**Modelling Methionine Requirements of Fast- and Slow-Growing Chinese Yellow-Feathered Chickens during the Starter Phase**

**Long Li 1,2,**†**, K.F.M. Abouelezz 2,3,**†**, Zhonggang Cheng 2, A.E.G. Gad-Elkareem 3, Qiuli Fan 2, Fayuan Ding 2, Jun Gao 4, Shouqun Jiang 2,\* and Zongyong Jiang 2,\***


Received: 21 January 2020; Accepted: 4 March 2020; Published: 6 March 2020

**Simple Summary:** In poultry production, consuming diets with low or excessive methionine levels leads to negative effects on growth performance. The requirements of methionine may differ among the fast and slow-growing breeds; therefore, the optimal dietary methionine level should be estimated for each. In this study, six dietary methionine levels were evaluated to estimate the optimal level for fast and slow-growing yellow feathered chicken breeds. The quadratic polynomial and exponential asymptotic regression showed that the optimal methionine requirements for maximal growth performance were 0.50% and 0.53% in the fast-growing breed, and 0.48% and 0.52% in the slow growing breed.

**Abstract:** Two experiments were carried out to investigate the dietary methionine requirement for fast and slow-growing Chinese yellow-feathered breeds during the starter phase, based on growth variables and regression models. In Experiment 1, a total of 2880 one-day-old Lingnan chicks (fast growing breed) were used to test the methionine requirement from 1 to 21 days of age for males and females separately. Of each gender, 1440 birds were allocated into 6 dietary methionine levels (0.28%, 0.32%, 0.37%, 0.43%, 0.50% and 0.63%), each with 6 pen replicates of 40 chicks. Experiment 2 had the same design with Guangxi chicks (slow growing breed) from 1 to 30 d of age. Results indicated that significant nonlinear or quadratic responses to increasing dietary methionine levels were observed in body weight, daily gain, feed intake and feed conversion ratio of both breeds. In summary, the quadratic polynomial regression showed that the optimal methionine requirements for maximal growth performance of Lingnan chickens were 0.52–0.58% in males, 0.51% in females, and 0.53% in mixed genders. The corresponding values for Guangxi breed were 0.53% in males by quadratic polynomial regression and 0.43% in females, and 0.48% to 0.49% in mixed sexes by exponential asymptotic models.

**Keywords:** amino acids; nutrient requirements; growth indices; modeling; Chinese yellow-feathered chickens

#### **1. Introduction**

With the rapid development of economy in China, the second largest worldwide producer of chicken meat, market demands for nutrient-rich and tasty meat have been in continuous increase, which is boosting the industry of Chinese yellow-feathered chickens. The contribution of such chicken type in production has been growing; 3.7 billion birds annually with more than 30% of whole chicken meat shares in recent years [1]. The indigenous chickens have a recent increase in their commercial importance due to their favorable meat color and flavor, which highlights the need for comprehensive studies to enhance their feeding standards. However, only few researches on their nutrient requirements were conducted up till now [2–5].

Some essential amino acids are often added as pure to poultry diets to ensure the optimal balance required for poultry in order to maximize the production efficiency. Globally, methionine (Met) is considered to be the first limiting amino acid for poultry fed on typical corn–soybean meal-based diets [6]. Met is an essential amino acid doing numerous vital biological activities in animals' body [7,8]; it has an important role in protein synthesis in addition to enhancing the antioxidant capacity of the organism via participating as a precursor of glutathione that eliminates the reactive oxygen species in the body cells [9,10]. Additionally, the Met is required for the polyaminase synthesis that mediates cell and nucleus division process, and is considered as the most common donor of methyl group consumed in the DNA methylation process [11–14]. Met, therefore, is required for ensuring normal growth performance in poultry. The dietary supplementation of Met in poultry is commonly used to fulfil the bird requirement of Met and to achieve rapid correction of any deficiency of some nutrients; Met is a precursor of cysteine, succinyl-CoA, creatine, homocysteine and carnitine. Additionally, optimizing the Met level in poultry diets can contribute in reducing the nitrogen emission of nitrogen into the surrounding environment [13,14].

Chinese yellow-feathered chicken is a general name, which consists of quite a few breeds, mainly referring to Chinese local breeds and the improved breeds. Chinese yellow-feathered chickens grow slowly and are reared for a longer time compared with white-feathered broilers. The Lingnan yellow-feathered chicken breed is classified according to its growth rate to fast (1.47–2.30 kg, marketable at 8–10 weeks), medium (1.00–2.27 kg BW, marketable 9–14 weeks), and slow growing (1.06–1.88 kg BW, marketable 12–25 weeks) [3,15,16]. The Guangxi yellow-feathered chicken is a native, slow growing and light-body type breed with good meat quality in China (1.02–1.6kg BW, marketable 13–22 weeks). The nutrient requirements of broilers differ among the different growth stages, which are divided into three periods in Lingnan yellow-feathered broilers: starter (1–21 days old), grower (22–42 days old), and finisher (>42 days), and four phases in Guangxi yellow feathered broilers: starter (1–30 days), grower (31–60 days), early finisher (61–90 days), and later finisher (>90 days old) phases [15,16].

The Met requirement of feeding standard of chicken (CNY/T33-2004) is mainly for mediumgrowing yellow-feathered broilers [12]. As the dietary Met requirement for the yellow-feathered meat type chickens has not been fully determined or optimized, the present study aimed to estimate it for males and females of Lingnan (a typical fast-growing yellow feathered) and Guangxi (a slow-growing yellow-feathered) chicken breeds during the starter period.

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

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

Two experiments were carried out to estimate the Met requirement for Lingnan (Exp1) and Guangxi (Exp2) yellow-feathered chicken breeds following the same experimental design. Before trial, the Met content of the basal diet ingredients used in both experiments was determined by ion-exchange chromatography on an automatic amino acid analyzer (L8800, Hitachi, Japan), according to the procedures described by Xi et al. [17]. In each experiment, a total of 2880 one-day-old chicks (50% males + 50% females) were randomly assigned to 6 dietary Met levels, each contained 12 identical floor pens of 40 birds, of which 6 pens were males and 6 were females (n = 240 birds from each

sex/treatment). The average initial body weights (g) were 33.88 ± 0.26, 38.75 ± 0.32, and 36.32 ± 0.28 for male, female and mixed sexes of Lingnan chicks, and 31.50 ± 0.23, 31.02 ± 0.25, and 31.20 ± 0.24 for male, female, and mixed sexes of Guangxi chicks. All birds were housed in one environmentally controlled room with dry wooden shaving flooring, continuous artificial lighting was provided from incandescent lamps, and room temperature was maintained at 30 ◦C in the 1st week and gradually reduced to 25 ◦C on the 4th week. All chicks were managed according to the Animal Care and Use Committee of Guangdong Academy of Agricultural Sciences Management Guide (GAASIAS-2016-017). The experimental diets were offered from d 1 to d 21 for the Lingnan breed (Exp1) and from d 1 to d 30 for the Guangxi breed (Exp2). Dietary treatments included a Met unsupplemented corn–soybean meal basal diet (Table 1) and the basal diets supplemented with 0.04%, 0.09%, 0.15%, 0.22%, and 0.35% Met in the form of DL-Met (Evonik Industries AG, Hanau-Wolfgang, Germany); the final dietary Met concentrations were 0.28%, 0.32%, 0.37%, 0.43%, 0.50%, and 0.63%.

Pelleted feed and drinking water were freely available to chicks. The different diets were prepared three weeks prior to the trial to allow time for checking content homogeneity, in terms of dry matter, ash, crude protein, ether extract, crude fiber, and amino acid concentrations.


**Table 1.** Composition of the basal diet (air-dry basis, %).

<sup>1</sup> Totally provided per kg of diet: 1500 IU vitamin A; 200 IU vitamin D3; 10 IU vitamin E; 0.5 mg vitamin K3; 1.8 mg thiamin; 3.6 mg riboflavin; 3.5 mg pyridoxine; 0.01 mg cyanocobalamin; 10 mg pantothenic acid; 30 mg niacin; 0.55 mg folic acid; 0.15 mg biotin; 500 mg choline; 80mg Fe; 8 mg Cu; 80 mg Mn; 60 mg Zn; 0.35 mg I; 0.3 mg Se. <sup>2</sup> Values were calculated based on the data provided by Feeding Standard of Chicken (Ministry of Agriculture, China, 2004). <sup>3</sup> Analyzed values.

#### *2.2. Growth Performance*

Birds were weighed at the beginning (day 1) and end of each experiment to record the initial and final body weights (FBW), which were used to calculate the average daily gain (ADG). Average daily feed intake (ADFI) was measured on a per pen basis for the entire experimental period, and the feed conversion ratio (FCR) was calculated. Mortality was checked daily and dead birds were weighed in order to adjust the feed intake calculations.

#### *2.3. Statistical Analysis*

Data were subjected to one-way ANOVA using the GLM procedure of SAS (version 9.3, SAS Inst., Cary, NC, USA, 2014). Tukey–Kramer test was used for means comparison, and pairwise comparisons among the means were assessed by Duncan's multiple-range tests at *p* < 0.05. All data were expressed as means and SEM, derived from ANOVA error mean square. When the main effect was significant (*p* < 0.05), linear and quadratic effects of Met content were determined. For key performance variables, the dietary methionine requirement of the birds was estimated using quadratic polynomial (QP) or exponential asymptotic (EA) models by the NLIN procedure of SAS (SAS Institute, 2014).

QP model:

$$\mathbf{Y} = \mathbf{c} + \mathbf{b}\mathbf{X} + \mathbf{a}\mathbf{X}^2 \tag{1}$$

where a = quadratic coefficient, b = linear coefficient, c = intercept. The requirement of Met was defined as Met = −b/(2 × a).

EA model:

$$\mathbf{Y} = \mathbf{a} + \mathbf{b} \times (\mathbf{1} - \mathbf{E} \mathbf{X} \mathbf{P} \left( -\mathbf{c} \times (\mathbf{X} - \mathbf{d}) \right)) \tag{2}$$

where a = relative response to the diet containing the lowest Met (deficient diet); b = difference between the minimum and the maximum response obtained with dietary Met; c = curve slope coefficient; d = Met level of the deficient diet). The optimal Met was defined as Met = (-ln (0.05)/c) + d, using 95% of the asymptotic response, since the exponential curve never reaches the asymptotic point [18–20]. The suitability of the different models was evaluated by the correlation coefficient (R2), Akaike information criteria (AIC) and mean square error values (MSE).

#### **3. Results**

#### *3.1. Growth Performance of Lingnan Broilers Aged 1 to 21 Days (Exp 1)*

The growth performance traits of male, female and mixed male and female Lingnan yellow-feathered chickens fed different dietary Met levels between 1 and 21 days of age are shown in Tables 2–4, respectively. The increase in the dietary Met level showed linear and quadratic effects on the final BW, ADG, ADFI and FCR (linear, *p* < 0.01; quadratic, *p* < 0.01) of males; and final BW, ADG and FCR (linear, *p* < 0.01; quadratic, *p* < 0.01) of females; and final BW, ADG and ADFI (linear, *p* < 0.01; quadratic, *p* < 0.01) of mixed genders. According to the EA and QP regression, the optimal dietary Met level for the highest body weight were 0.54% and 0.55% in males, 0.47% and 0.51% in females, and 0.50% and 0.53% in mixed genders. The corresponding EA and QP values for the highest ADG were 0.54% and 0.55% in males, 0.47% and 0.51% in females, 0.50% and 0.53% in mixed genders. With regard to these results, it is worth mentioning that obtaining the same optimum Met requirement value for maximal final BW and ADG is logically expected; certainly, because these two variables are linearly correlated, where the ADG is calculated as the (final BW—initial BW) /days. This, therefore, led to the same values of R2 and the same calculated Met requirements for these two variables (Tables 2–4, Figures 1–3). Additionally, the EA and QA models showed that the optimal Met level for ADFI were 0.58% and 0.50% in males, and 0.51% and 0.47% in females, and those of daily feed intake estimated 0.50% and 0.52% in males, and 0.52% and 0.53% in mixed genders. Goodness-of-fit results for the growth performance functions are shown in Figures 1–3. The high R2, and lowest AIC and MSE values of QP model are shown in Figure 1A–C, Figure 2A,B, Figure 3A–C. The results indicated that the QP model is more adequate for predicting the optimal Met requirements for maximal growth performance in Lingnan broilers, which showed that the optimal Met requirements for Lingnan male, female and mixed sexes were 0.52% to 0.58%, 0.51%, and 0.53%, respectively.




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**Table4.**Effectsoftotaldietarymethioninelevelongrowthperformanceof mixedmaleandfemaleLingnanyellow-featheredchickensaged1–21days.




In the same row, means not sharing a similar superscript (a, b, c, d) differ significantly (*<sup>p</sup>* < 0.05), the number of replicates was used as the experimental unit (n = 6). 1 Standard error of the mean from ANOVA (n = 6). 2 Where Y is final body weight, average daily gain or average daily feed intake and X is total dietary content of methionine. 3 SSR = sum of squared residuals.

**Figure 1.** Regression models plot of growth performance as a function of total dietary Met level (0.28, 0.32%, 0.37%, 0.43%, 0.50%, and 0.63% Met) of male rapidly growing yellow-feathered chickens between 0 and 21 days of age. Correlation coefficient (*R2*), Akaike information criteria (AIC), and mean squares error (MSE) are indicators for evaluating model fitness. (**A**) The optimum response arrow pointing at 0.55% Met by quadratic polynomial (QP) model. (**B**) The optimum response arrow pointing at 0.55% Met by QP model. (**C**) The optimum response arrow pointing at 0.52% Met by QP model. (**D**) The optimum response arrow pointing at 0.58% Met according to the QP model.

**Figure 2.** *Cont*.

**Figure 2.** Regression models plot of growth performance as a function of total dietary Met level (0.28%, 0.32%, 0.37%, 0.43%, 0.50%, and 0.63% Met) of female rapidly growing yellow-feathered chickens between 0 and 21 days of age. Correlation coefficient (*R2*), Akaike information criteria (AIC), and mean squares error (MSE) are indicators used in evaluating fitness of models. (**A**) The optimum response arrow pointing at 0.51% Met by the best fitting regression model. (**B**) The optimum response arrow pointing at 0.51% Met identified by the best fitting model. (**C**) The optimum response arrow pointing at 0.51% Met.

**Figure 3.** Regression models plot of growth performance as a function of total dietary Met level (0.28%, 0.32%, 0.37%, 0.43%, 0.50%, and 0.63% Met) of mixed sex rapidly growing yellow-feathered chickens aged 0 and 21 days of age. Correlation coefficient (*R2*), Akaike information criteria (AIC) and mean squares error (MSE) are used as indicators for evaluation model fitness. (**A**) The optimum response arrow pointing at 0.53% Met according to QP model. (**B**) The optimum response arrow pointing at 0.53% Met by QP model. (**C**) The optimum response arrow pointing at 0.53% Met.

#### *3.2. Growth Performance of Guangxi Broilers Aged from 1 to 30 Days (Exp 2)*

The growth performance results of male, female and mixed genders of Guangxi yellow-feathered chickens as affected by dietary Met levels between 1 and 30 days of age are shown in Tables 5–7. The increase in dietary Met level showed linear and quadratic effects on the final BW (linear and quadratic, *p* < 0.01), ADG (linear and quadratic, *p* < 0.01) and ADFI (linear, *p* < 0.01; quadratic, *p* < 0.05) of males; final BW, ADG, and ADG (linear, *p* < 0.01; quadratic, *p* < 0.01) of females; and final BW (linear, *p* < 0.01; quadratic, *p* < 0.01), ADG (linear, *p* < 0.01; quadratic, *p* < 0.01), and ADFI (linear and quadratic, *p* < 0.05) of mixed genders. According to the EA and QP regression models, the optimal Met for the highest final BW were 0.51% and 0.53 in males, 0.43% and 0.51% in females, and 0.48% and 0.52% in the mixed males and females. The EA and QP indicated that the optimal Met for the maximal ADG were 0.51 and 0.53% in males, 0.43% and 0.51% in females, and 0.48% and 0.52% in mixed genders. Additionally, the QP model showed that 0.49% was optimal for the best FCR in mixed genders. Fitness of the two growth performance models are shown in Figures 4–6. QP models showed higher R2, lower AIC and MSE values, in estimating the optimal Met requirement (Figure 4a,b and Figure 6a,b) for growth performance of males, whereas the EA model in females and mixed sexes, had higher R2, and the lower AIC and MSE values. These results reveal that the QP model are more fitting to predict Met requirement for growing Guangxi males (0–30 days), but the EA function was better fitting for females and mixed sexes. The QP models predicted the requirement of Met for males as 0.53%, and the EA models predicted the requirements of Met for females and mixed genders as 0.43% and 0.48% to 0.49%, respectively.

