCR were both affected (*p* < 0.01) during the starter period. In fact, D1, D2, D3, D6, D7, and D8 were associated with the highest live body weight (*p* < 0.01) with mean values of 706.31, 745.50, 671.10, 733.67, 741.00, and 723.27 g/pen/21day, and consequently, the feed conversion ratio was the lowest (*p* < 0.01) during this period compared to the Control Diet (C).



methionine at 30% in excess of NRC, and L-carnitine at 15% in excess of NRC; D6 = control diet supplemented with lysine equal to NRC recommendations, methionine equal to NRC recommendations, and L-carnitine at 75% in excess of NRC; D7 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 75% in excess of NRC; D8 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC; SEM = standard error of the mean; \* *p* < 0.05, \*\* *p* < 0.01, NS = *p* ≥ 0.05; a, b: Means within the same row with common superscript letters are not significantly different (*<sup>p</sup>* ≥ 0.05).

#### *Animals* **2019**, *9*, 362

However, during the grower period, the live bodyweight of the broiler fed on D1 and D2 was not different from that of C (1241.29 g/pen/period and 1165.50 g/pen/period vs. 1207.10 g/pen/period) and was higher than those of D3, D4, D5, D6, D7, and D8 (*p* < 0.01). D1 and D2 were associated with the lowest feed conversion ratio (*p* < 0.01) with mean values of 2.33 and 2.48, respectively. During the finisher phase, feed consumption was affected (*p* < 0.05) by dietary treatment. Feed intake of broilers fed on C, D3, D6, and D7 were statistically similar (*p* > 0.05) (1851.90, 1862.00, 1945.10, and 1872.80 g/pen/d, respectively) and were higher (*p* < 0.05) than 1564.40 g/pen/day (D5).

However, live body weights were similar between dietary treatments (*p* > 0.05) with mean values of 976.53 g/pen/period and 949.27 g/pen/period, respectively. The FCR of this period was not affected (*p* > 0.05) by dietary treatment.

#### *3.2. Carcass Characteristics*

The results obtained for carcass parameters (economical carcass segments, body organ segments, and gut organs) are shown in Table 4.

Our results indicate that, with the exception of thigh weights, economical carcass segments were not affected by dietary treatment (*p* > 0.05). The neck weights of the bird given D1 and D8 were the highest (*p* > 0.05) with mean values of 64.50 g and 64.63 g, respectively, compared to 79.57 for the control diet. D2 was associated with the highest back thoracic vertebrae weight (*p* > 0.05) at 103.99 g versus 45.11 g for birds given the diet D8. However, all other body segments (heart, liver, gizzard, and abdominal fat) weights were not affected by dietary treatment (*p* > 0.05). With the exception of proventriculus weight, gut organ weights were not affected (*p* < 0.05) by dietary treatment. Our data showed that the dietary supplementation of lysine and methionine at 30% with a level of L-carnitine equal to NRC and of 15% of lysine, methionine, and carnitine increased (*p* < 0.05) proventriculus weight from 10.77 g to 11.26 and 11.43 g, respectively.



NRC L-carnitine at 75% in excess of NRC; D8 = control diet

SEM = standard error of the mean; \* *p* < 0.05; NS = *p* ≥ 0.05; a, b: Means within the same row with common superscript letters are not significantly different (*<sup>p</sup>* ≥ 0.05).

recommendations,

 and L-carnitine at 75% in excess of NRC; D7 = control diet

supplemented

 with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC;

supplemented

 with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and

#### *3.3. Intestine Segments*

The effects of dietary treatment on the length, weight, width, and diameter of small intestine segments (duodenum, jejunum, and ileum) in broilers are shown in Table 5.

The dietary supplementation of amino acid decreased (*p* < 0.05) duodenum weight and length. Birds given a supplemented diet with 30% of lysine and methionine in excess of NRC and 75% of L-carnitine in excess of NRC had the lowest weight, with a mean value of 12.99 g. The lowest duodenum length was recorded when birds were given 15% of lysine and methionine in excess of NRC plus 75% of L-carnitine in excess of NRC. A combination of L-carnitine (75%) and lysine-methionine (30%) increased the duodenum width significantly (*p* < 0.05) from 7.50 mm to 9.38 mm. Concerning the duodenum diameter, the dietary addition of lysine-methionine at a level equal to NRC plus 15% of L-carnitine in excess of NRC (D3), of lysine-methionine and L-carnitine at a rate of 15% in excess of NRC (D4), and of lysine-methionine and L-carnitine at a rate of 30% in excess of NRC plus 15% of L-carnitine in excess of NRC (D5) increased the diameter from 0.94 mm (C) to, respectively, 1.61 mm, 1.67 mm, and 1.68 mm.

Dietary treatments did not affect (*p* > 0.05) the jejunum weight and length. The dietary addition of lysine-methionine at a level equal to NRC plus 15% of L-carnitine in excess of NRC (D3) and of lysine-methionine and L-carnitine at a rate of 15% in excess of NRC (D4) increased the jejunum width (*p* < 0.05) from 9.26 mm (C) to 9.50 mm (D3) and 9.80 mm (D4). The dietary supplementation of amino acids significantly decreased (*p* < 0.05) the ileum weight from 9.10 g (C) to 2.67 g (D4) and 2.97 g (D7) and the length from 18.16 mm to 9.26 mm (D8) and 8.00 mm (D7). The ileum width of the broiler group fed with lysine-methionine at a level of 15% plus L-carnitine at 75% in excess of NRC was the highest (*p* < 0.05) compared to the control diet 7.33 mm versus 7.97 mm (D7). Dietary treatment did not affect (*p* > 0.05) ileum diameter.

Caecum weights were not different between treatments and ranged from 12.62 g (D5) to 17.13 g (C) (*p* > 0.05). The colon weight of broiler bird fed with lysine-methionine at a level equal to NRC plus 15% of L-carnitine in excess of NRC (D4) was the highest (*p* < 0.05), with a mean value of 2.36 g. Rectum weights were similar between all the treatments groups and ranged from 1.78 g (D6) to 2.32 g (D7) (*p* > 0.05).



(Control) = diet with lysine, methionine, and L-carnitine equal to NRC recommendations; D1 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine equal to NRC; D2 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine equal to NRC; D3 = control diet supplemented with lysine equal to NRC, methionine equal to NRC, and L-carnitine at 15% in excess of NRC; D4 = control diet supplemented control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 15% in excess of NRC; D5 = control diet supplemented lysine at 30% in excess NRC, methionine at 30% in excess of NRC, and L-carnitine at 15% in excess of NRC; D6 = control diet supplemented with lysine equal to NRC recommendations, methionine equal to NRC recommendations, and L-carnitine at 75% in excess of NRC; D7 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 75% in excess of NRC; D8 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC, SEM= standard error of the mean; \*\* *p* < 0.01, \* *p* < 0.05; NS = *p* ≥ 0.05; a, b, c, d: Means within the same row with common superscript letters are not significantly different (*<sup>p</sup>* ≥ 0.05).

of #### *3.4. Humoral Immune Response*

#### 3.4.1. Humoral Immune Response against Sheep Red Blood Cell (SRBC)

The dietary effect of amino acids supplementation on primary and secondary antibody responses are shown in Table 6. IgT and IgG titers against SRBC for both primary and secondary responses were not affected by dietary treatments (*p* > 0.05). Birds receiving diets supplemented with excesses of amino acids (D1, D2, D3, D4, D5, D6, D7, and D8) had significantly lower titers of IgM than that of those receiving C for primary response (*p* < 0.05), with mean values of 2.33 log10 versus 2.00 log10 (D1), 1.66 log10 (D2), 1.33 log10 (D3), 1.33 log10 (D4), 1.00 log10 (D5), 1.33 log10 (D6), 1.33 log10 (D7), and 2.00 log10 (D8). Dietary treatment did not affect (*p* > 0.05) IgM titer after a secondary challenge.



C (Control) = diet with lysine, methionine, and L-carnitine equal to NRC recommendations; D1 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine equal to NRC; D2 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine equal to NRC; D3 = control diet supplemented with lysine equal to NRC, methionine equal to NRC, and L-carnitine at 15% in excess of NRC; D4 = control diet supplemented control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 15% in excess of NRC; D5 = control diet supplemented lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 15% in excess of NRC; D6 = control diet supplemented with lysine equal to NRC recommendations, methionine equal to NRC recommendations, and L-carnitine at 75% in excess of NRC; D7 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 75% in excess of NRC; D8 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC, SEM = standard error of the mean; \* *p* < 0.05, NS = *p* ≥ 0.05; a, b: Means within the same column with common superscript letters are not significantly different (*p* ≥ 0.05).

#### 3.4.2. Humoral Immune Response against Bronchitis, Newcastle and Gumboro Diseases

The influence of different amino acids dietary supplementations on serum antibody titer in chickens vaccinated against IB, NCD, and Gumboro virus are shown in Table 7. Serum antibody titers in broilers vaccinated against bronchitis were not affected (*p* > 0.05) by dietary treatment on day 23. However, a significant increase (*p* < 0.05) in serum antibody titers in broilers vaccinated against NCD and Gumboro on the 27th and the 30th days was observed from, respectively, 3.66 log10 (C) to 6.00 log10 (D3) and from 3.38 log10 (C) to 3.59 log10 (D6). Chickens fed with 30% of lysine-methionine in excess of the NRC and of L-carnitine equal to the NRC supplemented diet had the heaviest thymus (*p* < 0.01) with a mean value of 24.60 g versus 15.59 for the control diet. However, chickens fed on L-carnitine (15%) and lysine-methionine (15%) in excess of the NRC had the lightest thymus with a mean value of 7.17 g. Bursa of fabricius and spleen weights were not affected (*p* > 0.05) by dietary L-carnitine and lysine-methionine supplementation.



C (Control diet) with lysine, methionine, and L-carnitine equal to NRC recommendations; D1 control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine equal to NRC; D2 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine equal to NRC; D3 = control diet supplemented with lysine equal to NRC, methionine equal to NRC, and L-carnitine at 15% in excess of NRC; D4 = control diet supplemented control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 15% in excess of NRC; D5 = control diet supplemented lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 15% in excess of NRC; D6 = control diet supplemented with lysine equal to NRC recommendations, methionine equal to NRC recommendations, and L-carnitine at 75% in excess of NRC; D7 = control diet supplemented with lysine at 15% in excess of NRC, methionine at 15% in excess of NRC, and L-carnitine at 75% in excess of NRC; D8 = control diet supplemented with lysine at 30% in excess of NRC, methionine at 30% in excess of NRC, and L-carnitine at 75% in excess of NRC; SEM = standard error of the mean; \*\* *p* < 0.01, \* *p* < 0.05, NS = *p* ≥ 0.05; a, b, c, d: Means within the same row with common superscript letters are not significantly different (*<sup>p</sup>* ≥ 0.05).

#### **4. Discussion**

It has been observed that L-carnitine supplementation can enhance broiler productivity [26]. However, the effect of administrating an excess of L-carnitine may depend on the magnitude of its endogenic biosynthesis from lysine and methionine [20,29–31]. In the current study, we are reporting the effect of dietary supplementation of different levels of L-carnitine with or without an excess of lysine-methionine on growth performance, carcass traits, and some humoral immunity markers of broiler chicken.

#### *4.1. Growth Performance*

The results relative to the growth performance show that a combination of lysine and methionine at 15% and 30% in excess of the NRC recommendations was without impact on the feed intake, live body weight gain, and FCR of broiler chicken during the whole experiment period.

Live body weights at the end of the experimental trial varied from 2600.70 g to 2886.20 g. These values are consistent with those reported by Bouyeh and Gevorgyan [39], who showed that dietary supplementations of lysine-methionine at 0, 1.1, 1.2, 1.3, or 1.4% higher than NRC recommendations [11] led to body weight gains from mean values of 2960 g to 2920 g (1.1%), 2850 g (1.2%), 2970 g (1.3%), and 2730 g (1.4%), respectively.

On the other hand, Hickling et al. [12] showed that broilers fed with diets supplemented with methionine at a level as suggested by NRC and at a level 112% in excess with respect to the NRC recommendations weighed at six weeks 2221 g and 2248 g, respectively, and had feed conversion efficiencies of 1.81 (NRC) and 1.79 (112% of methionine in excess of the NRC), respectively. Birds fed with four levels of lysine—equal to the NRC recommendations and 106%, 112%, and 118% in excess of the NRC recommendations—weighed 2221 g, 2227 g, 2234 g, and 2238 g, respectively, at six weeks of age and had feed conversion efficiencies of 1.81 (NRC), 1.81 (106% of lysine in excess of the NRC), 1.80 (112% of lysine in excess of the NRC), and 1.79 (118% of lysine in excess of the NRC). Mukhtar et al. [50] found that dietary increasing of lysine from 53% to 78% and of methionine from 36% (control diet) to 61% had a significant effect on feed intake, body weight gain, and feed conversion. Mukhtar et al. [50] also reported a significant improvement of feed intake, average body weight gain, and feed conversion ratio when broiler chicken were fed with five diet: diet A (1.2% lysine + 0.49% methionine) without a broiler supper concentrate, used as control, diet B similar to diet A but with a broiler supper concentrate, diet C (1.3% lysine + 0.56% methionine), diet (D) (1.4% lysine + 0.6% methionine), and diet (E) (1.5% lysine + 0.63% methionine). Body weight gain increased from 1080.76 g (A) to 1806.75 g (B), 1828.31g (C), 1834.93 g (D), and 1940.0 g (E). Feed conversion ratio decreased from 2.32 (A) to 1.97 (B), 1.95 (C), 1.94 (D), and 1.93 (E). More recently, Bouyeh and Gevorgyan [51] reported that the dietary incorporation of lysine and methionine at 0, 10%, 20%, 30%, and 40% in excess of the NRC recommendations [11] did not affect the body weight gain at 42 days of age but that the feed conversion ratio was affected by dietary treatment at 21 days of age.

With regard to the L-carnitine dietary supplementation, our results show that dietary inclusion of L-carnitine at 75% or 15% in excess of NRC recommendations increased the live body weight, feed intake, and FCR of 308 Ross broilers. In this respect, our data are in line with those obtained by Celik et al. [40]. These authors reported that the growth performance of broilers was improved by L-carnitine supplementation at a level of 50 mg/L in drinking water.

As far as L-carnitine was concerned, the results reported here are consistent with those reported by Rodehutscord et al. [52] and by Farrokhyan et al. [32], although in both studies, carnitine supply was not supplied alone but in combination with other nutrients or additives. Indeed, Rodehutscord et al. [52] studied the effect of adding 80 mg of L-carnitine per kg of the diet with two dietary levels of fat (namely 4 and 8%) on growth performance of broiler chickens. At the end of the trial, on day 21, the live body weight averaged 853 g and feed conversion was improved by almost 5% in chicken groups receiving L-carnitine supplemented diets. Farrokhyan et al. [32] also examined the effect of dietary combinations of 0, 150, and 300 mg/kg of L-carnitine with or without 1 g/kg or 2 g/kg of gemfibrozil, on the growth

performance of broilers. It has been observed that, as dietary L-carnitine increased, weight gain and birds' feed intake increased and FCR decreased. However, our results are not in agreement with those of other studies. Barker and Shell [53] showed that the dietary addition of L-carnitine at 0, 50 or 100 mg/kg diet did not affect the weight gain or feed efficiency of broiler chicken. Lien and Horng [54] demonstrated that diets supplemented with 160 mg L-carnitine/kg for 6 weeks did not affect broilers' feed intake, body weight gain, and feed conversion ratio. Corduk et al. [55] also reported that the dietary addition of L-carnitine at 100 mg/kg did not influence body weight gain, feed intake, and feed conversion ratio of broiler chickens.

Our data show that the dietary combination of L-carnitine, lysine, and methionine had a significant effect on growth performance. In fact, L-carnitine is an amine compound biosynthesized primarily in the liver from the amino acids lysine and methionine. It is involved in energy metabolism, where it is required for the transport of long-chain fatty acids into the mitochondrial matrix for β-oxidation by the fatty acid oxidation complex [3]. One the other hand, Murray et al. [56] found that the addition of synthetic amino acids like lysine and methionine at high levels to the diet can stimulate insulin secretion from pancreas by aggregating in plasma which, in turn, releases amino acids and fatty acids [57] from the bodily saved sources and leads to protein synthesis.

Adding 75% of L-carnitine plus 15% or 30% of lysine-methionine or 15% of L-carnitine plus 15% of lysine-methionine had a significant effect on feed intake and live body weight at the end of the experimental trial. Furthermore, adding 15% of L-carnitine plus 30% of lysine-methionine had no benefits, and actually reduced feed intake and body weight and increased FCR significantly compared to the control.

To our knowledge, no information related to the effect of supplementing L-carnitine in combination with lysine-methionine on broiler growth performance is reported in the literature until now.

#### *4.2. Carcass Characteristics*

With the exception of drumsticks, neck, back thoracic vertebrae, and proventriculus weights, all other economical carcass segments weights were not affected by the dietary supplementation of L-carnitine and lysine-methionine. With respect to lysine-methionine supplementation, Mukhtar et al. [50] studied the effect of lysine and methionine on broilers' carcass characteristics and reported that the dietary inclusion of A (1.2% lysine + 0.49% methionine) without broiler supper concentrate, used as control, B similar to diet A but with a broiler supper concentrate, C (1.3% lysine + 0.56% methionine), D (1.4% lysine + 0.6% methionine), and E (1.5% lysine + 0.63% methionine) increased the eviscerated carcass weight from 1036.46 (A) to 1761.75 (B), 1783.21(C), 1790.93 (D), and 1894.6 (E) and the yield of commercial cuts (breast and drumstick). The percentage of meat in the drumsticks increased from 72.17% (A) to 76.54% (B), 81.76% (C), 78.67% (D), and 82.35% (E). Concerning the percentage of meat of the breast, it increased from 77.01% (A) to 80.33% (B), 85.84% (C), 86.55% (D), and 86.89% (E). However, Bouyeh and Gevorgyan [39] found that the dietary supplementation of lysine-methionine at levels of 0, 10, 20, 30, or 40% more than the NRC [11] recommendation did not affect the thigh and leg percentage to carcass weight. However, it had a significant effect on breast meat yield, carcass traits, and abdominal fat pad, and liver and heart weights. Concerning the effect of L-carnitine supplementation on carcass traits, previous studies have shown that the dietary inclusion of L-carnitine did not affect abdominal fat, heart, and liver weights [36,53,54,58]. On the other hand, Farrokhyan et al. [32] found that the dietary supplementation of L-carnitine (300 mg/kg) reduced abdominal empty carcass from 1826.6 g/chiks to 1793.3 g/chiks and breast weight from 1566.6 g/chiks to 1563.3 g/chiks. This dietary supplementation did not affect wing weight. The limited effect of L-carnitine observed in the present study could be attributed to a limited intestinal absorptive capacity of L-carnitine. Another possible explanation is that L-carnitine is easily degraded by intestinal microflora as suggested by Xu et al. [37].

#### *4.3. Intestine Segments*

In the present study, the dietary supplementation of lysine-methionine and L-carnitine had a significant effect on all intestine segments with the exception of jejunum weight and length, ileum diameter, and caecum and rectum weight. As far as we know, the effect of simultaneous dietary supplementation with lysine-methionine and L-carnitine on intestine segments is not documented. Some studies underline the relationship to villi surface area to better feed utilization, higher nutrient absorption, body weight gain, and growth performance [59,60]. However, Saki et al. [61] reported that the dietary addition of 0.36% of methionine in broilers feed did not affect intestinal villi characteristics on the 21st and 42nd days of age.

#### *4.4. Humoral Immune Response*

IgM primary response against the SRBC of birds fed with supplemented diets with L-carnitine and lysine-methionine was significantly lower than that of birds receiving the unsupplemented diet. No significant differences have been observed among dietary treatments for IgT and IgG titers against SRBC during both primary and secondary antibody responses. Latshaw [62] reported that antibodies are proteins. Therefore, any deficiency of essential amino acids, particularly during the growth of chickens, results in poor immune competence. Lysine is one of the amino acids that can influence the magnitude of antibody response [63,64]. This could be the reason that the lowest immune response was observed in the control diet where lysine was not supplemented. Therefore, a 15% of lysine broiler chicken diet was sufficient to stimulate optimum antibody production, and thus, a lower immune response was observed when lysine was added at 30% and 75% in excess of the NRC.

Reports on the effect of methionine supplementation on broiler chickens' humoral immune response are lacking. However, as far as L-carnitine was concerned, Deng et al. [65] found that the dietary addition of 0 (control), 100 mg/kg, or 1000 mg/kg of L-carnitine did not affect primary antibody responses to SRBC at the 4th week but that birds fed on a diet with 1000 mg of L-carnitine had a higher primary antibody response against SRBC than broilers in other groups at the 12th week. Moghaddam and Emadi [66] reported that there was a tendency for an increase in IgG, IgM, and IgA antibody titers as dietary threonine increased from 0.8% to 0.87%. However, IgG, IgM, and IgA antibody titers decreased when threonine was administrated at levels of 0.94% and 1.01%. The titers of IgG, IgM, and IgA antibodies for a secondary response were higher than those for a primary response.

#### **5. Conclusions**

The findings of this study suggest that the dietary supplementation of L-carnitine solely or in combination with excesses of lysine or methionine (with respect to NRC recommendations) did not affect body weight gain, feed conversion, and economical carcass segments of broiler chickens. By contrast, the diet with around 30% of lysine-methionine content increased the back thoracic vertebrae and the proventriculus weights. A combination of lysine-methionine (level equal to NRC recommendations) with L-carnitine (15% and 75%) improved the immune response of broiler chickens against Newcastle and Gumboro diseases by stimulating the antibody production.

**Author Contributions:** Conceptualization, A.S. (Alireza Seidavi) and A.S. (Antonello Santini); data curation, S.M.G., B.O., M.D., M.L., A.D., and R.v.d.H.; formal analysis, B.O., R.C., M.B., M.D., and R.v.d.H.; investigation, A.S. (Alireza Seidavi) and A.S. (Antonello Santini); Methodology, S.M.G., R.C., M.B., M.D., and R.v.d.H.; supervision, A.S. (Alireza Seidavi) and A.S. (Antonello Santini); validation, B.O., M.L., and A.D.; writing—original draft, S.M.G., A.S. (Alireza Seidavi), M.L., A.D., and A.S. (Antonello Santini).

**Funding:** Financial support by Rasht Branch, Islamic Azad University, grant number 4.5830 is gratefully acknowledged. We are grateful to the Rasht Branch, Islamic Azad University, Rasht, Iran for support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Application of Moringa (***Moringa oleifera***) as Natural Feed Supplement in Poultry Diets**

#### **Shad Mahfuz and Xiang Shu Piao \***

State Key laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

**\*** Correspondence: piaoxsh@cau.edu.cn; Tel./Fax: +86-1062733688

Received: 30 May 2019; Accepted: 28 June 2019; Published: 9 July 2019

**Simple Summary:** The wide application of in-feed antibiotics in poultry production has created public health hazards. A driving force for the interest of using natural herbs is to establish the antibiotics alternative in poultry production that has been reported in the literature. Therefore, the objective of the current review is to determine the effects of moringa (*Moringa oleifera*) tree leaves, seeds and their extracts on chickens' performance and health status. Based on previous findings, *M. oleifera* as natural feed supplement has sustained the production performance and improved the health status in chickens.

**Abstract:** Application of natural herbs with a view to enhancing production performance and health status has created an important demand in poultry production. With the increasing concerns on this issue, greater attention paid to alternatives to antibiotics for organic meat and egg production has led to a great demand. This study was conducted with view to assessing the possible role of *M. oleifera* as a natural feed supplement in poultry ration. Various scientific findings and published research articles were considered concerning issues including the study background, objectives, major findings, and conclusions of the review. *M oleifera* is known as a miracle tree because of its wealthy resource of various nutrients with high biological values. *M. oleifera* has been used as a growth promoter, immune enhancer, antioxidant, and has a hypo-cholesterol effect on chickens. It has both nutritional and therapeutic values. However, there is still much confusion in past published articles involving the major roles of *M. oleifera* in production performance and health status of chickens. Taking this into account, the present study highlights an outline of the experimental uses of *M. oleifera* on growth performance, egg production performance, egg quality, and health status in broilers and laying hens justified with the past findings to the present. The knowledge gaps from the past studies are considered, and the feasibility of *M. oleifera* in poultry ration is suggested. The findings have motivated further study on *M. oleifera* to find out the most active ingredients and their optimal doses in both broiler and laying hen rations. Finally, the present study highlights that supplementation of *M. oleifera* may play a role in the immunity, sound health, and production performance in poultry.

**Keywords:** *Moringa oleifera*; poultry; growth performance; laying performance; health status

#### **1. Introduction**

The human population is increasing globally day by day. Meeting the increasing demand of animal protein and providing safe food for human beings that is free from antibiotics by using herbal feed resources is a great challenge for the animal scientists in the future. The issue considering antibiotic resistance has created an augmented force to reduce antibiotic uses in livestock and poultry production [1,2]. Dietary inclusion of herbs and their extracts has growth-promoting roles in poultry [3]. Furthermore, different natural medicinal plants and their extracts as feed supplements have been used as a substitute for antibiotics in poultry production [4,5]. In addition, Mahfuz et al. [6] reported

that poultry scientists are now dedicated to applying unconventional natural feed supplement, which may play a role in possible therapies to improve the health as well as production performance of chickens.Thus, poultry researchers are searching for potential natural feed resources that will be both environmentally friendly and safe for human society [7,8].

*Moringa oleifera* is a well-known cultivated species in the genus *Moringa*, (family Moringaceae) under the order Brassicales. The common names of *Moringa oleifera* include moringa, drumstick tree, horseradish tree, and ben oil tree or benzoil tree or miracle tree [9–11]. The *M. oleifera* tree is native to South Asia, especially India, Sri Lanka, Pakistan, Bangladesh, Afghanistan; North Eastern and South Western Africa, Madagascar, and Arabia [12–15]. The moringa seed and leaves have a broad use in the food industry and therapeutic issues [12]. It is popular for its seeds, flowers and leaves inhuman food and as herbal medicine [16]. The different parts of the *M. oleifera* tree are used as a good source of human nutrition and in traditional diets in different countries of the world [17,18]. Furthermore, the seed powder of *M. oleifera* contains polyelectrolytes, which are the most important active ingredients for water purification [18,19].

*Moringa oleifera* is very useful as a feed supplement for animals, as its leaves are highly nutritious. The leaves of *M. oleifera* are the most nutritious part, being a significant source of vitaminB complex, vitamin C, pro-vitamin A as beta-carotene, vitamin K, manganese, and protein among other essential nutrients [20]. *Moringa oleifera* leaves have antimicrobial roles and are rich with fats, proteins, vitamins, and minerals [18,21]. The extracts from leaves of *Moringa oleifera* contain low amounts of polyphenols, which might have effects on blood lipid metabolism [20,22]. *Moringa oleifera* can be used as a source of micronutrient and as a dietary supplement in poultry [23,24]. In addition, *Moringa oleifera* leaf powder has anti-septic and detergent properties due to presence of different phytochemicalsin the leaves [25]. *Moringa oleifera* was reported to be an excellent source of vitamins and amino acids that reportedly boost immune systems [17]. The seed extracts of moringa are rich in polyunsaturated fatty acid [26,27]. *Moringa oleifera* exhibits anti-oxidant properties that can suppress formation of reactive oxygen species (ROS) and free radicals [27,28].

Until the present day, the application of *M. oleifera* in farm animals to improve the production performance and health status has been limited. Even though it was established that *M. oleifera* has medicinal importance for the health of chickens, unfortunately the inclusion levels of *M. oleifera* in poultry ration and their mode of actions are still under consideration. Taking this into consideration, the present study focuses on uses of *M. oleifera* as a natural feed supplement as well as an alternative to antibiotics that can improve the performance and health status of chickens.

#### **2. Biological Role of** *M. oleifera*

The *M. oleifera* tree is globally known for its economic and therapeutic roles (Figure 1). Ithas been honored as the "Botanical of the Year 2007" by the National Institute of Health (USA), [11]. The tree is also known as "never die" or "miracle tree"to the people of Africa [11]. Now the application of *M. oleifera* leaves in preparing foods is receiving great attention. Peoples from Ghana, Nigeria, Ethiopia, East Africa, and Malawi are consuming the moringa tree leaves directly in their diets [29]. Furthermore, *M. oleifera* leaves have been used for making soups, foods, breads, cakes, and yoghurts [30–33].

**Figure 1.** *Moringa oleifera* tree, tree leaves, and leaves powder.

#### *2.1. Antioxidant Properties of M.oleifera*

*M. oleifera* tree leaves possess various phytochemicals that have antioxidant properties and roles in controlling a wide range of diseases, like diarrhea, asthma, and various cancers [11]. The leaves of *M. oleifera* have also been reported to hold extensive amounts of total phenols, proteins, calcium, potassium, magnesium, iron, manganese, and copper [33]. They also contain rich sources of different phytonutrients, such as carotenoids, tocopherols, and ascorbic acid, which are good sources of dietary antioxidants [34,35]. A significant increase in activities of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), and a decrease in lipid peroxide (LPS) content were found in moringa leaf extracts [11]. In addition, leaves extract from *M. oleifera* could improve the superoxide dismutase (SOD), catalase, glutathione, and peroxidase levels and reduce lipid peroxidation in albino mice [36]. Furthermore, total phenolic, flavonoid, and flavonol content in leaf extracts was found to be 120 mg/g of gallic acid equivalents (GAE), 40 mg/g of GAE, and 12.12 mg/g of GAE, respectively [37,38].

#### *2.2. Therapeutic and Antimicrobial Properties of M.oleifera*

*M. oleifera* leaf extracts have been distinguished as having anticancer, cytotoxic, anti-proliferative, anti-leukemia, anti-hepatocarcinoma, and chemo-protective properties [39–41]. The antitumor function of leaf extracts of *M. oleifera* is associated with the antioxidant and apoptosis inducing properties [42,43]. The antimicrobial properties of *M. oleifera* are well established. The extracts derived from *M. oleifera* tree leaves have been reported to be potential antibacterial and antifungal functions against various bacterial and fungal species [11,44,45]. Oluduro et al. [46] and Pandey et al. [47] have highlighted that *M. oleifera* exhibited 4-(α-L-rhamnopyranosyloxy) benzyl isothiocyanate, methyl N-4-(α-Lrhamnopyranosyloxy) benzyl carbamate, and 4-(α-D-glucopyranosyl-1→4-α-L-rhamnopyranosyloxy) benzyl thiocarboxamide that were able to play antimicrobial properties. The antimicrobial activities of the *Moringa oleifera* may be due to presence of lipophilic compounds and different metabolites (carboxylic acid, 2,4-diacetyl phloroglucinol, enzymes, and chitinases) in plant cell walls [48].

#### *2.3. Immune Stimulating and Hypocholesterolemic Properties of M.oleifera*

The immune functions of *M. oleifera* are also established by several in vitro studies [11]. Various biochemical ingredients, like quercetin, different glycosides, various isothiocyanate, kaempferol glucosides, that possess anti-inflammatory properties have been demonstrated from the extract of various parts of *M. oleifera* [49,50]. Different protein and various peptides' (isothiocyanates, glycoside cyanides etc.) presences in *M. oleifera* leaf extracts were able to modify the immune response positively [51,52]. An investigation was carried out to detect the immunomodulatory activity of *M. oleifera* on mice model. Chronic administration of *M. oleifera* significantly increased white blood cell (WBC) count and percent of neutrophils in experimental mice [51]. The exact mechanism of action of moringa leaves on stimulating the humoral and cellular immunity is not clear yet [51]. *M. oleifera* leaf extracts are reported to possess ahypo-cholesterolemic function [53]. β-sitosterol and 4-[α-(L-rhamnosyloxy) benzyl]-o-methyl thiocarbamate (trans) are two important active substances presence in the leaf extracts of *M. oleifera* that exhibit cholesterol lowering activities. These compounds could reduce the intestine uptake of dietary cholesterol in rats [49,54]. Furthermore, plasma cholesterol was decreased and fecal cholesterol was increased in rats fed with moringa leaf extracts [49,53]. In addition, another two components, moringine and moringinine, have been recently identified from *M. oleifera* leaves, which have roles in anti-hypoglycemic functions [49,55].

#### *2.4. Nutritional Properties of M.oleifera*

*M. oleifera* is also very popular for its nutritional values. It is reported as a good source of six major nutrients: Carbohydrate, especially dietary fibers; proteins; vitamins; minerals; lipids; and water. The unique features of *M. oleifera* are its richness in proteins, carbohydrates, and fibers with low fat. The leaves have been reported to enclose a range of essential amino acids and are a good source of

alpha linoleic acid [56]. *M. oleifera* leaves have been seen to exhibit high contents of vitamin A, C, and E [33]. The relative bioavailability of folate originated from *M. oleifera* leaves were about 82% in a rat model, which confirmed the fact that *M. oleifera* leaves exhibit rich source of dietary folate [57].

The nutritional composition of *M. oleifera* leaves (dry matter basic) showed dry matter (DM) about 93.63% to 95.0%, crude protein (CP) 17.01% to 22.23%, carbohydrate 63.11%to 69.40%, crude fiber (CF)6.77% to 21.09%, crude fat (EE) 2.11% to 6.41%, ash (total mineral) 7.96% to 8.40%, gross energy 14.790 (MJ/kg), and fatty acid 1.69% to 2.31% [58–60]. In addition, estimated calcium (Ca) was 1.91%; potassium (K) was 0.97%; sodium (Na) was 192.95, iron was (Fe) 107.48, manganese (Mn) was 81.65, Zinc (Zn) was 60.06, and phosphorus (P) was 30.15 parts per million (ppm) [59]. Magnesium (Mg) was 0.38%, and copper (Cu) was 6.1%, tannins 21.19%, phytates 2.57%, trypsin inhibitors 3.0%, saponins 1.60%, oxalates 0.45%, and cyanide 0.1% was also reported by Ogbe and John [59]. The leaves of the plant are enriched with methionine, phosphorus, calcium, and iron [11]. It is believed that the leaves of *M. oleifera* contain more calcium and twice as much protein than milk, higher vitamin C than oranges, higher potassium and iron than bananas, and higher vitamin A than carrots [10,61], and thus the plant is considered unique in nature [62]. Niaziridin, an active component that was identified from *M. oleifera*, can improve the absorption of different vitamins, minerals, and other micro nutrients in gastrointestinal tract of the host [50]. The nutritional composition of *M. oleifera* leaves are presented in Tables 1 and 2.


**Table 1.** Chemical compositions of *Moringa oleifera* leaves †.

† All values are in 100 g per plant material. References: [10,56,63].


**Table 2.** Amino acid contents in *Moringa oleifera* leaves †.

† References: [15,56,64,65].

It was thought that the moringa contains different anti-nutritional factors, such as tannins, phytates, oxalates and cyanide, which may affect normal digestion and metabolism of nutrients in animals [66]. In moringa, tannins and phytates are 12 and 21 g kg−<sup>1</sup> of DM, respectively, which can be neutralized by different feed processing techniques, including chopping, socking, heat steaming, and fermentation with beneficial organisms [65]. Considering the health benefit effects of moringa, it is a unique plant due to its enriching minerals with lower anti-nutritional components [65].

#### **3. Application of** *M. oleifera* **on Performance in Chickens**

In most of the feeding experiments in poultry, the fresh, green, and undamaged mature *M. oleifera* leaves were properly air-dried, and then the dried leaves were ground to a fine powder in a hammer mill and considered as moringa leaf powder or leaf meal. Similarly, fresh mature moringa seeds were air-dried and ground and considered as moringa seed meal. In some experiments, the ground particles were then soaked into distilled water for 24 h, and the filtered aqueous solution was considered as moringa extract. Due to the rich nutrient content, especially the high amount of crude protein (CP), vitamins, and minerals, *M. oleifera* leaves can be used as a useful resource of dietary supplementation for livestock as well as poultry [65–67]. In addition, Briones et al. [68] stated that moringa leaves can be applied as a dietary supplement in layers and broilers due to high production performance and improved eggs quality. However, still there are many debates on the chicken's performance with different doses of *M. oleifera* in the previous studies. There are also many variables on doses and part of plant used, such as leaves, extract, sods, or seeds. Finally, many scientists agreed that *M. oleifera* plant might have a positive role in improving the production performance and health status in chickens. Further studies are still needed to detect the actual doses of application for optimum performance in chickens.

#### *3.1. E*ff*ects of M. oleifera on Growth Performance in Broilers*

The major findings on the role of *Moringa oleifera* on performance in broilers are summarized in Table 3. Alabi et al. [69] applied aqueous *M. oleifera* leaf extracts on the performance in broiler chickens. This study demonstrates that average daily body weight gain and final body weight were higher in 120 mL/L extract-supplemented groups than the control. Feed intake was highest in birds on positive control (having antibiotics) and lowest in birds that consumed 90 mL/liter of leaf extracts. Feed conversion ratio (FCR) was lower in birds on 90 mL/L and 120 mL/L of leaf extracts fed groups. Collectively, the authors suggested that moringa leaf extracts can be added up to 90 mL/L in broiler chickens for optimum performance. The higher body weight and lower FCR in this study might be related to the presence of different bioactive components in moringa leaf extracts that may play a role in improved nutrient utilization in supplemented birds. Similarly, higher body weight was also recorded by Khan et al. [70] who used moringa leaf powder as dietary supplement with 1.2% levels in broilers. Abdulsalam et al. [71] conducted an experiment with moringa leaf meal in broilers and found that supplemented diets could enhance the growth performance at finisher period. The authors finally stated that moringa leaf meal can be applied as a natural source of protein in broiler diets. Similarly, inclusion of *Moringa oleifera* leaves at higher levels (15% and 20%) in broiler diets resulted in a higher growth rate and better health status in broilers [14]. In addition, dietary supplementation of *M. oleifera* leaves at 5% to 20% level showed higher growth performance in broilers [66]. Final live weight, average weight gain, and FCR were higher in 10% moringa leaf meal supplemented diets than the control through a 35-day trial period [72].Furthermore, feeding with *M. oleifera* leaf powder could improve live weight, body weight gain, dressing percentage, and FCR in broilers [73].

In contrast, no significant differences were observed on growth performance and economic parameters in broilers fed with *Moringa oleifera* leaf meal, according to Onunkwo and George [18]. Finally, the authors stated that *Moringa oleifera* leaf meal may be used at the level of 10% with view to reducing the production cost [18]. Similarly, feeding with moringa leaf meal in broilers led to a lower feed intake with higher FCR, as reported by Gakuyaet al. [74], which was due to presence of anti-nutritional factors in moringa leaves used in the experiment diets as row basis. No significant differences were observed on final live weight and dressing percentage by feeding moringa seed powder in broilers [75]. Gadzirayi et al. [76] applied *Moringa oleifera* leaf meal as supplementing part of conventional soybean meal in broiler diets at 0%, 25%, 50%, 75%, and 100% level. The author did not find any significant differences on feed intake and body weight gain between control and 25% level of moringa supplementation. However, significantly lower FCR was observed in moringa leaf meal fed groups. Finally, the study suggested using moringa leaf meal at a 25% level to promote growth in broilers. In addition, Ayssiwede et al. [77] noted that dietary application of moringa leaf meal up to a level of 24% had no adverse effects on body weight, average daily weight gain, FCR, mortality, and the weight of organs in broilers compared to the control diet. Olugbemi et al. [78] stated that average daily growth rate was lower with *Moringa oleifera* leaf meal at the inclusion level below 5% in diets, and the authors suggested to use maximum level of 5% without any harmful effects on growth performance and FCR in broilers. These findings confirmed the fact that feeding with moringa leaves had no deleterious effects on normal physiology and growth in the experimental broilers. However, collectively, some authors suggested that use of the *Moringa oleifera* leaf meal up to a 10% level would not have any adverse effects in broilers [78–80]





#### *3.2. E*ff*ects of M. oleifera on Meat and Bone Quality in Broilers*

Dietary manipulation is an important way to improve the meat quality in poultry [2]. The meat derived from broiler chickens is an excellent source of protein, vitamins, minerals, and lower fat and has created a great demand among consumers [88]. Meat pH, tenderness, color (lightness, redness, and yellowness),and water holding capacity are very important meat quality characteristics to the consumers. An experiment on supplementation of *Moringa oleifera* leaf powder on the quality of meat and bone in broilers was conducted by Rehman et al. [81]. This study noticed that supplementation of leaf powder at 12 g/kg level could increase pH, water holding capacity, and muscle fiber diameter in the breast muscle of experimental broilers. In addition, higher weight, ash percentage, and the density of tibia bone in broilers fed with moringa leaf meal were also recorded in their studies [81]. In this study, authors hypothesized that higher muscle pH values in experimental groups were due to the stabilization of the myofibrils by activating antioxidant properties and preventing free radicals. Higher breast muscle weight could be the result of increased protein deposition in moringa-supplemented groups. The higher tibia bone weight and ash percent may be due to the presence of phytoestrogen flavonoids in moringa leaves powder. In contrast, Nkukwana et al. [82] found that *Moringa oleifera* leaf meal had no effects on tibia bone characteristics but could improve body weight gain and FCR. These differences might be related with inclusion levels and types of incorporation of moringa in broiler diets. However, it is a popular belief that dietary antioxidants can modify the meat color, minimize the rancidity, and retard lipid peroxidation, resulting in a well-maintained meat quality. The oxidative status of meat muscle is directly related to meat quality and has negative effects on cooking loss, drip loss, meat color, and pH [89]. Therefore, dietary supplementation of antioxidant-enriched moringa leaves would be a potential strategy to improve the meat quality in broilers. Moreover, it was reported that phytosterols could reduce malondialdehyde (MDA) content and increase glutathione (GSH) concentration in the breast muscle of experimental broiler chickens [88]. The inclusion of moringa leaf meal could improve fatty acid profile and could reduce lipid oxidation in breast muscle of broilers [84]. The authors assumed that improved fatty acid profile was due to the presences of saturated fatty acids in moringa leaves.

#### *3.3. E*ff*ects of M. oleifera on Health Status in Broilers*

Alnidawi et al. [14] has conducted an experiment with a view to examining the effects of *Moringa oleifera* leaf on health status in broilers.This study ensured that total cholesterol content was lower with higher level (at 15% and 20%)of *M. oleifera* fed in broiler diets. Similarly, high-density lipoprotein cholesterol (HDL) content in serum was increased and low-density lipoprotein cholesterol (LDL) was decreased with higher level of supplementation of *M. oleifera* in broilers. It was hypothesized that higher amounts of natural fiber in moringa leaves may have a role in lowering cholesterol level by increasing lipid metabolism in the host body. In addition, the blood parameters, like hemoglobin percent, total red blood cells number, and total packed cell volume, were found to be higher at 20% supplementation levels than the control diet [14]. *M. oleifera* leaf powder was considered as dietary supplement with 0.6%, 0.9%, 1.2% and 1.5% levels in broilers on growth performance and intestinal microarchitecture [70]. The intestine's morphological characteristics in chickens are vital for nutrient utilization and an indicator of sound physiology. The length and empty weight of small intestine were found higher in broilers fed with 1.2% leaves powder. In addition, higher villus height (duodenum, jejunum, ileum), villus surface area (duodenum), and villus height/crypt depth (ileum) were observed in 1.2% leaves powder fed group than the control. Higher villi suggest better absorption of nutrients due to enlarged surface area, which is a good indicator of gut system. Furthermore, the improvement of villus height and villus height/crypt depth ratio may be linked with high content of crude fiber in moringa-supplemented diets. This study further observed that total goblet cells of duodenum were higher in broilers fed with all levels of *M. oleifera* leaf powder in comparison with control group. The findings indicate enhanced mucosal protection with *M. oleifera* supplementation in broiler diets. Goblet cells are essential elements of innate gut immune system in poultry. Bursal follicle count was

also found to be higher in 1.2% *M. oleifera*-fed group than non-supplemented control diet. Finally, the authors concluded that dietary supplementation of *M. oleifera* at 1.2% level could modulate the intestinal structure and acidic mucin production without any adverse effects on growth performance in broilers [70].

The extract from the leaves of *Moringa oleifera* has apotential role as an anti-bacterial and antioxidant functions [22]. The roles of *Moringa oleifera* leaf meal at 10% and 15% level on the hematological parameters in broilers were examined by Ebenebe et al. [72]. Feeding *Moringa* leaf meal in broilers resulted in increased red blood cell (RBC), packed cell volume (PCV), and hemoglobin (HB) values in both levels of diets. Finally, the authors stated that *Moringa oleifera* leaf meal should be used within the 10% level in broiler diets. *Moringa oleifera* is known to a potential antioxidant with some antioxidant properties due to the presence of vitamins C and E, carotenoids, flavonoids, and selenium [15]. *Moringa oleifera* leaves contain various phytochemicals (carotenoids, flavonoids, chlorophyll, phenolics, xanthins, cytokines, alkaloids, etc.) that might have a role in improving health status [90].

#### *3.4. E*ff*ects of M. oleifera on Egg Production, Performance, and Egg Quality in Laying Hens*

The major findings on the role of *M. oleifera* in performance in laying hens are summarized in Table 4. The egg quality parameters, including egg size, shape, color, shell thickness, and egg yolk cholesterol, directly and indirectly influence egg consumers. In a recent study by Voemesse et al. [91], *M. oleifera* leaf meal was used in layer chickens' diet from 1 day old to 55 weeks of age to investigate the effects of moringa leaf meal on growth performance, egg production performance, and blood parameters. *M. oleifera* leaf meal was used at three different levels (0%, 1%, and 3%). In the growing period from 1 day to 20 weeks of age, this study did not find any significant differences on feed intake, but average daily body weight gain, final body weight, and FCR were improved in *M. oleifera*-supplemented groups. In the laying period, from 21 weeks to 55 weeks, feed intake was lower in moringafed groups, but the laying percent and FCR were higher in supplemented fed groups than the non-supplemented group. The higher body weight gain and egg production may be related to improved digestibility in supplemented groups due to different active components in moringa leaves. The author concluded that feeding moringa leaf meal at 1% level had positive effects on the growth and egg production in laying hens.In addition, *Moring oleifera* at 10% levels showed higher egg production in laying hens [66]. According to Abouz-Elezz et al. [92], *M. oleifera* supplementation could improve the egg production, egg mass, and egg yolk color scores compared with the non-supplemented groups. The improvement of yolk color scores could be due to high carotene content in moringa leaves. Higher feed intake, crude protein intake, weight gain, FCR, and protein efficiency ratios were recorded in laying chicks where *Moringa stenopetala* was the experimental supplement [93]. This is because of readily available proteins with their essential amino acids in the moringa leaf meal. The authors finally concluded that *Moringa stenopetala* leaf meal at up to 6% levels can be applied in growing chicks' ration.

In contrast, *Moringa oleifera* seed meal at 0%, 1%, 3% and 5% levels were used to examine the effects of egg production performance, egg quality, and egg fatty acid profile in Hy-Line laying hens [94]. Lower feed intake, egg production percent, egg mass, feed intake, and body weight were observed in moringa seed meal-fed groups than the control. Higher egg yolk color scores with higher linolelaidic acid in egg yolk were found in moringa seed meal supplemented groups than the non-supplemented diets [94]. The moringa seeds may contain different anti-nutritional factors, which may have deleterious effects on production performance in this study. In addition, Ahmad et al. [95] also reported that the decrease in production performance of layer chickens was due to high fiber and different anti-nutritional factors' presences in moringa pod meal. However, this study found a significant positive role in improving β-carotene, quercetin, and selenium levels in egg yolk with moringa pod supplementation. Moringa pods are naturally enriched with carotenoids and different flavonoids, which possess natural antioxidants that could modify the β-carotene and quercetin levels in egg yolk [74]. Egg yolk cholesterol was significantly lower in moringa pod meal fed groups than the control group, which may be due to presence of natural antioxidants in the experimental diets containing moringa pod meal in this study. In addition, the nutrient profile of egg yolk was higher with the supplementation of moringa pod meal in Hy-Line layers [95]. In another study, Lu et al. [96] found that *M. oleifera* leaf meal had no effects on egg production, egg weight, and feed intake in Hy-Line Grey commercial layers, but birds fed with moringa leaf meal at 15% levels showed deeper egg yolk color than the non-supplemented fed group. Similarly, the albumen height and Haugh unit were higher in moringa-supplemented groups during storage of eggs at 4 ◦C and 28 ◦C for 4 weeks. Finally, the author stated that 5% moringa leaves meal can be included in laying hens' ration without adverse effects on egg production and egg quality. Similarly, Abou-Elezz et al. [80] found that *Moringa oleifera* leaf meal could improve egg yolk color scores and albumen percentage. This study further observed the lower egg laying percentage and egg mass in laying hens fed with moringa leaf meal. However, this study did not find any significant differences on final body weight and on other egg quality parameters (yolk percent, shell percent, and shell thickness). Finally, the author stated that 10% moringa leaf meal can be incorporated into the diets of Rhode Island Red laying hens. Feed intake, feed conversion ratio, and laying percentage were not influenced by adding moringa leaf meal at a 10% level, which was noticed by Olugbemi et al. [78]. However, inclusion of 10% moringa leaf meal could increase higher egg Roche color score [78]. A similar report on decreased egg mass and egg production percent with moringa leaf meal supplementation at higher levels (at 10% and 20%) in laying hens was observed by Kakengi et al. [79]. Interestingly, *Moringa oleifera* leaf meal at 5% level increased the egg weight, but the decreased egg weight was found when inclusion level was at 20%. The authors assumed that higher feed intake, FCR with lower egg production percent, egg mass, and egg weight at a higher-level supplementation was due to poor digestibility of nutrients because of different anti-nutritional phytochemical presences in moringa leaves [79].

Improving the egg quality by means of increasing its anti-oxidative properties by supplementing natural unconventional resources has gained a significant interest in poultry research [97]. The synthesis antioxidants, like butylated hydroxyanisole and butylated hydroxytoluene, are commonly used in food processing. However, they are found to be carcinogenic to human health, therefore, discovering natural antioxidant products as safe and effective alternatives is a very crucial need [98–100].


**Table 4.** Role of *Moringa oleifera* on performance in laying hens.†.


**Table 4.***Cont.*

#### *3.5. E*ff*ects of M. oleifera on Health Status in Laying Hens*

Analyzing blood parameters is very important in detecting the health status of birds. According to Voemesse et al. [91], serum albumin level was higher in laying hens fed with 3% level of moringa leaf meal than the control group, but the number of white blood cells (WBCs),red blood cells (RBCs), lymphocytes, and the packed cell volume were lower in moringa-fed groups than the control diets. The authors assumed that lower WBCs and lymphocytes in moringa-fed chickens may be due to the antimicrobial activity of phytochemicals in the moringa leaves. It is well known that a high WBC count is related to an infection caused by bacteria in the host. Lower level of cholesterol content in serum with dietary supplementation of moringa pod meal were observed, which might be influenced by antioxidants (flavonoids and carotenoids) and high fiber presences in the moringa pod meal in the experimental diets [95]. However, this study did not find any significant differences on antibody response against Newcastle disease virus. Lower values for malondialdehyde (MDA) and higher glutathione peroxidase in the plasma of laying hens fed with moringa leaf meal indicated the higher antioxidant activities [96]. Plasma total protein levels were higher by dietary 5% for moringa leaf meal supplementation, which is a good indicator of the liver's synthetic function. Furthermore, lower plasma uric acid in supplemented groups indicated higher protein retention in laying hens [96]. The improved antioxidant enzyme activities and the reduced MDA levels in the plasma and egg yolk indicated the fact that dietary moringa supplementation could improve the antioxidant activities. *Moringa oleifera* is an effective phytobiotic and is known to possess broad-spectrum antibacterial properties and immuno-modulatory functions [70,81,102].

#### **4. Conclusions**

This review study highlights that *M. oleifera* could be fruitfully used as an effective natural growth promoter as well as an immune-boosting agent in chickens' ration. Although *M. oleifera* was used in the experimental diets of poultry, further study was recommended by various researchers regarding the doses of *M. oleifera* on optimum performance and sound health in chickens. Thus, the future study should beconducted ina proper way so that it will examine the uses of *M. oleifera* in reaction to a pathogen challenge as well as dosages. In this study, we suggest future research with *M. oleifera* as an alternative for antibiotics in chickens so that it may be used as an effective strategy for organic meat and egg production. It could be concluded that *M. oleifera* can be used as an environmentally friendly feed supplement in chicken ration. The present study will help future researchers to discover the important effects of *M. oleifera* on immunity and health status that the past studies were not able to explore. Thus, the supplementation with *M. oleifera* may be a new concept of research in chicken production. The inclusion level of *M. oleifera* up to 10% in both broilers' and laying hens' diet could be recommended.

**Author Contributions:** Conceptualization, X.S.P.; Preparation of Manuscript, S.M., and Scientific Editing, X.S.P. and S.M.

**Funding:** This research received no external funding.

**Acknowledgments:** The author would like to express their sincere gratitude to the College of Animal Science and Technology, China Agricultural University (CAU) for financially support from the National Natural Science Foundation of China (31772612) for his post-doctoral study under Xiang Shu Piao.

**Conflicts of Interest:** There is no conflict of interest relevant to this publication.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Impacts of Graded Levels of Metabolizable Energy on Growth Performance and Carcass Characteristics of Slow-Growing Yellow-Feathered Male Chickens**

### **K. F. M. Abouelezz 1,2,**†**, Y. Wang 1,**†**, W. Wang 1,3, X. Lin 1, L. Li 1, Z. Gou 1, Q. Fan <sup>1</sup> and S. Jiang 1,\***


Received: 13 June 2019; Accepted: 9 July 2019; Published: 19 July 2019

**Simple Summary:** Inadequate feed inhibits the potential performance of birds, and giving birds excess nutrients or levels higher than the requirement reduces production profits and may lead to negative effects on performance. Although recently there has been an expanding market worldwide for slower growing chickens to meet the consumer demand for a better tasting meat, little effort has gone into optimizing their dietary nutrient levels. Using fiv e different dietary energy levels, this study evaluated the optimal requirement of dietary energy for maximal growth rate, feed:gain ratio, meat quality indices, and blood metabolites of a Chinese yellow-feathered breed.

**Abstract:** A dose-response study was conducted to investigate the metabolizable energy (ME) requirement for Lingnan chickens from 9 to 15 weeks of age. One thousand two hundred 8-week-old slow-growing yellow-feathered male chickens were allotted to five dietary ME levels (2805, 2897, 2997, 3095 and 3236 kcal/kg). The results revealed that the daily metabolizable energy intake increased (*p* < 0.01), whereas the feed intake and feed:gain ratio decreased linearly (*p* < 0.01) with the increment in dietary ME level. The final body weight and daily gain of the highest ME treatment tended (*p* > 0.05) to be greater than those obtained with the lower ME levels. The fat content in breast muscle showed a quadratic response (*p* < 0.05) to the increase in dietary energy level. The shear force values of breast muscle in the 2897, 3095 and 3236 kcal/kg treatments were lower (*p* < 0.05) than those of the 2997 kcal/kg treatment. In conclusion, among the tested ME levels, 3095 kcal/kg was adequate for feed intake, shear force, and plasma uric acid, and 3236 kcal/kg tended to increase the body weight, body gain, and feed conversion ratio of Lingnan males between 9 and 15 weeks of age; further studies are still required for testing higher levels.

**Keywords:** energy requirement; meat quality; growth performance; slow-growing broilers; nutrient deposition

#### **1. Introduction**

In poultry production enterprises, feed cost accounts for around 70% of the total costs involved in production. Among the different feed-stuffs used in formulating poultry diets, the source of dietary energy resources is a major cost; 70% of the total poultry diet content are energy sources. Optimizing the dietary energy level, therefore, is important for lowering the feed cost per unit of poultry products [1]. Increasing dietary energy level provides fundamental benefits in the feed conversion ratio (FCR) of broilers, mostly by decreasing feed consumption [2–4]. On the other hand, using excessive energy or a level higher than the requirement can increase the deposition of undesirable abdominal fat in broiler carcass, considered to be an economic loss as it is often counted as a waste product [2]. The dietary energy can be optimized for both growth performance and for enhanced meat quality. Dietary nutrient levels alter meat color, energy content, and histological makeup as well as the metabolic characteristics of broiler muscles [5–7].

The optimal dietary energy for broilers has been estimated in several previous studies [8,9], but existing data require verification for modern genotypes [10]. In contrast, little effort has gone into optimizing the dietary energy level for slow-growing meat-type chickens. Recently, there has been an expanding market worldwide for slower growing meat-type chickens, giving them a place in contemporary production. This is mainly to meet the consumer demand for better tasting meat and for fulfilling organic production conditions [11], as well as avoiding some problems with the fast-growing broilers, such as sensitivity to environmental conditions, leg problems, metabolic failure, ascites, sudden death, and an increased mortality rate occurring during the finishing phase [12–14]. This relatively new interest in slow-growing meat chicken breeds is increasing worldwide, though it is associated with higher costs of production [11].

China is the second-largest global producer of chicken meat, almost half of which is from Chinese yellow-feathered breeds [15]; Chinese annual production of such breeds exceeds four billion birds. The distinct flavor and favorable color of the meat are highly desired by local consumers in China and in neighboring countries [16]. There are three types of such chickens [17], broadly classified as fast- (marketable around 8–10 weeks, 1.47–2.30 kg weight), medium- (marketable 9–14 weeks, 1.00–2.27 kg weight), and slow-growing (marketable 12–25 weeks, 1.06–1.88 kg weight). The increasing commercial importance of these indigenous birds means that comprehensive work is needed to improve their feeding standards. As the dietary energy requirement for slow-growing yellow broilers has not been estimated or optimized, the present study has evaluated the effects of different dietary ME levels on growth performance, blood biochemical variables, carcass quality, body composition, rate of energy deposition, and fat content in breast and thigh muscles.

#### **2. Materials and Methods**

#### *2.1. Chickens, Diets and Management*

The experimental conditions were approved by the Animal Care and Use Committee of the Institute of Animal Science, Guangdong Academy of Agricultural Sciences, China, with the approval number GAASISA-2015-011. The yellow-feathered male chickens (Lingnan breed, a meat-type breed that originated in South China) were obtained from a commercial hatchery (Guangdong Wiz Agricultural Science and Technology Co., Guangzhou, China) and were raised from day 1 to 8 weeks of age on a common, typical diet, provided ad libitum. One thousand two hundred birds were weighed at 8 weeks of age and randomly allocated to 30 equally-sized (4.55 m2) floor pens of 40 birds, having a similar average body weight (BW) (771.25 ± 10.23 g). Five dietary treatments, each with six replicates, consisting of graded metabolizable energy (ME) levels (2900, 3000, 3100, 3200 and 3300 kcal ME/kg, calculated), were pelleted and provided ad libitum, as was water. These experimental diets (Table 1) were formulated to provide the nutrient requirements of Chinese yellow-feathered broilers [18], except for the ME level. The gross energy of the diets was analyzed according to the guidelines of Association of Official Analytical Chemists [19], and the ME was determined and calculated according to the methods and the equation of Jiang et al., [20], which showed 2805, 2897, 2997, 3095 and 3236 kcal/kg, respectively. The 2997 kcal/kg was considered to be the control dietary energy level diet according to the previously determined value [18]. The birds were raised under artificial lighting providing 18 h light:6 h dark. Relative humidity and average room temperature were approximately 70.0% and 18 ◦C throughout the 7-week experimental period (9–15 weeks of age).


**Table 1.** Composition and nutrient levels of the experimental diets (%, as fed basis).

<sup>1</sup> Supplied per kilogram of diet: VA 5000 IU, VD3 500 IU, VE 20 IU, VK 0.5 mg, VB1 2.4 mg, VB2 4.0 mg, VB6 3.5 mg, VB12 0.01mg, niacin 30 mg, D-calcium pantothenate 10 mg, folic acid 0.55 mg, biotin 0.15 mg, choline chloride 1200 mg, Fe 80 mg, Zn 65 mg, Cu 7 mg, Mn 60 mg, I 0.35 mg, Se 0.3 mg. The vitamins and minerals in the diet were supplied exactly as stated by the Ministry of Agriculture of the People's Republic of China [18]. <sup>2</sup> Values were calculated from data provided by the Feed Database in China [21]. <sup>3</sup> Analyzed values.

#### *2.2. Growth Variables*

The amounts of provided and refused feed were measured weekly on a replicate basis to calculate the average daily feed intake (ADFI), including adjustments for any dead birds. Mortality of birds was recorded daily. The initial BW, final BW (FBW), average daily body weight gain (ADG), and feed:gain ratio (g/g) (FCR) were measured on a per replicate basis. Metabolic BW was calculated according to the following equation: [(Initial weight + final weight)/2] 0.75.

#### *2.3. Sampling*

At 15 weeks of age, after 12 h of feed-withdrawal, blood samples were collected in 5 mL heparinized tubes from the jugular vein of 12 birds per treatment (2/replicate) who had BW values within ± 10 g of the average; plasma was obtained by centrifugation at 1000× g for 15 min at 4 ◦C. The birds were slaughtered by approved methods for subsequent analyses. The right and left breast muscles were separately sampled, clear of observable connective tissues, and stored at −20 ◦C until analyses; the right breast muscle (*Pectoralis major* and *minor*) was sampled for meat quality determinations, and the left muscle was used in measuring the chemical composition.

#### *2.4. Carcass Trait Determinations*

Dressing percentage (bled and defeathered carcass weight (CW), including head and feet, expressed as a percentage of BW), semi-eviscerated (CW minus weights of trachea, crop, esophagus, intestine, pancreas, spleen, gallbladder, gonads, contents of the proventriculus, and gizzard lining, expressed as a percentage of BW), and eviscerated proportions (semi-eviscerated weight minus neck, head, liver, heart, gizzard, shank, abdominal fat, and proventriculus, expressed as a percentage of BW) were

calculated. In addition, the relative weights of de-boned thigh muscle, breast muscle, and abdominal fat, expressed relatively to BW, were calculated following the methods of the Chinese National Poultry Breeding Committee [22]. The breast and thigh muscles were placed in polyethylene bags and stored at −22 ◦C until chemical analysis.