**Figure 4.** Regression models plot of growth performance as a function of total dietary Met level (0.28%, 0.32%, 0.37%, 0.43%, 0.50%, and 0.63% Met) of male slowly growing yellow-feathered chickens between 0 and 30 days of age. Correlation coefficient (*R2*), Akaike information criteria (AIC) and mean squares error (MSE) are indicators used to evaluate fitness of models. (**A**) The optimum response arrow pointing at 0.53% Met by the best fitting model. (**B**) The optimum response arrow pointing at 0.53% Met by the best fitting regression model.



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**Table 6.** *Cont*.


3

2



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**Figure 5.** Regression models plot of growth performance as a function of total dietary Met level (0.28%, 0.32%, 0.37%, 0.43%, 0.50%, and 0.63% Met) of female slowly growing yellow-feathered chickens between 0 and 30 days of age. Correlation coefficient (*R2*), Akaike information criteria (AIC) and mean squares error (MSE) are indicators used in evaluating fitness of models. (**A**) The optimum response arrow pointing at 0.43% Met by the best fitting model. (**B**) The optimum response arrow pointing at 0.43% Met identified by the best fitting regression model.

**Figure 6.** Regression models plot of growth performance as a function of dietary total Met level (0.28%, 0.32%, 0.37%, 0.43%, 0.50%, and 0.63% Met) of mixed sexes of slowly growing yellow-feathered chickens between 0 and 30 days of age. Correlation coefficient (*R2*), Akaike information criteria (AIC) and mean squares error (MSE) are indicators of models fitness. (**A**) The optimum response arrow pointing at 0.48% Met by the best fitting model. (**B**) The optimum response arrow pointing at 0.48% Met identified by the best fitting model. (**C**) The optimum response arrow pointing at 0.49% Met.

#### **4. Discussion**

Diets having low or excessive Met levels could engender important influences on poultry performance [21]. An optimal dietary Met concentration could significantly improve growth performance of broiler chickens when the Met level was lower in the diet [4,5,22–24]. The results of the present study confirm that increasing the dietary Met level improved growth performance of Liangnan and Guangxi chicks. This improvement in growth performance is attributable to the important roles of Met in animal's body, as mentioned in the introduction. Tsiagbe et al. [25] demonstrated that the Met has direct influences on growth performance and immunity in meat-type chickens. In a like manner, Carew et al. [26] reported that Met deficiency reduces the growth and development of lymphoid organs, which have negative effects on growth. The excess of Met, higher than the requirement, is used to cover requirement of some important amino acids such as cystine; two molecules of Met are used in synthesizing one molecule of cystine [27], whereas Met requirement can be covered only by dietary methionine. Additionally, the results here indicated that an excess of dietary Met level did not synchronously improve growth performance of birds and even showed a tendency of negative effects, which was consistent with the findings of Jamroz et al. [28]. Excessive levels of dietary Met can have negative effects on growth. The results of D'Mello and D'Mello [29] indicated that the dietary addition of 20 or 40 g/kg of excess methionine decreased feed intake and reduced body weight gain. Edmonds and Baker [30] found that using an excess of Met at 4% of a corn-soybean meal diet containing 23% protein reduced body weight gain by 92%, whereas similar excesses of lysine, tryptophan, and threonine were far less toxic.

The Met requirement of Chinese yellowed-feathered chicks (unsexed) was 0.46% at the starter phase, according to the old estimations of [16]. According to the results here, however, the Met requirement of Chickens differed between males and females. These results agree with previous findings [4,5,17], which indicated that chicken males and females have different Met requirements. In the present study, the estimated Met requirements of male and female Lingnan, and male Guangxi chickens, determined according to EA and QP models, were higher than the old recommended level (0.46%) of FSC [16]; but the estimated Met requirement of female Guangxi chicken was lower than the recommended value [16]. According to NRC [27], the requirements of Met and Met + Cystine for commercial broiler chickens during the starter period (0–3 weeks) are 0.5% and 0.9% of the diet. The obtained Met requirements here were obtained in the presence of 0.26% cystine (Table 1) in the basal diet. The different Met requirements between breeds (fast and slow growing) or between both sexes is logically attributed to differences in growth rates and genetic potential [31]. Kalinowski et al. [24], reported that Ross 308 broilers optimized final BW (794 g) with dietary Met of 0.50% during 0–21 days; whereas Xi et al. [5], found that the yellow-feathered chicken only needed 0.433–0.435% Met (male) and 0.445–0.454% Met (female) for optimal 21 day BW (male: 351.12 g; female:314.37 g) in the starter phase. According to the available information on the FSC [16] and feeding management regulations of the yellow feathered-chicken [15], the Lingnan chicken breed is classified as a fast-growing breed, and the Guanxi chicken is a slow-growing breed; this can explain the differences in Met requirements obtained here between the two breeds. The different starter phase durations of the Lingnan (0–21 days), and Guangxi (0–30 days) are originally dependent on their growth rate and genetic potential, which have caused the differences in the optimal Met requirement of the two breeds. Similar results were reported by Kalinowski et al. [24] and Dozier et al. [32]. Additionally, several studies indicated that evaluating the response of more than one variable to a dietary nutrient makes it difficult to determine a unique value of the nutrient requirement, i.e., the optimal level of any nutrient required for obtaining the best result of growth rate, body weight, feed intake, feed conversion ratio, meat quality, or immunity indices can differ [2,3,33]; this can explain the different values of optimal Met level for the different growth variables here.

#### **5. Conclusions**

Supplementation of graded Met levels in basal diets of yellow feathered broilers during the starter period showed beneficial influences on their growth performance indices. The results indicated that the optimal Met requirement differed between the fast (up to 21 d of age) and slow-growing (up to 30 days of age) yellow-feathered chicken breeds as well as between males and females of each breed. The QP regression model was more appropriate for estimating the optimal Met requirements for the best body weight, average daily gain, feed intake and FCR indices of Lingnan (fast-growing) males (0.52% to 0.58%), females (0.51%), and mixed genders (0.53%), as well as for Guangxi (slow-growing) males (0.53%), whereas the EA model was found to be better for estimating the optimal Met requirement of Guangxi females (0.43%) and mixed genders (0.48% to 0.49%).

**Author Contributions:** L.L., K.F.M.A., Z.C., A.E.G.G.-E., Q.F., F.D., and J.G., participated in acquisition of data, and analysis and/or interpretation of data; L.L., K.F.M.A., A.E.G.G.-G., S.J., and Z.J., participated in drafting the manuscript; S.J. and Z.J. participated in fund acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by National Key R&D Project (2018 YFD0500600), National Natural Science Foundation of China (31802104), Presidential Foundation of the Guangdong Academy of Agricultural Sciences, P. R. China (201810B), 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. The Outstanding Talents Training Program of Guangdong Academy of Agricultural Sciences; Operating Funds for Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition (2014B030301054; Supporting Program for Guangdong Agricultural Research & Development Center of Livestock and Poultry Healthy Breeding.

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

#### **References**


© 2020 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*

### **Ameliorative E**ff**ects of Antibiotic-, Probiotic- and Phytobiotic-Supplemented Diets on the Performance, Intestinal Health, Carcass Traits, and Meat Quality of** *Clostridium perfringens***-Infected Broilers**

**Elsayed O.S. Hussein 1,\*, Shamseldein H. Ahmed 2, Alaeldein M. Abudabos 1, Gamaleldin M. Suliman 1, Mohamed E. Abd El-Hack 3, Ayman A. Swelum 1,4 and Abdullah N. Alowaimer <sup>1</sup>**


Received: 26 February 2020; Accepted: 10 April 2020; Published: 12 April 2020

**Simple Summary:** Necrotic enteritis is considered the most important economic problem for the poultry industry due to the sudden death rates of up to 50%. However, there is limited information concerning the ameliorative role of probiotic and/or phytobiotic compounds in the prevention of *Clostridium perfringens* infections in broilers. Hence, this trial is conducted to evaluate the influence of some antibiotic, probiotic and phytobiotic compounds (Maxus, CloStat, Sangrovit Extra, CloStat + Sangrovit Extra, and Gallipro Tect) on the growth performance, carcass traits, intestinal health, and meat quality of broiler chicks. The obtained in vivo results highlight that a probiotic- and/or phytobiotic-supplemented diet has many positive effects on the performance, organ weight, and meat quality of broilers. Besides, a notable reduction in the lesion score is observed with a combined probiotic and phytobiotic diet.

**Abstract:** The poultry industry needs efficient antibiotic alternatives to prevent necrotic enteritis (NE) infections. Here, we evaluate the effects of probiotic and/or prebiotic dietary supplementation on performance, meat quality and carcass traits, using only an NE coinfection model, in broiler chickens. Three hundred and twenty-four healthy Ross 308 broiler chicks are allocated into six groups. Taking a 35 d feeding trial, the chicks are fed a basal diet with 0.0, 0.1, 0.5, 0.12, 0.5 + 0.12, and 0.2 g Kg−<sup>1</sup> for the control (T1), Avilamycin (Maxus; T2), live probiotic (CloStat (*Bacillus subtilis*);T3), natural phytobiotic compounds (Sangrovit Extra (sanguinarine and protopine); T4), CloStat + Sangrovit Extra (T5), and spore probiotic strain (Gallipro Tect (*Bacillus subtilis* spores); T6) treatments, respectively. Occurring at 15 days-old, chicks are inoculated with *Clostridium perfringens.* The obtained results reveal that all feed additives improve the performance, feed efficiency, and survival rate, and reduces the intestinal lesions score compared with the control group. The T6 followed by T3 groups show a significant (*p* < 0.05) increase in some carcass traits, such as dressing, spleen, and thymus percentages compared with other treatments. Also, T5 and T6 have significantly recorded the lowest temperature and pHu values and the highest hardness and chewiness texture values compared to the other treated groups. To conclude, probiotics combined with prebiotic supplementation improves the growth, meat quality, carcass characterization and survival rate of NE-infected broiler chickens by modulating gut health conditions and decreasing lesion scores. Moreover, it could be useful as an ameliorated NE disease alternative to antibiotics in *C. perfringens* coinfected poultry.

**Keywords:** probiotic; *Clostridium perfringens*; phytobiotic; broiler

#### **1. Introduction**

*Clostridium perfringens* is a Gram-positive bacterium which is common within ecosystems and healthy intestinal microflora [1]. *C. perfringens* is responsible for several diseases in humans, wildlife, and farm animals [2], and is the leading cause of necrotic enteric (NE) disease in farm animals, especially in poultry [3]. Regarding birds, NE disease is caused by specific strains of *C. perfringens*. It costs the global poultry industry over two billion dollars annually, mainly due to the high costs of antibiotics and inactive feed conversion [4,5]. Several studies have reported that *C. perfringens* bacteria could produce more than 15 different toxins [6,7]. Although all *C. perfringens* types can induce α-toxin [3], this kind of toxin causes serious enteric and intestinal diseases in animals and humans [8]. Infected birds show severe lesions of the jejunum and ileum, with the small intestine presenting a degenerated mucosa and is distended by gases produced by *C. perfringens* [9]. Signs of infection include depression, reduced mobility, and diarrhoea, which is the most visibly obvious symptom [10].

Several strategies are commonly used to alleviate the symptoms of enteritis in broilers, including the use of probiotics [11]. It has been recognized that administering antibiotics as feed additives can avoid mortalities induced by NE [12]. Avilamycin is an antibiotic of the oligosaccharides family and is widely active against Gram-positive bacteria [13]. Moreover, Avilamycin has been shown to have potent bactericidal effects on *C. perfringens* in vitro [11]. Similar results were found by Paradis et al. [11] and Mwangi et al. [14], who reported linear relationships between the level of Avilamycin in the feed and a reduction in NE mortalities, NE lesion scores, and intestinal *C. perfringens* count.

A probiotic is a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance [15–17]. Probiotics can interact with the host to improve immunity and intestinal morphology or stimulate the metabolism, thus reducing the risk of infection by opportunistic pathogens [18]. Probiotic bacteria also have been shown to produce molecules with antimicrobial activities, such as bacteriocins, which target specific pathogens or inhibit the adhesion of pathogens or the production of pathogenic toxins [19,20]. Moreover, beneficial bacteria can act as competition against pathogenic strains within the host [21]. A large number of studies have described the isolation of some strains belonging to the genera *Bacillus* and *Lactobacillus*, which exhibit anti*-C. perfringens* activity in vitro [22,23]. The supplementation of animal feed with *Bacillus* spores (*B. licheniformis*) also was tested and proven to be an efficient alternative to antibiotics when used in larger amounts and for longer periods [24]. When 20 day-old chicks, inoculated with low amounts of *C. perfringens*, were given a single dose of 109 *B. subtilis* spores, the colonization and persistence of *C. perfringens* were abolished. However, the *B. subtilis* strain alone was unable to affect *C. perfringens* in vitro [25]. Also, Sokale et al. [26] reported that using *Bacillus subtilis* as a feed supplement in broiler chicks increased the performance and reduced mortality in the chicks treated with *C. perfringens*. The supplementation of *B. subtilis* not only controlled *C. perfringens*-induced NE, but also improved the intestinal health of the broilers [27].

Several natural products such as herbs, spices and essential oils are categorized under the term botanic, phytobiotic or phytogenic compounds [28–30]. Phytobiotics are well identified for their antibacterial and pharmacological effects and, thus, are commonly used in broiler feed as growth promoters and alternative medicines [17]. A huge number of in vitro and in vivo studies have approved a varied range of activities for phytobiotics in poultry nutrition, like stimulation of feed intake, or antimicrobial, coccidiostatic, anthelmintic and immunostimulating actions [31]. Abudabos et al. [32] reported that using Sanguinarine as a feed supplement in broiler chicks (Ross 308) challenged with *Clostridium perfringens* enhanced performance, carcass traits and some blood biochemical parameters. Also, El–Sheikh et al. [33] informed that the prebiotic supplemented (Sanguinarine) diet could be an effective treatment alternative to antibiotics for controlling necrotic enteritis diseases in broilers. Diverse types of additives include phytobiotics (primary or secondary components of plants that contain bioactive compounds that exert a

positive effect on the growth and health of animals) which could be a beneficial strategy that regulates the gastro-intestinal microbial community and improve broiler health [34].

Found in the literature, the positive effects of both *B. subtilis* and phytobiotic compounds have been reported. However, their synergistic effects have not been described yet. The present study aims to evaluate the ameliorative effects of probiotic and phytobiotic compounds alone or in a combined form of two different types of *B. subtilis* on the performance, carcass traits, meat measurements, and intestinal health of *C. perfringens*-infected broilers during the starter and finisher phases.

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

The experiment was performed in cage pens under similar managerial and hygienic conditions in an environmentally controlled poultry unit at the Animal Production Department, College of Food Science and Agriculture Science, King Saud University. All protocols were chosen according to the experimentation guidelines of the Animal Use Ethics in Research Committee of King Saud University (approval number: SE-19-150).

#### *2.1. Experimental Design and Feeding Regime*

Altogether, 324 day-old broiler chicks (Ross 308 strain) were randomly distributed into six groups. Each group contained nine replicates, with six birds per replicate, and were used for a 35 d feeding trial period. Each group was assigned to one of the following dietary treatments: T1; basal diet (control), T2; diet supplemented with 0.1 g kg<sup>−</sup><sup>1</sup> of Maxus (antibiotic), T3; diet supplemented with 0.5 g kg<sup>−</sup><sup>1</sup> of Clostat (natural probiotic strain), T4; diet supplemented with 0.12 g kg<sup>−</sup><sup>1</sup> of Sangrovit (phytobiotic), T5; diet supplemented with 0.5 g kg−<sup>1</sup> of Clostat combined with 0.12 g kg−<sup>1</sup> of Sangrovit, and T6; diet supplemented with 0.2 g kg−<sup>1</sup> of Gallipro Tect (spore probiotic). The chicks were fed a starter diet between days 0–14, which was then switched to a grower diet between days 15–35 (Table 1). After 14 d, all treated groups were inoculated with *C. perfringens* bacteria. Birds were fed ad libitum, and water was available at all times during the experimental period.