#### *2.5. Meat Quality Determinations*

Meat pH, color (a\* redness, b\* yellowness and L\* lightness), and drip loss were measured following the methods of Jiang et al. [23]. Meat pH was measured in the major right *Pectoralis* using a portable pH meter (version HI8424; Beijing Hanna Instruments Sci. & Tech. Co., Ltd., Beijing, China). Three readings of breast meat color were scored with a Chroma Meter (CR-410; Minolta Co., Ltd., Suita, Osaka, Japan) at different, but consistent, locations on the medial side of each muscle then averaged. Meat color scores, using L\* a\* b\* color scales, were measured; L\* is lightness (0 = black to 100 = white), a\* is green (a\*) to red (+a\*), and b\* is blue (b\*) to yellow (+b\*). Drip loss was estimated following a method modified from Shang et al. [24]. Briefly, about 11 g (fresh weight) of regular-shaped muscle section (4 cm (length) × 2 cm (width) × 1.5 cm (thickness)) cut from the same location in the breast muscle was weighed and suspended on a steel wire hook, without any contact, in a plastic bag inflated with air and stored at 4 ◦C for 24 h. The muscle samples were re-weighed to evaluate the drip loss percentage, according to the following equation: [(initial weight − final weight)/initial weight] × 100%. Finally, the shear force of cooked breast muscles was measured according to the methods described by Jiang et al. [23], using an Instron Universal Mechanical Machine (Instron model 4411, Instron Corp, Canton, MA, USA).

#### *2.6. Composition of Body, Breast and Thigh Muscles, and Deposition Rate of Energy and Protein*

The frozen samples of left breast and thigh muscles were dissected into small pieces and finely homogenized in a blender at −10 ◦C. To measure the fat and protein content, deposition rate of energy and protein in the whole body, ten birds at the age of 8 weeks (at the beginning of this experiment) and two additional birds per replicate at the age of 15 weeks were selected and prepared according to the methods of Zhou et al. [25] and Xi et al. [26]. Contents of crude protein (CP), crude fat, and gross energy were analyzed according to the guidelines of AOAC [19]. The deposition rate of protein and energy was estimated following the methods of Xi et al. [26].

#### *2.7. Blood Biochemical Variables*

The plasma contents of uric acid (UA), triglycerides (TG), and cholesterol (CHOL) were measured colorimetrically using a spectrophotometer (Biomate 5, Thermo Electron Corporation, Rochester, NY, USA) and commercial kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China).

#### *2.8. Statistical Analysis*

Each pen (replicate) served as the experimental unit. The effects of dietary ME levels were examined for each variable by ANOVA (JMP Ver. 8.0.2, 2009; SAS Institute Inc., Cary, NC, USA). Whenever significant effects of treatment were detected, Duncan's multiple range tests were used to compare the means. Where appropriate, orthogonal polynomial contrasts were used to estimate the linear and quadratic effects of the increasing levels of ME, and a probability level of 0.05 was applied to test significance (SPSS software version 17.0.1., IBM, Armonk, NY, USA). Based on the key indices (ADFI, feed:gain ratio, daily ME intake, uric acid, fat content of breast muscle, and fat content of thigh muscle), quadratic regression equations were used to determine the optimal dietary ME requirement of Chinese yellow-feathered chickens [27]. Data are expressed as means for each diet.

#### **3. Results**

#### *3.1. Growth Performance*

Daily ME intake increased, but ADFI and FCR decreased as linear responses to the increment in dietary energy level. The FBW, ADG, metabolic BW, and mortality rate were not affected (*p* > 0.05) by the dietary ME level, but the 3236 kcal/kg diet tended to have greater FBW and ADG than those of the lower ME levels (Table 2).

#### *3.2. Carcass Quality*

The tested dietary ME levels did not exhibit any significant effect on the carcass quality traits in terms of dressing percentage, eviscerated and semi-eviscerated proportions, relative weights of breast muscle, thigh muscle, and abdominal fat (Table 3).

#### *3.3. Composition of Body, Breast and Thigh Muscles*

As shown in Table 4, the fat content in thigh muscle increased linearly (*p* < 0.05) with the increase in dietary energy level, whereas the fat content in breast muscle showed a quadratic response (*p* < 0.05), and the highest value was obtained with the level 2997 kcal/kg. The protein, fat and energy content in the whole body as well as the energy and protein deposition were not affected by the dietary ME level. According to the regression model, the highest fat contents (%) in the breast and thigh muscles were obtained with diets containing 3047 and 3135 kcal/kg (Table 5).

#### *3.4. Breast Meat Quality*

The results of breast meat quality as affected by the dietary ME level are shown in Table 6. The 2897, 3095 and 3236 kcal/kg diets resulted in lower shear force values (*p* < 0.05) than those of the control diet, and those of the 2805 kcal/kg diet had an intermediate value (*p* > 0.05). The pH value, drip loss percentage, and meat color grades L\*, a\* and b\* did not differ (*p* > 0.05) among the tested diets.

#### *3.5. Blood Biochemical Variables*

The results shown in Table 7 indicated that plasma UA decreased linearly (*p* < 0.01) with the increase in dietary ME level. The CHOL and TG concentrations were not affected by the diets. The regression model indicated that the optimal plasma UA was obtained with a diet containing 3200 kcal/kg (Table 3).


**Table 2.** Effect of dietary metabolizable energy level on average daily metabolizable energy intake and the performance of Chinese yellow-feathered chickens from

Means within a row with different superscripts differ significantly (*<sup>p</sup>* < 0.05). SEM = pooled standard error mean. Metabolic body weight = [(Initial weight + final

**Table3.**Effectsofdietarymetabolizableenergylevelonthecarcass qualityofChineseyellow-featheredchickensat15weeksofage.


SEM = pooled standard error mean.


**Table 4.** Effect of dietary metabolizable energy levels on the compositions of body, breast and thigh muscles, and deposition rates of energy and proteinslow-growing

**Table 5.** Dose-response regressions for Chinese yellow-feathered chickens fed diets with different metabolizable energy levels from 9–15 weeks of age.


1 QP = quadratic polynomial; QP model = Y = α + β × X + γ × X2, where Y is the response variable, X is the dietary metabolizable energy (ME), α is the intercept; β and γ are the linear and quadratic coefficients, respectively. 2 Regression equations obtained using the analyzed metabolizable energy in the diets (2805, 2897, 2997, 3095 and 3236 Kcal/kg). 3 The response was obtained by −β/(2 × γ).

 in


**Table6.**Effectsofdietarymetabolizableenergylevelsonthebreastmeatqualityofslow-growingChineseyellow-featheredchickensat15weeksof age.

Means within a row with different superscripts differ significantly (*<sup>p</sup>* < 0.05). SEM = pooled standard error mean.

**Table 7.** Effects of dietary metabolizable energy levels on plasma variables of slow-growing Chinese yellow-feathered chickens at 15 weeks of age.


Means within a row with different superscripts differ significantly (*<sup>p</sup>* < 0.05). SEM = pooled standard error mean.

#### **4. Discussion**

#### *4.1. Growth Performance*

The present study tested five dietary ME levels (kcal/kg), consisting of a control level (2997), two lower levels (2805 and 2897), and two higher levels (3095 and 3236), respectively. The increase in dietary energy level did not affect the FBW or ADG of the slow-growing male yellow-feathered chickens, but the highest ME treatment (3236 kcal/kg) tended to result in greater FBW and ADG than lower ME levels. The daily ME intake increased, whereas ADFI and FCR decreased as linear responses to the increment in dietary energy level. Birds typically eat to fulfil their energy requirement [11,28], which can explain the reduced ADFI for the highest two dietary energy levels. The improved FCR for the highest ME level is attributable to the reduced ADFI and the relatively increased ADG. Supporting results were reported by Infante-Rodríguez et al. [4], indicating that BW and ADG were not affected by the dietary energy; however, ADFI was reduced by a high caloric level, and FCR was improved with a moderate increase in dietary energy. The present results were consistent with the findings of Kim et al. [29], who observed a reduced ADFI with higher energy levels than the standard diet. Other studies differed [30], where final BW and FCR in broilers increased with higher energy levels (2994 to 3013 and 3081 to 3111 kcal/kg ME, starter and finisher phases). Contrary to the present results, Houshmand et al. [31] found that broilers fed low-energy diets were heavier than those fed a standard diet. The results of Ferreira et al. [3] showed that a dietary energy close to 3000 kcal/kg did not affect BW in broilers, but a lower caloric level reduced BW, and a higher caloric level reduced ADFI. These varied responses to dietary energy levels in previous studies result from using different genotypes at different ages. Kim et al. [29] reported different responses to energy level with different strains of broilers. The results obtained here with slow-growing Chinese yellow chickens favor the increase in energy level over the control (2997 kcal/kg) and lower levels; the highest calorie intake occurred with the most energy-dense diet. Touchburn et al. [32] similarly noted that caloric intake increased as dietary ME level increased.

#### *4.2. Carcass Characteristics*

For the slow-growing Chinese yellow chickens studied here, dietary ME level had no significant effect (*p* > 0.05) on the dressing percentage, eviscerated and semi-eviscerated proportions, relative weights of breast muscle, thigh muscle nor abdominal fat. Supporting results were reported by Infante-Rodríguez et al. [4], who tested dietary energy levels (2960 to 3160) close to those used here; there was no influence on carcass weight, breast, drumstick and thighs, wings and back fat weight or carcass yields. Rosa et al. [33] used diets with 2950, 3200 and 3400 kcal/kg ME, but observed no effect on breast weight, carcass yield or back fat, despite the increase in energy concentration depressing the yield of thigh and drumstick and increasing abdominal fat. A preliminary study of Waldroup et al. [34] indicated no effect of dietary caloric level on growth performance or abdominal fat, although a higher energy level increased dressing percentage in females, but not in males. The present results with male chickens are consistent with that of the latter study. Others [35,36], similarly, found no effect of dietary energy level on carcass yield and abdominal fat. In contrast, Zhao et al. [37] found that dressing percentage, breast and thigh muscles, and abdominal fat content were greater with dietary energy and lysine levels higher than those in their controls. Marcu et al. [38] reported an improved growth performance and carcass yield for the main cuts of broiler chickens fed diets with high energy and protein contents. The preponderant previous findings on the effect of dietary energy level in broilers were inconclusive, but the results of the latter two studies showed that increased dietary energy along with increased CP or amino acids may result in a higher meat yield.

#### *4.3. Composition of Whole Body, Breast and Thigh Meat*

In the present study, dietary energy level did not influence the protein, fat or energy content in the whole body, but the fat content in thigh muscle increased linearly with increased caloric level, whereas the fat content showed a quadratic response and the highest value was obtained with the 2997 kcal/kg diet. Other studies showed similar results, with dietary energy level having no effect on the chemical composition of broiler's carcass muscles [39,40]. Ferreira et al. [3] indicated that using reduced dietary energy levels lowered the intramuscular fat in broilers. The present results are in partial agreement with those of Infante-Rodríguez et al. [4], who found that increased dietary energy had no effect on CP content in breast muscle, although the lower ME levels (2960 and 3040 kcal/kg) resulted in more lipids in breast meat than with higher caloric levels (3080 and 3160 kcal/kg). In another study, Marcu et al. [41] found that decreasing dietary ME level reduced CP and increased the lipid content of broiler breast and thigh muscles. The results here showed that the fat (Table 5) content in the whole body was not affected by the dietary energy. The latter results agree with our results, which suggest that increasing dietary energy content for broilers may not increase meat lipids in the thigh and pectoral muscles.

In commercial Ross 308 broilers, Rosa et al. [33] reported that increasing the dietary ME level reduced carcass CP and increased its lipid content. Marcu et al. [42] found that increasing dietary energy and protein levels increased breast weight and muscle mass, and reduced fat content, but reducing nutrient level decreased protein content and elevated fat content in pectoral muscle. The discrepant results could be attributable to using different strains in the previous studies. Díaz et al. [43] and Rosa et al. [33] reported different changes in meat quality and carcass composition among different genetic groups fed graded levels of dietary energy.

#### *4.4. Breast Meat Quality*

The color of raw broiler meat is highly affected by dietary nutrient factors [5]. Meat color is an important attribute for consumers; the greater a\* score of meat indicates better meat quality and the lower L\* and b\* scores implies less pale meat. Boulianne and King [44] reported that pale fillets have higher L\* and b\* values, and a lower a\* value than normal fillets. No available information could be found on possible effects of dietary energy level on breast meat color. The most important finding in the present study is related to the shear force measured on the breast muscle, which decreased in the 2897, 3095 and 3236 kcal/kg diets. Increased shear force is associated with increased connective tissue and decreased fat content in meat [45,46]. This implies a reduced content of connective tissue in the breast meat of birds fed the 2897, 3095 and 3236 kcal/kg diets. The measured fat contents for the 3095 and 3236 levels were consistent with this interpretation to some extent. The control level (2997) had relatively higher (*p* > 0.05) breast fat; however, it unexpectedly showed a high shear force value. The reason behind this increased shear force with this energy level is not clear, or it might imply a high content of connective tissue in this treatment. Low drip loss and shear force indicate higher meat quality. Higgins et al. [47] and Min and Ahn [48] reported that increased drip loss and decreased meat color a\* score reflects lipid peroxidation, leading to loss of pigments and deterioration of meat quality. Drip loss, meat color, and meat pH were not affected here by the dietary caloric levels used.

#### *4.5. Blood Biochemical Variables*

The evaluation of blood biochemistry in poultry shows metabolic alterations due to a number of factors, such as the physiological status, feeding standards, weather change, genetic type, age, housing conditions, and exposure to diseases [49–51]. The modification of dietary nutrient concentration can initiate stresses that induce dramatic changes in blood biochemistry [51,52]. In the present study, the increase in dietary ME level led to a linear decrease in plasma UA concentrations, likely reflecting changes in protein catabolism in the body [52]. The plasma UA values obtained here were comparable to those of Wang et al. [53], with the same chicken breed. According to the regression model, the lowest plasma UA was obtained with the 3200 kcal ME/kg diet, suggesting therefore that this level was optimal

or most adequate for the efficient use of protein. This confirms that high caloric levels (3236 kcal/kg) are more adequate than the lower levels, which led to higher plasma UA content. This is consistent with Rosebrough, McMurtry, & Vasilatos-Younken [54,55], who found that reducing dietary energy increased broiler's plasma UA content, and partially agreed with Rezaei and Hajati [52], who found in broilers at 21 d of age that a 40% reduction in dietary nutrients increased the concentrations of plasma UA; in contrast, they also found reduced plasma concentrations of CHOL and TG.

#### **5. Conclusions**

The increase in dietary energy level showed some benefits, with lowered ADFI, FCR, plasma UA, and shear force; without any adverse effect on the other meat quality variables, i.e., meat yield, nutrient deposition, mortality rate, or abdominal fat content. Under the conditions of this study, the 3095 kcal/kg diet was adequate for the best feed intake, shear force, and plasma uric acid, and the 3236 kcal/kg diet tended to increase the body weight and daily gain and reduce the feed conversion ratio of Lingnan males between 9–15 weeks of age; further studies are still required for testing higher ME levels. The regression analyses revealed that the optimal dietary ME levels for plasma UA, fat content in breast muscle, and fat content in thigh muscle were 3200, 3047, and 3135 kcal/kg, respectively.

**Author Contributions:** K.F.M.A., Y.W., W.W., X.L., L.L., Z.G. and Q.F. participated in the acquisition of data, and the analysis and/or interpretation of data; K.A., Y.W. and S.J. participated in drafting the manuscript.

**Funding:** This study was supported by National Key R&D Project (2018 YFD0500600), the "Twelve-Five" National Science and Technology Support Program (2014BAD13B02), China Agriculture Research System (CARS-41-G10) from the Ministry of Agriculture, Scientific and Technological Project (2017B020202003) from the Department of Science and Technology of Guangdong Province, and Grant No. 201804020091 from Guangzhou Science Technology and Innovation Commission, Presidential Foundation of the Guangdong Academy of Agricultural Sciences (201620, 201805, 201807B, 201809B and 201908), China.

**Acknowledgments:** The authors thank W. Bruce Currie (Emeritus Professor, Cornell University, Ithaca, NY, USA) for his valuable suggestions on the presentation of this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

### **E**ff**ects of Dietary Xylanase and Arabinofuranosidase Combination on the Growth Performance, Lipid Peroxidation, Blood Constituents, and Immune Response of Broilers Fed Low-Energy Diets**

### **Ahmed A. Saleh 1,\*, Abeer A. Kirrella 1, Safaa E. Abdo 2, Mahmoud M. Mousa 1, Nemat A. Badwi 1, Tarek A. Ebeid 1,3, Ahmed L. Nada 1,4 and Mahmoud A. Mohamed <sup>5</sup>**


Received: 15 June 2019; Accepted: 14 July 2019; Published: 22 July 2019

**Simple Summary:** Arabinoxylans (AXs) constitute the major non-starch polysaccharides (NSPs) existent in maize and soybean meal, comprising about 52% and 65% of the total NSP. Previous works have illustrated that the incorporation of arabinofuranosidase (Abf; GH51) plus xylanase (Xyl; GH11) enhanced the dry matter digestibility of maize and wheat in vitro, in comparison with Xyl alone. In broilers, the combination of dietary Xyl and Abf (Rovabio® Advance) enhanced energy, fat, fiber, and protein utilizations. This study shows the effect of feeding low-energy diets with or without Rovabio® Advance, including high concentrations of Xyl and Abf, on the growth performance, nutrient digestibility, lipid peroxidation, blood constituents, and immune response of broilers. Our results confirm the improved growth, digestibility, and immunity obtained by enzymes supplementation. Furthermore, diets supplemented with enzymes caused a higher antibody titer against the Newcastle disease virus. Moreover, they enhanced plasma lipid profiles and antioxidation.

**Abstract:** The present study was conducted to examine that impact of dietary xylanase (Xyl) and arabinofuranosidase (Abf) supplementation on the performance, protein and fat digestibility, the lipid peroxidation, the plasma biochemical traits, and the immune response of broilers. A total of 480, un-sexed, and one-day-old broilers (Ross 308) were randomly divided into three treatments with eight replicates, where chicks in the first treatment were fed basal diets and served as the control, chicks in the second treatment were fed diets formulated with reductions of 90 kcal/kg, and chicks in the third treatment were fed the same formulated diets used in the second group as well as the Xyl and Abf combination (Rovabio® Advance). Feed intake was decreased by the low energy diet, leading to an enhancement in feed efficiency enzyme supplementation in the low energy diet (*p* < 0.015). Both protein and fat digestibility were improved (*p* < 0.047) due to enzyme supplementation. Moreover, enzyme supplementation increased muscle total lipids content and decreased muscle thiobarbituric acid retroactive substance content. Furthermore, diets supplemented with Xyl and Abf exhibited an increase in antibody titers against the Newcastle disease virus (*p* < 0.026). In addition, enzyme supplementation increased gene expression related to growth and gene expression related to fatty acid synthesis. It could be concluded that dietary Xyl and Abf supplementation had beneficial impacts on growth, nutrient digestibility, lipid peroxidation, immune response, and gene expressions related to growth and fatty acid synthesis in broiler chickens fed low-energy diets.

**Keywords:** xylanase; arabinofuranosidases; broilers; nutrient utilization; growth performance; immunity

#### **1. Introduction**

Nowadays, in the broiler production industry, the total price of energy ingredients is about 65–70% of the total costs of the broiler diets. Additionally, these ingredients are usually imported from outside Egypt. Therefore, diverse experiments have been conducted to decrease the cost by reducing the rate of these energy ingredients in broiler feed, along with animating the performance of broilers [1]. One scenario involves adding enzymes to the broiler diets, which promotes such growth performance parameters as feed efficiency and body weight gain [2]. Indeed, the presence of soluble non-starch polysaccharides (NSPs) has reduced nutrient utilization and consequently minimized growth performance in broilers. These carbohydrates cannot be digested by birds, as they do not have the capability to produce these enzymes. Therefore, NSP enzymes are functional when they are added to cereal-based diets, e.g., wheat, soybean, barley, and maize. Exogenous enzymes like xylanase (Xyl), amylase, and protease are produced using a microbial source [3]. Almirall et al. found that the feed conversion ratio (FCR) was enhanced by exogenous enzyme supplementation in broiler diets, and this effect was connected with improving the digestibility and minimizing the viscosity of intestinal contents [4]. Furthermore, Abdel-Latif et al. and Saleh et al. illustrated that the improvement of growth performance due to NSP enzyme addition might be explained by their participation in reducing the digesta viscosity and amendment of gut microbiota by improving the beneficial microbes [3,5]. Worldwide broiler production, including in Egypt, farmers depend on corn and soybean meal for feeding birds, as these are the available ingredients; however, the level of NSP is 29% in soybean and 9% in corn [6]. Broilers do not produce enzymes for the hydrolysis of these NSPs present in the cell walls of the grains [7,8]. Exogenous enzymes which can hydrolyze NSPs are abundantly found in the feed given to birds [9].

Arabinoxylans (AXs) exemplify the major NSP existent in maize and wheat, comprising about 4.7% and 7.3% of the dry matter and 65% and 52% of the total NSP, respectively [10]. Previous studies showed that a mixture of arabinofuranosidase (Abf) and Xyl improved the dry matter digestibility of maize and wheat in vitro, in comparison with Xyl alone [11]. In fact, endoxylanases support the degradation of AXs by hydrolyzing the xylan backbone. Additionally, arabinose substitutions minimized the activity of Xyl in yellow corn and its correlating byproducts [10]. Abf could split the xylose backbone in arabinose and give access to endoxylanase activity [12]. Moreover, Cozannet et al. illustrated that dietary Rovabio® Advance (including high concentrations of Xyl and Abf) had a positive effect on the energy utilization and digestibility of starch, protein, fat, and insoluble and soluble fibers [11]. Furthermore, Ravn et al. documented that the addition of an enzyme combination (Xyl and Abf) improved the growth performance and gut morphology in broilers [13]. It could be hypothesized that the supplementation of a combination of Xyl and Abf to broiler diets might be involved in improving the utilization of nutrients of a low-energy diet and could consequently enhance the growth performance and immune responsiveness of broilers. The objective of this study was to examine the impact of dietary Xyl and Abf (Rovabio® Advance) supplementation on the growth performance, fat and protein digestibility, lipid peroxidation, biochemical plasma traits, and immune response of broilers fed low-energy diets.

#### **2. Materials and Methods**

#### *2.1. Animals and Experimental Design*

The study was approved by the Ethics Committee of Local Experimental Animals Care Committee and conducted in accordance with the guidelines of Kaferelsheikh University, Egypt (Number 4/2016 EC). A total of 480 un-sexed one-day-old broilers (Ross 308) were housed in bins (stocking density

was 10 birds/m2) and randomly divided into 3 experimental treatments with 8 replicates (20 birds each) to equalize the average body weight in each group. The control group was fed basal diets as commercial feed formulated according to the strain requirements, the second experimental group of chicks was fed diets formulated with reductions of 90 kcal/kg AME and 3% digestible amino acids, and the third experimental group was fed the same formulated diets used in the second group as well as Xyl and Abf. The composition and chemical analysis of the experimental diets (starter, grower, and finisher) are shown in Table 1. The Xyl and Abf (Rovabio® Advance) were kindly given by the Adisseo company, France S.A.S. Antony Parc 210, Place du Général de GaulleF-92160 ANTONY, France. ® This enzyme was industrially created by the fermentation of *Talaromyces versatilis* (IMI378536 and DSM26702; Adisseo France S.A.S. proprietary strains), and the main enzyme activity in Enz comes from Xyl and Abf. The enzyme was added to the premix mixture, which is one of the basic ingredients in the all diets. The diets were provided to the birds ad libitum; starter diets were in crumble form. However, the grower and finisher diets were pellet form. The trail was managed in an open-door house with a 23 h light–1 h dark cycle. Daily temperature and humidity inside the house were controlled at 24–26 ◦C and 60–70%, respectively. The experimental diets were offered from 1 day to 35 days of age. Bird body weight was measured individually every week. However, feed intake was measured daily (on a group basis per pen) throughout the experimental period. At 32 days, all birds were weighed individually and sorted from the smallest to the heavy weight. Then, 12 male birds/treatment have the same average weight were transferred to special batteries containing individual cages to enact the digestibility experiment. Then, these birds were slaughtered and dissected to gauge the weights of the breast muscle, thigh muscle, liver, gizzard, heart, spleen, abdominal fat, and bursa of Fabricius. All organs were weighed and described as a ratio of the body weight. Blood samples were collected from the wing vein immediately before slaughtering, gathered into heparinized test tubes, and then rapidly centrifuged (3000 rpm for 20 min at 5 ◦C) to separate the plasma. Plasma was stored at −20 ◦C pending analysis.


**Table 1.** Composition of the experimental starter, grower, and finisher diets.


**Table 1.** *Cont.*

\* Hero mix® (Hero pharm, Cairo, Egypt). Composition (per 3 kg): Vitamin A 12,000,000 IU, vitamin D3 2,500,000 IU, vitamin E 10,000 mg, vitamin K3 2000 mg, vitamin B1 1000 mg, vitamin B2 5000 mg, vitamin B6 1500 mg, vitamin B12 10 mg, niacin 30,000 mg, biotin 50 mg, folic acid 1000 mg, pantothenic acid 10,000 mg, manganese 60,000 mg, zinc 50,000 mg, iron 30,000 mg, copper 4000 mg, iodine 300 mg, selenium 100 mg, and cobalt 100 mg. Diets ingredients and final feed diets were analyzed by chemical analysis in the Adisseo company lab, Antony, France.

#### *2.2. Nutrient Digestibility*

In the last three days of the experiment, excreta were gathered and weighted from 12 males per treatment, where broilers were housed individually in special metabolic cages (40 × 40 × 50 cm) for digestibility tests. During these three days, the birds and feed intake were weighted daily, and extracted faces were collected, weighted, and stored in a freezer. After the digestibility experiment period, all samples were dried in a drying oven at 60 ◦C for 24 h. The whole dried samples were then homogenized. Samples were taken and finely ground for analysis according to the Association of Official Analytical Chemists (AOAC) [14]. The crude protein concentration in the diet and excreta was gauged to determine nitrogen digestibility using the Kjeldahl method, and crude fat was gauged by the Soxhlet method (AOAC 945.38 F and 920.39 C, respectively). The calculation was as follows: Nitrogen digestibility (%) = (total nitrogen intake − total nitrogen excreted)/total nitrogen intake × 100.

#### *2.3. Biochemical Analysis*

Triglycerides (TG), total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, glutamic oxaloacetic transaminase (GOT), glutamate pyruvate transaminase (GPT), glucose, creatinine, total protein, albumin, and globulin were measured colorimetrically using commercial kits (Diamond Diagnostics, Egypt) according to the procedure outlined by the manufacturer. Muscle total lipid content, fatty acid profile, and amino acid analysis were measured using gas liquid chromatography (GLC) according to the method of Saleh [15]. The muscle thiobarbituric acid retroactive substance (TBARS) concentration was measured by the process of Ohkawa et al. [16].

#### *2.4. Serum Antibody Titers*

Serum antibody titers against Newcastle disease (ND) and avian influenza (H9N1) were determined by means of the hemagglutination inhibition test using standard methods qualified by OIE [17].

#### *2.5. RNA Analysis*

Each breast muscle sample was homogenized, and the total RNA was extracted using a total RNA purification kit following the manufacturer's protocol (Fermentas, K0731, Thermo Fisher Scientific, Waltham, MA, USA). The extracted total RNA (5 μg per sample) was reverse transcribed into cDNA using Revert Aid H Minus Reverse Transcriptase as described by the manufacturer (Fermentas, EP0451, Thermo Fisher Scientific, Waltham, MA, USA). Following amplification, PCR products were electrophoresed, and the expression level of different bands was analyzed using the ImageJ gel analysis program [18].

#### *2.6. Statistical Analysis*

The differences between the experimental treatments and the control were analyzed with a General Liner model using SPSS Statistics 17.0 (Statistical Packages for the Social Sciences, SPSS Inc., Chicago, IL, USA, released 23 August 2008). Tukey's multiple comparison test was used to identify which treatment conditions were significantly different from each other.

#### **3. Results and Discussion**

One of the major objectives of the current study was to evaluate the impacts of feeding low-energy diets supplemented with or without Xyl and Abf enzymes on the growth performance, nutrient digestibility, lipid peroxidation, blood plasma biochemical traits, immune response, and gene expressions related to growth and fatty acid synthesis in broilers. The inclusion of Xyl and Abf enzymes in low-energy diets in the present study improved the growth performance in broilers, and this improvement might be related to the enhancement of nutrient digestibility by Xyl and Abf enzyme supplementation. This supposition is in harmony with Nortey et al., who suggested that dietary exogenous enzyme addition had a beneficial effect on nutrient digestibility in swine specimens [19]. Moreover, Slominski et al. reported that the inclusion of a debranching enzymes mixture improved the overall enzyme effectiveness and, consequently, enhanced the nutrient digestibility and the alleviation of the negative impacts of NSPs [20]. Recently, Ravn et al. stated that the addition of an enzyme combination (Xyl and Abf) improved duodenum villi length, which was probably involved in enhancing the growth performance, including the body weight and FCR, in broilers [13].

The data presented in Table 2 show that feeding low-energy diets decreased body weight gain significantly compared to the control group, while, dietary supplementation with Xyl and Abf enzymes increased body weight gain and improved FCR, crude protein digestibility, and crude fat digestibility significantly (*p* < 0.05). No significant differences were detected in the feed intake. These findings are in correspondence with Cozannet et al., who demonstrated that a dietary combination of Xyl and Abf had a positive effect on the energy utilization and digestibility of protein, starch, fat, and insoluble and soluble fibers [11]. Additionally, Cowieson and Ravindran reported improvements in crude protein and amino acid digestibility when a multiple enzyme mixture including protease, Xyl, and amylase was employed to supplement corn–soybean diets [21]. Similarly, Rutherfurd et al. found enhancements in crude protein and amino acid digestibility in broilers fed commercial diets supplemented with a multiple enzymes complex, including amylase, β-glucanase, and Xyl [22]. Cowieson and Ravindran reported that the mechanisms that enhanced the amino acid utilization due to the addition of exogenous enzymes are connected with minimizing endogenous losses related to a decreased secretion of endogenous enzymes [23]. Moreover, Meng et al. stated that dietary enzymes take off the nutrient encapsulating effect of NSP [24], thus enhancing the nutrient availability to endogenous enzymes and improving the overall nutrient digestibility and intestinal microbial environment [11,21].


**Table 2.** Effect of non-starch polysaccharide (NSP) enzyme supplementation on growth performance in broilers.

a,b Mean values with different letters in the same raw differ significantly at *p* < 0.05. Values are expressed as means <sup>±</sup> standard error. The NSP enzyme used in this experiment was Rovabio® Advance. FCR: Feed conversion ratio.