Maxus was manufactured by BIOFERM CZ, spol. Sro. Banskobystrická 461, 621 00 Brno-Reˇ ˇ ckovice a Mokrá Hora, Czechia, as a source of Avilamycin antibiotics (each 1000 g, containing 100 g of Avilamycin) in feed diets, while, CloStat products were manufactured by KERMIN Ind., Inc., 2100 Maury Street Des Moines, IA 50317 USA (each 1 g, containing 2 <sup>×</sup> 107 CFU/g *Bacillus subtilis*). The Sangrovit Extra used phytobiotic compounds (extracts of Benzophenanthridine alkaloids (sanguinarine) and protopine) produced by Albitalia s.r.L., Co., Milano, Italy. Gallipro Tect were used as a source of a highly-selected strain (DSM17299) of *Bacillus subtilis* (*B. subtilis* 4 <sup>×</sup> 109 CFU/g DSM 17299), and was provided by Boege Alle Co., Hoersholm, Denmark.

#### *2.2. Challenge with Clostridium perfringens Bacteria*

The *C. perfringens* challenge model was performed as described by Prescott [35]. All treated groups received a *C. perfringens* challenge at a rate of 4 <sup>×</sup> 10<sup>8</sup> CFU g−<sup>1</sup> via oral gavages on day 14, as recommended by Olkowski et al. [36], using the defined B positive bacteria *C. perfringens* isolated from a local farm. The identified bacterium was previously confirmed to be sensitive to antibiotics (Avilamycin) using the broth dilution method (MIC testing) [37]. An inoculum was equipped to contain nearly 10<sup>8</sup> cells of *C. perfringens* per ml and was processed at a ratio of 1:1.5 ration-to-broth [11]. The challenge feed was mixed with the treatment diet. After the administration of the challenged material, dead birds were counted as study mortalities. These birds underwent a necropsy evaluation to estimate the cause of death/disease.


**Table 1.** Composition of starter and finisher diets.

<sup>1</sup> V–M premix; 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.

#### *2.3. Growth Performance Parameters*

During the starter (0–14-days-old) and finisher (15–35-days-old) periods, the growth and feed efficiency parameters of the broiler chicks were estimated. The daily feed intake was calculated by subtracting the quantity of feed rejected from the feed offered. Additionally, live body weight was estimated at biweekly intervals, while the final body weight and total feed consumption were determined at the end of each trial period. The body weight gain (BWG) was measured by calculating the difference between the live body weight and final body weight for each trial period. The feed conversion ratio (FCR) was computed for each group, as mentioned by Abudabos et al. [32], using the following formula:

$$\text{FCR} = \text{Feed intake/Weight gain} \tag{1}$$

Meanwhile, the production efficiency factor (PEF) was calculated as suggested by Griffin [38], using the following formula:

$$\text{PEF} = \text{(Liavisibility} \times \text{Live weight (kg))} \newline \text{(Age in days} \times \text{FCR)} \times 100 \tag{2}$$

During the feeding trial, the number of deaths was counted to calculate the survival rate, as mentioned by Hussein et al. [39], using the following formula:

$$\text{Survival rate (SR)}\% = \text{(the number of the surrounding providers/the initial number of broilers)} \times 100\tag{3}$$

#### *2.4. Carcass Measurements and Lesion Scores*

Ten broiler chicks were randomly collected from each group to estimate the organ weights (heart, gizzard, liver, bursa, spleen, thymus, small and large intestine, and ceca). Chicks were weighed before sacrifice. All internal organs were weighed immediately after slaughter. The gizzard was weighed after its content was removed. The small intestine was measured by determining the distance between

the site of the duodenum emergence from the gizzard and the beginning of the ceca. The organ weight was calculated relative to the live body weight.

According to the Hofacre et al. [40] procedure, two birds per pen were examined for gross intestinal lesions. The characteristic of necrotic enteritis was defined using the description of the Long et al. [41] study. The lesion scores were outlined as follows: 0 = none, 1 = mild, 2 = moderate and 3 = marked (severe) [42].

#### *2.5. Meat Characteristics*

At the end of the finisher period (35-days-old chicks), three birds were randomly selected per pen to estimate the meat characteristics followed by the Chen et al. [43] method. After euthanasia, the jugular vein was cut, the feathers, heads, and shanks were removed, and the remaining carcasses were dissected. The left and right breast from each bird were used for the quality measurements. The breast samples were stored at −80 °C until further analysis. At the time of the analysis, frozen muscles were thawed overnight in a chiller at 4 °C.

The pH of the breast muscle was measured twice (after slaughtering and 24 h postmortem), using a microprocessor pH-Meter (Model PH 211, Hanna Instruments, Padova, Italy). The core temperature in the breast muscle was measured after slaughtering with a portable digital thermocouple (Eco Scan Series, Temp JKT, Eu tech Instruments, 7 Gul Circle, level 2M, Keppel Logistic Building, Singapore 629563). The colour values of the CIELAB Color System (L\*(lightness), a\* (redness), and b\* (yellowness)) were determined for the breast muscles 15 min and 24 h after slaughtering, using a Chroma meter (Konica Minolta, CR-400-Japan) following the method used by Castellini et al. [44].

The myofibril fragmentation index (MFI) of the breast muscle was determined by multiplying the absorbance value at 540 nm, as described by Culler et al. [45]. The water-holding capacity (WHC) was determined based on the technique described by Hamm [46] and following the modification performed by Wilhelm et al. [47], using the following equation:

$$\text{WHC} = 100 - \left[ (\text{Wi} - \text{Wf}/\text{Wi}) \times 100 \right] \tag{4}$$

where, Wi and Wf are the initial and final sample weights, respectively.

The drip loss (DL) was determined as a percentage based on the initial sample weight. The cooking loss (CL) was determined as the difference between the initial and final weights. Then, cooked samples were used to evaluate the shear force according to the procedure described by Wheeler et al. [48], under a 200 mm/min crosshead speed. The texture profile analysis (TPA) values were estimated using a Texture Analyzer (TA–HD–Stable MicroSystems, Golborne, Warrington WA3 3GR, England) equipped with a compression-platen attachment. The variables determined included the hardness (maximum force needed to compress the sample), cohesiveness (ratio between the total energy required for the first and second compression), springiness (the ability of a sample to recover to its original form after the removal of the compressing force), and chewiness (a resultant of springiness × hardness × cohesiveness).

#### *2.6. Statistical Analysis*

The data underwent a one-way ANOVA using a completely randomized design. Before analysis, the data were examined for normality of distributions and homogeneity of variance. Percentage data were subjected to arcsine transformation before analysis. SPSS 22 analysis software was used for all statistical analyses. Data are expressed as the mean ± SEM, with a statistical significance level of *p* ≤ 0.05. Significant differences between means were determined using Duncan's Multiple Range [49].

#### **3. Results**

#### *3.1. Growth Performance and Intestinal Lesions Score*

The effects of some feed additives on the total feed intake (TFI), body weight gain (BWG), feed conversion ratio (FCR), and protein efficiency ratio (PEF) of broilers in the starter and finisher periods are shown in Table 2. During the starter period, our results revealed no significant differences between all treatment and non-treatment groups. T4 produced the lowest TFI values, while T3 and T1 exhibited the highest. Regarding the FCR values, both the T4 and T5 treatments had lower values than those of the other treatments. The dietary supplementation of probiotic bacteria increased the PEF (except for T3) compared with the control group.

**Table 2.** Effects of some feed additives on the growth performance, survival rate, and intestinal lesion score of broilers challenged with *C. perfringens* during the starter and finisher feeding periods.


IBW, initial body weight; BW 14 d, body weight at 14 days of age; FBW, final body weight; TFI, total feed intake; BWG, bodyweight gain; FCR, feed conversion ratio; PEF, protein efficiency ratio; and SR, survival rate. SEM, mean values of the standard error. Mean values of three replicates with deferent letters (a,b,c) in the same row are significantly different (*p* < 0.05). Sig, significance; NS, non-significance; \*\*\* significance at *p* < 0.001.

During the finisher period, all experimental additives significantly increased (*p* < 0.01) the FBW, BWG, FCR, PEF, survival rate (SR), and decreased the intestinal lesions score compared with those of the control group (Table 2). However, the TFI was not significantly affected by the supplemented diets. The T3, T4 and T5 groups had higher BWG values compared with those of the other groups. Additionally, the T3-treated group exhibited the best FCR values, while the different treatment groups produced intermediate FCR values. Regarding the PEF values, compared with the control, all experimental additives were significantly enhanced (*p* < 0.05). The SR (%) and lesions score were enhanced considerably by the dietary treatments at 15–35 days of age (Table 2).

#### *3.2. Carcass Measurements*

The effects of supplemented diets on carcass traits after a 35-day feeding trial are presented in Table 3. Regarding the T6 treatment group, the dressing percentage showed significant (*p* < 0.05) increases. Also, the T1 and T3 treatment groups recorded the highest significant thymus values, while the lowest values were recorded for T4. Meanwhile, the highest significant (*p* < 0.05) spleen weight percentage value was recorded in the T5 group.


**Table 3.** Effects of some feed additives on the carcass traits of broilers challenged with *C. perfringens* during the starter and finisher feeding periods.

DP, dressing percentage. SEM, mean values of the standard error. Mean values of three replicates with different letters (a,b,c) in the same row are significantly different (*p* < 0.05). Sig, significance; NS, non-significance; \*, significance at *p* < 0.05; \*\*, significance at *p* < 0.01.

#### *3.3. Meat Characteristics*

The effects of feed additives on broiler meat quality parameters are presented in Table 4. The T3 and T5 treatments resulted in the lowest pHi values, with the highest values observed in T2. Conversely, the pHu was significantly reduced by the treatments, with the lowest values recorded in T5 and T6. Furthermore, the Temperature was significantly reduced in T6 followed by T5-treated groups compared with other treated groups. However, no significant differences were demonstrated among the other treatment groups concerning the colour component after slaughter (L15 and b15) and 24 h (L24, a24, and b24) at 35 days of age.


**Table 4.** Effects of some feed additives on the meat quality of broilers challenged with *C. perfringens* during a feeding trial period.

pHi, meat pH after slaughter; pHu, meat pH after 24 h; Temp., carcass temperature after slaughter; L15, a15, and b15, colour components after slaughter; and L\*24, a\*24, and b\*24, colour components after 24 h. SEM, mean values of the standard error. Mean values of three replicates with different letters (a,b,c) in the same row are significantly different (*p* < 0.05). Sig, significance; NS, non-significance; \*\*, significance at *p* < 0.01; \*\*\*, significance at *p* < 0.001.

The results presented in Table 5 reveal the effects of feed additives on meat characteristics, such as the Cooking Loss (CL), Water Holding Capacity (WHC), Myofiber Fragmentation Index (MFI), Shear Force (SF) and texture profile analysis (TPA) at 35 days of age under a *C. perfringens* challenge test. All treatment groups showed no significant differences concerning the CL, WHC, MFI, and SF. The T6 and T5 treatments exhibited significantly increased (*p* < 0.001) hardness and chewiness values compared with those of other treatment groups, respectively. Although the dietary supplementation showed no significant differences in Springiness and Cohesiveness values between all treated groups.


**Table 5.** Effects of some feed additives on the meat characteristics of broilers challenged with *C. perfringens* during a feeding trial period.

CL, cooking loss; WHC, water holding capacity; MFI, Myofiber Fragmentation Index; SF, shear force; and TPA, texture profile analysis. SEM, mean values of the standard error. Mean values of three replicates with different letters ( a,b,c) in the same row are significantly different (*p* < 0.05). Sig, significance; NS, non-significance; \*\*\*, significance at *p* < 0.001.

#### **4. Discussion**

Necrotic enteritis (NE) has become one of the most critical problems in the poultry industry [50]. Feed additives, including antibiotics, prebiotics, and probiotics, have frequently been used for improving the health, growth, and feed efficiency parameters of animals [51]. Recently, interest in incorporating probiotics and antibiotics into broiler treatments has been rapidly increasing [52]. Several studies have shown that the addition of probiotics has positive effects on the growth rate, feed utilization, feed efficiency, and mortality rate [53,54]. However, the efficacy of probiotics depends upon the selection of more efficient strains, manipulation of genes, a combination of several strains, and the combination of probiotics and synergistically=acting components [55]. The use of multi-strain probiotics seems to be the best way of potentiating the efficacy of probiotics, as it beneficially affects the host by improving growth-promoting bacteria with competitive antagonism against pathogenic bacteria in the gastrointestinal tract [56].

#### *4.1. Growth Performance*

The performance results obtained in the starter period and the finisher period are presented in Table 2. The IBW, TFI, BWG, and FCR of all groups receiving feed-supplemented diets and basal diets had no significant differences among them. However, the BWG, FBW, PEF, FCR, and SR during the finisher phase, at 15–35 days-old, were significantly (*p* < 0.05) improved in all supplemented diets compared to the control group. These results are similar to those of Khaksefidi and Ghoorchi [57], who found that the BWG of birds fed a diet supplemented with 50 mg/kg of *Bacillus subtilis* was significantly higher during the finisher period (22–42 d) than birds fed the control diets. Additionally, the feed conversion ratio of birds fed a diet supplemented with probiotics significantly reduced from 22 to 42 d compared with birds fed the control diets. Consequently, Patel et al. [55] indicated that the dietary supplementation of combined probiotics and prebiotics (Protexin) at 100 g/ton of feed significantly enhanced the BWG, along with an improved FCR, and benefitted without any adverse effects on the feed intake, mortality, or carcass characteristics. Also, Anjum et al. [58] and Singh et al. [59] observed similar results. The improvement of all performance parameters may be due to the biological role of probiotics in altering the intestinal pH, which modifies both the microbial population and nutrient absorption, ultimately improving the efficiency of feed utilization [60]. Moreover, increased feed intake and digestive enzyme secretions also are detected in animals' fortified phytobiotic-supplemented feed [61]. Growth enhancement through the use of phytobiotics probably depends on the synergistic effects among complex active molecules present in phytobiotics [39,62].

The mortality rate in this study was low (finisher period), representing the positive effect of feed additives on the mortality rate (Table 2). These findings were similar to those found by Abdel–Hafeez et al. [63] and Riad et al. [64], who indicated that the addition of probiotics as feed additives decreased the mortality rate. The reduction in mortality was attributed to the inhibitory effects of these additives toward enteric microorganisms via modifying the intestinal pH [63]. Compared to a wide range of antibiotics (including Avilamycin), a significant decrease in mortality was seen for treated broilers with 2 g/kg Bio–Mannan-oligosaccharides (Bio–MOS) as a source of prebiotic, depending on the age of birds [65,66]. Moreover, there are major modes of action by which broiler performance is improved by proposed oligosaccharide prebiotics, such as control of type-1fimbriae pathogenic bacteria (mannose-sensitive lectin), immune modulation effects, and modulation of intestinal morphology and expression of mucin and brush border enzymes [67].

#### *4.2. Carcass Traits*

The mean values for the carcass characteristics, dressing percentage, and organ weight (%) relative to the bodyweight of the broilers are presented in Table 3. The dressing percentage was significantly (*p* < 0.05) increased in the T6 group compared with those of the other treatments. However, non-significant differences were observed in the other carcass measurements amongst the treatment groups. Some organ weight (spleen and thymus) significantly increased in probiotic supplemented diets alone or in combination with prebiotics compared with other treated groups. Generally, the inclusion of probiotics in broiler rations had no extra additional benefits for the organ weight or carcass yield [55]. Also, Salamkhan et al. [68], Panda et al. [69], and Patel et al. [55] reported that the dressing percentage did not differ significantly (*p* ≤ 0.05) between the probiotics-fed broilers and control broilers. To contrast, Banday and Risam [70] and Kabir et al. [71] observed a significant (*p* ≤ 0.05) improvement in the dressing percentage in groups supplemented with probiotics. Nevertheless, Sarangi et al. [72] reported a highly significant increment in breast yield in birds supplemented with prebiotics. Jin et al. [73] attributed the lower fat deposition in birds treated with probiotics to how the product could interfere in the availability of fat for lipogenesis in the birds.