The bursa of Fabricius relative weight was significantly increased by feeding low-energy diets supplemented with Xyl and Abf enzymes, while the abdominal fat relative weight was significantly decreased in both low-energy diets and control. However, the carcass, thigh, liver, gizzard, heart, and spleen relative weights were not affected by low-energy diets supplemented with or without a combination of Xyl and Abf enzymes (Table 3). These findings are in agreement with previous reports [25,26]. However, Farran et al. found that breast muscle, pectoralis major, thigh, and drum yields were not affected by the inclusion of enzyme preparations [27]. Garipoglu et al. reported that the dressing percentage was reduced but abdominal fat weight was not influenced by feeding diets supplemented with multienzymes [28]. Similarly, Kocher et al. found that found that a Xyl, amylase and protease addition to the corn–soybean meal did not affect abdominal fat weight [29]. Contrarily, Garcia et al. reported that the Xyl and β-glucanase supplementation of barley–wheat-based diets elevated the abdominal fat content in broilers [30]. In the present study (Table 3), the lower abdominal fat relative weight noted in low-energy diets, with or without enzyme supplementation, might be attributed to the fact that the lower energy diets caused less fattening and were also connected with reducing the feed intake.

**Table 3.** Effect of NSP enzyme supplementation on organ weights (g/100 g BW) in broilers.


a,b Mean values with different letters in the same raw differ significantly at *p* < 0.05. Values are expressed as means <sup>±</sup> standard error. The NSP enzyme used in this experiment was Rovabio® Advance. BW: Body weight.

Table 4 shows the effect of low-energy diets supplemented with or without Xyl and Abf enzymes on blood biochemical parameters. Plasma globulin and HDL-cholesterol were significantly increased due to dietary supplementation in comparison with the control group. However, plasma total cholesterol was significantly reduced by the addition Xyl and Abf enzymes to low-energy diets compared with the control group; however, plasma GOT, GPT, albumin, triglycerides, glucose, LDL-cholesterol, and creatinine were not significantly affected. Interestingly, serum antibody titers against ND were significantly increased by the enzyme group, while the antibody titer against avian influenza (H9N1) was enhanced insignificantly in comparison with the control group (*p* = 0.11). However, there was an

insignificant increase in the antibody titer against avian influenza (H9N1) due to the dietary Xyl and Abf enzyme supplementation to low-energy diets. These results supported our findings of the bursa of Fabricius relative weight, which was significantly increased due to the dietary Xyl and Abf enzyme supplementation. These results are coincident with our previous findings, which noted that ND and infectious bronchitis disease (IBD) antibody titers were improved by enzyme addition to broiler diets, and this may be regarded as an improvement in protein digestibility because of these enzyme mixtures and peptide transporter 1(PEPT1) gene expression, which enhanced absorption [3]. Different authors have reported the impacts of supplementing AX or arabinoxylooligosaccharides in a broiler diet, and they have observed that oligosaccharides with a polymerization score of less than five encourage the propagation of beneficial bacteria and enhance microbiota variety [31–33]. The increment of arabinoxylooligo-saccharides (AXOS) presented in wheat or soybean AX improves the proliferation of bifidobacteria in ceca without influencing the body weight of birds. The inclusion of AXOS encourages beneficial bacteria and protects against pathogenic bacteria [34,35], which consequently enhances gut health. Lei et al. documented that dietary Xyl, Abf, and feruloyl esterase improved gut health [36]. Pettey et al. also indicated that adding 0.05% β-mannanase and arabinoxylooligosaccharides led to an improved blood IGF-I concentration in growing and finishing pigs [37]. It might be speculated that the inclusion of Xyl and Abf in low-energy diets had a positive effect on gut health and nutrient digestibility, leading to improved growth performance, lymphoid organs weights, and immune response in broilers.

Liver function indicators (plasma GOT and GPT) and the kidney function indicator (plasma creatinine) were not significantly affected. These results are in harmony with Ahmad et al., who evaluated the effect of dietary Xyl addition on plasma biochemical constituents in broilers and illustrated that Xyl might be safe in poultry rations without negative effects on vital organ functions [38]. Additionally, Saleh et al. reported that the serum concentrations of GOT, GPT, and creatinine were not significantly affected by dietary enzyme supplementation [3].


**Table 4.** Effect of NSP enzyme supplementation on plasma parameters in broilers.

a,b Mean values with different letters in the same raw differ significantly at *p* < 0.05. Values are expressed as means ± standard error. Glutamic oxaloacetic transaminase (GOT); glutamate pyruvate transaminase (GPT); high-density lipoprotein (HDL); low-density lipoprotein (LDL). International Units (I/U). The NSP enzyme used in this experiment was Rovabio® Advance.

The data presented in Table 5 illustrate the effect of a low-energy diet supplemented with or without Xyl and Abf enzymes on the muscle content of fatty acids, amino acids, total lipids, and TBARS. Muscle TBARS content was significantly decreased, while muscle total lipids content was significantly increased by feeding a low-energy diet supplemented with Xyl and Abf enzymes compared with the control group. However, the muscle contents of lysine, methionine, oleic, linoleic, and linolenic acids were not significantly influenced by dietary treatments. These findings are in agreement with Cowieson and Ravindran, who found enhancements in the digestibility of lysine, methionine, cysteine, and threonine when a multiple enzyme mixture possessing protease, Xyl, and amylase was used

to supplement corn-based diets, but these improvements did not affect the amino acid contents in muscle [21]. Furthermore, Head et al. reported that dietary α-linolenic acids in the form of linseed resulted in a significant increase of hepatic omega-3 poly unsaturated fatty acids (n-3PUFA) [39]; however, the inclusion of a multiple enzyme complex of Xyl and amylase in a linseed-based diet resulted in a reduction in the n-6PUFA-like linoleic acid, but oleic and linolenic acids were not affected. Regarding lipid peroxidation, the data illustrate that the muscle TBARS concentration was decreased by reducing the energy in diets, and this agreed with Cho and Kim, who observed that muscle malondialdehyde (MDA) concentration was decreased in low-energy density diets supplemented with or without β-mannanase and Xyl supplementation in pigs [9].

**Table 5.** Effect of NSP enzyme supplementation on lipid peroxidation, fatty acids, and amino acids in breast muscle of broilers.


a,b Mean values with different letters in the same raw differ significantly at *p* < 0.05. Values are expressed as means ± standard error. Thiobarbituric acid reactive substance (TBARS). The NSP enzyme used in this experiment was Rovabio® Advance.

The mRNA expressions of the growth hormone receptor (GHR), insulin-like growth factor receptor (IGFR), and fatty acid synthesis (FAS) were significantly increased by adding Xyl and Abf enzymes to low-energy diets in comparison with the control group (Figure 1A–C). Meanwhile, acetyl-coA carboxylase (ACC) was increased by Xyl and Abf enzyme supplementation to a low energy diet but these differences were not significant in comparison with the control group (Figure 1D). These improvements are confirmed by the previous results of Guo et al., who demonstrated that the addition of Xyl upregulated the expression of the sodium–glucose cotransporter 1 and IGFR genes in broiler chickens [40]. Furthermore, Hosseini et al. reported that the inclusion of Xyl improved the expression of GHR and IGFR genes [41]. Moreover, ACC and FAS encode a biotin-dependent enzyme which is involved in the biosynthesis of fatty acids via the catalyzation of the irreversible carboxylation of acetyl-CoA for malonyl-CoA production [42]. In the present study, ACC and FAS gene expressions were significantly elevated in the low-energy diet supplemented with the Xyl and Abf enzyme combination. This impact was connected with the freedom of blocked macronutrients and, consequently, elevated lipogenesis due to the eternal energy adequacy [43]. Indeed, FAS and ACC, which play important roles in the lipogenic passage, are also key determinants for the maximal ability of a muscle tissue to synthesize fatty acid; they are extremely expressed in tissues such as those of the liver and muscles [3,44]. In common physiological cases, nutritional factors such as high-fat feed and hormones could organize the enzyme activity and gene expression of the FAS and ACC [39,45,46].

**Figure 1.** Effect of NSP enzyme supplementation on the gene expression of the growth hormone receptor (GHR) (**A**), insulin-like growth factor receptor (IGFR) (**B**), fatty acid synthesis (FAS) (**C**), and acetyl-coA carboxylase (ACC) (**D**) in broilers. a,b Mean values with different letters in the same column differ significantly at *p* < 0.05. Values are expressed as means ± standard error. The NSP enzyme used in this experiment was Rovabio® Advance.

#### **4. Conclusions**

It could be concluded that dietary Xyl and Abf (Rovabio® Advance) supplementation had positive effects on the growth performance, protein and fat digestibility, plasma lipid profiles, lipid peroxidation, immune response, and gene expression related to growth and fatty acid synthesis in broiler chickens fed low-energy diets.

**Author Contributions:** A.A.S., A.A.K., A.L.N. and M.A.M.; methodology, A.A.S.; software, A.A.S.; validation, A.A.S., A.A.K., S.E.A. and A.L.N. ; formal analysis, A.A. S., M.M.M., N.A.B.; investigation, A.A.S.; resources, A.A.S. and A.L.; data curation, A.A.S., A.A.K., T.A.E.; writing—original draft preparation, A.A.S., T.A.E. and M.A.M.; writing—review and editing, A.A.S.; visualization, A.A.S.; supervision, A.A.S. and A.L.N.; project administration, A.A.S. and A.L.N. and M.A.M.; funding acquisition, A.L.N. and M.A.M.

**Funding:** This research was funded by Orkila Egypt Chemicals Company and Adisseo France Company.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Does in Ovo Injection of Two Chicken Strains with Royal Jelly Impact Hatchability, Post-Hatch Growth Performance and Haematological and Immunological Parameters in Hatched Chicks?**


Received: 24 June 2019; Accepted: 23 July 2019; Published: 25 July 2019

**Simple Summary:** The present investigation examined improvements in egg hatchability and the growth performance of hatched chicks of two strains upon injection with increasing concentrations of royal jelly (RJ). The results showed positive effects of RJ injection on all parameters. Limited impacts of the different chicken strains were observed on the tested parameters. The study revealed that varying the chicken strain could alter the response to the in ovo injection with RJ.

**Abstract:** The hypothesis of the present work was that the effects of in ovo injection may differ in different chicken strains. The influence of in ovo royal jelly (RJ) injection on hatching, growth and blood parameters in two chicken strains (Dokki-4 and El-Salam as example for different strains) was evaluated. A total of 1080 eggs were used. On the seventh day of incubation, the eggs were randomly allocated into six experimental groups in a 2 × 3 arrangement that included the two chicken strains and three concentrations of RJ (0, 0.25 and 0.5 mL RJ/egg). Injection with 0.5 mL RJ/egg improved hatchability compared to the other treatments. The El-Salam strain exhibited significantly higher body weight and body weight gain than the Dokki-4 strain. Injection with 0.5 mL RJ/egg significantly (*p* < 0.05) improved chicken body weight and daily weight gain compared to the control treatment. RJ injection decreased blood lipid profile parameters and the numbers of monocytes and eosinophils and increased total protein, globulin, haemoglobin (Hb) and lymphocyte levels compared to the control treatment. The Dokki-4 strain showed significantly higher antibody titres against avian influenza virus (AIV) (*p* < 0.05) and sheep red blood cells (SRBCs) (*p* < 0.0001) than the El-Salam strain and RJ injection enhanced antibody titres against AIV, Newcastle disease virus (NDV) and SRBCs. Therefore, the Dokki-4 strain was superior to the El-Salam strain for the tested parameters and injection with 0.5 mL RJ/egg produced the best hatching parameters, growth performance and health-related traits. RJ in ovo injection was much more effective in the Dokki-4 strain than in the El-Salam strain, which supported the hypothesis of the study that varying the chicken strain could alter the response to the in ovo injection with RJ.

**Keywords:** strain; in ovo injection; royal jelly; chicken; growth; hatchability; blood

#### **1. Introduction**

Embryonic growth in poultry can be manipulated through in *ovo* administration of nutrients and natural bioactive compounds [1–7]. In ovo injection of such substances influences the pre- and post-hatching physiological status of broiler embryos, leading to improved hatchability, superior nutritional status of hatchlings, greater vigour and higher post-hatch growth [8,9].

Royal jelly (RJ), a honeybee secretion fed to larvae and queen bees, is a rich dietary supplement for humans. RJ in fresh form consists of water (60–70%), protein (9–18%), carbohydrate (7–18%), fat (3–8%), mineral salts (calcium 1.5%), 10-hydroxy-2-decenoic acid (1.4%), fructose (3–13%), glucose (4–8%), sucrose (0.5–2.0%) and Ash (0.8–3.0%). While the lyophilised form contains <5% water, 27–41% protein, 22–31% carbohydrate and 15–30% fat [10,11].

RJ is the richest known natural source of vitamin B5 [12]. The other components of RJ include gamma globulins, mostly immunoglobulins, which powerfully strengthen the immune system; 10-hydroxy-Δ2-decanoic acid, which is a powerful antibacterial and anti-fungal agent [13] that keeps RJ sterile; gelatine, the precursor of collagen in skin, tendon, ligaments; and up to 1 mg/g acetylcholine, of which RJ is the richest natural source and which important in nerve transmission and the production and release of glandular secretions [14].

RJ has been reported to have several pharmacological properties, such as antioxidant [15], hypocholesterolaemic [16], anti-inflammatory [17], anti-malignant [18], antibacterial [19] and anti-ageing [20] properties, in animals. Additionally, RJ in ovo injection has been concluded to improve chick body weight [21], internal organ weight and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion without adverse effects on hatchability [22]. Furthermore, in ovo injection of RJ has been found to promote feed intake in broiler chicks with no effect on immunity against Newcastle disease virus (NDV) [23], egg quality parameters or erythrocyte counts [24].

Different strains of chickens differ in their productivity, reproductive performance and immune responses [25–28]. Furthermore, strain differences affect feed intake, feed conversion ratios (FCRs) [29] and carcass traits [30,31].

Several studies have reported the effects of in ovo injection on the pre- and post-hatching performance of chickens; however, interactions between strain and in ovo injection have not been investigated. Therefore, the present study aimed to analyse the effects of in ovo injection of different levels of RJ, strain differences and interactions between strain and RJ treatment levels on hatching, growth rates, blood chemistry, haematology and immunological parameters.

#### **2. Materials and Methods**

This study was carried out at the Poultry Farm, Faculty of Agriculture, Kafr El-Sheikh University, Egypt. All procedures and experiments were performed in accordance with the ethical guidelines of the Committee of Local Experimental Animal Care and were approved by the Faculty of Agriculture, Kafr El-Sheikh University, Egypt (KFS2018-0078). All efforts were made to minimize the suffering of the animals.

A total of 1080 incubating eggs produced by two local chicken strains (Dokki-4 and El-Salam) were used. The Dokki-4 strain was developed by El-Itriby and Sayed [32] from a cross between a Fayoumi cock and Barred Plymouth Rock females (used as a dual purpose, meat and egg production) and the El-Salam strain was developed by Abd El-Gawad et al. [33] from crosses between Nichol sires and Mamourah dams (used as a dual purpose, meat and egg production).

The eggs were collected from the Experimental Research Station, Sakha, of the Animal Production Research Institute, Agricultural Research Centre, Ministry of Agriculture, Egypt. The eggs were stored at 15 ± 1 ◦C with 70–75% relative humidity for 3 days. The eggs were immediately cleaned with a dry, clean cloth; then, the surface of each egg was sprayed with a disinfectant solution and the shell was

wiped dry with clean paper. The eggs were incubated at 37.6 ± 1 ◦C with a relative humidity of 57 ± 2%. All eggs were set large end up in an automatic turner. The incubation equipment included an incubator (for the first 18 days of incubation) and a hatcher (for the remaining 3 days until hatching). The eggs from each experimental group were set in separate and marked containers in both the incubator and the hatcher.

On day 7 of incubation, the eggs were randomly divided into six groups in a 2 × 3 factorial design that included the two chicken strains (Dokki-4 and El-Salam) and three levels of in ovo injections (0, 0.25 and 0.5 mL/egg). Each group had six replicates of 30 eggs each. The RJ solution was prepared by dissolving 1 g of pure RJ (YS Organic Bee Farms "2774 N 4351st Rd, Sheridan, IL 60551, USA," item #69313, 3x freeze-dried RJ; equivalent to 1500 mg of fresh RJ) in 2 mL of normal saline solution in a water bath at 37 ◦C for 15 min.

A hole was made in the broad end of each egg using an automatic needle (syringe); 0.5 mL of RJ solution was then warmed to 30 ◦C and injected through the hole using a 23–gauge needle. The injection site was disinfected with 70% ethanol prior to injection. The pinhole was sealed with sterile paraffin wax immediately after injection and the eggs were returned to the incubator and set large end up to complete the incubation process. At the end of 18 days of incubation, the eggs were transferred to hatching trays at 37.2 ◦C under 70% humidity for the following 3 days or until hatching. After 21 day of incubation, the live hatched chicks were graded and counted, while the un-hatched eggs were broken to estimate the percentage of fertility. Hatchability was calculated as a percentage of fertile eggs using the following equations:

$$\text{Ferrility } (\text{\textquotedblleft} \text{\textquotedblright}) = (\text{\textquotedblleft} \text{\textquotedblright} \text{\textquotedblleft} \text{\textquotedblright} \text{\textquotedblleft} \text{\textquotedblright}) \times 100\tag{1}$$

### *Hatchability o f the f ertile eggs* (%) = (*hatched chick*/ *f ertile eggs*) × 100 (2)

Twenty hatched chicks for each replicate were sexed, wing-banded and reared until 12 weeks of age and were supplied with standard feed for local chicken strains. The basal diet contained 19% crude protein (CP), 2834 kcal/kg metabolizable energy (ME), 3.019% ether extract, 3.906% crude fibre, 1.018% calcium, 0.348% available phosphorus, 0.857% lysine, 0.360% methionine and 0.699% methionine and cystine. The birds were vaccinated against most epidemic diseases in Egypt; they were vaccinated with Hitchner B1 (HB1) and Gumboro vaccines at 7 and 10 day of age, respectively, via eye drops and killed NDV, Reo, Gumboro and infectious bronchitis (IB) vaccines were injected intramuscularly at 13 day of age. Killed avian influenza virus (AIV; H5N2) vaccines were injected intramuscularly at 15 day of age, while Gumboro and Lasota vaccines were given at 22, 32 and 42 day of age via eye drops. Later, booster doses of Lasota vaccines were given at 50 day of age and then biweekly via eye drops. Feed and drinking water were offered ad libitum. All birds were reared under the same environmental, managerial and hygienic conditions. Body weight was recorded to the nearest 0.1 g. Feed intake was recorded at 0, 4, 8 and 12 weeks of age and the FCR was then calculated.

At 7 weeks of age, the birds received a single intramuscular injection of 0.1 mL of a 0.25% sheep red blood cell (SRBC) suspension. After 5 days, ten blood samples (1 mL each) from each group were collected from the wing vein with a sterile syringe and 0.5 mL of each sample was transferred into a heparinized tube. Plasma antibodies were measured by the microtitre haemagglutination method [34]. The titres are expressed as the log2 of the reciprocal of the highest dilution in which there was haemagglutination. The remaining 0.5 mL of each blood sample was allowed to coagulate in sterile tubes, after which the serum was collected to assess the antibody titre against NDV.

At 12 weeks of age, ten birds (5 males and 5 females) were selected randomly from each treatment, weighed and then sacrificed by decapitation. The carcasses and giblets (gizzard, heart and liver) were individually weighed to the nearest 0.1 g. The studied carcass traits were recorded as the percentage of the live body weight. Ten blood samples were collected in heparinized tubes to determine the complete blood count (CBC) and white blood cell (WBC) differentiation. After overnight clotting at 4 ◦C, the samples were centrifuged for 20 min at 4000× *g*.

The separated serum was transferred to a private laboratory for analysis of biochemical parameters. The total lipids (mg/dL), cholesterol (mg/dL), triglycerides (TGs; mg/dL), high-density lipoprotein (HDL, mg/dL), low-density lipoprotein (LDL, mg/dL), total protein (g/dL) and albumen (g/dL) levels were determined spectrophotometrically according to the methods of Akiba et al. [35], using commercial diagnostic kits from Biodiagnostic Company, Giza, Egypt. Additionally, the antibody titres against AIV and NDV were estimated.

The data were subjected to ANOVA with the generalized linear model (GLM) procedure of SAS software, USA [36] according to the following model:

$$X\_{\rm rtk} = \mu + O\_{\rm r} + B\_{\rm f} + I\_{\rm r\&t} + E\_{\rm rtk} \tag{3}$$

where *Xrtk* = the value of any observation, μ = the population mean *Or* = the in ovo injection effect (of 0, 0.25 or 0.5 mL RJ/egg), *Bf* = the strain effect (El-Salam or Dokki-4), *Ir* <sup>×</sup> *<sup>t</sup>* = the interaction between the treatment and the chicken strain and *Ertk* = random error.

#### **3. Results**

#### *3.1. Hatching Parameters*

As shown in Table 1, the Dokki-4 strain presented significantly higher fertility than the El-Salam strain (*p* < 0.05). No differences were recorded in the hatchability of the total eggs and fertile eggs (*p* < 0.05) between the two studied strains. Regarding the effect of in ovo RJ injection, the 0.5 mL RJ/egg dose significantly increased the hatchability of the total incubated eggs (*p* < 0.0001) compared to both the 0.25 mL RJ/egg dose and the control dose (0 mL RJ/egg) and improved the hatchability percentages of fertile eggs (*p* < 0.05) compared to the control dose.

**Table 1.** Fertility and hatchability percentages of two chicken strains (El-Salam and Dokki-4) injected in ovo with two different levels of royal jelly (RJ; 0.25, 0.5 mL/egg) compared to counterpart control strains (0 mL RJ/egg) and the interaction between strain and treatment level.


SEM—standard error of the mean. In the same column and within the same effect, means with different superscripts ( a, b) differ significantly (*p* < 0.05).

#### *3.2. Growth Performance and Carcass Parameters*

The results of chicken performance according to strain and in ovo injection level, as well as their interactions, are presented in Table 2. Daily weight gain was not significantly affected (*p* ≥ 0.05) by strain. The results of the interaction between strain and in ovo inoculation were not significant (*p* ≥ 0.05).

**Table 2.** Effect of in ovo injection of royal jelly (RJ) at two levels (0.25, 0.5 mL/egg) on growth performance and carcass parameters of two chicken strains (El-Salam, Dokki-4) as compared to counterpart control chicks (0 mL/egg) and the interaction of strain and treatment levels.


SEM—standard error of the mean. In the same column and within the same effect, means with different superscripts ( a, b) differ significantly (*p* < 0.05).

Feed consumption and conversion ratios did not differ significantly between the El-Salam and Dokki-4 strains, except for feed consumption at 0–12 weeks of age (*p* < 0.0001), which were significantly improved in the El-Salam strain. No variation in dressing percentages was observed between strains (*p* ≥ 0.05) but Dokki-4 had higher (*p* < 0.01) giblet percentages than the El-Salam Strain. For the in ovo RJ injection effects, chickens from eggs inoculated with 0.5 mL RJ consumed more feed (*p* < 0.05) compared to injection with 0.25 mL RJ or the control at 0–12 weeks of age; chickens from eggs inoculated with 0.5 mL RJ also displayed higher (*p* < 0.05) dressing percentages compared to the control group.

#### *3.3. Serum Lipid and Protein Profiles*

Strain clearly had no effect on serum lipid and protein profiles (Table 3). In contrast, in ovo injections with 0.5 mL RJ/egg decreased serum total lipids and increased (*p* < 0.001) globulin and total protein levels (*p* < 0.05) compared to the other injection. Both levels of RJ injection (0.25 and 0.5 mL RJ/egg) resulted in significantly reduced serum levels of cholesterol, TGs, HDL and LDL. No significant interactions between the strain and treatment effects were recorded for the serum lipid and protein profiles.


*Animals* **2019** , *9*, 486


*Animals* **2019** , *9*, 486

and within the same effect, means with different superscripts (a,

 c) differ significantly (*<sup>p</sup>* < 0.05).

#### *3.4. Complete Blood Count (CBC)*

The effects of strain, RJ in ovo injection level and their interaction on CBCs are listed in Table 4. Strain had no significant effect (*p* > 0.05) on red blood cell (RBC) count; packed cell volume (PCV); haemoglobin (Hb); WBC count; heterophil and lymphocyte numbers; the heterophil/lymphocyte (HL) ratio; or monocyte, eosinophil and basophil numbers. With regard to the in ovo RJ injection levels, both 0.25 and 0.5 mL RJ/egg resulted in increased Hb levels and lymphocyte counts compared to 0 mL RJ/egg, whereas significant reductions were observed in the numbers of monocytes and eosinophils. No significant interactions were recorded between the strain and treatment effects for any of the blood parameters analysed.

#### *3.5. Immunological Parameters*

As illustrated in Table 5, the Dokki-4 strain had significantly higher antibody titres against the AIV vaccine (*p* < 0.05) and SRBCs (*p* < 0.0001) than the El-Salam strain but no significant differences (*p* > 0.05) were recorded between the two strains for NDV titres. Regarding the impact of in ovo RJ injection on the immunity of the chickens, both levels of RJ (0.25 and 0.5 mL RJ/egg) increased (*p* < 0.0001) the antibody titre against the AIV vaccine, while 0.5 mL RJ/egg increased (*p* < 0.0001) the antibody titres against NDV and SRBCs compared to the other treatments. Regarding the interaction between the strain and in ovo RJ injection effects, no significant differences in antibody titres against AIV and NDV were recorded between the different groups; however, in ovo injection with 0.25 or 0.5 mL RJ/egg increased immunity (*p* < 0.0001) against SRBCs in the Dokki-4 strain compared to the El-Salam strain.


**Table 5.** Effect of in ovo injection of royal jelly (RJ) at two levels (0.25, 0.5 mL/egg) on Antibody titre against AIV, NDV and SRBCs of two chicken strains (El-Salam, Dokki-4) as compared to counterpart control (0 mL/egg) and the interaction of strain and treatment levels.

AIV—avian influenza virus; NDV—Newcastle disease virus; SRBCs—sheep red blood cells; SEM—standard error of the mean. In the same column and within the same effect, means with differ superscripts (a, b, c, d) differ significantly (*p* < 0.05).

#### **4. Discussion**

The data listed in Table 1 shows that the in ovo RJ injection (0.5 mL RJ/egg) improved the hatchability percentages of fertile eggs (*p* < 0.05) compared to the other groups. The improvement in hatchability may be due to the enriched nutritive values of RJ, which contain vitamins and essential amino acid that enhance chick embryonic growth and hatchability. However, our results disagree with those obtained by Moghaddam et al. [21] who reported that in ovo RJ injection significantly decreased hatchability (46.7%) compared to saline injection (68.3%). Moreover, Moghaddam et al. [22] found significantly lower hatchability percentages with RJ compared to saline phosphate antibiotic injection. Conclusively, RJ in ovo injections (0.5 mL RJ/egg) improved hatchability percentage of chicken eggs.

Our results showed that in ovo RJ injection with 0.5 mL RJ/egg improved the hatchability percentages of fertile eggs (*p* < 0.05) compared to the other injections. The improvement in hatchability may have been due to the high nutritive value of RJ, which contains vitamins and essential amino acids that enhance chick embryonic growth and hatchability. However, our results disagree with those obtained by Moghaddam et al. [21], who reported that in ovo, RJ injection significantly decreased hatchability (46.7%) compared to saline injection (68.3%). Moreover, Moghaddam et al. [22] found significantly lower hatchability percentages in eggs injected with RJ than in eggs injected with a saline phosphate antibiotic. Conclusively, RJ in ovo injections (0.5 mL RJ/egg) improved the hatchability percentages of chicken eggs in this study.

The results presented in Table 2 indicate that the El-Salam strain had significantly greater body weight and daily weight gain (*p* < 0.05) than the Dokki-4 strain, which may be attributable to its genetic makeup [28]. Injecting eggs with 0.5 mL RJ/egg significantly (*p* < 0.05) improved chicken body weight and daily weight gain compared to injecting eggs with saline. In agreement with our results, Ahangari et al. [23] reported that in ovo RJ injection elicited a significant positive effect on the body weight of broiler chicks at 21 days of age. RJ plays an important role in bee colonies, stimulating and increasing larval growth and metabolism [37] and some RJ bioactive components can affect crucial physiological processes [13,38]. Additionally, increased body weight has been observed after injection or ingestion of RJ in experimental animals [37]. The differences in body weight between the two strains at 8 weeks of age can be explained by the fact that at this age, this quantitative trait in chickens is affected by complex physiological mechanisms and multiple genetic factors [39]. Overall, RJ in ovo injections (0.5 mL RJ/egg) had limited beneficial effects on the body weights and weight gain of the chickens.

Consistent with the findings of Rondelli et al. [29], our results revealed improved feed consumption at 8–12 weeks of age (*p* < 0.05) and total feed consumption (0–12 weeks of age) (*p* < 0.0001) in the El-Salam strain compared to the Dokki-4 strain. No significant improvement in the feed conversion ratio (FCR) was recorded upon in ovo RJ injection. Ahangari et al. [23] found that in ovo RJ injection increased feed consumption and reduced the FCR. Seven et al. [40] reported that propolis and RJ enhanced growth performance measured as body weight, feed intake and FCR; these effects could be attributed to enhanced intestinal health, digestion and absorption due to the antimicrobial effects of the RJ and propolis components. The significant increases in dressing percentage upon treatment with 0.5 mL RJ/egg are consistent with results obtained by Moghaddam et al. [21], who found that in ovo RJ injection significantly increased dressed carcass percentages and heart and liver weights compared to a control treatment. There are several possible explanations for these results. First, RJ can increase oxygen metabolism and animal activity by increasing the concentration and use of blood glucose [41] and can also promote respiration and oxidative phosphorylation, increasing tissue oxygen consumption and, consequently, performance and endurance [42]. Furthermore, RJ also exhibits antioxidant properties [42] in addition to containing many dietary proteins with a wide range of functional and biological properties; some of these properties are attributable to biologically active peptides (of 2–20 amino acid residues) that are inactive when part of a protein but are activated when digested in vivo [43]. Feed consumption and giblet percentages were substantially affected by strain differences but dressing percentages were increased significantly in the group treated with 0.5 mL RJ/egg compared to the other groups.