#### *4.3. Meat Quality Parameters*

The effects of antibiotic-, probiotic-, and phytobiotic-supplemented diets on the measurements of meat characteristics under *C. perfringens* infection in broilers are presented in Table 4. The change in pH is one of the most significant changes that can occur during rigor mortis and can affect meat quality characteristics, such as texture, colour, and WHC [74]. Fletcher et al. [75] reported that post-mortem pH measurements are a good indicator of meat characteristics. Generally, a rapid post-mortem pH decline in breast meat can lead to protein denaturation, which may result in a pale colour and low WHC [76]. During this trial, the temperature and pH values obtained after slaughter and 24 h post-slaughter showed significant (*p* ≤ 0.05) enhancements for all supplemented diets compared with the group fed the basal diet. These results are similar to those found by Battula et al. [77] and Corzo et al. [78] where the bacterial challenge had negative impacts on the temperature and pH of the breast muscle.

Additionally, the cooking loses (CL) is a measure of the percentage of water lost during cooking as a result of shrinkage. Consequently, the degree of shrinkage upon cooking is directly correlated with a loss of juiciness. During this trial, the CL, WHC, and SF showed non-significant differences between the control and treatment groups. During a subsequent study, Pelicano et al. [79] examined the effects of *Lactobacillus-* and *Bacillus subtilis*-based probiotics on SF and reported non-significant values compared with the control group. However, the MFI of the breast muscle was influenced by the treatments (*p* ≤ 0.05), as shown in Table 4. The MFI decreased in T2 and T3 compared with the other treatment groups. Myofibrillar fragmentation is the extent of myofibril destruction caused by homogenization [80]. Olson and Stromer [81] reported that MFI values are correlated with other muscle indices, such as SF and tenderness. Therefore, it can be concluded that probiotic supplementation causes less damage to the myofibrils [76].

TPA is an instrumental measurement of the sensory attributes of chicken breasts which imitates the conditions to which food is subjected to in the mouth [76]. The TPA, which includes hardness, cohesiveness, springiness, and chewiness, is shown in Table 5. The treatments had no significant effects on any TPA variable (*p* ≤ 0.05). All tested groups were very tender, except for the T6 and T5 supplemented groups, which had hardness values that were significantly (*p* ≤ 0.001) higher than those of the other treatment groups. The data presented in this trial aligns with that of Angelovicova et al. [82] who noticed that probiotics (CloStat) moderately affect the hardness, cohesiveness, springiness, and chewiness of cooked breast meat. Also, Lisowski et al. [83] investigated a beneficial effect of the prebiotic on breast muscle weight and carcass percentage. It could be supposed on the one hand, that using prebiotics in broiler diets has a positive influence on muscle weight [84].

#### **5. Conclusions**

To conclude, fortified feed diets with 0.2, 0.5, 0.12 g kg−<sup>1</sup> of *Bacillus subtilis* spores (Gallipro Tect), live *Bacillus* strain (CloStat) and phytobiotic natural compounds (Sangrovit Extra) respectively, alone or in combined form, could promote growth and reduce the NE mortality rates. Additionally, using these probiotic and phytobiotic compounds as an alternative for antibiotics in feed diets could be a useful strategy to ameliorate the harmful effects of *C. perfringens* bacterium on broilers, in terms of the performance, feed efficiency, meat quality and carcass characteristics regarding necrotic enteritis infections.

**Author Contributions:** Conceptualization, E.O.S.H. and S.H.A.; methodology, A.M.A. and G.M.S.; software, E.O.S.H., A.M.A. and G.M.S.; investigation, M.E.A.E.-H.; data curation, M.E.A.E-H. and A.A.S.; writing—original draft preparation, review and editing; A.N.A., visualization. All authors have read and agreed to the published version of the manuscript.

**Funding:** Authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through Research Group Project No. RGP-267.

**Conflicts of Interest:** Authors declare no conflict of interest.

#### **References**


© 2020 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*

### **Olive Cake Meal and** *Bacillus licheniformis* **Impacted the Growth Performance, Muscle Fatty Acid Content, and Health Status of Broiler Chickens**

### **Ahmed A. Saleh 1, Bilal Ahamad Paray <sup>2</sup> and Mahmoud A.O. Dawood 3,\***


Received: 19 February 2020; Accepted: 14 April 2020; Published: 16 April 2020

**Simple Summary:** The extraction of oils from olives usually results in large quantities of olive cake meal (OCM), which has a high nutritional value. The OCM is used successfully in livestock and poultry feeding, but due to the high fiber content, alternative methods of treating OCM must be considered. To increase the efficiency of OCM in broiler chickens' diet, it can be mixed with suitable microorganisms with beneficial effects. Hence, the current study investigated the influence of OCM and *Bacillus licheniformis* (BL) on the growth, nutrient utilization, blood chemistry, and muscle fatty acid profile of broilers. Birds were divided into six experimental groups (control, OCM (2%), OCM (4%), BL, OCM (2%)/BL, and OCM (4%)/BL groups). The results revealed that the inclusion of BL with OCM diets improved the fat utilization and, accordingly, increased the growth, nutrient utilization, and antioxidative response in broilers.

**Abstract:** Olive cake meal (OCM) is characterized by its high nutritional value and is used as an alternative source of protein and fats in poultry diets. However, due to the high percentage of fiber in OCM, beneficial bacteria cells are used to improve the digestion rates. Therefore, the influence of OCM and *Bacillus licheniformis* (BL) on the growth, nutrient utilization, blood chemistry, and muscle fatty acid profile of broilers was exclusively examined in this study. Three hundred and sixty birds were randomly divided into six experimental groups (6 replicates/10 birds each): Control, OCM (2%), OCM (4%), BL, OCM (2%)/BL, and OCM (4%)/BL groups. Although feed intake was not meaningfully influenced by dietary treatments, weight gain was enhanced and feed conversion ratio was reduced (*p* < 0.05). The abdominal fat was lowered in broilers fed OCM (2%), OCM (4%), OCM (2%)/BL, and OCM (4%)/BL diets without a difference to those fed BL only (*p* < 0.05). Interestingly, blood total protein, albumin, Newcastle disease (ND) titer, and high-density lipoprotein (HDL) cholesterol were significantly increased, while total cholesterol was decreased by the mixture of OCM and BL (*p* < 0.05). Muscle oleic and linoleic acids, as well as vitamin E, increased significantly in broilers fed both OCM (4%) and BL, while linolenic acid increased in all groups except those fed BL and control diets (*p* < 0.05). Liver malondialdehyde (MDA) was decreased by feeding BL or both OCM at 2% or 4% and BL (*p* < 0.05). In conclusion, the inclusion of BL to OCM diets resulted in improved fat utilization and, accordingly, enhanced growth, nutrient utilization, and antioxidative response in broilers. Based on the obtained results, it is recommended to use BL to improve the nutritional value of OCM and to increase the feed utilization of OCM by broilers.

**Keywords:** broilers; alternative ingredients; probiotics; growth; lipid peroxidation

#### **1. Introduction**

As a result of increasing demand, limited supply, and a dramatic increase in the prices of feed ingredients, suitable alternative sources for poultry feed have recently been intensively studied [1,2]. Feed cost may account for more than 70% of the total production costs of broilers [3,4]. Any reduction in feed costs, which still preserves the health status of broilers, is bound to have a direct positive effect on the profitability of poultry production. A considerable effort has been applied to find alternative and sustainable protein sources to be included in broilers diets [5]. In this context, among the available plant protein alternatives, olive cake meal which has high nutritional value (lipids, 13–15%, and proteins, 9–10%), with a high level of non-starch polysaccharides (NSP) (xyloglucan and xylan-xyloglucan complexes) [3,6–8]. The extraction of oils from olives usually results in large quantities of olive cake meal. The olive cake meal is available in several countries around the world at reasonable prices and can be used as a plant ingredient in the feed of broilers [7]. Potential problems in olive cake meal feeding exist due to the existence of fiber and high levels of unsaturated fatty acids, which can cause high fatty acid pre-oxidation, malnutrition, and lower palatability [3]. Olive cake meal was used successfully in poultry diets of up to 10% of the total ration [3,9]. To increase the efficiency of olive cake meal in broilers' diet, it can be mixed with suitable microorganisms to obtain beneficial effects.

A probiotic is defined as "live strains of strictly selected microorganisms which, when administered in adequate amounts, confer a health benefit on the host" [10]. Using probiotic-enriched diets is an inexpensive practice that can be adopted by both small- and large-scale farmers, and which can offer several benefits from increasing broilers' growth to increasing immune parameters and disease resistance [11,12]. The use of probiotics has increased due to its remarkable beneficial effects on microbiota and gut health in swine [13], poultry [14], and rabbits [15–17]. In parallel, the significant role of probiotic bacterium on growth, intestinal microbiota, and immunological responses in broilers has been demonstrated [14,18,19]. Indeed, lactic acid bacterial species are unique strains of probiotics authorized by the Food and Drug Administration for administration in animals [10,20]. Furthermore, dietary *Bacillus licheniformis* has been shown to increase growth performance and feed efficiency due to the secretion of digestive enzymes that can increase the digestibility of nutrients in the animal's gut [21–25].

This offers a new topic for researchers, where a combination of the plant by-products and the probiotics mixture is used in poultry feeds. Al-Harthi [3] concluded that there was no adverse effect on the performance of broilers when fed a dietary olive cake meal and *Saccharomyces cerevisiae* blend. Sateri et al. [9] were also able to include up to 8% olive cake meal with a digestive enzyme mixture in the diet of broilers. To date, no data are available about the use of olive cake meal mixed with *B. licheniformis* in the diet of broilers.

With the continued increase in broiler production, it is necessary to find non-traditional alternative ingredients for use in the preparation of feed [2]. Therefore, the objective of the current study was to evaluate the effects of olive cake meal mixed with *B. licheniformis* on the performance parameters, the muscle fatty acid content, and the blood parameters of broilers.

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

#### *2.1. Birds and Experimental Design*

All of the experimental procedures in this study followed the guidelines set by the Institutional Animal Care and Use Committee at Kafrelsheikh University (Number 4/2016 EC). A total of 360 one-day-old male broiler chickens (45.7 g) were placed inside a room equipped with 36 floor bins (10 birds each) (6 treatments/6 replicates each, stocking density was 10 birds/m2) with a chain feeder system and automatic nipple cup drinker. The bins were arranged by placing the first replicate of each treatment then the second replicate of each treatment until the sixth replicate of each treatment to follow the completely randomized design (CRD). The first group served as control and was fed basal diets without any additives. The second and third groups were fed diets containing 2% and 4% olive cake meal (OCM; Al-Sabeel Al-Gadidah Company, Tanta, Al-Gharbia, Egypt); the fourth group was fed control diets with probiotic (*Bacillus licheniformis* (BL), Dutch State Mines Company, DSM 17236; the recommended inclusion level was 100 g/ton of feed to achieve a target inclusion of <sup>8</sup> <sup>×</sup> <sup>10</sup><sup>12</sup> colony-forming units (CFU)/kg); the fifth and sixth groups were fed diets containing 2% and 4% OCM with BL. The compositions of the experimental diets are presented in Table 1. *B. licheniformis* is a commercial probiotic product called GalliPro® Tech DSM 17236, and this probiotic strain was isolated from soil and is a non-GMO; the recommended dose, as provided by Interpharma® Company, Egypt, was used. The diets were presented to the birds ad libitum. The photoperiod was maintained as a 21 h light/3 h dark cycle. After the brooding period, the room temperature was kept between 24 and 26 ◦C, with relative humidity from 50% to 60% throughout the experiment.



640

\* The basal diet fed

vitamin A 30,000 mg, biotin 50

cobalt 100 mg). \*\*\* Olive cake meal (OCM) analysis (crude protein (CP; 9%),

13.7%, fiber; 11.6%).

metabolizable

 energy (ME; 3320 Kcal/kg), Ca; 0.021%, available phosphorus (Avp.; 0.29%), ether extract;

 100 mg, and

 (per 3 kg):

 10 mg, niacin

#### *2.2. Growth Performance and Carcass Parts*

Bird body weight was measured individually every week. However, feed intake was measured daily (on a group basis per pen) throughout the experimental period. The feed conversion ratio per bird was calculated. At 35 days, all birds were weighed individually and sorted from the smallest to the heaviest in weight. Then, 36 birds (1 bird per replicate; 6 birds per treatment) were slaughtered and then dissected to measure the weights of the breast muscle, the thigh muscle, the liver, and the abdominal fat.

#### *2.3. Blood Samples and Plasma Biochemical Analysis*

At 35 days, blood samples from 36 birds (1 bird per replicate; 6 birds per treatment) 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. Plasma total cholesterol, high-density lipoprotein (HDL), glutamic oxalacetic transaminase (GOT), total protein, albumin and globulin, and uric acid were measured calorimetrically by using a commercial chickens' kit (Diamond Diagnostics, Cairo, Egypt), according to the procedure outlined by the manufacturer, using spectrophotometric analysis. Serum antibody titers against Newcastle disease (ND) were determined using the hemagglutination inhibition (HI) test using standard methods qualified in the manual of the World Organisation for Animal Health (OIE) [26].

#### *2.4. Muscle Biochemical Analysis*

The analysis of muscle fatty acids was conducted in 36 birds (1 bird per replicate; 6 birds per treatment) from the breast muscle (pectoral superficial muscle) by gas-liquid chromatography (GLC) according to the procedure of Saleh [27]. The concentration of muscle vitamin E and liver malondialdehyde (MDA) was determined according to Ohkawa et al. [28].

#### *2.5. Statistical Analysis*

The differences between the treatment groups and the control group were analyzed with a General Linear model using SPSS (version 17.0: SPSS Inc., Chicago, USA). Two-way ANOVA was applied to determine the effects of *B. licheniformis* supplementation (BL), olive cake meal inclusion (OCM), and their interaction (BL × OCM) (2 × 3 factorial design). Duncan's new multiple range tests were used to identify which treatment conditions were significantly different from each other at a significance level of *p* < 0.05.

#### **3. Results**

#### *3.1. Growth Performance and Organ Weight*

Body weight gain (WG), feed conversion ratio (FCR), and abdominal fat were significantly influenced by OCM, BL, and their interaction (*p* < 0.05) (Table 2). Body WG showed higher (*p* < 0.05) levels in broilers fed OCM (4%), OCM (2%)/BL, and OCM (4%)/BL than those fed the control, while no difference (*p* > 0.05) was observed between the other groups (Table 2). Broilers fed OCM at 2% or 4% with BL showed a reduced FCR (Table 2). The highest WG and the lowest FCR were observed in birds fed both OCM (4%) and BL (Table 2).

The abdominal fat was decreased (*p* < 0.05) in broilers fed OCM (2%), OCM (4%), OCM (2%)/BL, and OCM (4%)/BL diets without a difference to those fed OCM (2%) (Table 2).


**Table 2.** Effects of feeding *Bacillus licheniformis* (BL) or/and olive cake meal (OCM) on growth performance and organ weights in broilers.

#### *Animals* **2020** , *10*, 695

#### *3.2. Biochemical Parameters*

Blood total protein, albumin, total cholesterol, HDL cholesterol, and ND titer were significantly influenced by OCM, BL, and their interaction (*p* < 0.05) (Table 3). Blood total protein increased by feeding both BL and OCM at 2% or 4% when compared with those fed the control or BL without a difference to OCM at 2% or 4% (*p* > 0.05) (Table 3). The albumin content increased in the BL and OCM (4%) groups with regard to the control (*p* < 0.05). Interestingly, the ND titer was influenced by OCM, BL, and their mixture (*p* < 0.05). Blood total cholesterol decreased in those fed OCM (4%), OCM (2%)/BL, and OCM (4%)/BL, while HDL cholesterol increased in those fed OCM (2%), OCM (4%), OCM (2%)/BL, and OCM (4%)/BL (*p* < 0.05) (Table 3). However, GOT and uric acid were not affected by the test diets (*p* > 0.05) (Table 3).