Our results showed that there were no strain effects on the tested serum parameters. However, in ovo RJ injection decreased lipid profile parameters and increased total protein and globulin content compared to control saline injection (Table 3). A hypocholesterolaemic effect of RJ has also been reported by Kashima et al. [44], who suggested that the major identified RJ proteins (MRJP1, MRJP2 and MRJP3), as bile acid-binding proteins, significantly decreased the micellar solubility of cholesterol. Pavel et al. [45] confirmed the ability of RJ to reduce blood cholesterol and several studies have demonstrated the efficacy of RJ in lowering and controlling blood TGs and cholesterol levels [46,47]. Vittek [48] showed that administration of 50–100 mg RJ/d lowered serum total cholesterol levels by 14% and total lipids by 10%. A different study reported that ingestion of 6 g RJ/d for 4 weeks led to reduced serum total cholesterol and LDL but had no effect on HDL or TGs content [16,49]. The increased total protein, albumen and globulin in the RJ-treated groups may be attributable to a direct promoting impact of RJ on haemopoietic tissue in addition to a stimulatory anabolic effect on liver tissues that favours protein synthesis. In addition, RJ has been proven to protect against degeneration of body protein [50]. Our results are similar to those recorded by Mahmoud [51], who found that feeding of RJ to Ross broilers under different stocking densities increased serum total protein, albumen and globulin levels. Collectively, RJ in ovo injection (0.5 mL RJ/egg) had a hypocholesterolaemic effect on chickens in addition to a role in increasing total serum protein.

The results presented in Table 4 show that in ovo RJ injection increased the Hb percentage and lymphocyte count and decreased monocyte and eosinophil numbers compared to control saline injection. The increased lymphocyte count indicates that in ovo RJ injection improved the chicken response to stress. However, contradictory results were obtained by Ahangari et al. [23], who reported that RJ injection resulted in decreased lymphocyte counts and increased heterophil counts and heterophil/lymphocyte (HL) ratios. Additionally, Rabie et al. [52] reported that dietary or drinking water supplementation of Cobb 500 broiler chicks with propolis significantly increased Hb concentrations compared with a control treatment. However, Morita et al. [53] reported that there is a lack of articles that interpret the effect of RJ on anaemia. Recently, Bhalchandra et al. [54] reported that intraperitoneal injection of RJ in rats improved Hb concentrations and mean corpuscular haemoglobin (MCH) and suggested that honey and RJ exert protective effects against blood cell damage via preservation of cellular integrity. Our findings suggest that RJ feeding might have an anti-anaemic effect.

The results revealed a higher antibody titre against AIV (*p* < 0.05) and SRBCs (*p* < 0.0001) in the Dokki-4 strain than in the El-Salam strain (Table 5). The better immune response of Dokki-4 chickens to AIV and SRBCs may be attributable to the genetic potential inherited from the Fayoumi strain, a pure Egyptian native strain known globally for its strong immunity [28]. The effects of in ovo RJ injection on chicken immunity were unique (Table 5). RJ contains several forms of free amino acids at levels ranging from 0.6–1.5% and most are L-series amino acids, such as lysine and proline [55]. Administration of these amino acids influences the immune responses of poultry against disease, during which body proteins are broken down and the resultant amino acids are used for critical protein synthesis rather than growth, with consequent enhancement of defence against certain diseases [56]. Ahangari et al. [23] reported non-significant effects of RJ injection on day 14 of the experiment, although a significant increase in antibody titre against NDV was observed on day 28 of the trial. Generally, the Dokki-4 strain had a better immune response against AIV and SRBCs than the El-Salam strain; moreover, RJ in ovo injection (0.5 mL RJ/egg) improved the immunity of chickens against AIV, NDV and SRBCs.

Finally, with regard to the feasibility of RJ application in poultry production, RJ can be injected into eggs at trace concentrations (not exceeding 0.5 mg/egg). Poultry production depends on both input and output; the marked improvements in bird growth characteristics in the present study as a result of RJ treatment suggest that even if the price of RJ is high, the economic return will cover the cost of its use and even yield a good profit margin for chicken keepers.

#### **5. Conclusions**

On the basis of our results, the study hypothesis was accepted that varying the chicken strain could alter the response to the in ovo injection with RJ (the Dokki-4 strain was superior to the El-Salam strain for the tested parameters). RJ injection into the yolk sac elicited significant positive effects on hatching parameters, growth performance, blood chemistry, haematology and immunological parameters. Among the injected doses, the 0.5 mL dose of RJ resulted in the best hatching parameters, growth performance and immune and health-related traits.

**Author Contributions:** A.E.T., O.A.A., K.M.A., R.E.A.E.-K. and M.E.A.E.-H. Designed the Study Plan, Collected Literature and Drafted the Manuscript, M.A.E.-E. Helped in Conducting the Research Work, A.A.S., I.M.S. and E.O.S.H. Provided Technical Help in Writing the Manuscript and Conducting Data Analysis. All the Authors Read and Approved the Final Manuscript.

**Funding:** The authors extended their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through College of Food and Agriculture Sciences Research Center.

**Acknowledgments:** The authors extended their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through College of Food and Agriculture Sciences Research Center. The authors thank the Deanship of Scientific Research and the Researchers Support and Services Unit (RSSU) at King Saud University for their technical support. The authors extend thanks to their respective institutes and universities.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Dietary Chitooligosaccharide Inclusion as an Alternative to Antibiotics Improves Intestinal Morphology, Barrier Function, Antioxidant Capacity, and Immunity of Broilers at Early Age**

#### **Jun Li, Yefei Cheng, Yueping Chen, Hengman Qu, Yurui Zhao, Chao Wen and Yanmin Zhou \***

College of Animal Science and Technology, Nanjing Agricultural University, No. 6, Tongwei Road, Xuanwu District, Nanjing 210095, China

**\*** Correspondence: zhouym6308@163.com; Tel.: +86-25-8439-6067; Fax: +86-25-843953

Received: 9 July 2019; Accepted: 25 July 2019; Published: 27 July 2019

**Simple Summary:** At early an age, broilers are susceptible to exterior stressors and therefore have a higher disease incidence rate. Antibiotic growth promoters have been forbidden in animal production by the European Union and other countries since their usage has caused potentially adverse effects such as antibiotic residues in livestock, environmental pollution, and the generation of drug-resistant bacteria. The search for safe and environmentally friendly alternatives to antibiotics to prevent disease and promote growth has become necessary in poultry production. Chitooligosaccharide (COS), a natural alkaline polymer of glucosamine with a number of bioactive groups, is easily obtained by chemical and enzymatic hydrolysis of chitosan, which is the second most abundant carbohydrate polymer in nature. Our results indicated that dietary supplementation with chitooligosaccharide, at a dosage of 30 mg/kg, enhanced the feed conversion ratio, benefited the intestinal morphology and barrier function, and improved antioxidant capacity and immunity in broilers at 21 days of age. These effects were similar with those observed as a result of chlortetracycline inclusion. Therefore, dietary COS supplementation can be used as a potential alternative to antibiotics in broilers.

**Abstract:** This study aimed to investigate the effects of chitooligosaccharide (COS) inclusion as an alternative to antibiotics on growth performance, intestinal morphology, barrier function, antioxidant capacity, and immunity in broilers. In total, 144 one-day-old Arbor Acres broiler chicks were randomly assigned into 3 groups and fed a basal diet free from antibiotics (control group) or the same basal diet further supplemented with either chlortetracycline (antibiotic group) or COS, for 21 days. Compared with the control group, inclusion of COS reduced the feed to gain ratio, the jejunal crypt depth, the plasma diamine oxidase activity, and the endotoxin concentration, as well as jejunal and ileal malondialdehyde contents, whereas increased duodenal villus height, duodenal and jejunal ratio of villus height to crypt depth, intestinal immunoglobulin G, and jejunal immunoglobulin M (IgM) contents were observed, with the values of these parameters being similar or better to that of the antibiotic group. Additionally, supplementation with COS enhanced the superoxide dismutase activity and IgM content of the duodenum and up-regulated the mRNA level of claudin three in the jejunum and ileum, when compared with the control and antibiotic groups. In conclusion, dietary COS inclusion (30 mg/kg), as an alternative to antibiotics, exerts beneficial effects on growth performance, intestinal morphology, barrier function, antioxidant capacity, and immunity in broilers.

**Keywords:** chitooligosaccharide; intestinal integrity; antioxidant capacity; immunity; broiler

#### **1. Introduction**

At an early age, broilers are susceptible to exterior stressors and, therefore, have a higher disease incidence because of their weak physiological status, including their small size, undeveloped organs, and poor immune function [1]. Antibiotics have excellent therapeutic effectiveness and growth promotion properties and were used as feed additives for livestock for several decades [2,3]. However, their usage has caused potential adverse effects, such as antibiotic residues in livestock, environmental pollution, and the generation of drug-resistant bacteria. The European Commission has therefore banned the use of antibiotics as growth promoters in animal production since 2006 [4]. A wide array of functional substances are currently being tested as substitutes for antibiotics to prevent disease and promote growth in livestock production, and these substrates include probiotics, prebiotics, plant extracts, and other agents [5,6]. Chitooligosaccharide (COS), as a functional prebiotic, is a natural alkaline polymer of glucosamine with a number of bioactive compounds and it is easily obtained by chemical and enzymatic hydrolysis of chitosan, which is the second most abundant carbohydrate polymer in nature [7]. Presently, many researchers tend to use chitosan in its oligosaccharide form since COS has a low molecular weight, good solubility, and low viscosity [8]. It is reported that COS could exert an antibacterial effect, regulate lipid metabolism, and promote antioxidant capacity and immunity in *in vitro* studies [9–11]. These properties of COS led to its application in livestock, especially pig production. Previous studies have shown that COS can be an alternative to antibiotics [5], promote growth [12,13], improve intestinal morphology and barrier function [14–17], and enhance antioxidant capacity and immunity in pigs [16,18]. In broilers, improved immunity and nutrient digestibility have been reported after inclusion of COS [19–21]. However, information is scarce concerning its effects on intestinal morphology and barrier function, as well as its antioxidant capacity, in broilers, although other functional oligosaccharides, such as fructooligosaccharide and mannan oligosaccharide could improve intestinal integrity and antioxidant ability in broilers [22–24]. In consideration of the similar biological functions among oligosaccharides and the application effects of COS in pigs, we hypothesized that dietary COS inclusion may be an alternative to antibiotics and may induce beneficial consequences in broiler chickens. Therefore, we investigated the effects of dietary COS supplementation, used as an alternative to antibiotics, on the growth performance, intestinal morphology, barrier function, antioxidant capacity, and immunity of broilers.

#### **2. Materials and Methods**

#### *2.1. Animals, Diets, and Experimental Design*

All procedures related with management and care of chickens in this experiment were approved by the Nanjing Agricultural University Animal Care and Use Committee (Certification No.: SYXK (Su) 2017-0007).

A total of 144 one-day-old male Arbor Acres broiler chicks with similar birth weights (42 ± 0.2 g) were used in this experiment. The chicks were randomly assigned to three dietary treatments of 6 replicate pens/cages per treatment, with eight broilers per pen. Broilers in the three treatments were fed a basal diet free from antibiotics (control group) or the same diet further supplemented with either 50 mg/kg of chlortetracycline (by effective content, antibiotic group) or 30 mg/kg of COS (COS group) for 21 days. The composition and nutrient contents of the basal diet are shown in Table 1. The broilers had free access to mash feed and water with continuous lighting in three-layer cages (120 cm × 60 cm × 50 cm) in a temperature-controlled room. The ambient temperature of the room was maintained at 32–34 ◦C for the first 3 days and then reduced by 2–3 ◦C per week to a final temperature of 26 ◦C. Body weight was recorded at 21 days of age after feed deprivation for 12 h and feed intake was determined from the difference between the offered and residual feed, on a cage basis, to calculate the average daily gain (ADG), the average daily feed intake (ADFI), as well as the feed/gain ratio (F/G). The COS dosage used in this study was according to the manufacturer's recommendation (Zhongkerongxin

Biotechnology Co., Ltd., Suzhou, Jiangsu, P.R. China). The average molecular weight of COS ranged from 1000 to 2000 Daltons (Da) and its purity was higher than 90%.


**Table 1.** Composition and nutrient level of the basal diet (g/kg, as fed basis unless otherwise stated).

<sup>1</sup> Premix provided per kilogram of diet: Vitamin A (trans-retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 3,000 IU; vitamin E (all-rac-α-tocopherol), 30 IU; menadione, 1.3 mg; thiamin, 2.2 mg; riboflavin, 8 mg; nicotinamide, 40 mg; choline chloride, 400 mg; calcium pantothenate, 10 mg; pyridoxine·HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; vitamin B12 (cobalamin), 0.013 mg; Fe (from ferrous sulfate), 80 mg; Cu (from copper sulphate), 8.0 mg; Mn (from manganese sulphate), 110 mg; Zn (from zinc oxide), 60 mg; I (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.3 mg.

#### *2.2. Sample Collection*

On day 21, one bird per pen, that was close to the average body weight of the pen, was selected and weighed after a 12-h fasting. Whole blood samples were collected into both anti-coagulant tubes coated with EDTA and non-heparinized tubes via jugular venipuncture and kept at −20 ◦C until analysis. Serum was then obtained from the blood samples after centrifugation at 4450 × *g* for 15 min at 4 ◦C, and it was immediately stored at −20 ◦C for further determination. After blood collection, broilers were euthanized by cervical dislocation and necropsied immediately. Then, the immune organs including the thymus, spleen, and bursa of Fabricius were quickly excised and weighed to calculate the relative immune organ weight, which was expressed as g/kg live body weight. Approximately two-centimeter segments of the mid-duodenum, mid-jejunum, and mid-ileum were harvested, flushed several times with ice-cold phosphate-buffered saline (pH 7.4), fixed with 10% paraformaldehyde, and kept at 4 ◦C for evaluation of the mucosal morphology. The duodenum, jejunum, and ileum mucosa were scraped off using a sterile glass slide, which was frozen in liquid nitrogen rapidly and stored at −80 ◦C for further assessment.

#### *2.3. Intestinal Morphological Examination*

The preserved intestinal segments were dehydrated, cleared, and embedded in paraffin. Serial sections were performed at 5 μm thickness and stained with hematoxylin and eosin. The villus height and crypt depth were measured on the stained sections under a microscope with a Nikon ECLIPSE 80i light microscope equipped with a computer-assisted morphometric system (Nikon Corporation, Tokyo, Japan). A total of 10 well-oriented and intact villi were measured for each intestinal sample.

#### *2.4. Evaluation of Serum Biomarkers of Intestinal Permeability*

The activity of serum diamine oxidase (DAO) was determined by a corresponding reagent kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, P.R. China). Serum D-lactate acid levels were measured using a colorimetric assay kit (catalogue no. K667-100; BioVision Inc., Shanghai, China). Assays for serum endotoxin were carried out as described by the manufacturer's method of instruction (Xiamen Bioendo Technology Co., Ltd., Xiamen, Fujian, China).

#### *2.5. Determination of Intestinal Antioxidant Capacity and Mucosal Immunity*

Intestinal mucosal samples were homogenized (1:9, wt/vol) with ice-cold 154 mmol/L sterile sodium chloride solution using an Ultra-Turrax homogenizer (Tekmar Co., Cincinatti, OH, USA). Then, the mixture was centrifuged at 4450 × *g* for 15 min at 4 ◦C to obtain the supernatant, which was stored at −20 ◦C for the determination of the anti-oxidative and immune parameters. The anti-oxidative parameters, including total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde (MDA) level, were assayed following the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). Immunoglobulin G (IgG), immunoglobulin M (IgM), and secretory immunoglobulin A (sIgA) were measured by enzyme-linked immunosorbent assay (ELISA) using chicken-specific IgG, IgM, and sIgA ELISA quantitation kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

#### *2.6. Messenger RNA Quantification*

The intestinal mucosal RNA was isolated using TRIzol reagent (Takara Biotechnology Co. Ltd., Dalian, Liaoning, China). Then, the RNA quality was analyzed in agarose gels stained with ethidium bromide and the total RNA concentration was determined from OD260/280 readings (ratio > 1.8) using a NanoDrop ND-1000 UV spectrophotometer (Nano Drop Technologies, Wilmington, DE). After determining the quality and purity, the resultant cDNA was synthesized using the PrimeScriptTM RT reagent kit (Takara Biotechnology Co. Ltd, Dalian, Liaoning, China), according to the manufacturer's instructions, and stored at −20 ◦C for real-time PCR. The primer sequences, including occludin (OCLN), claudin 2 (CLDN2), claudin 3 (CLDN3), and zonula occludens-1 (ZO-1), used real-time PCR and their gene bank ID numbers are presented in Table 2. The reaction mixture was prepared using a TB GreenTM Premix Ex TaqTM kit (Takara Biotechnology Co. Ltd., Dalian, Liaoning, P.R. China) and gene expression levels were subsequently determined by a real-time quantitative PCR using an ABI PRISM 7500HT Detection System (Applied Biosystems, Foster City, CA, USA). The reaction was performed as follows: One cycle pre-run at 95 ◦C for 30 s, 40 cycles of denaturation at 95 ◦C for 5 s, and a 60 ◦C annealing step for 30 s. The expressions of relative genes were expressed as 2−ΔΔCT [25] and the results were normalized according to the expression of β-actin.


**Table 2.** Sequences for real-time PCR primers.

<sup>1</sup> OCLN, occludin; CLDN2, claudin 2; CLDN3, claudin 3; ZO-1, zonula occludens-1.

#### *2.7. Statistical Analysis*

A complete randomized design was used in this study and one-way ANOVA was performed using SPSS (Version 20.0, SPSS Inc., Chicago, IL, USA) with pen (cage) as the experimental unit. Differences among treatments were detected by Tukey's multiple range tests. Results were expressed as means with their pooled standard errors. Probability values less than 0.05 were considered significant.

#### **3. Results**

#### *3.1. Growth Performance*

The effect of dietary COS supplementation on growth performance in broilers is presented in Table 3. Broilers that received the COS supplemented diet had a lower F/G (*p* < 0.05) when compared with those offered the basal diet, with the value of F/G being similar between COS and antibiotic groups (*p* > 0.05). However, the ADG and ADFI were not affected by treatments (*p* > 0.05).

**Table 3.** Effect of COS supplementation on growth performance in broilers.


a, b Means within a row with different superscripts differ significantly at *p* < 0.05. <sup>1</sup> ADG: average daily gain; ADFI: average daily feed intake; F/G: feed to gain ratio. <sup>2</sup> Control group, basal diet; Antibiotic group, basal diet supplemented with 50 mg/kg chlortetracycline; COS group, basal diet supplemented with 30 mg/kg chitooligosaccharide; SEM, standard error of means (*n* = 6).

#### *3.2. Relative Immune Organ Weights*

Compared with the control group (Table 4), COS dietary supplementation tended to increase the thymus relative weight (*p* = 0.095) and the value of this parameter did not differ between the COS and antibiotic groups (*p* > 0.05). Relative spleen and bursa of Fabricius weights were similar among the groups (*p* > 0.05)


**Table 4.** Effect of COS supplementation on relative immune organ weight in broilers (g/kg).

<sup>1</sup> Control group, basal diet; Antibiotic group, basal diet supplemented with 50 mg/kg chlortetracycline; COS group, basal diet supplemented with 30 mg/kg chitooligosaccharide; SEM, standard error of means (*n* = 6).

#### *3.3. Intestinal Morphology*

Compared with the control group (Table 5), COS dietary supplementation increased duodenal villus height (*p* < 0.05) and the ratio of villus height to crypt depth (*p* < 0.05) in the duodenum and jejunum, whereas it caused the depression of crypt depth in jejunum (*p* < 0.05), with the values of these parameters being similar to antibiotic groups (*p* > 0.05). Moreover, the values of crypt depth in the ileum were also found to be decreased in response to antibiotic supplementation when compared with the control and COS groups (*p* < 0.05).

#### *3.4. Serum Biomarkers of Intestinal Permeability*

As was shown in Table 6, the activity of DAO and endotoxin concentration were found to decrease in response to COS or antibiotic supplementation (*p* < 0.05). Furthermore, the inclusion of COS or antibiotic had a tendency to reduce the serum D-lactate level (*p* = 0.061). In addition, there were no significant difference between the COS and antibiotic groups regarding these parameters (*p* > 0.05).


**Table 5.** Effect of COS supplementation on intestinal mucosal morphology in broilers.

a, b Means within a row with different superscripts differ significantly at *p* < 0.05. <sup>1</sup> Control group, basal diet; Antibiotic group, basal diet supplemented with 50 mg/kg chlortetracycline; COS group, basal diet supplemented with 30 mg/kg chitooligosaccharide; SEM, standard error of means (*n* = 6).

**Table 6.** Effect of COS supplementation on serum markers of intestinal permeability in broilers.


a, b Means within a row with different superscripts differ significantly at *p* < 0.05. <sup>1</sup> DAO, diamine oxidase. <sup>2</sup> Control group, basal diet; Antibiotic group, basal diet supplemented with 50 mg/kg chlortetracycline; COS group, basal diet supplemented with 30 mg/kg chitooligosaccharide; SEM, standard error of means (*n* = 6).

#### *3.5. Intestinal Antioxidant Capacity*

Compared with the control and antibiotic groups (Table 7), dietary COS inclusion increased SOD activity in the duodenum of broilers (*p* < 0.05). Furthermore, broilers receiving the COS supplemented diet had a lower ileal MDA content (*p* < 0.05), when compared with those fed basal diet, and the value of this parameter was intermediate in the antibiotic group (*p* > 0.05). Additionally, the supplementation with COS caused depression of jejunum MDA content (*p* < 0.05), with the value of this parameter being similar between the COS and antibiotic groups (*p* > 0.05). However, the remaining values of anti-oxidative parameters were not affected by the incorporation of antibiotics or COS (*p* > 0.05).

**Table 7.** Effect of COS supplementation on intestinal oxidant status in broilers.


a, b Means within a row with different superscripts differ significantly at *p* < 0.05. <sup>1</sup> T-AOC, total antioxidant capacity; SOD, superoxide dismutase; MDA, malondialdehyde. <sup>2</sup> Control group, basal diet; Antibiotic group, basal diet supplemented with 50 mg/kg chlortetracycline; COS group, basal diet supplemented with 30 mg/kg chitooligosaccharide; SEM, standard error of means (*n* = 6).

#### *3.6. Immunoglobulin Concentration in the Intestine*

The IgG levels (Table 8) of duodenum, jejunum, and ileum, as well as jejunal IgM level, were observed to be higher in response to COS supplementation (*p* < 0.05), with the values of these parameters being similar between COS and antibiotic groups (*p* > 0.05). Additionally, dietary supplementation with COS resulted in a higher duodenal IgM content when compared with the control and antibiotic groups (*p* < 0.05).

**Table 8.** Effect of COS supplementation on intestinal immunoglobulins in broilers (μg/mg protein).


a, b Means within a row with different superscripts differ significantly at *p* < 0.05. <sup>1</sup> IgG, immunoglobulin G; IgM, Immunoglobulin M; sIgA, secretory immunoglobulin A. <sup>2</sup> Control group, basal diet; Antibiotic group, basal diet supplemented with 50 mg/kg chlortetracycline; COS group, basal diet supplemented with 30 mg/kg chitooligosaccharide; SEM, standard error of means (*n* = 6).

#### *3.7. Gene Expressions Related to Intestinal Barrier Function*

Compared with control and antibiotic groups (Table 9), the supplementation of COS upregulated the mRNA expression of CLDN3 in the jejunum and ileum (*p* < 0.05). However, treatments didn't alter the mRNA abundance of intestinal OCLN, CLDN2, and ZO-1 (*p* > 0.05).


**Table 9.** Effect of COS supplementation on intestinal gene expression in broilers.

a, b Means within a row with different superscripts differ significantly at *p* < 0.05. <sup>1</sup> OCLN, occludin; CLDN2, claudin 2; CLDN3, claudin 3; ZO-1, zonula occludens-1. <sup>2</sup> Control group, basal diet; Antibiotic group, basal diet supplemented with 50 mg/kg chlortetracycline; COS group, basal diet supplemented with 30 mg/kg chitooligosaccharide; SEM, standard error of means (*n* = 6).

#### **4. Discussion**

#### *4.1. Growth Performance*

Previous studies have reported that the function of chitosan is closely associated with its molecular weight and Vila et al. [26] showed that chitosan with a molecular weight greater than 100000 Da could only serve as an adhesive or a carrying agent. The COS could modulate immune responses and reduce establishment of pathogens in the intestine when its molecular weight is between 1000 to 10000 Da [27]. In the present study, the COS had an average molecular weight between 1000 and 2000 Da and our results indicated that broilers receiving the COS supplemented diet had a lower F/G, with the value being similar to antibiotic group. This finding suggests that the inclusion of COS could be used as an alternative to antibiotics for improving the growth performance of broilers. Consistent with our results, Huang et al. [21] observed that dietary COS supplementation improved the ADG and F/G in broilers by acting as an antibiotic. Likewise, Li et al. [28] reported that the inclusion of COS improved ADG, ADFI, and F/G in broilers. In weaning pig, both a higher ADG and feed conversion ratio (FCR) were observed in response to COS supplementation [16]. Additionally, Yin et al. [29] found that supplementation with COS improved the ADG and F/G in early-weaned piglets. There are several possible mechanisms that could explain the positive effect of COS on the growth performance in livestock, including enhancement of the nutrient digestibility [21,28], increment of growth hormone or IGF-1 concentration [30], and the improvement of intestinal integrity and antioxidant capacity, as well as immunity [16].

#### *4.2. Relative Immune Organ Weights and Intestinal Immunoglobulin Levels*

Relative organ weight could reflect the growth and development of organs in some degree [31]. The effects of COS on relative immune organ weight have already been studied, however, the results were inconsistent. Li et al. [32] found that COS promoted the development of immune organs in broilers. Similarly, Deng et al. [20] reported that COS supplementation increased the spleen, thymus, and bursa index of broilers on day 21. However, it has also been reported by Zhou et al. that COS did not affect the immune organ index in broilers [33]. The discrepancy is likely due to the degree of polymerization, deacetylation level, dosage, and purity of COS [32]. In the current study, supplementation with COS tended to increase the thymus relative weight, coupled with simultaneously increased IgG and IgM contents, which further indicated that dietary COS supplementation could exert a positive effect on immune function. These beneficial consequences are likely due to that COS could regulate cytokine secretion, promote the proliferation of T and B lymphocytes, and inhibit lymphocyte apoptosis in immune organs [20,32]. Huang et al. [19] also showed that supplementation with COS enhanced serum IgG and IgM contents of broilers. Similar results were found by Deng et al. [20], who demonstrated that broilers receiving the COS supplemented diet had a higher circulating IgM content. Additionally, Wu and Tsai [34] showed that COS improved the IgM secretion of human hybridoma HB4C5 cells, indicating that COS could improve immunity. These changes were likely attributed to the alteration in the microenvironment caused by COS supplementation [35] and were consistent with the results of intestinal morphology and barrier function observed in our study. Additionally, COS may also inhibit pro-apoptotic pathways via improving the capacity of free radical clearance in the immune organs, thus benefitting the immune function [32].

#### *4.3. Intestinal Morphology and Barrier Function*

The structure of the intestinal mucosa can reveal some information about gut health, and a shortening of villus height is associated with a decrease in the surface area for nutrient absorption [36]. On the other hand, a large crypt indicates fast tissue turnover and increased nutrient requirements for new tissue are needed, which contribute to poor nutrient absorption [37]. The ratio of villus height to crypt depth is a useful criterion to estimate the nutrient digestion and absorption capacity of the small intestine [38]. In the current study, dietary COS supplementation could exert a positive effect on intestinal morphology, as evidenced by the increased villus height and ratio of villus height to crypt depth, as well as the decreased crypt depth. Liu et al. [14] also reported that broilers fed a diet supplemented with COS had a higher villus height and ratio of villus height to crypt depth in the jejunum and ileum in weanling pigs. Similarly, it is reported that dietary COS supplementation could attenuate compromised intestinal morphology in weanling pigs challenged by *Escherichia coli* through

increasing villus height to crypt depth ratio [39]. Previous researches have shown that the N-acetyl glucosamine, the main component of COS, may bind to certain types of bacteria and therefore interfere with their adhesion to the gut tissue of host [40–42]. Additionally, Mourão et al. [43] reported that an increase in villus height in the ileum of weaned rabbits was correlated with a decreased intestinal microflora. The possible explanation for improved intestine structure in the present work was that the N-acetyl glucosamine abundance in COS may create a more favorable intestinal microbial environment.

DAO is an enzyme synthesized primarily in the gastrointestinal mucosal cells of mammalian species and distributed primarily in the cytoplasm and blood DAO levels are increased when the mucosa is damaged and DAO enter into the bloodstream [15]. Plasma D-lactate acid is produced by the intestinal microflora and the content of D-lactate acid in the serum may increase if the small intestine mucosa is injured as a result of dysfunction in the intestinal barrier [44]. Serum DAO activity and D-lactate acid level are useful biomarkers for evaluating the integrity of the gastrointestinal tract [45]. In the present study, dietary supplementation with COS decreased serum DAO activity and endotoxin content. Similarly, previous literature showed that COS dietary supplemented pigs had lower DAO activity and endotoxin concentration than pigs in the control group after 14 days of supplementation [16]. In addition, a lower DAO activity in the serum and a higher activity of DAO in the jejunum mucosa were found in piglets on day seven, postweaning, in response to COS supplementation [15]. Tight junction, the multi-protein complex, are made up of transmembrane proteins, peripheral membrane proteins and regulatory molecules including kinases, among which CLDN family proteins and ZO family proteins are crucial to tight-junction assembly [46]. It was reported that the permeability of the leak pathway can be acutely regulated by the cytoskeleton via mechanisms that involve ZO-1 and OCLN [47]. In the current study, the mRNA expression of CLDN3 was found to be higher in response to COS supplementation in broilers, coupled with the simultaneously decreased circulating DAO activity and endotoxin level, further indicated that COS could improve intestinal barrier function in broilers at an early age. Likewise, Alizadeh et al. [48] reported that the mRNA expressions of various tight junction proteins, including CLDN1, ZO-1, ZO-2, and OCLN, were up-regulated in the intestines of piglets fed a galacto-oligosaccharides diet. However, Xiong et al. [49] showed that the COS inclusion decreased the mRNA expressions of OCLN and ZO-1 in the intestines of weaned piglets, indicating that dietary COS supplementation compromised the intestinal barrier integrity in weaned piglets. These discrepancies may be also attributed to the polymerization level, purity, and dosage of COS, as well as the animal species. Further studies are necessary to examine the effects of COS on intestinal barrier function.

#### *4.4. Intestinal Antioxidant Capacity*

Oxidative stress is observed when production of reactive oxygen species (ROS) exceeds the capacity of cellular antioxidant defenses to remove these toxic species [6,50]. The SOD is an important antioxidant enzyme in scavenging the oxygen free radical [51] and the content of MDA is the main end product of lipid peroxidation by ROS [52]. In the present study, the supplementation of COS improved the activity of SOD, whereas it decreased the intestine lipid peroxidation biomarker MDA level. Likewise, Li et al. [53] reported that dietary COS supplementation enhanced the activities of T-AOC, glutathione peroxidase (GSH-Px), and SOD, whereas it decreased the MDA content of the ileum mucosa in broilers. Similar results were also observed by Zhao et al. [16], who demonstrated that the inclusion of COS enhanced circulating T-AOC and GSH-Px activities and decreased plasma MDA content, simultaneously, in weaned piglets. Available literature indicated that COS with average molecular weight below 5000 Da can be regarded as a potential antioxidant due to its ROS scavenging properties [10,54]. It can be concluded that the improved antioxidant capacity observed in our research is primarily attributed to the antioxidant characteristics of COS. In addition, the improved antioxidant capacity may also be closely related with the improved intestinal integrity and immunity observed in this study.