**Table 3.** Effects of feeding *Bacillus licheniformis* (BL) or/and olive cake meal (OCM) on plasma parameters in broilers.

high-density

 lipoprotein (HDL).

#### *3.3. Muscle Fatty Acid Profiles*

Muscle oleic, linoleic, and linolenic acids were influenced by OCM, BL, and their interaction (*p* < 0.05) (Figure 1; Table 4). Muscle oleic and linoleic acids increased significantly in broilers fed both OCM (4%) and BL, while linolenic acid increased in all groups except those fed BL and control diets (*p* < 0.05) (Figure 1). However, arachidonic acid was not affected by the test diets (*p* > 0.05).

**Figure 1.** Effects of feeding *Bacillus licheniformis* (BL) or/and olive cake meal (OCM) on muscle fatty acids (oleic acid, arachidonic acid, linoleic acid, and linolenic acid) in broilers.

**Table 4.** Two-way ANOVA (*p*-value) of the muscle fatty acids (oleic acid, arachidonic acid, linoleic acid, and linolenic acid) in broilers fed *Bacillus licheniformis* (BL) or/and olive cake meal (OCM).


#### *3.4. Muscle Vitamin E and Liver MDA*

Muscle vitamin E and liver MDA were significantly influenced by OCM, BL, and their interaction (*p* < 0.05) (Figure 2; Table 5). Vitamin E was increased by feeding BL or both OCM (4%) and BL (*p* < 0.05) (Figure 2A). Liver MDA was decreased by feeding BL or both OCM at 2% or 4% and BL (*p* < 0.05) (Figure 2B). The highest MDA level was found in broilers fed the control or OCM at 2% (*p* < 0.05) (Figure 2B).

**Figure 2.** Effects of feeding *Bacillus licheniformis* (BL) or/and olive cake meal (OCM) on muscle vitamin E and liver malondialdehyde (MDA) in broilers.

**Table 5.** Two-way ANOVA (*p*-value) of the muscle vitamin E and liver MDA in broilers fed *Bacillus licheniformis* (BL) or/and olive cake meal (OCM).


#### **4. Discussion**

Most of the recent studies concluded that growth performance was not affected by including up to 10% of OCM in poultry diets [3,6–9]. This study revealed that OCM was successfully included in the diet of broilers at 4%. Moreover, by adding 4% of OCM, broilers obtained better weight gain compared to the control group. The inclusion of up to 4% of OCM in the diet did not impair broiler feed efficiency (FI and FCR). As the above-mentioned level of OCM inclusion would not yet be sufficient for today's scenarios, the intensification of using a blend of the plant by-products has made it necessary to formulate the most cost-effective balanced feed with sound nutrition. This agrees with numerous studies that tested OCM in the diets of broilers [3,6,7,9]. The results showed enhanced growth parameters when broilers were fed OCM at 4%. However, chicks fed a diet with a high level of OCM (4%) with BL showed better growth performance and feed utilization. In the case of a high inclusion level (4%) with BL, the body weight was significantly higher than the other groups, which may result in a reduced feed conversion ratio (FCR). High digestive enzyme activity and feed palatability are also other factors that could increase feed efficiency and, accordingly, the growth of broilers [29]. Zhao et al. [11] reported that low feed intake and a low feed efficiency ratio (increased FCR) resulted from BL supplementation in the broilers' diet. In parallel, a significant role of this

probiotic bacterium on the growth, intestinal microbiota, and immunological responses in chicks has been demonstrated [14,18]. Probiotics may increase the growth and feed efficiency by increasing the secretion of amylase, protease, and lipase, which can increase the digestibility of nutrients in the animal's gut [21–24]. The improvement of growth performance by feeding OCM with BL appears to result from an increase in the feed efficiency of broiler chickens and metabolizable energy (ME) from the diet. The reason for this increase in ME could be due to the digestion of either raw starches or soluble and insoluble non-starch polysaccharide content in OCM, as this probiotic possesses the ability to digest raw starches and to produce cellulase and xylanase, which are required for the digestion of insoluble non-starch polysaccharides [30,31]. In addition, probiotics could improve the nutritional quality of soybean meal because the trypsin inhibitor contained in unprocessed soybean is degraded by BL [32].

The abdominal fat was lowered by including OCM and/or BL in the current study. Similarly, Al-Harthi [3] stated that abdominal fat was decreased by using OCM and/or yeast as probiotics in the broilers' diet. In this study, broilers' feed may elevate the body fat bulk, which may be the reason for the increasing abdominal fat level in broilers fed the control diet in comparison to OCM and/or BL. Probiotics are well known for their function in facilitating the gut absorption of essential nutrients to improve the growth and, accordingly, the general health status, which means reducing the accumulation of nutrients in the gut such as abdominal fat [33].

Blood biochemical indicators relate to some enzymes activity and protein levels in the blood [34] which can reflect the physiological and immunological status of the organism [35]. GOT levels in serum can reflect liver function. When the liver is damaged, the activity will be higher than the normal range [36]. The result of this study revealed that GOT was not influenced by test diets, which indicates the safe function of OCM and/or BL in the broilers' diet. Albumin is the highest protein in serum, which refers to increased antibody and immunity responses [37]. Compared to the control group, after the addition of OCM and/or BL, the changes of biochemical indices and blood routine indices were within the normal range, indicating that OCM and/or BL had no adverse effects on the liver, kidney, and other organs and muscles, as well as on the protein metabolism of broilers. This also proves that the addition of OCM and/or BL had no adverse effect on animal health. Furthermore, dietary OCM and/or BL supplementation in the present investigation elevated the level of total protein and ND titer, which can be attributed to the improved immunity of broilers. Olive oil is a potent immunomodulator that can improve immunity and generate more pathogen resistance [5].

By feeding OCM and/or BL, plasma HDL cholesterol concentration was increased, while plasma total cholesterol was decreased in broilers. Unfortunately, similar investigations concerning the inclusion of OCM and/or BL in broilers' diet are very scarce. Generally, OCM has been reported to decrease the total cholesterol and to increase HDL lipids due to its content of unsaturated and polyunsaturated fatty acids [3,38,39]. Similarly, using OCM in broilers' feed has been shown to result in low levels of total cholesterol [3,40]. The obtained results also revealed that the effect of BL on blood cholesterol levels depends on the level of OCM inclusion. The influence of probiotics in reducing the total cholesterol can be attributed to its role in breaking down the total lipids and bile acids to avoid the re-synthesis of cholesterol [24,33]. Similar results were obtained when chicks fed diets supplemented with different probiotic strains [3,41]. These studies stated that diets containing probiotic bacteria have a negative effect on plasma total cholesterol, but a positive effect on plasma high-density lipoprotein cholesterol (HDL-C) in chickens. Saleh et al. [3,41] reported that the mechanism underlying the cholesterol-lowering effect of probiotics could be due to the inhibition of 3-hydroxyl-3-methylglutaryl-coenzyme (HMG-CoA) reductase. In addition, probiotics might affect fat deposition by influencing the activities of hormone-sensitive lipase and malate dehydrogenase enzyme in adipose tissues [42].

The obtained results also revealed improved ND titer in broilers fed OCM and/or BL, which indicates an improved immunity and, consequently, resistance against infectious diseases. Balanced diets supplemented with reasonable functional feed additives usually can keep high immunity status and resistance against infectious diseases in broilers [1,2]. Probiotics, on the other hand, are beneficial microorganisms that compete with the harmful bacteria in the animals' gut and provides the host high resistance against infectious diseases [43]. The muscle oleic, linoleic, and linolenic acids as unsaturated fatty acids were increased by OCM and/or BL in the broilers' diet in this study. The increased levels of muscle oleic, linoleic, and linolenic acids can be attributed to the high content of OCM from unsaturated and polyunsaturated fatty acids [38,39]. The increase in oleic, linoleic, and linolenic acids in the muscle is probably due to the intestinal activities of the probiotic. Srianta et al. [44] reported that probiotics produce linolenic acid. Furthermore, BL has the ability to produce desaturase, which changes saturated fatty acids to unsaturated fatty acids [45].

Regarding the muscle vitamin E, feeding OCM and/or BL increased the level of vitamin E in the muscle of broilers, which might be involved in reducing the lipid peroxidation process in broilers and, accordingly, in reducing the oxidation [24]. Oxidative emphasis normally happens when the creation and elimination of free radicals (ROS) are unbalanced, since the oxidative damage of cultured species is directly related to the quality of the diet [46,47]. Superoxide dismutase, glutathione peroxidase, and catalase are important scavengers of ROS, protecting the body tissues from oxidative stress damage [48]. Malondialdehyde (MDA) is a product of lipid peroxides and high levels of ROS, which can cause damage to the cell's DNA, protein, and cytoplasm [49,50]. Interestingly, broilers fed OCM and/or BL showed reduced MDA, confirming that the known antioxidant properties of this probiotic are not lost when administered orally in broilers. Like the current study, earlier reports revealed improved antioxidant response by feeding probiotics [11,51].

#### **5. Conclusions**

In conclusion, feeding olive cake meal with *B. licheniformis* improved growth performance, modified plasma lipid, and fatty acid profiles, as well as enhanced the health status of broiler chickens—these factors were probably influenced through improved feed efficiency and antioxidative response. Based on the results obtained, the use of BL to improve the nutritional value of OCM and to increase the feed utilization of OCM by broilers is recommended.

**Author Contributions:** Conceptualization, A.A.S. and M.A.O.D.; Formal analysis, A.A.S. and M.A.O.D.; Funding acquisition, M.A.O.D. and B.A.P.; Investigation, A.A.S., M.A.O.D., and B.A.P.; Methodology, A.A.S. and M.A.O.D.; Project administration, A.A.S.; Supervision, A.A.S.; Writing—Original draft, A.A.S., M.A.O.D., and B.A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Researchers Supporting Project Number (RSP-2019/144), King Saud University, Riyadh, Saudi Arabia.

**Acknowledgments:** The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP-2019/144), King Saud University, Riyadh, Saudi Arabia.

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

#### **References**


© 2020 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 Supplementation of Biological Curcumin Nanoparticles on Growth and Carcass Traits, Antioxidant Status, Immunity and Caecal Microbiota of Japanese Quails**

### **Fayiz M. Reda 1, Mohamed T. El-Saadony 2, Shaaban S. Elnesr 3, Mahmoud Alagawany 1,\* and Vincenzo Tufarelli 4,\***


Received: 27 March 2020; Accepted: 22 April 2020; Published: 26 April 2020

**Simple Summary:** Nanoparticles such as nano-curcumin are easier to pass through cell membranes in organisms and interact rapidly with biological systems. Therefore, using nano-curcumin is one of the recommendations for improving the bioavailability of curcumin, which would increase its absorption. Thus, this study focused on effects of nano-curcumin levels on the growth, carcass yield, blood chemistry and caecal microbiota of growing quails. From our results, supplemental nano-curcumin had beneficial impacts on growth, lipid profile, antioxidant, immunity of quail, and reduction in pathogenic bacteria.

**Abstract:** This study was planned to evaluate the impact of different nano-curcumin levels on the growth rate, carcass, blood chemistry and caecal microbes of growing quail. A total of 270 Japanese quails at one-week-old were distributed to six equal groups; each group consisted of 45 unsexed birds with five replications (nine quails each). The 1st group was fed a basal diet, whereas the 2nd, 3rd, 4th, 5th and 6th groups were fed diets containing nano-curcumin (0.1, 0.2, 0.3, 0.4 and 0.5 g/kg diet, respectively). Nano-curcumin levels significantly increased (*p* ≤ 0.0001) body weight at 3 weeks and 5 weeks of age. Body weight gain during 1–3, 3–5 and 1–5 weeks of age was significantly increased (*p* < 0.0001) in groups treated with nano-curcumin levels (except at 0.3 g/kg; 1–3 weeks) compared to control. During 1 to 5 weeks, feed intake was decreased (*p* < 0.0001) in birds receiving nano-curcumin (0.1, 0.3 and 0.4 g/kg) diets. The best values of feed conversion ratio were recorded for the 0.4 g nano-curcumin-treated group. Carcass traits were not affected Nano-curcumin levels. The inclusion of nano-curcumin (0.2, 0.3 or 0.5 g/kg) significantly increased serum TP (*p* = 0.0004), albumin (*p* = 0.0078) and globulin (*p* < 0.0001). Quails fed with nano-curcumin (0.2 g/kg) exhibited the highest SOD and GSH activities, serum IgG and IgM concentrations and complement values compared to control. The addition of any level of nano-curcumin in the quail diet also significantly improved the lipid profile. In conclusion, supplemental nano-curcumin had beneficial impacts on growth, lipid profile, blood constituents, antioxidant indices, and immunity of growing quail, as well as increasing counts of lactic acid bacteria and reducing pathogenic bacteria.

**Keywords:** biological nano-curcumin; growth; diet; immunity; antioxidant; pathogens; quail

#### **1. Introduction**

The general trend in the poultry industry is to provide a safer feed, to enhance physiological and productive indicators [1]. The effect of natural products on the capability of nutrients absorption in the gut is a major rationale for recent research. Several investigations have stated that plant derivatives included in poultry feeds deliver useful effects on performance, health, immune response and product quality [2–5]. One of these plant materials is curcumin. Curcumin is the principle active constituent of *Curcuma longa*. Curcumin has long been used in poultry feeds, owing to its favorable effects, including antimicrobial, antioxidant, anti-inflammatory, anti-inflammatory and immunostimulant properties [6,7]. Curcumin shows pharmacological efficacy and safety and contributes to the treatment of several diseases. It also improves the endogenous secretion of digestive enzymes [8] and reduces lipid peroxidation [9].

Curcumin can be used with nanotechnology that potentiates its useful effects. Nanoparticles are easier to pass through cell membranes in organisms and interact rapidly with biological systems [10]. Thus, using nano-curcumin is one of the recommendations for enhancing the bioavailability of curcumin, leading to an increase in its absorption [11]. It has been established that nano-curcumin displays improved bioavailability and distribution in the tissues [12]. The dietary supplementation of nanocurcumin displayed a significantly positive effect on performance [13]. Sayrafi et al. [14] clarified that the declined the liver enzyme activity following supplementation with nanocurcumin may be due to its antioxidant properties. Curcumin has an antioxidant function and adjusts the intestinal microbial composition [15]. Furthermore, Partovi et al. [16] stated that nanocurcumin at a level of 300 mg/kg diet can be used in poultry production as a good feed additive. However, the inclusion of nano-curcumin in quail diets during the growth period is still limited.

Thus, the purpose of the current study was to determine the effects of different nano-curcumin levels on the growth, carcass yield, lipid profile, blood constituents, and antioxidant and immunological indices, as well as the caecal microbiota, of growing quails.

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

All experimental procedures of the study were performed according to the Local Experimental Animal Care Committee and approved by the ethics of the institutional committee of Department of Poultry, Faculty of Agriculture, Zagazig University, Zagazig, Egypt.

#### *2.1. Source of Curcumin Nanoparticles*

Curcumin nanoparticles in this study were synthesized from *Bacillus subtilis* LA4, which was isolated from soil samples that were collected from different sites next to the plant rhizosphere in Sharqia Governorate, Egypt [17–19]. Under the optimum conditions of temperature, pH, incubation time, and other parameters, curcumin nanoparticles were produced. The characterization of the curcumin nanoparticles using modern devices and technologies was also performed to learn the properties of the curcumin nanoparticles obtained from the *Bacillus subtilis* LA4 bacteria.

#### *2.2. Biosynthesis of Curcumin Nanoparticles*

For the biofabrication of curcumin nanoparticles using the tested bacterium, 250 mL conical flasks containing 20 mL of supernatant from bacterial culture were separately mixed with 30 mL of 100 mg (0.27 mM) aqueous solutions of filtered sterilized curcumin, following the method of [20,21] with some modification. Then, the reaction mixture flasks were placed at 160 rpm in a shaker incubator at 30 ◦C for 72 h to allow the reduction process to occur. Furthermore, a set of flasks containing 20 mL of NB and 30 mL of 0.27 mM curcumin solution were prepared to confirm that the biotransformation of curcumin nanoparticles was only mediated by the use of bacterial cell-free extract [21].

#### *2.3. Antibacterial Activity of Curcumin Nanoparticles*

Fresh LB medium was used in all experiments to recover bacteria by sub-culturing. A tiny part from an inoculum of each bacterium was mixed in 5 mL of nutrient broth and kept overnight at 37 ◦C. The pathogenic bacteria *Staphylococcus aureus* MTTC 1809, *Bacillus subtilis* MTCC 430, *Salmonella enterica* MTCC 1253 and *Pseudomonas aeruginosa* MTCC 741 strains were gained from Egyptian Microbial Culture Collection, Microbiological Resource Center (The Cairo MIRCEN: Ain Shams University, Cairo, Egypt), cultured on a nutrient agar plate, and kept in the NA slants at 4 ◦C. Overnight cultures in the nutrient broth were used for the laboratory studies. The antibacterial activity of curcumin nanoparticles was estimated using the disc diffusion method [22], which was presented by the National Committee for Clinical Laboratory Standards (NCCLS). The zone of inhibition was measured after a day of incubation at 30 ◦C or 37 ◦C. Bactericidal effects of curcumin nanoparticles were detected using a modified version of the method shown by NCCLS. The diluted bacterial culture (0.1 mL) was extended on the sterile NA plate. Dried discs of 6 mm diameter of Whatman filter paper No. 1 that had been previously soaked in curcumin nanoparticles were placed on the seeded plates against Gram-negative and Gram-positive bacteria [23–25]. The estimation of the MIC was obtained through the determination of the turbidity of the bacterial growth after a day of incubation. The inhibited concentration was 99% of bacterial growth, which was considered as the MIC estimate [24,26]. According to the standard method, the MBC values of the particles were measured, and the MBC estimate was determined through sub-culturing the MIC dilutions onto sterile Muller-Hinton agar plates incubated at 37 ◦C for one day.

#### *2.4. Experimental Design and Diets*

The study was carried out at the Poultry Research Farm, Department of Poultry, Faculty of Agriculture, Zagazig University, Egypt. At one week of age, we used 270 Japanese quails with an average body weight of 26.1 ± 0.08 g. Quail chicks were haphazardly distributed across six equal groups, each group consisting of 45 unsexed birds with five replications (nine birds each). Quails were reared in common type cage (90 × 40 × 40 cm) under the same conditions with 23 h light:1 h dark. Feed and water were opened throughout the experiment (five weeks). Birds received feeds in mash form according to their treatment. The dietary treatments were as follows: the 1st group was fed a basal diet without any supplementation (0 g/kg diet), whereas the 2nd, 3rd, 4th, 5th, and 6th groups were fed diets supplemented with 0.1, 0.2, 0.3, 0.4 and 0.5 g/kg of nano-curcumin, respectively. The basal diet was based on corn-soybean meal and contained 24% CP, 12.53 MJ/kg, 0.8 Ca, and 0.45 P, according to NRC [27].

#### *2.5. Growth Performance and Carcass Measurements*

All growth parameters [body weight (BW), body weight gain (BWG) feed intake (FI) and feed conversion ratio (FCR = g feed/g gain)] were measured at 1, 3 and 5 weeks of age. At 5-weeks-old, 24 quails were used for carcass examinations. All edible parts (gizzard, liver, heart, and carcass) were weighed and expressed as a % of the live BW before slaughter.

#### *2.6. Microbiological Analysis*

We collected the samples (~10 g) from the cecum content (five samples per each treatment) and separately transported them to a 250 mL Erlenmeyer flask containing 90 mL of sterile peptone (0.1% peptone) saline solution (0.85% NaCl) and blended the mixture well. The total bacterial count (TBC), total yeasts and molds count (TYMC), *Enterococci*, lactic acid bacteria count, Coliform, *E. coli* and *Salmonella* were recorded according to [28,29].

#### *2.7. Blood Chemistry*

After slaughter by sharp knife to complete bleeding, we collected the blood samples from 24 quails in sterilized tubes. We used the centrifuge (Janetzki, T32c, 5000 rpm, Wall-hausen, Germany) at 2000× *g* 15 min to separate the plasma. Using commercial kits from Biodiagnostic Company (Giza, Egypt), we determined the level of albumin (ALB), total protein (TP), globulin (GLOB), A/G ratio, and the activity of alanine transaminase (ALT), lactate dehydrogenase (LDH), aspartate transaminase (AST), urea, creatinine, total cholesterol (TC), triglycerides (TG), very low-density lipoprotein (VLDL), high-density lipoprotein (HDL), and low-density lipoprotein (LDL). The levels of immunological parameters (IgG) and M (IgM) as well as complement (C3) were determined using kits from Spectrum Company (Cairo, Egypt). For the antioxidant parameters, using commercial kits and a spectrophotometer (Shimadzu, Japan), the content of reduced glutathione (GSH) and malondialdehyde (MDA), and the activity of superoxide dismutase (SOD) were determined in quail plasma.

#### *2.8. Statistics*

All of the statistical analyses were carried out using the SAS software (SAS Institute Inc., Cary, NC, USA). Data regarding growth, carcass, blood chemistry and microbiology traits were analyzed with one-way ANOVA using the post-hoc Tukey's test (*p* < 0.05).

#### **3. Results**

The antibacterial activity of synthesized curcumin nanoparticles was tested against *Staphylococcus aureus* MTTC 1809, *Bacillus subtilis* MTCC 430, *Salmonella enterica* MTCC 1253 and *Pseudomonas aeruginosa* MTCC 741. The antibacterial activity against G+ and G- bacteria at different concentrations of curcumin nanoparticles was performed using the disc diffusion method. The bacterial growth culture at 24 h (0.1 mL) was spread aseptically onto nutrient agar plates. Discs (6 mm) which were impregnated with one of each curcumin nanoparticle concentration were dispensed with a sufficient separation from each other so as to avoid the overlapping of the inhibition zones. The diameters of the inhibition zone around each disc were estimated. They showed good antimicrobial activity against all the tested bacteria, though the effect of curcumin nanoparticles was found to be more pronounced with *Staphylococcus aureus* MTTC 1809 and *Bacillus subtilis* MTCC 430 (Table 1) compared to other bacteria. The data in Table 1 show the susceptibility of four bacterial strains to five concentrations of curcumin nanoparticles, namely 100, 200, 300, 400 and 500 μg/mL. The data confirmed that, with increasing concentrations of curcumin nanoparticles, halo diffusion increased regardless of the bacterial strains tested—giving a maximum diameter of 31 mm for *Staphylococcus aureus* MTTC 1809 when 500 μg/mL was used. From the obtained results, it was also observed that curcumin nanoparticles display more efficient as antibacterial activity compared to normal curcumin. The MIC values were 90, 100, 200 and 220 μg/mL, respectively, when *Staphylococcus aureus* MTTC 1809, *Bacillus subtilis* MTCC 430, *Salmonella enterica* MTCC 1253 and *Pseudomonas aeruginosa* MTCC 741 were used. The MBC rates were 180 μg/mL, 200 μg/mL, 400 μg/mL and 440 μg/mL, respectively, for the same pathological bacterial isolates mentioned earlier (Table 2).



a–e: Means in the same row with no superscript letters after them or with a common superscript letter following them are not significantly different (*p* < 0.05).


**Table 2.** The MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) of the curcumin nanoparticles.

#### *3.1. Growth Performance and Carcass Yield*

Results for growth performance are shown in Table 3. It was found that the nano-curcumin levels significantly increased (*p* < 0.0001) body weight at 3 weeks and 5 weeks of age. It was reported that the diet enriched with nano-curcumin at levels of 0.2 or 0.4 g/kg resulted in the best body weight. Body weight gain during all periods (1–3, 3–5 and 1–5 weeks of age) was increased (*p* < 0.0001) in the groups treated with nano-curcumin levels, except the BWG of the group fed nano-curcumin (0.3 g/kg), which did not significantly differ from the control between 1 and 3 weeks of age. Between 1 and 3 weeks, the FI was significantly lower (*p* < 0.0001) with the supplementation of nano-curcumin (0.3 g/kg) than in all other groups, while the highest FI was with the birds fed on a diet containing nano-curcumin (0.2 g/kg). During the period of weeks 3–5, quails fed 0.1, 0.2, 0.3 and 0.4 g nano-curcumin-treated diets consumed less feed (*p* < 0.0001) than the others. Between 1 and 5 weeks, the FI was decreased (*p* < 0.0001) in the birds that received nano-curcumin (0.1, 0.3 and 0.4 g/kg) diets compared with that of the control and other groups. In all periods, the quails fed nano-curcumin had better FCR (*p* < 0.0001) than the control quails, except those quails fed nano-curcumin (0.5 g/kg) did not significantly differ from the control between 3 and 5 weeks of age. Generally, the best value in FCR was recorded for the 0.4 g nano-curcumin-treated group. As shown in Table 4, the carcass traits of Japanese quail were not affected by variation in nano-curcumin levels.


**Table 3.** Growth performance of Japanese quail as affected by dietary nanocurcumin.

a–d: Means in the same row with no superscript letters after them or with a common superscript letter following them are not significantly different (*p* < 0.05).


**Table 4.** Carcass traits and relative organs of growing Japanese quail as affected by dietary nano-curcumin.

#### *3.2. Blood Chemistry*

The effects of dietary nano-curcumin on the liver and kidney function of quail are shown in Table 5. The inclusion of nano-curcumin (0.2, 0.3 or 0.5 g/kg) increased serum TP (*p* = 0.0004) and globulin (*p* < 0.0001) compared to the control and other groups. The group fed nano-curcumin (0.3 g/kg) had the highest serum albumin level (*p* = 0.0078). The A/G ratio in the group fed 0.2, 0.3 and 0.5 g nano-curcumin/kg diet was lower than that in the control and other groups (*p* < 0.0001).

**Table 5.** Liver and kidney function of growing Japanese quail as affected by dietary nano-curcumin.


a–d: Means in the same row with no superscript letters after them or with a common superscript letter following them are not significantly different (*p* < 0.05). † TP: total protein; ‡ Alb: albumin § GLOB: globulin; ¶ A/G: albumin/globulin ratio; †† AST: aspartate aminotransferase and ‡‡ ALT: alanine aminotransferase. \* LDH: lactate dehydrogenase.

The AST activity in the serum decreased (*p* = 0.0116) with the addition of dietary nano-curcumin (0.1, 0.3 or 0.4 g/kg) to the feed. Furthermore, the serum ALT activity of the birds fed nano-curcumin (0.3 g/kg) was lower (*p* < 0.0001) than of those in the control and the other groups. The LDH values of the birds fed rations enriched with nano-curcumin (0.1, 0.2 or 0.4 g/kg) were lower (*p* = 0.0105) than of those in the other groups. There was no significant difference in the serum urea and creatinine values between birds supplemented with nano-curcumin at all levels and the control group.

The response of quails to dietary nano-curcumin levels on the lipid profile is presented in Table 6. The addition of various levels of nano-curcumin in the quail feed significantly decreased the TC and LDL in the serum (*p* < 0.0001) compared to the control. The highest values of HDL (*p* = 0.0308) were recorded with the group fed a diet containing nano-curcumin (0.4 g/kg). The TG and VLDL values were significantly decreased (*p* < 0.0001) with the addition of nano-curcumin (0.2, 0.4 and 0.5 g/kg) compared with the control and other groups, but the highest values were recorded for the 0.1 g/kg level of nano-curcumin.


**Table 6.** Lipid profile of growing Japanese quail as affected by dietary nano-curcumin.

a–d: Means in the same row with no superscript letters after them or with a common superscript letter following them are not significantly different (*p* < 0.05). † TC: total cholesterol; ‡ TG: triglycerides; § HDL: high density lipoprotein; ¶ LDL: low density lipoprotein; ¶¶ LDL: very low density lipoprotein.

The results of the antioxidant and immunity indices are presented in Table 7. Quails fed with nano-curcumin (0.2 g/kg) exhibited the highest SOD and GSH activities. However, MDA concentrations of serum were decreased by the addition of dietary nano-curcumin levels. In Table 7, serum IgG concentrations were increased by nano-curcumin (0.1, 0.2 and 0.5 g/kg) supplementation compared to those of the control. However, serum IgM concentrations were also increased by nano-curcumin (0.1, 0.2, 0.3 and 0.4 g/kg) supplementation compared to those of the control. The values of complement 3 were significantly augmented in the group fed diets containing nano-curcumin (0.2, 0.3, 0.4 and 0.5 g/kg).

**Table 7.** Antioxidant and immunological indices of growing Japanese quail as affected by dietary nano-curcumin.


a–d: Means in the same row with no superscript letters after them or with a common superscript letter following them are not significantly different (*p* < 0.05). † SOD: superoxide dismutase; ‡ MDA: malondialdehyde; § TAC: total antioxidant capacity; ¶ GSH: reduced glutathione; †† GPX: glutathione peroxidase; ‡‡ C3: complement 3; §§ IgG: immunoglobulin G.

#### *3.3. Microbiological Aspects*

Table 8 presents the effect of nano-curcumin on the caecal microbiota of quail. The significant reduction in TBC, TYMC and *Enterobacter* in the caecal microbiota of quail was observed following the supplementation of nano-curcumin levels. The coliform count in the caecal microbiota of quail was significantly decreased in those groups fed a diet containing nano-curcumin (0.1, 0.4 and 0.5 g/kg) compared to in the other groups. Furthermore, the supplementation of nano-curcumin (0.2, 0.4 and 0.5 g/kg) led to a reduction in the caecal *E. coli* count compared to the control and other groups. Quails fed diets supplemented with nano-curcumin (0.2, 0.3 and 0.4 g/kg) exhibited higher lactic acid bacteria colonization than those in the control and other groups. The *Salmonella* counts in the caecal microbiota of quails were significantly decreased in all of the groups fed diets containing nano-curcumin. Finally, the best caecal microbiota were observed in the groups fed nano-curcumin at levels of 0.2 and 0.4 g/kg.


**Table 8.** Caecal microbiota of growing Japanese quail as affected by dietary nano-curcumin.

a–d: Means in the same row with no superscript letters after them or with a common superscript letter following them are not significantly different (*p* < 0.05); TBC: Total bacterial count; TYMC: total yeast and molds count.

#### **4. Discussion**

Concerning the antimicrobial activity of curcumin nanoparticles against tested pathogenic bacteria, it was found [24] that when studying the effect of curcumin nanoparticles on four bacterial isolates, Gram-positive bacteria are more affected than Gram-negative bacteria. Furthermore, it was reported [24] that the antibacterial activity of nanocurcumin against *S. aureus*, *B. subtilis*, *E. coli*, and *P. aeruginosa* demonstrated a broad-spectrum inhibitory effect against all microorganisms. The MICs of nano-curcumin for *S. aureus*, *B. subtilis*, *E. coli*, and *P. aeruginosa* were 100, 75, 250, and 200 μg/mL, respectively.

The trend of using nano-curcumin in poultry feed has recently been discussed and may be a possible approach to enhance the physiological and productive performance, and the health status, of poultry. The enhancement of curcumin bioavailability using nanotechnology techniques can increase its absorption [30], in turn boosting the poultry performance and health. Nanocurcumin can be used as a safe and natural feed additive to increase nutritional value [16]. Supplemental nanocurcumin displayed a positive impact on BW and FCR, which is in accordance with previous studies [13,31], which validated the affirmative effect of curcumin on the growth performance of birds. As for the present findings, it was indicated [32] that the addition of 10 mg of nano-encapsulated curcumin/kg diet improved the FCR of quail. It was also illustrated [33] that the addition of nano-curcumin in the drinking water of broiler chicks improves the body growth and FCR. Curcumin, when used as a functional molecule, can act as a growth promoter in poultry, and as a strong natural antioxidant in the improvement of performance [34]. Additionally, using curcumin in the diet encourages the secretion of bile acids and stimulates the proteases lipase, amylase trypsin and chymotrypsin enzymes [35]. The favorable impacts of curcumin on the growth of broilers might be due to boosted secretions of these enzymes. The improvement in the growth of birds fed diets containing curcumin may be due to improvements in the intestinal morphology of the birds [13]. Furthermore, this positive effect of curcumin might be attributed to its well-reported antibacterial, antioxidant and anti-inflammatory effects [36]. Finally, nanocurcumin can be used in the poultry industry as a potential, promising feed additive.