#### **5. Conclusions**

The results of our study indicated that dietary supplementation with COS at a dosage of 30 mg/kg can improve FCR, benefit the intestinal morphology and barrier function, and improve antioxidant capacity and immunity in broilers at an early age. These effects were similar with that observed after dietary chlortetracycline inclusion. Therefore, dietary COS supplementation can be used as a potential alternative to antibiotics in broilers.

**Author Contributions:** Formal Analysis, J.L., Y.C., H.Q., and Y.Z.; Writing–Original Draft Preparation, J.L.; Editing, Y.C., C.W., and Y.Z.; Supervision, Y.Z.

**Acknowledgments:** The technical assistance of colleagues in our laboratories is gratefully acknowledged.

**Conflicts of Interest:** The authors declare no conflict of interest in the present work.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **E**ff**ects of Dietary Betaine on Growth Performance, Digestive Function, Carcass Traits, and Meat Quality in Indigenous Yellow-Feathered Broilers under Long-Term Heat Stress**

**Wenchao Liu 1, Yilin Yuan 1, Chenyu Sun 1, Balamuralikrishnan Balasubramanian 2, Zhihui Zhao 1,\* and Lilong An 1,\***


Received: 29 May 2019; Accepted: 30 July 2019; Published: 31 July 2019

**Simple Summary:** Heat stress, one of the major problems in tropical and subtropical regions, adversely affects poultry production. This study was designed to evaluate the effects of dietary betaine on growth performance, digestive function, carcass traits, and meat quality in indigenous yellow-feathered broilers subjected to long-term heat stress. The results demonstrated that long-term heat exposure reduced the growth performance, digestive function, and carcass yield, and dietary betaine supplementation partially alleviated the adverse effects of heat stress on these parameters. These findings are useful for development of anti-heat stress feed additives in indigenous yellow-feathered broilers.

**Abstract:** Heat stress has a profound effect on poultry health and productivity. The present study evaluated whether feeding betaine could ameliorate long-term heat stress-induced impairment of productive performance in indigenous yellow-feathered broilers. A total of 240 five-week-old male broilers were randomly allocated to five treatments with six replicates of eight broilers each. The five treatments included a thermoneutral zone control group (TN, fed basal diet), a heat stress control group (HS, fed basal diet), and an HS control group supplemented 500, 1000, 2000 mg/kg betaine, respectively. The TN group was raised at 26 ± 1 ◦C during the whole study, HS groups exposed to 32 ± 1 ◦C for 8 h/day from 9:00 am to 17:00 pm. The results showed that heat stress decreased the body weight gain (BWG) and feed intake of broilers during 1–5, 6–10, and 1–10 weeks (*p* < 0.05). Dietary betaine tended to improve the BWG and feed intake of broilers under 5 weeks of heat stress (linear, *p* < 0.10), and betaine supplementation linearly increased the BWG and feed intake during 6–10 and 1–10 weeks (*p* < 0.05). Additionally, nitrogen retention was reduced by 5 weeks and 10 weeks of heat stress (*p* < 0.05), whereas dietary betaine could improve nitrogen retention in heat stressed broilers after both 5 and 10 weeks of heat stress (linear, *p* < 0.05). Moreover, this study observed that the trypsin activity of jejunum was decreased by 5 weeks of heat stress (*p* < 0.05), whereas betaine supplementation had quadratic effects on trypsin activity of jejunum in heat stressed broilers (*p* < 0.05). Furthermore, 10 weeks of heat stress induced a reduction of villus height of the duodenum and jejunum (*p* < 0.05), and decreased the villus height to crypt depth ratio of the jejunum (*p* < 0.05). Supplementation with betaine ameliorated the adverse effects of heat stress on these parameters (*p* < 0.05). Compared with the TN group, 10 weeks of heat stress reduced carcass and breast yield (*p* < 0.05) and betaine supplementation improved carcass and breast yield of heat stressed broilers (linear, *p* < 0.05). In conclusion, dietary supplementation of betaine could reduce the

detrimental effects of long-term heat stress on growth performance, digestive function, and carcass traits in indigenous yellow-feathered broilers.

**Keywords:** broilers; digestive function; heat stress; indigenous yellow-feathered breed

#### **1. Introduction**

With global warming, the deleterious effects of heat stress induced by high ambient temperature on poultry productivity have been of great concern all over the world, especially in tropical and subtropical regions. Heat stress has a profound effect on broilers' health and production, and leads to multiple physiological disturbances, such as endocrine disorders, systemic immune dysregulation, and electrolyte imbalance [1]. Heat stress also causes a disruption in the intestinal structure and function, including reduced regeneration and integrity of the intestinal epithelium [2,3], which in turn suppresses the growth rate and feed efficiency of birds. In addition, heat stress impairs carcass traits and meat quality through affecting energy-substance metabolism and redox status, resulting in decreased meat yield and increased abdominal fat rate in broilers [4,5]. Reducing the house temperature to the thermoneutral zone is a direct strategy to eliminate heat stress of poultry, and the thermoneutral zone can maximize the growth potential [1]. However, the cost of cooling equipment is relatively high in broiler production. It has previously been reported that nutritional manipulation could be a viable option to minimize the adverse impacts of heat stress on broilers [6], including supplementation of functional feed additives, such as probiotics, prebiotics, and natural active substances.

In recent years, special attention has been paid to the use of natural plant extracts in animal science. Betaine is a trimethyl derivative of the amino acid glycine and widely found by a variety of plants in nature. There is increasing evidence that it is a highly valuable feed additive and can produce positive effects on animal performance [7–9]. Betaine is known to have two major functions in the body, as a methyl group donor and an organic osmolyte. On the other hand, betaine has been shown to protect cells from osmotic pressure and allow them to continue normal metabolic activities under conditions that inactivate cells [10]; thus, the use of betaine may improve broiler tolerance to heat stress. Furthermore, it has been suggested that betaine could be used as a natural antioxidant and had the ability to improve meat quality of broilers [11]. Based on the above properties of betaine, previous studies have demonstrated that dietary betaine could improve the heat stressed broilers' growth performance, physiology, carcass criteria [12], lipid metabolism [13], immune response [14], and intestinal barrier function [15]. However, the findings of one study were inconsistent [16], which revealed that betaine supplementation had no significant effects on carcass traits and intestinal morphology of broilers under heat stress. The variable results suggested that further research and development is still required in this regard. Meanwhile, due to the good meat quality, yellow-feathered broilers are increasingly favored by Chinese consumers. Huaixiang chicken is a famous Chinese indigenous yellow-feather broiler breed and is widely farmed in southern China [17]. However, there is extremely limited information about the effects of betaine on these indigenous yellow-feathered broilers under long-term heat stress. Therefore, the current experiment was conducted to investigate the adverse effect of long-term heat stress on growth performance, digestive function, carcass traits, and meat quality in indigenous yellow-feathered broilers (Huaixiang chicken), and to evaluate whether feeding betaine could ameliorate long-term heat stress-induced impairment of these parameters.

#### **2. Materials and Methods**

#### *2.1. Animal Ethics*

The present study was carried out at the College of Agriculture, Guangdong Ocean University, Zhanjiang, China. The protocol of this experiment was approved by the Animal Care Committee, College of Agriculture, Guangdong Ocean University, Zhanjiang, China (SYXK-2018-0147).

#### *2.2. Experimental Design, Animals, and Diet*

A total of two hundred and forty 5-week-old yellow-feathered male broilers (indigenous breed, Huaixiang chickens) were randomly allocated to five treatments, each of which was replicated six times with eight broilers per replicate. The experimental period lasted 10 weeks. The five treatments were thermoneutral zone control group (TN, fed basal diet); heat stress control group (HS, fed basal diet); heat stress treatment group 1 (basal diet +500 mg/kg betaine); heat stress treatment group 2 (basal diet +1000 mg/kg betaine); heat stress treatment group 3 (basal diet +2000 mg/kg betaine). The betaine was obtained from a commercial Chinese company (anhydrous betaine, 99% purity, Shandong Jianchuan Biotechnology Co., Ltd., Shandong, China). The ingredient composition and nutrient content of basal diets are presented in Table 1. Basal diets were formulated to meet or exceed requirements suggested by the Chinese Chicken Feeding Standard (NY/T33-2004) [18]. The diet was given to the birds in the form of mash, and betaine was mixed into the diet before feeding. To ensure that the betaine was thoroughly mixed into the diet, firstly, betaine was mixed with 1 kg of feed by hand, and then the premix was mixed with the remaining feed by using a blender. The crude protein, lysine, cystine, methionine, calcium, and phosphorus of the diet were determined according to the methods of AOAC (2000) [19]. Birds had free access to feed and water. The broilers in TN group were raised at 26 ± 1 ◦C during the whole study. Other groups, designed as HS groups, were subjected to cyclic heat stress by exposing them to 32 ± 1 ◦C for 8 h/day from 9:00 am to 17:00 pm, the temperature of rest time is consistent with TN groups. Relative humidity was controlled at 65–75% among all groups during the entire experimental period. The birds in the TN and HS groups were housed in different facilities, the temperature and relative humidity of the TN and HS groups were measured three times a day. Continuous artificial light was used to illuminate the interior space for the whole period. The chicken houses were equipped with environmental control equipment, and the size of cage is 90 (length) × 70 (width) × 40 (height) cm.

#### *2.3. Sampling and Measurements*

The cage was considered as the experimental unit. Broilers were weighed on a cage basis (*n* = 30) initially and after 5 and 10 weeks of heat stress. The feed consumption was recorded weekly based on the cage (*n* = 30). Body weight gains (BWG), feed intake, and feed conversion ratio (FCR) were then calculated using this information for each phase.

After 5 and 10 weeks of heat stress, one bird from each replicate was randomly selected and moved to metabolic cages for metabolic testing (one bird per cage). The metabolic test lasted 4 days, and the nutrient retention was analyzed as average data by cage during the 4 days (*n* = 30). The total excreta collection method was used for determination of nutrient retention. During the test, feed intake and excrements were recorded daily, and the excreta were collected. The nitrogen, ash, gross energy, Ca, and phosphorus contents in the feed and excreta were then analyzed based on the method of AOAC (2000) [19]. The crude fat contents were analyzed by using a fat analyzer (Hua Bei Experimenting, Co., Ltd., Hebei, China) based on the Soxhlet extraction method, and ether was used as the solvent. The nutrient retention was calculated by the following formula:

Nutrient retention (%) = (feed intake × Nf- excretion amount × Ne)/(feed intake × Nf) × 100

where Ne = nutrient concentration in excreta (% DM), Nf = nutrient concentration in feed (% DM).

After 5 and 10 weeks of heat stress, six birds per treatment (one bird per replicate was randomly selected) were slaughtered (*n* = 30) by severing the jugular vein, respectively. Small intestine was then separated and samples of the contents of the duodenum, jejunum, and ileum were immediately collected for the determination of digestive enzyme activity by using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Subsequently, approximately 2 cm segments of the duodenum, jejunum, and ileum at the middle position were collected immediately. The intestinal samples from each section were fixed in 10% buffered formalin until analyzed. Each intestinal segment was embedded in paraffin. A 7 μm section of each sample was placed onto a glass slide and stained with alcian blue/haematoxylin and eosin for examination with a light microscope. Villus height and crypt depth of the small intestine were measured at 40× magnification using computer software (Sigma Scan, Jandel Scientific, San Rafael, CA, USA), then villus height to crypt depth ratio was calculated.


**Table 1.** Basal diet composition (as-fed basis).

<sup>1</sup> Premix provided per kilogram of diet: 5000 IU of vitamin A, 1000 IU of vitamin D3, 10 IU of vitamin E, 0.5 mg of vitamin K3, 3 mg of thiamin, 7.5 mg of riboflavin, 4.5 mg of vitamin B6, 10 μg of vitamin B12, 25 mg of niacin, 0.55 mg of folic acid, 0.2 mg of biotin, 500 mg of choline, and 10.5 mg of pantothenic acid. 60 mg of Zn, 80 mg of Mn, 80 mg of Fe, 3.75 mg of Cu, and 0.35 mg of I. <sup>2</sup> Nutrient levels on DM basis; except for metabolic energy (ME), others are measured values; ME calculated according to Chinese feed ingredient database.

At the end of the experiment (after 10 weeks of feeding trial), the carcass traits and meat quality of the slaughtered broilers were determined (one bird per replicate was randomly selected, *n* = 30). The carcass traits, included: slaughter rate (%) = (slaughter weight/live weight) × 100; semi-eviscerated carcass rate (%) = (semi-eviscerated weight/live weight) × 100; eviscerated carcass rate (%) = (eviscerated weight/live weight) × 100; leg muscle yield (%) = (leg muscle weight on both sides/live weight) × 100; breast muscle yield (%) = (breast muscle weight on both sides/live weight) × 100; abdominal fat rate (%) = (abdominal fat weight/live weight) × 100. Subsequently, cooking loss was measured by using approximately 20 g of meat sample from the left breast and leg muscle according to the method described by Honikel [20]. The shear force of breast and leg muscle was detected by using C-LM3 digital display tenderness meter (kgf, Northeast Agricultural University, Harbin, China). Duplicate pH values of leg and breast muscle for each sample at 45 min and 24 h after slaughtered were measured using a pH meter (PHSJ-5, Leici, Shanghai Yidian Scientific Instrument Co., Ltd., Shanghai, China).

#### *2.4. Statistical Analysis*

All data were analyzed by using SAS 9.4 (SAS Institute Inc., Cary, NC, USA). The growth performance was analyzed during 1–5 weeks, 6–10 weeks, and 1–10 weeks of heat stress. Nutrient retention, digestive enzyme activity, and intestinal morphology were analyzed after 5 and 10 weeks of heat stress. Carcass traits and meat quality were investigated after 10 weeks of heat stress. Data were expressed as means. Differences among means were tested by using Tukey's test. Orthogonal polynomial contrasts were used to test the linear, quadratic, and cubic effects of the increasing levels of dietary betaine among HS groups. Variability in data was expressed as standard error of means (SEM), *p* < 0.05 was considered to be statistically significant, 0.05 ≤ *p* < 0.10 was considered to be a tendency.

#### **3. Results**

#### *3.1. Growth Performance*

The results of growth performance were shown in Table 2. During 1–5 weeks, heat stress reduced the BWG and feed intake (*p* < 0.05), whereas it increased the FCR (*p* < 0.05). Dietary betaine supplementation tended to improve the BWG (linear, *p* = 0.078) and feed intake (linear, *p* = 0.075) of broilers under heat stress. During 6–10 weeks, heat stress decreased the BWG and feed intake (*p* < 0.01). Supplementation of graded levels of betaine improved the BWG and feed intake (linear, *p* < 0.05) of broilers under heat stress. During the whole experimental period (1–10 weeks), heat stress reduced the BWG and feed intake (*p* < 0.01) and dietary inclusion of betaine increased BWG and feed intake (linear, *p* < 0.05) of broilers under heat stress.

**Table 2.** Effects of heat stress and dietary betaine on growth performance of yellow-feathered broilers \*.


\* BW, body weight; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio; TN, thermoneutral zone; HS, heat stress; SEM, standard error of means; TN vs. HS, TN group vs. HS control (0 mg/kg betaine) group.

#### *3.2. Nutrient Retention*

Nitrogen retention was significantly reduced by 5 weeks of heat stress (Table 3, *p* < 0.05), and 5 weeks of heat stress tended to decrease the P retention (*p* = 0.065). Supplementation of betaine increased the nitrogen and P retention (linear, *p* < 0.05). After 10 weeks of heat stress, decreased nitrogen retention was observed (*p* < 0.05). Dietary betaine could improve nitrogen retention in heat stressed broilers (linear, *p* < 0.05).


**Table 3.** Effects of heat stress and dietary betaine on nutrient retention of yellow-feathered broilers \*, %.

\* CF, crude fat; TN, thermoneutral zone; HS, heat stress; SEM, standard error of means; TN vs. HS, TN group vs. HS control (0 mg/kg betaine) group.

#### *3.3. Digestive Enzyme Activity*

As presented in Table 4, after 5 weeks of heat stress, the trypsin activity of the jejunum was decreased by heat stress (*p* < 0.05). Detary supplementation of betaine had quadratic effects on trypsin activity of jejunum in heat stressed broilers (*p* < 0.05). Additionally, after 10 weeks of heat stress, dietary betaine supplementation improved the trypsin activity of the duodenum in heat stressed broilers (linear, *p* < 0.05).


**Table 4.** Effects of heat stress and dietary betaine on digestive enzyme activity of yellow-feathered broilers\*, U/mg protein.

\* TN, thermoneutral zone; HS, heat stress; SEM, standard error of means; TN vs. HS, TN group vs. HS control (0 mg/kg betaine) group.

#### *3.4. Intestinal Morphology*

As shown in Table 5 and Figures 1 and 2, after 5 weeks of heat stress, as compared with the TN group, the heat stress control group had lower villus height (*p* < 0.05) and tended to decrease the villus height to crypt depth ratio of duodenum (*p* = 0.057). Supplemental betaine had a tendency to increase the villus height to crypt depth ratio (linear, *p* = 0.057), and had a trend of quadratic effect on villus height (*p* = 0.084) and villus height to crypt depth ratio (*p* = 0.056) of the duodenum in heat-stressed treatments. After 10 weeks of the feeding trial, heat stress induced reduction of villus height of the duodenum and jejunum (*p* < 0.05), and decreased the villus height to crypt depth ratio of the jejunum (*p* < 0.05). Supplementation with betaine improved the villus height and villus height to crypt depth ratio of the jejunum (linear, *p* < 0.05) and had quadratic effects on villus height and villus height to crypt depth ratio of the duodenum (*p* < 0.05) in heat stressed broilers.


**Table 5.** Effects of heat stress and dietary betaine on intestinal morphology of yellow-feathered broilers \*.

\* VH:CD, villus height to crypt depth ratio; TN, thermoneutral zone; HS, heat stress; SEM, standard error of means; TN vs. HS, TN group vs. HS control (0 mg/kg betaine) group.

**Figure 1.** Photomicrographs of the effects of dietary betaine on intestinal morphology of yellowfeathered broilers after 5 weeks of heat stress (Stained with hematoxylin and eosin; TN, thermoneutral zone; HS, heat stress).

**Figure 2.** Photomicrographs of the effects of dietary betaine on intestinal morphology of yellowfeathered broilers after 10 weeks of heat stress (Stained with hematoxylin and eosin; TN, thermoneutral zone; HS, heat stress).

#### *3.5. Carcass Traits*

After 10 weeks of heat stress, broilers in the heat stress control group had lower eviscerated carcass rate and breast muscle yield than those in TN group (Table 6, *p* < 0.05). Additionally, heat stress tended to reduce semi-eviscerated carcass rate (*p* = 0.085). Dietary betaine improved the semi-eviscerated carcass rate, eviscerated carcass rate, and breast muscle yield of heat stressed broilers (linear, *p* < 0.05).


**Table 6.** Effects of heat stress and dietary betaine on carcass traits of yellow-feathered broilers \*, %.

\* TN, thermoneutral zone; HS, heat stress; SEM, standard error of means; TN vs. HS, TN group vs. HS control (0 mg/kg betaine) group.

#### *3.6. Meat Quality*

The results of meat quality were presented in Table 7. Heat stress challenge had no effects on cooking loss, shear force, and pH of breast and leg muscle (*p* > 0.05). In heat stress treatments, dietary betaine supplementation had no significant effects on investigated meat quality parameters (*p* > 0.05).


**Table 7.** Effects of heat stress and dietary betaine on meat quality of yellow-feathered broilers \*.

\* TN, thermoneutral zone; HS, heat stress; SEM, standard error of means; TN vs. HS, TN group vs. HS control (0 mg/kg betaine) group.

#### **4. Discussion**

#### *4.1. Growth Performance*

It has been well documented that heat stress causes a series of drastic changes in broilers' physiological function, including decreasing the feed intake, disturbing the intestinal function and electrolyte balance, and adversely affecting blood metabolites and hormonal secretions, which results in impairment of productive performance [2,4]. In this study, expectedly, heat stress induced a reduction in BWG and feed intake of indigenous yellow-feathered broilers. This was in agreement with the reports by Zhong et al. [21,22], who observed that heat stress suppressed the average daily gain of yellow-feathered broilers under similar experimental conditions. The lower growth rate in heat stressed broilers may be attributed to the decreased feed intake, which is a defense mechanism to reduce the heat increment of bodies [23]. In addition, the heat stressed broilers consume more energy to adapt to high ambient temperature, thereby reducing energy for growth and leading to a lower growth performance [1].

Betaine is a functional active substance from a variety of plants, which can act as methyl group donor and organic osmolyte, and has the ability to improve growth performance in animals [7–9]. Meanwhile, according to the previous studies, betaine could be used as an effective antistress additive in broilers. For instance, He et al. [13] demonstrated that betaine improved the BWG and feed intake of Arbor Acres broilers under 32 ◦C heat stress. Chand et al. [14] found that dietary supplementation of 1.5% and 2% betaine increased the feed intake and BWG and reduced the FCR of fast-growing broilers exposed to heat stress. Similar findings have been reported in the studies of Sakomura et al. [16] and Singh et al. [24], who observed a significant increase in feed intake and BWG of heat stressed Cobb broilers fed with diet contained betaine. However, the studies related to the effects of betaine on indigenous slow-growing broilers are very scarce. Attia et al. [12] reported that 0.5 or 1.0 g/kg betaine supplementation improved the BWG and feed intake, whereas it decreased the FCR of slow-growing white-feathered broilers under heat stress. To the best of our knowledge, no research has been reported to study the effect of dietary betaine on Chinese indigenous yellow-feathered broilers. Our data showed that dietary supplementation of betaine could mitigate the adverse effects of heat stress on feed intake and BWG in indigenous yellow-feathered broilers, indicating that betaine has potential as an anti-heat stress additive for slow-growing yellow-feathered broilers. Regarding the mechanism of action, it was assumed that the beneficial effects on growth performance of heat stressed broilers might be due to the osmoregulatory, methyl group donors, and antioxidative properties of betaine.

#### *4.2. Digestive Function*

During heat stress, the intestinal epithelial cells of broilers are subjected to osmotic stress, as high ambient temperature may lead to water imbalance and cell permeability changes through dehydration [25]. Additionally, the fluid transport in the gastrointestinal tract during heat stress may also cause changes in intestinal structure and digestive function [23]. Indeed, in the current study, heat stress groups had lower nitrogen retention and trypsin activity. This was supported by previous findings of Attia et al. [12] and Chen et al. [26], who demonstrated that heat stress decreased the nitrogen digestibility and digestive enzymes activity of broilers. The present study showed that dietary supplementation of betaine ameliorated the nitrogen retention and trypsin activity of the duodenum and jejunum in heat stressed yellow feather broilers. Similarly, Attia et al. [12] reported that 0.5 or 1.0 g/kg betaine supplementation recovered the crude protein digestibility coefficients from the adverse effects of heat stress on slow-growing chicks. Eklund et al. [27] revealed that supplementation of betaine in broilers' diet could improve the apparent nutrient digestibility, including protein, methionine, and crude fat. However, because the available data regarding the effect of betaine on nutrient digestibility and digestive enzymes activity in heat stressed yellow-feathered broilers is limited, no more comparisons could be made. On the other hand, according to the results obtained by Wang et al. [28], betaine supplementation increased the activities of amylase, lipase, trypsin, and chymotryps of the small intestine in stressed rats. Pollard and Wyn Jones [29] also found that betaine protected against stress inhibition of enzymes. It has been suggested that betaine could promote the activity of key cellular enzymes, and the effects of betaine involved universal water–solute–macromolecule interactions [28]. Betaine possesses an osmotic effect and attaches to the surface of biopolymers and helps proteins fold more compactly [30]. In the same study, they also noted that this protective effect may be limited to certain enzymes. This was in agreement with our results, which only show an increase in the activity of trypsin.

The intestinal villus height and villus height to crypt depth ratio were decreased by heat stress in our experiment, suggesting that heat stress induced deterioration of intestinal morphology. These findings were in accordance with previous reports of Quinteiro-Filho et al. [2] and Burkholder et al. [3]. Animals have mechanisms to regulate body temperature as well as changes in physiological status. When the ambient temperature exceeds the thermoneutral zone, the body temperature raises, and peripheral blood flow increases as a response to heat stress, meaning that the blood flow of turbinate, nasal mucosa, myocardium, and respiratory muscles is higher than that of the intestine [2]. Ischemia and hypoxia of the intestine can cause epithelial shedding, leading to a deeper crypt depth and shorter villus height [3]. This study showed that the intestinal epithelial morphology was revived by the inclusion of betaine. Several possible mechanisms could explain the positive response of heat stressed broilers to dietary betaine: the methyl group donor nature of betaine might promote the proliferation of intestinal epithelial cells; the osmotic effect of betaine could improve the intestinal environment; and the antioxidant activity of betaine could alleviate intestinal oxidative damage induced by heat stress [28]. However, most studies to date have only investigated the effects of betaine on intestinal morphology of fast-growing broilers or rats. For instance, Kettunen et al. [31] discovered that dietary betaine supplementation increased the villus-crypt ratio of intestine in broilers. Eklund et al. [27] reported that betaine could maintain gut villi integrity and consequently promote better nutrient digestibility and absorption in broilers. Wang et al. [28] also demonstrated that betaine supplementation enhanced villus heights and villus height to crypt depth ratio of the duodenum, jejunum, and ileum in stressed rats. One study on the effects of betaine on heat stressed broilers obtained a contrary finding [16], revealing that the morphometrics of the intestinal crypts and villi in heat stressed broilers were not influenced by supplementation of betaine. The extent and duration of heat stress, species of broilers, growth stages, and the type of diet could help to explain these inconsistencies. Overall, betaine favorably affected the intestinal structure and digestive function could account for the boosted growth performance in this study.

#### *4.3. Carcass Traits and Meat Quality*

The present study discovered that the eviscerated carcass rate and breast muscle yield were reduced in response to heat exposure. This was supported by previous findings of Lu et al. [32], who reported that the carcass parameters were negatively affected by chronic heat stress in broilers. However, Sakomura et al. [16] did not find any significant impacts of heat stress on carcass, leg, and breast yield. The possible reasons of these results might be due to the experimental conditions and genetic background of broilers. In that study, thermoneutral zone groups were held at 28 ◦C from day 22 to day 45, and they used fast-growing (Cobb) broilers; these differences could lead to inconsistent findings. Betaine is often considered as a carcass modifier due to methyl group donor property, which causes a higher availability of methionine and cystine for protein deposition, thus contributing to improving the carcass lean percentage [33]. In this study, when supplemented with betaine in heat stressed broiler groups, the carcass traits were subsequently improved. Consistent with our results, Attia et al. [12] observed an improvement in the carcass traits of heat stressed slow-growing chicks by dietary betaine. Nofal et al. [34] found that inclusion of 0.2% betaine increased carcass weight and breast muscle yield in growing broilers under heat stress conditions. Similar results in thermoneutral conditions were obtained by Rao et al. [7] and Zhan et al. [35], who reported that dietary betaine supplementation enhanced the breast muscle yield of male broiler chickens.

Regarding meat quality, even though some studies have suggested that chronic heat stress had adverse effects on the meat quality of broilers, such as drop loss, cooking loss, shear force, pH, and meat color [4–6], our study failed to show any significant impacts of heat stress on cooking loss, shear force, or pH of breast and leg muscle. The different broiler breeds used might explain the difference of these results. Feeding betaine ameliorated heat stress-induced impairment of meat quality according to Attia et al. [12], who suggested that dietary betaine improved dry matter composition and water holding capacity of meat in slow-growing broilers. Also, Alirezaei et al. [11] indicated that betaine could act as an antioxidant agent and improve broilers' meat quality. However, there were no significant differences in meat quality criteria between treatments in this study. This might be because the number of observations was insufficient, or the detected criteria of meat quality were limited. Increasing the sample size of the experiment, and investigating other criteria related to meat quality, such as TBARS, intramuscular fat, lactic acid, etc., are essential in future studies.

#### **5. Conclusions**

To summarize, the current results indicated that long-term heat stress induced inferior growth performance, injured digestive function, and lower carcass yield in indigenous yellow-feathered broilers. Dietary supplementation of betaine was effective in improving growth performance, digestive function, and carcass traits in indigenous yellow-feathered broilers subjected to heat stress.

**Author Contributions:** Conceptualization, W.L. and B.B.; Methodology, W.L. and C.S.; Analysis, C.S. and Y.Y.; Data curation, W.L. and C.S.; Writing—original draft preparation, W.L.; Writing—review and editing, W.L., Z.Z. and B.B.; Supervision, Z.Z. and L.A.; Project administration, W.L.; Funding acquisition, W.L. and L.A.

**Funding:** This research was funded by Talent Research Start-up Project of Guangdong Ocean University (R18007); Innovative Strong School Engineering Youth Talent Project (2017KQNCX090) and Key Platform Projects (2018302) by Department of Education in Guangdong Province; Natural Science Foundation of Guangdong Province (2018A030307023); Pandeng Program of Guangdong Province (pdjh2019b0234); and National Research Foundation grant (2018R1C1B5086232) funded by Korean Government (MEST).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Responsiveness Expressions of Bitter Taste Receptors Against Denatonium Benzoate and Genistein in the Heart, Spleen, Lung, Kidney, and Bursa Fabricius of Chinese Fast Yellow Chicken**

#### **Enayatullah Hamdard, Zengpeng Lv, Jingle Jiang, Quanwei Wei, Zhicheng Shi, Rahmani Mohammad Malyar, Debing Yu and Fangxiong Shi \***

College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China **\*** Correspondence: fxshi@njau.edu.cn; Tel./Fax: +86-25-84399112

Received: 2 July 2019; Accepted: 29 July 2019; Published: 6 August 2019

**Simple Summary:** In chickens, bitter taste is the most significant biological taste disrupter; it is believed to protect chickens against consuming poisonous/toxic materials and considered a warning signal prior to ingestion. The bitter taste receptors' extraoral expression information is deficient in chicken, and denatonium benzoate is extensively used as a bitter taste receptor agonist in different cells. Our results found that qRT-PCR showed a high level of dose-dependent expressions of ggTas2Rs in the starter and grower stages in the heart, spleen, lungs, and kidneys, while the dose-dependent expressions were lower in the bursa Fabricius. The growth performance of the selected organs significantly (and unexpectedly) improved upon the administration of denatonium benzoate 5 mg/kg and genistein 25 mg/kg treatments, while the gains in organ weights were impaired in the groups given denatonium benzoate 20 mg/kg and 100 mg/kg, respectively.

**Abstract:** The present study was conducted to investigate the responsiveness expressions of ggTas2Rs against denatonium benzoate (DB) and genistein (GEN) in several organs of the Chinese Fast Yellow Chicken. A total of 300 one-day-old chicks that weighed an average of 32 g were randomly allocated into five groups with five replicates for 56 consecutive days. The dietary treatments consisted of basal diet, denatonium benzoate (5 mg/kg, 20 mg/kg, and 100 mg/kg), and genistein 25 mg/kg. The results of qRT-PCR indicated significantly (*p* < 0.05) high-level expressions in the heart, spleen, and lungs in the starter and grower stages except for in bursa Fabricius. The responsiveness expressions of ggTas2Rs against DB 100 mg/kg and GEN 25 mg/kg were highly dose-dependent in the heart, spleen, lungs, and kidneys in the starter and grower stages, but dose-independent in the bursa Fabricius in the finisher stage. The ggTas2Rs were highly expressed in lungs and the spleen, but lower in the bursa Fabricius among the organs. However, the organ growth performance significantly (*p* < 0.05) increased in the groups administered DB 5 mg/kg and GEN 25 mg/kg; meanwhile, the DB 20 mg/kg and DB 100 mg/kg treatments significantly reduced the growth of all the organs, respectively. These findings indicate that responsiveness expressions are dose-dependent, and bitterness sensitivity consequently decreases in aged chickens. Therefore, these findings may improve the production of new feedstuffs for chickens according to their growing stages.

**Keywords:** denatonium benzoate; genistein; chicken; ggTas2Rs; bitter taste receptors

#### **1. Introduction**

In chickens, bitter taste is one of the most significant senses for choosing and consuming feeds, alongside their olfactory and visual senses [1–3]. Taste signals have been associated to food recognition

and avoidance, as well as feed or liquid intake in different species of animals [4–7]. Bitter taste provokes an aversive reaction and is assumed to protect chickens against consuming poisons and harmful toxic substances. The age effects in humans were found to be almost exclusively generic and taste sensitivity was found to decline with age, although the level of bitterness differs depending on taste quality and is never compound-specific within a taste [8]. Chickens demonstrate bitter taste sensitivity despite having only three bitter taste receptors: ggTas2R1, ggTas2R2, and ggTas2R7. It has been shown that chickens have a well-developed sense of taste and only three of the aforementioned bitter receptors have been investigated [9]. In chickens, an insufficient number of studies have been performed to investigate growth-related taste loss and its subsequent effect on the animal's production. Additionally, behavioral experiments were conducted and found that day-old and immature chicks were more susceptible against salt and sour taste qualities than the adults [10]. It is meaningful to elucidate the bitter taste sensitivity of chicken because of the different nutrient requirements and prerequisites during their growing stages. Bitter molecules detected by the ggTas2R family of G-protein-coupled receptors (GPCRs) were involved in perceiving potentially toxic compounds [11–13]. The commercial feed factories produce several categories of chicken feed according to their growth stages such as starter, grower, and finisher. When the taste buds are counted, birds have few compared to humans and other mammals. For example, humans have approximately 9000 taste buds, chickens have between 250–350 taste buds, and pigeons have only 37–75 [14–16]. Furthermore, in chickens, the gustatory and extra-gustatory mechanisms of involving taste signaling have been shown recently [1,17–19].

Denatonium benzoate (DB) is intensely bitter and non-toxic, which can be detected by human taste receptors [20]. Denatonium benzoate has been demonstrated widely as a bitter taste agonist and used to activate bitter taste receptors on many cell types, including tastes cells [21]. A previous study indicated that denatonium increased cholecystokinin release through Ca2<sup>+</sup> influx in enteroendocrine STC-1 cells [22]. After oral glucose administration in diabetic mice, a prior gavage of denatonium attenuated blood glucose levels through the increased secretion of glucagon-like peptide-1 [23]. In addition, exposure to denatonium quickly suppressed the ongoing intake and delayed gastric emptying in rodents [24,25]. Apparently, genistein is mainly derived from soy products, which contain a phytochemical with isoflavone structure that is found in an extensive variety of foods, legumes, animal forages, and particularly in soybeans. Genistein (GEN) has protective effects against atherosclerosis, cardiovascular risk, and type 2 diabetes, which are attributed to its antioxidant activity; furthermore, dietary GEN can enhance the growth performance of livestock [26–29].

In recent years, the majority of reports on the expression of extra-gustatory taste receptors obviously suggested that their role is not restricted to taste perception in the mouth and gastrointestinal tract. Taste receptors have additionally been recognized in the respiratory system [30,31] and gastrointestinal tracts of mammals, in the male reproductive system, and in the brain, as well as in the heart [32,33]. Currently, the direct involvement of the human bitter taste receptor TAS2R38 in the detection of minimum bacterial sensing molecules was suggested [34]. One of the most recent additions of scientific investigations is the expression of bitter taste receptors in human and animal hearts [32,35]. Remarkably, the whole heart cDNA of neonatal rats analyzed by qRT-PCR, the seven bitter taste receptors genes, and two genes encoding the umami receptor subunits, Tas1R1 and Tas1R3, were identified as expressed in hearts. However, many research studies on the influence of bitter taste receptors materials on cardiac tissue observe need to be warranted [36].

Taste sensitivity in chickens is lower compared to that of mammals. It has been reported that chickens respond to several tastants, and these responses are conserved from post-hatching until adulthood [10,37–41]. Nevertheless, the relationship between taste sensitivity and number of taste buds in various chicken breeds remain unclear. It has been clarified recently that Tas2Rs receptors are also expressed in the extraoral tissues of the chicken [35,42]. The molecular mechanism of bitter molecules by their receptors is slightly complicated and less studied. However, bitter taste receptors (Tas2Rs or T2Rs) are developing as novel regulators of native immunity in the respiratory tract [43].

Recent studies findings indicate that T2Rs are extensively expressed in several parts of the human body, and have been identified to be involved in respiratory system physiology, the gastrointestinal tract, and the endocrine system, and T2Rs may play regulatory roles in the mentioned areas of the body [35,41,44,45]. In contrast, GPCRs facilitate the sensations of bitter, sweet, and umami tastes in mammals and chickens [46]. Moreover, the gene expression compilation collection (http: //www.ncbi.nlm.nih.gov/geo) also confirmed that T2Rs are widely expressed in other human tissues such as the heart, brain, skeletal muscle, endometrium, liver, omental adipose tissue, nasal cavity, lung, and different cell types (chemosensory cells, smooth muscle cells, endothelial cells, epithelial cells, and inflammatory cells) [47].

The detection of taste thresholds and their identification is crucial for studying the potential effects of Tas2Rs on chicken feeding behavior. Interestingly, the chicken's genome contains only three bitter taste receptors, which are responsible for bitterness identification, as described previously [48]. The presence of a minimum lower number of bitter taste receptors makes the chicken a significant minimalistic model for an understanding of vertebrate taste necessities [49]. However, there is limited knowledge about the expressions of bitter taste receptors, and no study has yet investigated the bitter taste responsiveness expressions in the extra-gustatory organs of chickens. Therefore, the objectives of this research were to investigate the bitter taste receptors' (ggTas2Rs) responsiveness expressions against different doses of denatonium benzoate, genistein and compare the ggTas2Rs mRNA expressions levels among different organs in the starter, grower, and finisher stages of Chinese Fast Yellow Chicken.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Denatonium benzoate 98% was purchased from Adamas Reagent Co Ltd. (Nanjing, China), Genistein 98% was purchased from Kai Meng. Co Ltd. (Xi'an, China) and stored at room temperature. RNAose (phenol 38%), trichloroethane, isopropyl alcohol, alcohol absolute, and DNA/RNA-ase free water were purchased collectively for RNA extraction from TAKARA BIO INC (Nojihigashi 7-4-38, Kusatsu, Shiga Japan), while PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time, Cat. # RR047A v201710Da) and TB Green™ Premix Ex Taq™ (Tli RNase H Plus, Cat. # RR420A v201710Da) were both purchased from TaKaRa (Dalian, China).

#### *2.2. Birds and Procedures*

The experimental protocol and procedures were designed and approved in accordance with the Guidelines for the Care and Use of Animals prepared by the Institutional Animal Care and Ethical Committee for Nanjing Agricultural University, Nanjing, China (Permit Number: SYXK (Su) 2019–0036). A total of three hundred (300), 1-day-old Chinese Fast Yellow chicks at the average weight of 32 ± 5 g were randomly allocated into five (5) groups with five replicates of 12 chicks in each. In order to find the dose-dependent comparison expressions of bitter taste receptor genes against DB, we designed the experiment with different doses of denatonium benzoate from low levels to high levels; the experiment groups were as follows: the control, denatonium benzoate 5 mg/kg (Low Dose), denatonium benzoate 20 mg/kg (Medium Dose), denatonium benzoate 100 mg/kg (High Dose), and genistein 25 mg/kg (GEN 25 mg/kg). All were reared under the ventilated chicken house in which the light remained 16-h light: 8-h dark, humidity was approximately 40–45% and formulated feed (Table 1) was offered ad libitum with freely available tap water for 56 consecutive days.


**Table 1.** Feed formulation for the entire period of the experiment (d 1–56).

Provided the following % per kilogram in completed diet: vitamin A, 12,500 IU; vitamin D3, 2500 IU; vitamin E, 30 IU; vitamin K3, 2.65 mg; vitamin B1, 2 mg; vitamin B2, 6 mg; nicotinic acid, 50 mg; pantothenic acid, 12 mg; vitamin B6, 4 mg; folic acid, 1.25 mg; vitamin B12, 0.025 mg; biotin, 0.25 mg; Fe, 50 mg; Zn, 75 mg; Mn, 100 mg; Cu, 8 mg; I, 0.35 mg; Co, 0.2 mg; and Se, 0.15 mg.

#### *2.3. Feed Formulation and Mixing Procedure*

We purchased the basal diet from ADM factory (Nanjing, China) with respective ingredients (Table 1); then, we mixed the basal diet feed with the different treatments through an electric feed mixing machine available in the Nanjing Agricultural University animal house. According to the experimental design, five (5) types of treatments were prepared: the basal diet (Control), denatonium benzoate 5 mg/kg (Low Dose), denatonium benzoate 20 mg/kg (Medium Dose), denatonium benzoate 100 mg/kg (High Dose), and genistein 25 mg/kg. The chemicals and organic materials were mixed appropriately with the help of an electric mixer and provided ad libitum for the feeding of chicken until the end of the experiment, respectively.

#### *2.4. Organs Weight Measurements*

A total of 10 chickens in each group (two chickens/replicate) were used in each stage of killing to collect and measure the heart, spleen, lung, kidney, and bursa Fabricius weights at Day 7 (starter stage), Day 28 (grower stage), and Day 56 (finisher stage), respectively (Table 3).

#### *2.5. Sample Collection and RNA Extraction*

On days 7, 28, and 56 (the starter, grower, and finisher stages) tissues from the heart, spleen, lung, kidney, and bursa Fabricius were collected and kept in an −80 ◦C freezer until RNA extraction. Afterward, the total RNA for RT-PCR and real-time PCR was extracted and purified from frozen collected tissues using RNAose (TAKARA BIO INC, Nojihigashi 7-4-38, Kusatsu, Shiga Japan), which includes gDNA Eraser (Perfect Real Time) for the elimination of genomic (g) DNA according to the manufacturer's protocols.

#### *2.6. Primer Design and RT-PCR*

Initially, first-strand cDNA was synthesized by reverse transcription (RT) with the application of 2.0 ug of total RNA with or without reverse transcriptase using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time, Cat. # RR047A v201710Da) in accordance with the manufacturer's instructions. Gene-specific primers for ggTas2R1 (Accession no. AB249766.1), ggTas2R2 (Accession no. AB249767.1), ggTas2R7 (Accession no. NM\_001080719.1) and the housekeeping gene (β-actin) (Accession no. NM\_205518.1) were generated with the aid of the nucleotide database of The National Center for Biotechnology Information (NCBI) [50], according to their published cDNA sequences (Table 2). The target genes and the housekeeping gene were synthesized by the Sangon company and applied for real-time PCR (Table 2). Amplicon lengths for real-time PCR were between 102–162 bp. The PCR mixture consisted of 2 μL of a cDNA template diluted in a ratio of 1:3, 10 μL of TB Green premix Ex Taq (Tli RNase H Plus) (2×), 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 0.4 μL of ROX Reference Dye 1 or Dye 2 (50×) and 6.8 μL of DNA/RNA enzyme-free water in a final volume of 20 μL (TaKaRa, Dalian, China). Entire PCR reactions were performed in 96-well reaction plates on a 7500 Real-time PCR instrument (Applied Biosystems, ABI, USA), and all the genes were repeated six times under the following conditions: ha old stage (95 ◦C for 30 s), a PCR stage (40 cycles of 95 ◦C/2 min, 60 ◦C/34 s) apparently to verify the amplification of a single product, while a stage with a temperature increment (melt curve stage) was conducted to generate a melting curve under the following conditions: (95 ◦C/15 s, 60 ◦C/1 min), followed by a temperature increment of 95 ◦C/15 s.



gg–Gallus Gallus; TasR-Taste Receptor, β-actin-Housekeeping gene.

#### *2.7. Housekeeping Gene for Internal Control (*β*-actin)*

We investigated the Ref-Finder online database (http://www.leonxie.com/rferencegene.php) to choose the most constant housekeeping gene as an internal control for the real-time PCR analysis. The mentioned database consists of various computational programs (geNorm, Norm-finder, Best-Keeper, and the comparative ΔΔCt method). We calculated the relative gene expression (arbitrary units) utilizing the 2-ΔΔCt method and normalized the relative abundance to tested candidate reference genes. Promising housekeeping genes were statistically tested for significant differences among various tested tissues, developmental time points, and their interaction using JMP Pro 10 software (SAS Institute, 2006, Cary, NC) [49,51]. The geometric average of β-actin was found to be the most stable and significant reference gene with no significant differences (*p* > 0.05) among the target organs (heart, spleen, lung, kidney, and bursa Fabricius) on days 7, 28, and 56 using one-way ANOVA for analysis.

#### *2.8. Determination of mRNA Expression by Real-Time PCR Using the Comparative* ΔΔ*Ct Method*

To confirm and validate the target gene expressions for the first time, the data were subjected to new ΔΔCt fold-change calculations [52], and statistical analysis were carried out to compare the expression of the ggTas2Rs target genes in heart, spleen, lung, kidney, and bursa Fabricius in the starter, grower, and finisher stages of the chicken. Finally, the efficiencies of all the tested genes and the reference gene were calculated. Cycle threshold (Ct) values for every sample were calculated using the ΔCt (Δ cycle threshold) procedure [53]. Gene expression was normalized against the geometric average of β-actin. Changes in mRNA determination were analyzed by comparing the relative expression among the genes in the heart, spleen, lung, kidney, and bursa Fabricius in different denatonium benzoate treatments, GEN, and a control group. Each stage's relative expression data were analyzed separately, and consequently, each target gene in a single selected organ was compared across all of the growing stages, respectively. Primers and gene accession numbers are described in Table 3, as described in detail in previous publications [49,54].


**Table 3.** Live body weight and organ weights in the starter, grower, and finisher stages.

Heart, spleen, lung, kidney, and bursa Fabricius weight unit (g, n = 10); the table shows different treatments, DB 5 mg/kg (Low Dose), DB 20 mg/kg (Medium Dose), DB 100 mg/kg (High Dose), and GEN 25 mg/kg, values shown are mean ± SE. (Standard Errors), a–e means in a row with different superscript differ significantly (*p* < 0.05). The table also shows the interaction between stages and feed. DB: denatonium benzoate; GEN: genistein.

#### *2.9. Statistical Analysis*

#### 2.9.1. Organ Weight Measurements

Weight measurements for five (5) selected organs were described previously [51]. Significant differences between treatment groups and the control group were analyzed by one-way analysis of variance (ANOVA) followed by Duncan's post hoc test. A *p* < 0.05 was considered as statistically significant, and subsequently marked with (a, b, c, d). Value = MEAN ± SEM, weight unit (g).

#### 2.9.2. Gene Expression

For relative gene expression analysis of the genes (ggTas2R1, ggTas2R2, and ggTas2R7), for each organ compared the chosen control gene (β-actin) in all the tissues (heart, spleen, lung, kidney, and bursa Fabricius) in different growing stages using one-way ANOVA. In addition, multiple comparison among means of ggTas2R1, ggTas2R2, ggTas2R7, and the β-actin gene in each group were calculated using Dunnett's test (marked with a, b, c, d, and e), and *p* < 0.05 was considered significant, as shown in the figures, respectively. An alpha level of 0.05 was set for all the tests. These statistical analyses were conducted with GraphPad Prism 6 and IBM SPSS Statistics, version 20 software (SPSS Inc., Chicago, IL, USA).

#### **3. Results**

#### *3.1. Organ Weight Measurements*

The results on the heart weight gained showed that the GEN and DB 5 mg/kg (Low Dose) groups significantly (*p* < 0.05) gained more weight compared to other DB and control groups (Table 3). Conversely, the live body weight and organ weights gained for the heart in the denatonium benzoate 100 mg/kg (High Dose) significantly (*p* < 0.05) decreased in the starter and grower stages, but not in the finisher stage (Table 3). However, no significant differences were observed among other treatments (Table 3). Meanwhile, the live body weight and spleen weight gained in the GEN group in all the growth stages significantly (*p* < 0.05) increased compared to the medium dose of DB 20 mg/kg. However, the

spleen weight in the denatonium benzoate 20 mg/kg (Medium Dose) group increased in the grower and finisher stages compared to the DB High-Dose group, respectively (Table 3). However, the spleen weight of the control group increased in the grower stage among all the groups (Table 3). Lung weight gaining also significantly (*p* < 0.05) increased in the GEN and DB Medium-Dose groups in the starter, grower, and finisher stages, while it significantly decreased in the DB High-Dose group in the starter stage and surprisingly increased in the DB Low-Dose group compared to the DB High-Dose group at the grower and finisher stages (Table 3). Furthermore, the kidney weight in the GEN group in all the growing stages increased significantly (*p* < 0.05) and decreased in the DB 100 mg/kg (High-Dose) group in the grower and finisher stages among the groups, while it increased in the DB 5 mg/kg (Low-Dose group) in the starter and grower stages compared to the other DB doses, respectively (Table 3). Finally, the weight gained for bursa Fabricius remarkably increased in the DB 5 mg/kg (Low-Dose group) in the grower and finisher stages, while it decreased in the GEN and DB 20 mg/kg (Medium-Dose) groups in all three stages of growing, respectively (Table 3). The interaction between feed and stages was declared in data analysis; there was lower interaction in the starter and grower stages, while higher interaction was observed in the finisher stage. Meanwhile, the live body weight and organ weights subsequently increased, while taste sensitivity decreased, respectively (Table 3).

#### *3.2. Detection of ggTas2Rs Responsiveness Expressions Against Denatonium Benzoate and Genistein*

#### 3.2.1. mRNA Responsiveness Expressions of ggTas2Rs in Chicken Heart

Real-time PCR analysis showed that the expressions of ggTas2R1, ggTas2R2, and ggTas2R7 in chicken hearts was significantly (*p* < 0.05) higher in the starter and grower stages in all the treated doses of denatonium benzoate and genistein compared to the finisher stage (Figure 1A–C). While comparing different growing stages, the ggTas2R1, ggTas2R2, and ggTas2R7 genes were dose-dependent and highly expressed in GEN and DB 100 mg/kg (High Dose) in the starter and grower stages among other treatments and consequently similarly lower expressed in the finisher stage (Figure 1A–C). However, the expressions of ggTas2R1, ggTas2R2, and ggTas2R7 in the DB 5 mg/kg (Low Dose), DB 20 mg/kg (Medium Dose), and GEN groups were almost similar in the starter and grower stages, except for ggTas2R2 in the GEN group in the grower stage (Figure 1B and Figure 1A–C). The expressions of ggTas2Rs in all the groups were lower in the finisher stage, and no significant differences were observed among them.

#### 3.2.2. mRNA Responsiveness Expression of ggTas2Rs in the Spleen of Chicken

The ggTas2Rs expressions in the spleens of chickens were quite highly and significantly (*p* < 0.05) expressed, and the responsiveness expressions were dose-dependent in the starter stage among all the growth stages, respectively (Figure 2A–C). For the comparison of the individual treatments, the ggTas2R1 and ggTas2R2 in DB 100 mg/kg (High Dose) in the starter stage was highly expressed among the other groups and growing stages (Figure 2A,B), whereas ggTas2R7 was highly expressed in the DB Medium-Dose group in the starter stage among all the other treatments in all the stages of growing, respectively (Figure 2C). Expressions of ggTas2Rs were dose-dependent to different DB doses and GEN in the starter stage (Figure 2A–C). However, it was also observed that the ggTas2R7 receptor was highly dose-dependent to the medium dose of DB in the grower stage, and the expressions for all the treatments subsequently decreased in the grower and finisher stages, respectively (Figure 2A–C). Furthermore, ggTas2Rs were gradually less expressed in the DB High-Dose and GEN groups in the grower stage and surprisingly increased in the finisher stage compared to other treatments within the stage. Finally, all the responsiveness expressions were absolutely descendant in the finisher stage, respectively (Figure 2A–C).

**Figure 1.** Real-time PCR analysis of (**A**) ggTas2R1, (**B**) ggTas2R2, and (**C**) ggTas2R7 of bitter taste receptors showing their relative mRNA expressions against different doses of DB and GEN in the hearts of chickens in different stages of growth (Day 7, Day 28, and Day 56). The relative mRNA abundance of ggTas2Rs in different growth stages with the heart serving as the control (relative expression set to 1; n = 6). Values are presented as the mean of relative expressions ± SEM. Differences between groups within a gene means those without a common letter differ significantly (*p* < 0.05); differences between the tested tissue (heart of chicken) and the control tissue (heart) within a gene means that those with marks (a, b, c) differ significantly (*p* < 0.05) from the control group by ANOVA.

**Figure 2.** Comparing expressions of bitter taste receptors ((**A**) ggTas2R1, (**B**) ggTas2R2, and (**C**) ggTas2R7), showing their relative mRNA expressions against different doses of DB and GEN in chicken spleens in different growth stages (Day 7, Day 28, and Day 56). The relative mRNA abundance of ggTas2Rs in different growth stages with the spleen serving as the control (relative expression set to 1; n = 6). Values are presented as the mean of relative expressions ± SEM. Differences between groups within a gene mean that those without a common letter differ significantly (*p* < 0.05); differences between the tested tissue (spleen) and the control tissue (spleen) within a gene means that those with marks (a, b, c) differ significantly (*p* < 0.05) from the control group by ANOVA.

#### 3.2.3. mRNA Responsiveness Expressions of ggTas2Rs in Chicken Lungs

The expressions of ggTas2Rs receptors in chicken lungs were significantly (*p* < 0.05) higher in the starter stage than in the grower and finisher stages, respectively (Figure 3A–C). The ggTas2R1 receptor in the DB 100 mg/kg group in the starter stage had potentially higher expression among the groups (Figure 3A). However, ggTas2R2 and ggTas2R7 were significantly (*p* < 0.05) comparably expressed in the grower and finisher stages within the treatments (Figure 3B,C). The responsiveness expressions of ggTas2Rs against DB different doses and GEN in three growing stages of Fast Yellow Chicken was almost equal and dose-dependent with negligible high expressions and variations in the group given DB 100 mg/kg (High-Dose). Therefore, the expressions subsequently decreased in the finisher stage for all the groups, respectively (Figure 3A–C).

**Figure 3.** Comparing expressions of bitter taste receptors ((**A**) ggTas2R1, (**B**) ggTas2R2, and (**C**) ggTas2R7), which are showing their relative mRNA expressions against different doses of DB and GEN in chicken lungs in different growth stages (Day 7, Day 28, and Day 56). The relative mRNA abundance of ggTas2Rs in different growth stages with the lung serving as the control (relative expression set to 1; n = 6). Values are presented as mean of relative expressions ± SEM. Differences between groups within a gene mean that those without a common letter differ significantly (*p* < 0.05); differences between the tested tissue (lung of chicken) and the control tissue (lung) within a gene mean that those with marks (a, b, c) differ significantly (*p* < 0.05) from the control group by ANOVA.

#### 3.2.4. mRNA Responsiveness Expressions of ggTas2Rs in Chicken Kidneys

The expressions of ggTas2Rs in chicken kidneys showed significantly (*p* < 0.05) high expressions against the GEN and DB Medium-Dose group with the exception of ggTas2R7, which was higher expressed in the DB High-Dose group in the starter stage, and then gradually less expressed than the other ggTas2Rs in the grower and finisher stages, respectively (Figure 4A–C). These results showed that ggTas2R1, ggTas2R2, and ggTas2R7 have contrary expressions levels in kidneys at different chicken growth stages (Figure 4A–C). For instance, it was found that the responsiveness expressions of ggTas2Rs were dose-dependent against DB different doses and GEN in the starter stage, while ggTas2R1 expressions were highly dose-dependent to DB 20 mg/kg in the starter stage and dose-independent in all the groups in the finisher stage (Figure 4A). However, ggTas2R2 response was highly dose-dependent to GEN in the starter stage and dose-independent in the grower and finisher stages for the mentioned gene (Figure 2B). The ggTas2R7 expression was approximately dose-dependent to all the treatments in the starter stage, but only to GEN in the grower stage (Figure 4C). In the finisher stage, neither of the expressions were dose-dependent and showed dose-independent responsiveness against all the treatments, respectively (Figure 4A–C).

**Figure 4.** Comparing expressions of bitter taste receptors ((**A**) ggTas2R1, (**B**) ggTas2R2, and (**C**) ggTas2R7) showing their relative mRNA expressions against different doses of DB and GEN in chicken kidneys in different growth stages (Day 7, Day 28, and Day 56). Relative mRNA abundance of ggTas2Rs in different growth stages with the kidney serving as the control (relative expression set to 1; n = 6). Values are presented as the mean of relative expressions ± SEM. The differences between groups within a gene mean that without a common letter differ significantly (*p* < 0.05); differences between the tested tissue (chicken kidney) and the control tissue (kidney) within a gene means that those with marks (a, b, c) differ significantly (*p* < 0.05) from the control group by ANOVA.

3.2.5. mRNA Responsiveness Expressions of ggTas2Rs in Chicken Bursa Fabricius

The expressions of ggTas2Rs in the bursa Fabricius of Fast Yellow Chicken were significantly (*p* < 0.05) lower expressed than the control group in different growth stages (Figure 5A–C). Regarding each gene's individual expressions level, the mRNA expressions in the GEN group were significantly (*p* < 0.05) highly expressed in all the growing stages except for ggTas2R1, which was highly expressed in DB 20 mg/kg (the Medium-Dose group) in the starter stage. The responsiveness expression against dose was independent in bursa Fabricius and had a relatively low response in the starter and grower stages, but all of the expressions subsequently decreased in the finisher stage (Figure 5A–C). The dose-dependent responsiveness in bursa Fabricius at the starter/grower stages was a result of the subjection to GEN (Figure 5A–C).

**Figure 5.** Comparing expressions of bitter taste receptors ((**A**) ggTas2R1, (**B**) ggTas2R2, and (**C**) ggTas2R7), and showing their relative mRNA expressions against different doses of DB and GEN in the bursa Fabricius of chicken at different growth stages (Day 7, Day 28, and Day 56). Relative mRNA abundance of ggTas2Rs in different growth stages with the bursa Fabricius serving as the control (relative expression set to 1; n = 6). Values are presented as mean of relative expressions ± SEM. Differences between groups within a gene mean that those without a common letter differ significantly (*p* < 0.05); differences between the tested tissue (chicken bursa Fabricius) and the control tissue (bursa Fabricius) within a gene mean that those with marks (a, b, c) differ significantly (*p* < 0.05) from the control group by ANOVA.

#### **4. Discussion**

Over the past years, tremendous progress has been made investigating the wide-ranging expression of bitter taste receptors (Tas2Rs) inside the vertebrate's various tissues and their bitter taste perception. Interestingly, numerous tissues in addition to gustatory and non-gustatory tissues have been identified to express taste receptor molecules. These findings bear imperative implications for the roles that taste receptors fulfill in vertebrates, which are currently envisioned much broader than previously thought [44]. The sense of taste facilitates the recognition of beneficial or potentially poisonous and harmful food ingredients prior to ingestion [55]. Bitter taste perception in vertebrates relies on the Tas2R genes, ranging from only three in chicken to over 50 in frogs. Possessing a low repertoire of Tas2Rs makes the chicken an appropriate candidate for a model animal in the study of different aspects regarding bitter taste. Furthermore, the agricultural reputation for finding bitter tastants in chicken feedstuff is countless, since their nutrition may be enhanced due to lack of aversiveness [17,43].

To our knowledge, this is the first research to evaluate the ggTas2Rs expression responses against different doses of denatonium benzoate and genistein in the heart, spleen, lung, kidney, and bursa Fabricius of chickens. Several studies reported that in vertebrates, the sensors for bitter compounds are taste receptors (T2Rs or Tas2Rs); basically, they are distributed in the taste receptor cells of taste buds of an oral cavity belonging to the G-protein-coupled receptors super family (GPCRs). In chickens, taste sensing research has mostly focused on taste bud morphological distribution, development, and various tastants' thresholds [17,35,43,45,46]. However, in mammals, it is well established that the expressions of Tas2Rs and their downstream signaling molecules and taste-related genes have been found in various extraoral systems such as the respiratory, digestive, and genitourinary systems, as well in brain and immune cells. These receptors are functional in different body locations with varied biological regulatory mechanisms [36]. The extra-gustatory Tas2Rs receptors have been concerned in diverse functions, representing cellular responses to poisons and toxins [56]. This suggests that bitter composition sensing has a physiological role beyond food evaluation and consumption. Furthermore, in chickens, the gustatory and extra-gustatory mechanisms of involving taste signaling have been recently shown [57]. In this study, the expression levels of different DB doses with GEN were varied, and a high dose of DB was comparatively higher expressed among different doses. Therefore, the study demonstrated the responsiveness expressions of ggTas2Rs (ggTas2R1, ggTas2R2, and ggTas2R7) against DB and GEN in five (5) essential organs of the local Chinese Fast Yellow Chicken.

Several studies reported that the expressions of taste-related genes demonstrated their involvement in gustatory and extra-gustatory tissues; furthermore, the expression was also evaluated as a taste transduction gene in chickens [58–60]. In the current experiment, these bitter taste-related genes, which were determined by qRT-PCR, were found at different expression levels in all the organs in the starter, grower, and finisher stages with varied responsiveness expressions against different doses of DB and GEN over 56 consecutive days of growing. Furthermore, the expressions were sufficiently higher at the starter and grower stages of growing, and then consequently decreased in the finisher stage. However, the organ weight gained adequately improved for all the treatments as predicted; beyond seven days, the chicken organ weights increased collaboratively with feed consumption, and bitterness sensitivity subsequently decreased. Therefore, the bitterness sensitivity in chickens is dependent on age. It was reported earlier that in chickens, bitterness susceptibility is dependent on the age of the chicken, as bitter taste receptors were highly expressed in zero to one-week-old chicks and dependently decreased in aged chickens, and these behavioral responses were conserved since hatching to the maturing period [19,61]. However, insufficient research has been done to investigate growth-related taste perceptions and their subsequent effect on the animal's growth [61,62]. Bitter molecules detected by the ggTas2R family of G-protein-coupled receptors (GPCRs) were involved in chickens perceiving potentially toxic compounds [63]. As described earlier, chickens in the initial period of growing are more susceptible against salt, sour, and bitterness than those entering the maturing stage [64,65]. Therefore, we also found that chicks in the starter/grower stages were more sensitive than those in the finisher stage, and the responsiveness expressions of bitter receptors were correlatively high in the

starter/grower stages than in the finisher stage of growing. In addition to this, it has been reported that the human bitter taste receptor hTAS2R39 seems to be a bitter receptor agonist for many dietary compounds, such as isoflavones from soy bean [66] and many other flavonoids from several plant sources and synthetic denatonium benzoate [67,68]. The birds fed with 40 to 80 mg/kg of genistein revealed the greater relative weight gains of thymus and bursa Fabricius; however, the spleen weight was not affected. Genistein supplementation not only improved growth performance, it also could beneficially affect immunological responses in broiler chicks [69]. Additionally, studies have shown that genistein improves kidney function and weight gain [70]. Therefore, our study also indicated that genistein improved the organ growth performance and may have a potential influence on the regulation of bursal immunity and kidney function.