The results of the current study on the carcass yield of quail were in agreement with previous articles that studied the effects of turmeric or its extract on carcass traits, which were not affected. In Durrani et al. [31], higher breast and thigh weights, and a higher dressing percentage, were noticed in broilers fed a diet enriched with 5 g turmeric powder/kg compared with the control. A previous study [37] observed no improvement in the gizzard or liver following the application of *Curcuma longa* in the diet. Furthermore, it was stated [38] that the addition of turmeric rhizome extract (TRE) (100–300 mg/kg) had no significant influence on the dressing %. Moreover, it was found [39] that the gizzard and dressing % were not significantly influenced by dietary turmeric treatments.

The levels of blood ALT and AST reflect the health status of the liver. In the present study, these enzymes were significantly decreased following the addition of dietary nano-curcumin. Looking at previous research [14], it was clarified that supplementation of nanocurcumin (200 mg/kg) declined the serum AST enzyme level, and they attributed this reduction to the antioxidant properties of nano-curcumin. Furthermore, one study [40] reported that chickens fed a diet containing curcuma powder (5 g/kg) had the highest level of LDH, implying that curcuma might have a positive impact on liver enzymes. Moreover, it was highlighted [41] that a nano-curcumin level of 400 mg/kg diet displayed affirmative and consistent influences on the serum biochemical parameters.

Serum TG, TC, HDL and LDL concentrations are viewed as diagnostic markers in lipid metabolism. The present results indicated that the addition of nano-curcumin levels in the quail feed significantly decreased the lipid profile. In agreement with previous studies [42,43] it was stated that curcumin reduced the serum LDL cholesterol and triglycerides levels and improved the liver function. Dietary curcumin lessened blood cholesterol levels and encouraged the digestion of fat [44,45]. Research [40] has revealed that the dietary supplementation of turmeric in broiler chickens significantly diminished LDL-cholesterol and augmented HDL-cholesterol, but did not affect triglyceride levels. Moreover, it was indicated [43,46] that curcumin caused a reduction in TC, perhaps due to the inhibition of enzyme hepatic 3-hydroxyl-3-methyglutaryl CoA-reductase (HMGCR) activity, which is responsible for the production of TC in the hepatic tissues [47]. Furthermore, curcumin may decline the activity of the enzymes that act as rate-limiting enzymes in lipogenesis, such as acetyl-CoA carboxylase (the rate-limiting enzyme in fatty acids synthesis) [48].

Antioxidant ability is the key to the health and growth of poultry. Curcumin, the major antioxidative molecule of curcuma longa, is a powerful damper of oxygen species [49]. The results of the current study show that the groups fed with nano-curcumin exhibited high SOD and GSH activities. As shown by the present findings, nano-curcumin possesses a better antioxidant and biological activity than curcumin [50]. The antioxidant ability against peroxyl radicals was augmented in the nanocurcumin-supplemented group compared to the control [32]. The curcumin can alleviate the oxidative stress by modifying the hepatic nuclear transcription factors and decreasing lipid peroxidation in the muscle and serum of quail [51]. Curcumin helps to maintain the antioxidant status of the cells through suppressing oxidative enzymes, scavenging free radicals and prompting de novo glutathione synthesis [52]. Zhai et al. [15] illustrated that curcumin compounds could reduce the oxidative injury and disruption of lipid metabolism through modifying the cecum microbiota of ducks. The dietary supplementation of turmeric rhizome extract augmented the enzymatic activities of SOD and GSH-PX, and reduced the malondialdehyde concentration [38]. As a result, dietary curcumin reduces the production of reactive free radicals, leading to an increase in the antioxidant metabolites concentration in the poultry body. The inclusion of curcumin in the diet decreased the malondialdehyde concentration and boosted the activities of CAT, T-AOC, SOD, and GSH-Px compared to the control group [53]. Thus, curcumin can alleviate the negative impact of any stressful environmental condition.

Curcumin has been found to have numerous pharmacological activities, including antimicrobial, anti-inflammatory, antifungal, antiviral and antioxidant activities [54]. The highest values of immunoglobulins in the present study were obtained from birds fed a diet containing nano-curcumin. Similar results have been observed in other studies, which pointed out that a diet with a nano-curcumin level of 400 mg/kg displayed the best immune response compared to the control group [41]. Emadi et al. [55] showed that the immunologlobulins (IgA, IgM and IgG) of chickens were significantly increased by dietary turmeric. Furthermore, the turmeric plant has been proven to be a powerful immunomodulatory factor that can improve the activation of B and T cells, neutrophils and macrophages cells [56]. Thus, it can be said that nano-curcumin can be an appropriate alternative to synthetic antioxidants, perhaps due to the improvement of the antioxidant metabolites of birds, which may boost the bird's immunity.

The present study found that the supplementation of nano-curcumin in quail diets reduced harmful bacteria and boosted useful bacteria. Curcumin, the main bioactive component of turmeric, was found to possess antibacterial activities [57]. Curcumin could modify the gut microbial balance, improving the intestinal integrity [58]. El-Rayes et al. [39] illustrated that the dietary supplementation of turmeric as source of curcumin led to an increase in counts of lactic acid bacteria and a reduction in pathogenic bacteria (*S. aureus*, *E. coli* and total coliform bacteria) compared to in the control group. Gupta et al. [59] described that the extracts of *C. longa* inhibited the growth of pathogenic bacteria. Curcumin had an inhibitory effect against many pathogenic bacteria and decreased the population of harmful gut bacteria [60].

#### **5. Conclusions**

The findings of our study demonstrated the positive effects of dietary nano-curcumin supplementation on the growth, lipid profile, blood constituents, immunity and antioxidant indices of quails. The best values in feed efficiency were achieved when quails were fed with 0.4 g nano-curcumin in their diets. Furthermore, the best values in immune response and antioxidant indices were observed in the 0.2 g nano-curcumin-treated group.

**Author Contributions:** Conceptualization, F.M.R. and M.A.; Data curation, M.T.E.-S.; Formal analysis, M.T.E.-S. and S.S.E.; Investigation, F.M.R. and S.S.E.; Writing—original draft, V.T.; Writing—review & editing, V.T. All authors have read and agreed to the published version of the manuscript.

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

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

#### **References**


© 2020 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*

### **Green Tea and Pomegranate Extract Administered During Critical Moments of the Production Cycle Improves Blood Antiradical Activity and Alters Cecal Microbial Ecology of Broiler Chickens**

### **Vera Perricone** †**, Marcello Comi** †**, Carlotta Giromini, Ra**ff**aella Rebucci, Alessandro Agazzi, Giovanni Savoini and Valentino Bontempo \***

Department of Health, Animal Science and Food Safety "Carlo Cantoni" (VESPA), Università degli Studi di Milano, Via dell'Università 6, 26900 Lodi, Italy; vera.perricone@unimi.it (V.P.); marcello.comi@unimi.it (M.C.); carlotta.giromini@unimi.it (C.G.); raffaella.rebucci@unimi.it (R.R); alessandro.agazzi@unimi.it (A.A.); giovanni.savoini@unimi.it (G.S.)

**\*** Correspondence: valentino.bontempo@unimi.it

† Perricone and Comi should be considered joint first author.

Received: 21 March 2020; Accepted: 28 April 2020; Published: 30 April 2020

**Simple Summary:** Since the European Union's (EU) antibiotic ban in 2006, interest in natural feed additives has largely increased. Natural feed additives are used to prevent diseases and promote growth in chickens, supporting animal health and modulating the development of the gut microflora during stressful situations. In the present study, a bioactive compound from plants belonging to the class of phytobiotics was assessed for its effects on production performance, antiradical activity and gut microflora in broiler chickens. The obtained results show how the tested compound is able to exert beneficial effects on the antiradical activity and gut microbial ecology of birds, even though the chickens' performance was unaffected.

**Abstract:** Phytobiotics are usually tested in feed and throughout the production cycle. However, it could be beneficial to evaluate their effects when administered only during critical moments, such as changes in feeding phases. The aim of the trial was to investigate the effect of a commercial plant extract (PE; IQV-10-P01, InQpharm Animal Health, Kuala Lumpur, Malaysia) on growth performance, blood antiradical activity and cecal microbiome when administered in drinking water to broiler chickens during the post-hatching phase and at each change of diet. In the experiment, 480 1-day-old male broiler chicks were assigned to two groups in a 50-day trial. Broilers received drinking water (C) or drinking water plus PE (T) at a rate of 2 mL/L on days 0 to 4, 10–11 and 20–21. PE did not affect performance and water intake, while total antiradical activity was improved (*p* < 0.05). A greater abundance of lactic acid bacteria (false discovery rate (FDR) < 0.05) was found in the T group and the result was confirmed at a lower taxonomic level with higher Lactobacillaceae abundance (FDR < 0.05). Our findings suggest that PE administration during critical moments of the production cycle of broiler chickens may exert beneficial effects at a systemic level and on gut microbial ecology.

**Keywords:** broiler chickens; phytobiotics; green tea; pomegranate; drinking water; antiradical activity; cecal microbiota

#### **1. Introduction**

In 2006, the European Union banned the use of antimicrobial growth promoters in animal nutrition [1]. This decision led to the result that antimicrobials, other than coccidiostats and histomonostats, were no longer allowed as feed additives [2]. As such, antibiotic alternatives designed to maintain productivity and health became the focus of much research [3,4]. At the present moment, different molecules, compounds, bioactive substances, and active principles have been investigated and are still under investigation [5]. Among them, several classes of feed additives are now available, including probiotics [6,7], prebiotics [8,9], organic acids [10,11], and phytobiotics [12]. Although the benefits of such additives have been proven in most cases, there is still a lack of clarity on their effects, as evidenced by some contrasting results in different trials.

Considering the available feed additive classes, the use of phytobiotics in poultry nutrition could represents a valuable tool [13]. Phytobiotics, also known as botanicals, are plant-derived products that are a natural source of bioactive compounds [14]. Supplementation with phytobiotics for broiler chickens has shown beneficial effects on animal production and the quality of animal-derived products [13]. However, their mechanism of action remains to be elucidated, and different hypotheses have been proposed, in which the antioxidant properties seem to play a major role [15]. Phytobiotics in fact are rich in polyphenolic compounds, which can support the antioxidative capacity by counteracting the harmful effects of free radicals generated during stressful situations, finally resulting in improved general health and better performance of the animals [16].

Phytobiotics were also found to be able to modulate gut microflora [14,17] and its development, which plays an important role in production performance and overall health [18]. It is indeed recognized that the first microbial population colonizing the gut could impact an animal's entire life span [19]. In this view, the chance to modulate the gut microflora in chickens via a nutritional approach is of particular interest, especially during critical moments of their life, such as the post-hatching phase.

Among phytobiotics, green tea and pomegranate extracts have been shown to improve broiler productivity and antioxidant status [20–22], as well as modulate the intestinal microflora [20,23]. Green tea (*Camelia sinensis*) has been widely studied in humans and animals due to its numerous biofunctional properties, including antioxidant, antiviral and anticoccidial activity [24,25]. Most of these properties are ascribed to the high levels of polyphenolic compounds, among which catechins are the most represented group [26]. Similarly, pomegranate (*Punica granatum*) also possesses biofunctional properties, such as antioxidant and anti-inflammatory properties, antimicrobial activity and anticancerogenic effects [27]. A recent in vitro study by Jain et al. [28] showed that the simultaneous use of different plant extracts, including green tea and pomegranate, led to a synergistic enhancement of antioxidant activity. However, combined administration of green tea and pomegranate has not yet been tested in animal nutrition. Green tea and pomegranate have also been demonstrated to affect the intestinal microbiota [23,25], promoting beneficial bacteria in the intestinal tract [29,30].

Until now, the majority of in vivo studies in poultry have investigated the effects of administering phytobiotics in the feed and for the entire rearing period, while the effect of their inclusion in drinking water was scarcely investigated. Phytobiotics supplementation in drinking water might represent a valuable way to perform targeted interventions, limited to the critical moments of the production cycle (e.g., limited number of days during post-hatching phase and transitions between feeding phases). This route of administration could sustain the health of animals when it needs to be supported and boosted, rather than being used for the whole rearing period through the feed. This could then be turned into a smaller amount of phytobiotics used per rearing cycle, with economic advantages in terms of production cost.

To the best of our knowledge, at the present moment no literature is available on the addition of pomegranate to drinking water for poultry. Only two studies considered the effects of including green tea in drinking water [31,32], and neither of these accounted for treatment only during specific critical moments of the rearing cycle. In both these trials, in fact, tea supplementation was performed consecutively from 3 to 10 weeks of age [32] or for a total of 42 days [31]. Kaneko et al. [32] outlined linearly reduced growth performance with an increasing concentration of tea extract, while Rowghani et al. [31] reported improved growth performance following supplementation with 3 mL/L of green tea extract.

The aim of the trial was to evaluate the effect of including a commercial plant extract based on green tea leaves and pomegranate rinds in drinking water on the growth performance, antiradical activity and cecal microbial ecology of broiler chickens during specific critical moments of the rearing cycle.

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

#### *2.1. Animals and Housing*

The trial was performed at the Animal Production Research and Teaching Centre of the Polo Veterinario, Università degli Studi di Milano (Lodi, Italy), using 1-day-old male broiler chicks (ROSS 308) obtained from a commercial hatchery (Avicola Alimentare Monteverde, Rovato, BS, Italy). At hatching, all chickens were vaccinated against Marek's disease, Newcastle disease, infectious bronchitis, and coccidiosis. The chickens were housed in floor pens (2.9 m2) on new shavings of white wood in two identical climate-controlled rooms. Water and feed were provided ad libitum. Room temperature was 35 ◦C for the first 3 days, then decreased weekly by 2 ◦C to a final temperature of 21 ◦C at the end of the trial. The study period lasted from the day of hatch until day 50. All procedures were reviewed and approved by the Animal Care and Use Committee of the University of Milan (OPBA\_92\_2016).

#### *2.2. Experimental Design*

A total of 480 1-day-old ROSS 308 male broiler chickens were randomly allocated to two experimental groups of 12 pens each at a stocking density of 20 birds/pen. Each experimental room housed six randomly distributed pens per treatment, in order to reduce any environmental effects.

All animals received the same diets (Table 1) formulated to meet the nutrient requirements established by the National Research Council (NRC, 1994).


**Table 1.** Feed ingredients and nutrient composition of basal diets (as-fed basis).

† Provided the following per kg of diet: vitamin A, 11,250 IU; vitamin D3, 5000 IU; vitamin E, 60 mg; MnSO4·1H2O, 308 mg; ZnSO4·1H2O, 246 mg; FeSO4·1H2O, 136 mg; CuSO4·5H2O, 39 mg; KI, 2.4 mg; Na2SeO3, 657 μg; 6-phytase EC 3.1.3.26, 750 FTU; endo-1, 4-beta-xylanase EC 3.2.1.8, 2250 U.

Diets were provided by Agricom International (Pognano, BG, Italy) according to a three-phase feeding program, in crumbled form for starter and grower phases (0–10 and 11–20 days, respectively), and pelleted form for finisher phase (21–50 days). All experimental diets were formulated and manufactured using the same lots of ingredients and without antibiotics or coccidiostats. Collected feed samples were analysed before the beginning of the trial to determine the content of dry matter (method 930.15), crude protein (method 984.13), ether extract (method 920.39A), ash (method 942.05), Ca (method 968.08), and P (method 946.06) following the relevant Association of Official Analytical Chemists methods of analysis [33].

Experimental treatments consisted of including (treated, T) or not including (control, C) a plant extract (PE) in the drinking water at a dosage of 2 mL/L. Treated birds received PE from 0 to 4 days of the trial and on days 10, 11, 20, and 21, corresponding to the beginning of the trial and the start of the second and third feeding phases. PE was included in one graduated tank for each pen to determine water intake during the treatment period.