In recent years, several reports on the extra-gustatory expression of taste receptors obviously suggested that their role is not only limited to taste perception [17,36,45,71]. Meanwhile, the expression of taste receptor genes and functions have been identified in the gastrointestinal and respiratory tracts of mammals, in the male reproductive system, as well as in the brain and heart [19,72–74]. Seemingly, the responsiveness expressions of the ggTas2Rs bitter receptors in the chicken organs were also found with distinguished expression levels in different stages of growth; these contributions have received the most attention from researchers in recent years. Bitter taste receptor expression is not restricted to the upper respiratory tract; it extends into the lower respiratory tract [75]. It should be acknowledged that Tas1r gene expression has been detected in the respiratory system of rodents [76]. qRT-PCR analyses showed that rat neonatal whole heart cDNA, the seven bitter taste receptor genes, as well as two genes encoding the umami receptor subunits, Tas1r1 and Tas1r3, were found to be expressed. Moreover, samples of ventricular tissue of failing hearts were tested and revealed the expression of more than half of all human Tas2Rs genes [13,32]. Remarkably, the mRNA responsiveness expressions of the chicken bitter taste receptor Tas2Rs against DB different doses and GEN, which are known to be extensively represented in the heart, spleen, and lungs of chickens. In comparison, the different DB doses with GEN, the responsiveness expressions against DB 100 mg/kg (High Dose) and GEN were highly dose-dependent in the heart, spleen, lung, kidney, and bursa Fabricius in the starter and grower stages. However, dose-independent lower responsiveness expressions were found in the finisher stage with some exceptions in bursa Fabricius. In summary, the results of the present study indicate the significantly higher expression of bitter taste responsive genes in the starter and grower stages among all the organs except for bursa Fabricius. The Tas2Rs receptors were highly expressed in the heart, spleen, and lung, but lower in the bursa Fabricius in the experimental period among the organs. These findings prove and suggest that bitterness sensitivity decreases as chickens age. However, baby chicks were found to be more sensitive to bitterness than adults. The mentioned findings may be useful in the production of new feedstuffs for chicken depending on their growth stages. Hereafter, further research studies are required to investigate bitter receptors' varied expressions in body organs, the physiological and functional effects of bitter taste receptors in the non-gustatory organs of the chicken, and the molecular mechanism pathways involved in bitter responsiveness and their role involved in the regulation of bronchodilation, heart functions, kidney functions, and the immunity of bursa Fabricius in chickens.

#### **5. Conclusions**

Our study demonstrated that the responsiveness expressions of ggTas2Rs (ggTas2R1, ggTas2R2, and ggTas2R7) against denatonium benzoate at different doses were higher in the lungs, spleen, and heart, but lower in bursa Fabricius among the organs. The responsiveness expressions were highly dose-dependent in the starter and grower stages of the heart, spleen, lungs, and kidneys, but dose-independent in the bursa Fabricius in different growing stages of local Chinese Fast Yellow Chicken; bitterness sensitivity decreased subsequently. However, organ weight gains were impaired in the group that received a high dose of denatonium benzoate, and the researchers observed that chickens have a lower tolerance for high-dose denatonium benzoate feed. These findings are valuable

for clinicians and pharmacologists because of ggTas2Rs-wide extraoral expressions, as taste biology is directly correlated to diseases, and may affect kidney and heart functions, the regulatory mechanisms of lungs, and the immunity pathways of bursa Fabricius. It may also help with nutritionist and benefit feed industries to improve the production of new feedstuffs for chicken according to their growing stages.

**Author Contributions:** Conceived and design the Experiment: the work was conceived by H.E. Performed the experiment: E.H., J.J., Z.S. & Z.L. Analyzed the data: data was analyzed by E.H. and R.M.M. Contributed reagents/materials/analysis tools: J.J., D.Y., Z.L., Q.W. and Z.S. Manuscript writing: Manuscript writing was performed by E.H. All authors reviewed and approved the manuscript.

**Funding:** This study was supported by the Agricultural Independent Innovation Project in Jiangsu Province, China CX (18)2002. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing Jiangsu China 210095.

**Acknowledgments:** We thank to the Agricultural Independent Innovation Project of Jiangsu Province for providing the fund for successful implementation of the project.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Review*

### **Use of Licorice (***Glycyrrhiza glabra***) Herb as a Feed Additive in Poultry: Current Knowledge and Prospects**

**Mahmoud Alagawany 1,\*, Shaaban S. Elnesr 2, Mayada R. Farag 3, Mohamed E. Abd El-Hack 1, Asmaa F. Khafaga 4, Ayman E. Taha 5, Ruchi Tiwari 6, Mohd. Iqbal Yatoo 7, Prakash Bhatt 8, Gopi Marappan <sup>9</sup> and Kuldeep Dhama 10,\***


Received: 22 July 2019; Accepted: 4 August 2019; Published: 7 August 2019

**Simple Summary:** The present review updates the current knowledge about the beneficial effect of licorice supplementation in poultry diets, particularly its positive effect on the treatment of high-prevalence diseases of the immune system, liver, and lungs.

**Abstract:** Supplementation of livestock and poultry diets with herbal plants containing bioactive components have shown promising reports as natural feed supplements. These additives are able to promote growth performance and improve feed efficiency, nutrient digestion, antioxidant status, immunological indices, and poultry health. Several studies have used complex herbal formulas with the partial inclusion of licorice. However, the individual use of licorice has been rarely reported. The major problem of the poultry industry is the epidemiological diseases, mainly confined to the respiratory, digestive, and immune systems. Licorice has certain bioactive components such as flavonoids and glycyrrhizin. The roots of this herb contain 1 to 9% glycyrrhizin, which has many pharmacological properties such as antioxidant, antiviral, anti-infective and anti-inflammatory properties. Licorice extracts (LE) have a positive effect on the treatment of high-prevalence diseases such as the immune system, liver, and lung diseases. Studies showed that adding LE to drinking water (0.1, 0.2, or 0.3 g/L) reduced serum total cholesterol (*p* < 0.05) of broiler chickens. Moreover, LE supplementation in poultry diets plays a significant role in their productive performance by enhancing organ development and stimulating digestion and appetite. Along with its growth-promoting effects, licorice has detoxifying, antioxidant, antimicrobial, anti-inflammatory, and other health benefits in poultry. This review describes the beneficial applications and recent aspects of the *Glycyrrhiza glabra* (licorice) herb, including its chemical composition and role in safeguarding poultry health.

**Keywords:** licorice; *Glycyrrhiza glabra*; beneficial effects; pharmaceutical; poultry; health

#### **1. Introduction**

Medicinal plants have gained great popularity for their several beneficial applications in animals, poultry, and humans [1,2]. Nowadays, the addition of feed additives and nutritional supplements, including prebiotics, plant extracts, and probiotics, in the diets of birds are gaining wide attention owing to their multiple beneficial applications while enhancing growth performances and production as well as safeguarding the health of poultry [1,3–5]. This review focuses on the use of herb licorice (*Glycyrrhiza glabra*) as a feed additive in poultry, a popular traditional medicinal plant that belongs to the legume family Fabaceae [6]. It is broadly used in the medicine sector, as a flavouring and food preservative agent and also for commercial purposes [7]. It is derived from the sweet root of various species of *Glycyrrhiza*; however, the cultivation and harvesting practices modify the composition of various biologically important components of the *Glycyrrhiza* plant [8]. Phytochemical analysis showed that the major fraction of licorice extract (LE) consisted of triterpene saponins (e.g., glycyrrhizin, glycyrrhetinic acid, and licorice acid), flavonoids (e.g., liquiritin, isoflavonoids, and formononetin), sugars, starch, amino acids, ascorbic acid, tannins, choline, coumarins, phytosterols, and some other bitter principles [7,9]. Importantly, numerous pharmacological effects have been described for LE and its isolated active principles in humans and animals [7]. Licorice represents a replacement candidate reported to be useful for its multiple beneficial health effects including immunomodulatory, antimicrobial, antioxidative, anti-inflammatory, antidiabetic, hepatoprotextive, antiviral, anti-infective, and radical-scavenging activities [7,8,10]. This review describes the beneficial applications and recent aspects of the licorice herb, including its chemical composition, health benefits, and useful applications for nutritionists, physiologists, scientists, pharmacists, veterinarians, pharmaceutical industries, and poultry breeders. Therefore, we can safely assess and get a new vision for further research on licorice benefits in poultry nutrition and its effects on the growth and productive performance and immune and antioxidant status of poultry.

#### **2. Chemical Composition and Structure**

Licorice is also known as Radix Glycyrrhizae or Liquiritiae Radix. It is the root of *Glycyrrhiza uralensis* Fisch. ex DC., *G. glabra* L. or *G. inflata* Bat., Leguminosae [10,11]. The roots of *G. glabra* (Figure 1) are widely used in preparing several pharmaceutical preparations. Phytochemical analysis of licorice root extract exhibited that it contained flavonoids (isoflavonoids, formononetin, and liquiritin), saponin triterpenes (liquirtic acid and glycyrrhizin), and other components such as sugars, coumarins, amino acids, starch, tannins, phytosterols, choline, and vitamins (e.g., ascorbic acid) [7,9,12]. Previous reports have shown that more than 20 triterpenoids and 300 flavonoids have been procured from licorice [13]. Glycyrrhizin constitutes up to 25% of the licorice root extract [14]. Glycyrrhizin consists of glucuronic acid (two molecules) and glycyrrhetinic acid (one molecule) [15]. Badr et al. [16] analyzed the raw form of licorice chemically and summarized its contents as follows: carbohydrate (47.11%), fiber (24.48%), protein (9.15%), silica (3.56%) and low fat content (0.53%). Moreover, the ash and moisture content values of the licorice root were found to be 7.70 and 6.80%, respectively. Additionally, the same authors reported that the calcium and phosphorus content values of the raw LE were 1720 and 78 mg/100 g, respectively, and the major components of amino acids that were found in LE were proline (1.02%), aspartic (0.88%), alanine (0.51%) and glutamic acid (0.50%).

**Figure 1.** Pictorial representation of the *Glycyrrhiza glabra* herb, its root and extracts.

The licorice root color is yellow because of its flavonoid components such as hispaglabridins and glabridin [17]. Additionally, the dried aqueous extracts of licorice contain approximately 4–25% glycyrrhizinic acid [18]. The main active ingredients of licorice are liquiritin, isoliquiritigenin, liquiritigenin, and glycyrrhetinic acid, glycyrrhiza polysaccharide, and this herb is rich in flavonoids and syringic, abscisic, trans-ferrulic, 2,5-dihydroxy benzoic, abscisic, and salicyclic acids [7,9,19]. Pharmacological activities are contributed to by glycyrrhizin, 18β-glycyrrhetinic acid, glabrin A and B, and isoflavones of *Glycyrrhiza glabra* Linn [7].

#### **3. Beneficial Role of Licorice**

In ancient times, *G. glabra* was used as a medicine and flavouring herb. It is a soothing herb that enhances various body functions, protects the liver and is used in various conditions, such as mouth ulcers and arthritis, and as a potent anti-inflammatory, immunomodulatory, hepatoprotective, detoxifying, anti-cancer, anti-aging, antioxidant, antimicrobial, with growth promoting effects [6,8,9]. The licorice herb has several biological activities and health promoting effects, as are discussed in the following sections.

#### *3.1. Antioxidant and Anti-Inflammatory Activities*

Previous phytochemical analyses have revealed that the bioactive components of the licorice root include flavonoids (isoflavonoids and liquiritin), glycyrrhizic acid, liquiritigenin, triterpenes (glycyrrhizin), and saponins, which have anti-inflammatory and antioxidant properties [20–23]. Various modes of action with regards to antioxidant and anti-inflammatory properties of licorice can be narrated as: licorice extracts inhibits the lipid peroxidation of mitochondria, decreases the oxidative rate and reactive substance formation of thiobarbituric acid; protects from scavenging free radicals; stimulates antioxidant enzyme activities; inhibits the activity of phospholipase A2 that acts as a critical enzyme in various inflammatory processes; Licochalcone inhibits lipopolysaccharide- induced inflammatory responses; Lico A derived from the licorice root inhibits the lipopolysaccharide-induced inflammatory responses in a dose-dependent manner by suppressing the activation of NF-κB and p38/ERK MAPK signaling; licochalcone A prevents cellular oxidation; licorice flavonoids renders a pro-inflammatory action; flavonoids might target the NF-κB signaling pathway to prevent the secretion of inflammatory cytokines; glycyrrhizic acid, liquiritigenin, and liquiritin can reduce the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the liver, and block the generation of several inflammatory mediators created by activated macrophages; glycyrrhizic acid directly inhibits prostaglandin E2 formation and cyclooxygenase activity and indirectly inhibits platelet aggregation and inflammatory factors [8,14,23–25]. Glycyrrhetinic acid might lead to the delayed secretion of cortisol with subsequent high levels of oxidation that resulted in increased heart weight in hens [26]. The proven and potent anti-inflammatory and antioxidant properties of the licorice herb need to be studied in poultry for the safeguarding of the heath of birds in poultry production.

#### *3.2. Immunomodulatory and Antiviral E*ff*ects*

Herbs have proven potent immunomodulatory and antiviral activities [2,25,27]. The extracts of licorice have a positive effect on the immune system of poultry. It can be used to optimize their immune response and improve the productive performance. The dietary supplementation of 0.1% LE improved the humoral immunity in broilers by inducing antibody titres against non-specific and specific antigens. An experiment was conducted to assess the effect of the supplementation of licorice root extracts on the immune profile of 54 commercial broiler chicks. The serum biochemical parameters, such as serum total protein, albumin, globulin, and albumin/globulin ratio, were estimated from three groups of chicks. Chicks were categorized into three groups; one in which the birds were provided with 1% *G. glabra* crude extract powder, the second one in which 0.1% *G. glabra* extract powder was given, and the third one in which no *G. glabra* extract was given. Humoral immunity was assessed by measuring the hemagglutination inhibition (HI) titre against the Ranikhet disease virus (LaSota) and the hemagglutination antibody (HA) titre against sheep red blood cells (SRBC) antigens, while the cell-mediated immune response was measured by estimating the total and differential white blood cell (WBCs) counts. The results of the study revealed that the chicks supplemented with 0.1% licorice extract powder showed considerable improvement in their immune responses [28]. Also, natural feed supplements are used as immunity enhancers because it increases WBC counts and ultimately boosts interferon levels [29]. Furthermore, Dorhoi et al. [30] stated that the addition of LE (50 μg/mL) to the diet of laying hens had some beneficial effects on their cellular immunity. The glycyrrhiza polysaccharide has a sturdy immune action and is widely involved in some features of immune regulation [31]. Additionally, LE increased the phagocytic capacity of mononuclear cells and granulocytes of chicken [30].

Notably, the addition of licorice in broiler diets improved the weight of immune organs, such as the spleen or bursa, thereby promoting immune efficacy and the situation of livability and health [32]. Glycyrrhetinic acid has several favourable pharmacological properties, such as immunomodulation and production of interleukins [1,2,12] with subsequent production of antibodies, gamma interferon, and T-cells, which indicates its antiviral activity [33]. However, Hosseini et al. [34] reported that the supplementation of the broiler diet with LE (2.5 and 5 g/kg diet) had no effect on immune organ weights.

The active components of licorice and its extracts have anti-inflammatory, immunomodulatory and antiviral functions, and thus can augment the immunity of poultry by modulating both humoral and cell-mediated immune responses, prevent viral diseases, and render supplementary treatment for viral diseases [35,36]. Omer et al. [35] reported that LE (60 mg/100 mL *Glycyrrhiza* extract) when used as a phytogenic feed additive exhibited antiviral activity against the Newcastle disease virus (NDV). Moreover, the broilers treated with glycyrrhizic acid (GRA) at the concentration of 60 μg /mL drinking water showed higher antibody titers against the ND virus as well as an enhanced cellular immune response, as indicated by an increase in blood lymphocyte and thrombocyte counts [36]. In an in vivo antiviral study, a dose of 300 μg/mL of *G. glabra* extract showed potent antiviral action against NDV. Survival rates were higher in embryonated egg groups inoculated with NDV and treated with extract, and no virus was recovered in allantoic fluids in such groups which indicated the effective control of the virus by the herbal extract [5]. Dziewulska et al. [37] stated that dietary LE (10% extract) supplementation inhibited paramyxovirus type 1 (PPMV-1) replication in pigeons, and the copy number of viral RNA in some organs, such as the kidney and liver, of the pigeons fed LE was lower compared with the control pigeons, suggesting that LE has antiviral effects. In an aqueous solution of LE when administered at a dose rate of 300 or 500 mg/kg body weight to pigeons inoculated with ppmV-1 for 7 days, the expression of the IFN-γ gene was found to be increased in all ppmV-1 inoculated and herbal extract treated pigeons. Expression of the CD3 gene was lowest at 7 dpi in treated birds. CD4 gene expression was higher in uninoculated and treated pigeons but was lower in extract treated pigeons inoculated with ppmV-1. The CD8 gene also showed a non-significant difference in inoculated and extract treated pigeons, and the percentage of IgM<sup>+</sup> B cells was also not affected [38]. The immunomodulatory and antiviral effects of licorice observed in pigeons also reveals

its potent application to be explored and applied in poultry and other avian species to counter viral diseases. However, Moradi et al. [39] reported that the antibody titres against Newcastle disease (ND) and avian influenza (AI) viruses, as well as liver and lymphoid organ (e.g., bursa of Fabricius, thymus and spleen) weights, were not affected by LE supplementation in broiler drinking water. Glycyrrhizin has been reported to act as an immune stimulant for ducklings, inhibits the cytopathic effect of duck hepatitis virus (DHV) in VERO cells, potentiates the production of higher antibody titer in a DHV vaccinated group, and demonstrates a pronounced lymphocytic proliferation response, indicating its antiviral effects [40]. Glycyrrhizin has also been reported to inhibit influenza A virus uptake into the cell, mediated by its interaction with the cell membrane, which, in turn, results in reduced endocytotic activity and decreased virus uptake [41]. Thus, several studies have confirmed the positive impact of licorice on the immune potential and anti-viral effects in poultry; however, further studies are recommended to optimize the inclusion levels of LE in poultry diets and to determine their possible physiological and protective effects, as well as economic value.

Regarding the protective role of licorice against aflatoxicosis in broilers, Al–Daraji et al. [42] reported that the addition of licorice to the aflatoxin (AF)-contaminated diet (at the concentrations of 150, 300, or 450 mg licorice/kg of diet) significantly recovered the adverse effects of AF on most carcass traits.

Saponins from *G. glabra* in combination with antigens from *Eimeria tennela* have shown the ability to serve as immune-stimulating complexes (ISCOMs) and provides immunity against avian coccidiosis, caused by *E. tennela* [43]. They protected birds from the experimental challenge of *E. tennela* and additionally, the antibody titer (IgG and IgM) against a homologous challenge was found to be increased. Saponins have also been identified to act as effective delivery units for antigens for vaccine development, have shown no toxicity and provided stronger immunity [44]. Being natural constituents of plants, saponins of *G. glabra* are unlikely to have any side effects and are comparable to *Aesculus hippocastanum*, *Gipsophila paniculata* or Quil-A saponins [43,44]. More recently, saponins derived from *G. glabra* (Glabilox) have shown better adjuvant potential and the ability to be used as immunostimulatory complexes along with antigens for vaccine purposes. *G. glabra* derived saponins were not found to be toxic or hemolytic as compared to Quil-A saponins, and could produce stable immunostimulatory complexes, hence were preferable as safe and effective vaccine adjuvants [33]. Glabilox induced strong humoral and cellular immune response against H7N1 influenza virus antigens on subcutaneous inoculation and provided 100% protection against homologous infection in chickens [33]

#### *3.3. E*ff*ect of Licorice on Some Blood Chemistry*

Broiler chickens given drinking water supplemented with LE (0.1, 0.2, or 0.3 g/L) showed reduced serum glucose, LDL cholesterol, and total cholesterol levels (*p* < 0.05) as well as reduced gall bladder weight [45]. The inclusion of *G. glabra* extract (0.5%) in broiler diets induced an increase in serum globulin concentration, which, in turn, led to an improvement in the humoral immune status [46]. However, the broilers fed 0.5 and 1 g licorice/kg during their growing period showed an increase in the number of WBCs (*p* <0.05) compared to the control. Furthermore, dietary licorice supplementation (0.5, 1, and 2 g/kg) did not have significant effects on the lymphocyte (L), heterophil (H), and monocyte percentages, heterophil to lymphocyte (H/L) ratio, and proliferation of red blood cells [47]. Moreover, the heterophil and lymphocyte percentages and H/L ratio were not affected by LE supplementation (0.1, 0.2, and 0.3 mg/L) in drinking water [39]. The licorice root enclosed phytoestrogens that boosted the rate of erythrocyte sedimentation and decreased the number of erythrocytes [48]. Additionally, the injected LE stimulated cell cycle and activity in lymphocytes [49]. Furthermore, Sharifi et al. [50] clarified that the licorice root supplementation in broiler diets (2 mg/kg diet) reduced some serum components, such as triglycerides, cholesterol, and LDL, and increased the high-density lipoprotein (HDL) levels. In another study, Sedghi et al. [47] concluded that the concentrations of cholesterol and LDL significantly declined in the birds fed diets containing licorice (0.5, 1, and 2 g/kg) compared to the control. This might be attributed to the inhibition of lipid peroxidation and lipoxygenase and cyclooxygenase

enzyme activities as well as reduction of LDL oxidation by licorice. The cholesterol-lowering effects of LE are attributed to the high secretion of cholesterol, bile acids, neutral sterols, and improvement in the content of hepatic bile acid. Besides, the active components of licorice (saponin) are able to reduce the levels of LDL-associated carotenoids, inhibit the formation of lipid peroxides, and enhancement of the rate of conversion of cholesterol to bile acids with subsequent hepatic clearance. However, the feeding of licorice (0.5, 1, and 2 g/kg) in the study of Sedghi et al. [47] did not have a significant influence on the concentrations of triglycerides, HDLs, VLDLs, and glucose in the blood serum of broilers. Al-Daraji [51] concluded that the high levels of LE (150 to 450 mg/L in water) augmented glucose concentrations in the serum of broiler chickens under heat stress.

The dietary supplementation of LE increased the HDL concentration and HDL/LDL ratio in serum because of its high concentrations of flavonoids and ascorbic acid [45]. Moreover, the inclusion of 0.4% LE in the drinking water of broilers increased the plasma HDL levels, but reduced the level of alanine aminotransferase (ALT) (*p* < 0.05) [52]. However, Shahryar et al. [53] concluded that the serum blood parameters of the laying hens supplemented with different concentrations of licorice powder (0.5, 1.0, 1.5, and 2.0%) did not significantly (*p* > 0.05) vary compared to the control group. Thus, the presence of saponins and phytosteroids in LE could be essential for removing cholesterol and increasing the content of hepatic bile acid in animals fed LE diets. *G. glabra* produced lower abdominal fat percentage (*p* < 0.05) in broilers given 0.3 g/L of LE in drinking water [6]. Moreover, the supplementation of LE at levels 0.1, 0.2, and 0.3% in drinking water decreased the concentrations of serum LDL, total cholesterol, and glucose (*p* < 0.05) [45].

#### *3.4. E*ff*ect of Licorice on Some Growth Parameters and Performance*

Currently, it is well established that the growth and laying performances of poultry are usually improved via supplementation of feed additives or growth promoters, which have a positive influence on their general health state and growth performance [1,3]. The inclusion of 0.4% LE in the drinking water of broilers increased (*p* < 0.05) the feed intake at 21 and 42 days, but did not affect the body weight at different ages [51]. However, Jagadeeswaran and Selvasubramanian [28] found that the inclusion of 1% LE to the basal diet of the broiler chickens improved their body weight and FCR at 42 days of age in comparison to the control group. In Japanese quails [54] it was reported that the inclusion of 200 ppm of licorice root extract containing 1% probiotic supplement to the quail diet improved the amount of daily feed intake and body weight gain. Furthermore, LE had positive effects on the productive performance of heat-stressed broiler chickens [55,56]. *G. glabra* diet supplementation in poultry positively affected their growth performance by enhancing the development of their organs. Furthermore, the digestion and appetite improved in broilers fed diets supplemented with 2.5 g/kg *G. glabra*. Moreover, the inclusion of up to 0.5% *G. glabra* in poultry diets during the pullet growing period enhanced the performance of laying hens [6].

Concerning the use of glycyrrhizic acid (GRA), the broilers supplemented with GRA (60 μg/mL in water) had higher body weight gain (BG), final body weight, better FCR, and the lowest mortality rate compared to the non-treated controls [36]. The feed intake of the laying hens fed 0.5, 1.0, 1.5, and 2.0% of licorice powder added to the basal diet was not affected [52]. Simultaneously, Hosseini et al. [34] used 5 g licorice/kg broiler diet and found no significant effect (*p* > 0.05) on body weight, feed intake, FCR, livability, and production index. Additionally, Moradi et al. [39] concluded that the inclusion of 0.1, 0.2, and 0.3 mg LE/L drinking water for broiler chicks had no significant effect on their body weight, feed intake, and FCR compared to the control group. Moreover, Sedghi et al. [47] used 0.5, 1, and 2 g LE/kg broiler diet and reported no effect on broiler weight, feed intake, and FCR compared to the non-supplemented group. However, another study reported different results when the percentage of licorice was modified and given in combination. This study was performed to determine an improvement in the productive traits of 180 one-day-old Ross 308 broiler chicks fed diets supplemented with different concentrations of licorice and garlic mixture powders. It was concluded that the diet supplemented with a mixture of garlic and licorice (at 0.25, 0.50, and 1% concentrations) improved the productive performance of broiler birds [57]. Another study carried out on 480 one-day-old male broiler chicks (Ross 308) showed the beneficial effects of 1% LE on the growth performance, immune system, and blood parameters of broilers, when supplemented along with the extracts of other plants, such as German chamomile, yarrow, eucalyptus, Iranian caraway, and garlic, and one antibiotic virginiamycin [58]. One more study performed on 400 unsexed (Cobb 500) broiler chicks advocated that LE reduced the abdominal fat of chicks without illustrating any adverse effects on their immune status and performance of broiler when receiving LE in drinking water for 42 days [59].

Experiments were performed to determine the effect of diet supplementation with thyme, peppermint, green tea, and licorice in 245 one-day-old broiler chickens on enhancing their growth performance, serum lipid profile, immune response, and carcass characteristics. An aqueous blend of 400 g thyme extract, 300 g peppermint extract, 200 g green tea extract, and 100 g LE, which finally provided 1.4% essential oils (0.4% thyme oil/0.9% peppermint oil), 25% polyphenols, 15% catechins, and 0.5% glycyrrhizinic acid as active principles, was used. A total of seven experimental groups were set up: five groups supplied with 100, 200, 500, 1000, and 2000 ppm of the aqueous blend and one negative (no supplementation) and one positive (antibiotic: oxytetracycline) control group. The results depicted an overall increase in the performance of chicks at 200 and 1000 ppm levels as compared to the negative control and a significant increase in humoral immunity as compared to the positive control. These findings recommended the inclusion of the plant extract blend (at 200 ppm concentration) in poultry diet to support the broiler performance and immune status, in addition to its use as a growth promoter and an alternative to antibiotics [60].

A study performed on hundred 40-week-old laying hens showed that the diets supplemented with LE improved the production of functional eggs and modulated the productive performance of laying hens by lowering the egg cholesterol level, some plasma parameters (reduction in LDL and egg-yolk cholesterol level with an increase in the HDL level and total antioxidant capacity of plasma [61]. Scientific literature has witnessed the global impact of this herb on the performance, carcass traits, and meat quality of poultry, when their diet is supplemented with licorice (*G. glabra*) either in feed and/or in drinking water [6]. In broiler chickens, probiotic and licorice extract (500 ppm) increased body weight gain of broilers exposed to high stocking density [62]. Drinking water supplementation of LE has been suggested to be an alternative to in-feed antibiotic growth promoter in broiler chickens [45]. Body weight gain (BWG) was improved in birds reared at high stocking density by licorice extract (500 ppm) but not the feed conversion ratio (FCR) [62]. This was further improved by the addition of probiotics (200 ppm).

An overview of the beneficial effects and modes of action of *Glycyrrhiza glabra* in poultry health and production is depicted in Figure 2.

#### **4. Conclusions and Future Prospects**

The extract of *G. glabra* might play an important role in the preparation of several pharmaceutical compounds for further use in the poultry industry. Licorice contains bioactive components, such as flavonoids and glycyrrhizin, which have pharmacological properties and medicinal applications. The licorice extract has been found to show immunogenic and antioxidant activities, which might improve the growth performance, feed efficiency, carcass traits, and blood biochemical indices of the poultry birds, and act as a potential solution for solving respiratory, digestive, and immune problems in poultry. The use of LE up to 0.4 g/L in the drinking water of poultry increased the feed intake, and improved the immune response and antioxidant parameters as well as lipid profile. The addition of LE at 50 μg/mL in the diet of laying hens has been found to produce some beneficial effects on their cellular immunity. A dose of 300 μg/mL of *G. glabra* extract showed potent antiviral action against NDV. Further studies need to be conducted to evaluate the beneficial effects of using the licorice herb as poultry feed additive, as well as to explore other properties of this medicinal plant that might enhance productivity and health in poultry. Efforts need to be made to enhance the delivery of this important

herb in poultry by exploring the nanodelivery and in ovo delivery techniques, thereby efficiently enhancing production and safeguarding the health of birds in a better way.

**Figure 2.** Beneficial effects and modes of action of *Glycyrrhiza glabra* in poultry health and production.

**Author Contributions:** All the authors substantially contributed to the conception, design, analysis and interpretation of data, checking and approving final version of the manuscript, and agree to be accountable for its contents.

**Funding:** This compilation is a review article written, analyzed and designed by its authors and required no substantial funding to be stated.

**Acknowledgments:** All the authors acknowledge and thank their respective Institutes and Universities.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **Abbreviations**


#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