The PE was composed of green tea leaves (*Camellia sinensis*) and pomegranate rinds (*Punica granatum*) (IQV-10-P01, InQpharm Animal Health, Kuala Lumpur, Malaysia). During the entire trial, water was provided ad libitum via automatic nipple cup drinker, except during the three treatment periods, when it was provided in graduated plastic tanks placed in each pen. During the trial, growth performance was evaluated at the beginning, at each feed change and at the end of the experiment. On day 50, one representative broiler chicken from each pen was selected and sacrificed; dressing percentage was calculated and blood and cecal content were collected for total antiradical activity assay and gene sequencing, respectively.

#### *2.3. Growth Performance and Water Intake*

Body weight (BW) and feed intake (FI) of the broilers were determined on a pen basis at 0, 10, 20, and 50 days of age. Mortality was recorded daily together with the BW of dead birds to calculate mortality percentage and correct productive performance results. Water intake was determined on a pen basis during PE administration on days 0–4, 10–11 and 20–21 as the difference between offered and residual water. At the end of the trial, one representative animal was selected from each pen based on pen average BW and sacrificed.

Dressing percentage was calculated by dividing eviscerated weight by live weight. Breast muscle was then removed and weighed, and breast muscle yield was calculated as percentage of eviscerated weight.

#### *2.4. Total Antiradical Activity*

Blood samples were collected from sacrificed broiler chickens on day 50 in 10 mL vacutainer tubes containing EDTA (Venoject®, Terumo Europe NV, Leuven, Belgium) and stored at 4 ◦C for determination of total blood antiradical activity. Blood samples were processed within 3 h of sampling and analysed in the next 24 h after collection by a Kit Radicaux Libres biological test (KRL, Laboratories Spiral, Dijon, France) following the user protocol. The results were expressed as time (in minutes) required to achieve 50% of maximal haemolysis (half-haemolysis time, HT50), which references whole blood and red blood cell (RBC) resistance to a standardized free-radical attack generated from the thermal decomposition of a 27 mmol/L solution of 2,2 -azobis (2-amidinopropane) hydrochloride (AAPH) at 37 ◦C [34–36].

#### *2.5. Cecal Microbiota*

Cecal contents were collected from sacrificed broiler chickens to perform 16S rRNA gene sequencing. Cecal contents were removed and placed into a sterile tube (Sarstedt, Nümbrecht, Germany), snap-frozen in liquid nitrogen and stored at –80 ◦C. Bacterial DNA was isolated from cecal contents using the ExgeneTM Stool DNA mini kit (Geneall Biotechnology Co., Ltd., South Korea) starting with 200 μg samples following the manufacturer's procedure. The extracted DNA was quantified using Synergy HTX (Biotek, Winooski, VT, USA) with a final concentration ranging from 3–10 ng/uL. Variable regions V3–V4 of the 16S rRNA were amplified by Polymerase Chain Reaction (PCR) with universal

primers for prokaryotes [37]. Amplicon sequencing was carried out on an Illumina MiSeq 300PE platform to obtain raw paired-end reads 2 × 300 bp (BMR Genomics, Padova, Italy). The 16S sequencing data were processed and analysed using CLC Genomics Workbench version 12.0 and CLC Microbial Genomics Module version 4.1 (CLC bio, Arhus, Denmark). The paired-end reads were merged into one high-quality representative by default settings of CLC Workbench (mismatch cost = 1, minimum score = 40, gap cost = 4, maximum unaligned end mismatches = 5). The CLC pipeline was used for primer and quality trimming (trim using quality scores = 0.05; trim ambiguous nucleotides: maximum number of ambiguities = 2; discard reads below length = 5). The SILVA reference database [38] was used for sequence alignment, and sequences were binned into operational taxonomic units (OTUs) based on 97% similarity. The OTU table was further filtered by removing OTUs with low abundance (minimum combined abundance = 10), to get a final abundance table for each sample. The phylogenetic tree was constructed using a maximum likelihood phylogeny tool based on multiple sequence alignment of the OTU sequences (100 most abundant OTUs) generated by the multiple sequence comparison by log-expectation (MUSCLE) tool [39] in the workbench. The maximum likelihood phylogeny tool determines the probability of sequences in the tree, using neighbour joining as the construction method and the Jukes–Cantor model as a nucleotide substitution model. The OTU table was used to calculate alpha diversity indices such as Chao1 and Shannon's indices.

#### *2.6. Statistical Analysis*

A completely randomized design was used. Growth performance was analysed using Statistical Analysis System software (SAS version 9.4; SAS Institute Inc., Cary, NC, USA) applying a MIXED procedure for repeated measurements and accounting for the effects of treatment, time and treatment × time interaction. Total weight gain (TWG), total feed intake (TFI), total feed conversion ratio (TFCR), water intake, carcass characteristics, and KRL measurements were analysed using one-way analysis of variance (ANOVA) to compare the means of the two groups using the GLM procedure of SAS. Mortality rate was analysed by PROC FREQ of SAS over the trial period.

The pen represented the experimental unit for growth performance parameters, while the broiler represented the experimental unit for carcass characteristics and KRL measurements. All numerical data in tables are presented as least-square means (LSMeans) accompanied by standard error of the mean (SEM) values. Differences between groups were considered statistically significant at *p* < 0.05, whereas a trend for a treatment effect was noted for 0.05 < *p* < 0.10.

To determine diversity shared among communities in the cecal microbiome of the samples, beta diversity (both weighted and unweighted UniFrac) was calculated in the CLC Workbench (CLC bio, Aarhus, Denmark) and significance was measured by permutational multivariate analysis of variance (PERMANOVA). MicrobiomeAnalyst tool [40] was used for further relevant statistical analysis. During the analysis, the OTUs that did not meet the following parameters were removed: minimum number of counts 1, 5% prevalence in the sample and 1% of samples below the standard deviation. Log transformation was used as a normalization method for downstream analysis, which also includes differential abundant analysis at different taxon levels, performed by the metagenomeSeq package (v3.10, Bioconductor) [41]. Differentially abundant taxa were determined at a false discovery rate (FDR) < 0.05.

#### **3. Results**

#### *3.1. Growth Performance, Water Intake, Carcass Characteristics and Total Antiradical Activity*

Body weight, weight gain, FI, and FCR are shown in Table 2. The administration of PE in drinking water did not affect growing performance of treated broilers during the different rearing phases (*p* > 0.05). In the same way, no significant differences were seen for mortality rate, dressing or breast percentage. Pen water intake was not influenced by the treatment in the first 4 days of hatching (C: 3.42 L vs. T: 3.36 L; *p* = 0.84), and on days 10–11 (C: 5.75 L vs. T: 7.72; *p* = 0.86) and 20–21 (C: 11.53 L vs. T: 11.75 L; *p* = 0.44) of the trial.


**Table 2.** Effects of plant extract supplementation on growth performance parameters and carcass characteristics of broilers. Data shown as LSMeans ± SEM.

Note: *p* < 0.05 considered significantly different, 0.05 < *p* < 0.1 considered tendency. SEM: standard error of the mean; BW: body weight; TWG: total weight gain; FI: feed intake; TFI: total feed intake; FCR: feed conversion ratio; TFCR: total feed conversion rate. C: animals receiving no supplementation; T: animals receiving 2 mL/L green tea and pomegranate extract in drinking water at days 0–4, 10–11 and 20–21. <sup>1</sup> Corrected for mortality; mortality and BW of dead birds were recorded daily to calculate mortality percentage and correct productive performance results. <sup>2</sup> One representative animal from each pen was selected based on pen average BW.

The effects of PE on total antiradical activity are shown in Table 3. Including PE in drinking water during critical moments of the broiler's rearing cycle significantly improved the total antiradical activity, in both whole blood (HT50 blood, *p* < 0.01) and RBCs (HT50 RBC, *p* < 0.05).

**Table 3.** Effects of plant extract supplementation on total antioxidant activity. Data shown as LSMeans ± SEM.


Note: *p* < 0.05 considered significantly different. HT50: time (minutes) required to achieve 50% of maximal haemolysis; RBC: red blood cell. C: animals receiving no supplementation; T: animals receiving 2 mL/L green tea and pomegranate extract in drinking water at days 0–4, 10–11 and 20–21. <sup>1</sup> One representative animal from each pen was selected based on pen average BW; blood samples were obtained at slaughter.

#### *3.2. Cecal Microbiota*

Sequencing of amplicons resulting from the amplification product of PCR for variable regions V3–V4 of the 16S rRNA by PCR was performed to investigate the treatment effect on cecal microbiome. Details of sequence read and OTU counts are provided in the supporting materials (Figure S1).

No statistical differences (*p* > 0.05) were seen in alpha diversity measured by bias-corrected Chao1 and Shannon's indices between C and T groups. Similarly, for beta diversity, no statistical differences (*p* > 0.05) were observed in PERMANOVA (unweighted and weighted UniFrac) between the experimental groups.

Relative abundance at different taxon levels (phylum, order, class) is shown in Figure 1. Firmicutes was found to be the most abundant phylum in both experimental groups, accounting for 69.47% in the C group and 68.65% in the T group. Bacteroidetes emerged as the second most abundant phylum, with 20.94% in the C group and 25.55% in the T group. Proteobacteria were the third phylum, with 8.49% in the C group and 4.84% in the T group. At the class level, Clostridia was the most abundant taxon in both experimental groups, followed by Bacteroidia, Gammaproteobacteria and Bacilli (Figure 1).

**Figure 1.** Relative abundance in control and treated groups at different taxon levels: (**A**) phylum, (**B**) class and (**C**) order. Classes and orders with counts <10 are merged and reported as "others".

Differential abundant analysis was performed to find the significantly different (FDR < 0.05) taxon between the two groups (C and T). No significant differences were found at the phylum level. At the class level, Bacilli were significantly higher in the T group with respect to the C group. Similarly, at the order level, Lactobacillales showed significantly (FDR < 0.05) greater abundance in T animals compared to C animals. At the family level, Lactobacillaceae and Peptococcaceae were significantly more abundant in the T group compared to the C group (FDR < 0.05). Clostridiaceae\_1 tended (FDR = 0.06) towards higher abundance in the T group compared to the C group. At the genus level, *Roseburia* was found to be significantly higher in the T group compared to the C group (FDR < 0.05). On the contrary, *Shuttleworthia* was found to be significantly (FDR = 0.04) higher in the C group. *Lactobacillus\_ambiguous\_taxa*, *Christensenellaceae\_R7\_ambiguous\_taxa* and *Tyzzerella\_3* tended (FDR = 0.06) to be higher in the T group compared to the C group. A list of significantly differentially abundant taxa based on *p*-value (<0.05) is given in Supplementary Table S1.

#### **4. Discussion**

Recently, phytobiotics gained increasing attention as a replacement for antimicrobial growth promoters to enhance growth performance and improve animal health [42,43]. The positive effects of phytobiotics have been associated with high polyphenolic content, which can counteract the effect of free radical generation [15], and their ability to modulate gut microflora composition, leading to increased performance [14,22].

In the present study, the lack of positive results as expected might be due to the administration route, the dosage applied, or the duration of supplementation. Generally, supplementation of poultry with green tea and pomegranate extracts was shown to improve broiler productivity [20–22]. However, nearly all studies reporting positive effects on growth performance administered the compounds in the feed and for the entire rearing period. To the best of our knowledge, only two studies investigated the single administration of green tea extract to broiler chickens in drinking water, while no data are available on pomegranate or combined supplementation. Rowghani et al. [31] outlined improved growth performance after administration of green tea extract in drinking water at a rate of 3 mL/L, while Kaneko et al. [32] observed a linear reduction of body weight and feed intake with increased concentration of Japanese tea from 6.25 g/L to 25 g/L. In our trial, the lower dosage of green tea and pomegranate mixture was chosen on the basis of the synergistic activity previously evidenced between green tea and other plants, including pomegranate that was able to enhance antioxidant activity in vitro [28]. Finally, the administration of PE for only a few days rather than the total length of the trial could have contributed to the lack of expected results. This is in contrast with the results we obtained in a previous study on post-weaning piglets, with a similar experimental design, which led to an increase in average daily weight gain during the last week of the experimental period [44].

Besides these aspects, a large body of literature highlights the high variability of the efficacy of phytobiotics in improving animal performance and carcass characteristics. This can be explained by the different biological potential of the phytobiotics tested, accounting for the extraction procedure, the part of the plant used, the geographic origin, and the harvest season [45]. According to our findings, Farahat et al. [46] observed no effect on carcass characteristics with different amounts of green tea extract in feed, while Erener et al. [47] and Hamady et al. [20] reported improved carcass characteristics following the administration of green tea and pomegranate extract, respectively.

The administration of PE significantly increased the total antiradical activity of whole blood and RBCs, confirming the beneficial effect of PE in improving antioxidant defences of animals. This result can be attributed to the high polyphenol content of both green tea and pomegranate extract, which is able to prevent reactive oxygen species (ROS) generation and the damage they induce. The proposed mechanism of action for polyphenols is that after being absorbed in the gut, they are bound by blood cells, mainly erythrocytes, leading to enhanced total antioxidant-scavenging capacity of the blood [48]. The antioxidant effects of PE were recently confirmed by Rao et al. [49], who observed reduced lipid peroxidation and increased glutathione peroxidase activity after supplementation with pomegranate peel meal in broiler chickens. Similarly, including green tea extract in the poultry diet increased the glutathione-reduced level in the liver and significantly decreased the malondialdehyde level of meat tissue [46].

The development of intestinal microbiota in poultry plays an important role in production performance and overall health [18], and phytobiotics, including green tea and pomegranate, have been proven to be effective in its modulation [20,23]. It is recognized that colonization of the gut microbiota in critical moments of life could have an impact on an animal's entire life span [19]. Among the critical moments, the post-hatching phase is one of the most important, since it is when the first gut colonization occurs [50]. Several studies have shown that early gut microflora modulation can affect health and productivity in later stages of a broiler's life [36,51]. The post-hatching phase, however, is not the only critical moment in defining the gut microbiota composition. The microbial population can also be affected by changes in the diet, with regard to the feed form or its chemical composition [52]. To the best of our knowledge, the present study is the first to investigate the effects of a targeted intervention with phytobiotics at critical moments of the production cycle. Our results show that the administration of PE during the post-hatching phase and changes in the feeding phase did not impact the cecal microbiota composition, keeping the microbial profile in line with the diet used in general practice. The gut microbial population observed in this study was indeed aligned with what was reported by Wei and colleagues [53]. In this review, the authors described the cecal microbial composition of adult birds, reporting Firmicutes as the most abundant phylum, followed by Bacteroidetes and Proteobacteria.

Although the microbial profile was not different between the two experimental groups, relative abundance differences were noted at different taxonomic levels (class, order and family), suggesting a beneficial modulation of gut microflora by PE. In accordance with our findings, Saeed et al. [29] observed higher relative abundance of Bacilli in the ileum and jejunum of broiler chickens following supplementation with L-theanine, an amino acid extracted from green tea [29]. In our study, animals receiving PE showed greater relative abundance of lactic acid bacteria compared to the control group. This result was confirmed at the family level, where Lactobacillaceae and Enterococcaceae were found to be more abundant in T broilers. Also, at the genus level, *Lactobacillus* showed a tendency to be higher in the T group. These findings are of particular interest because lactic acid bacteria are recognized for their beneficial effect in the intestine, regulating the composition of intestinal microflora, developing intestinal immunity and promoting gut health [54]. Lactobacilli can indeed protect against the colonization of pathogenic bacteria through the acidification of the lumen and the production of bacteriocins [55,56].

Besides modulating lactic acid bacteria, PE supplementation also determined some differences at the genus level. *Roseburia\_ambiguous\_taxa* was found to be significantly higher in animals receiving PE. *Roseburia* genus is a commensal saccharolytic bacteria that produces SCFAs and has been proposed in human medicine as probiotic for restoration of beneficial flora [57]. In addition, a lower abundance of *Shuttleworthia* was observed in the T group. Information about this genus is limited, but a study reported that enrichment of *Shuttleworthia* in the ceca of male broiler chickens was associated with high body weight [58], which was not evidenced in our study.

#### **5. Conclusions**

The administration of PE in drinking water during the post-hatching phase and at changes between feeding phases can improve total blood antiradical activity and may positively affect the gut microbial ecology of adult broiler chickens by increasing the relative abundance of lactic acid bacteria, with no effect on performance parameters.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-2615/10/5/785/s1. Figure S1: (A) Mean number of 16S rRNA sequence reads and (B) OTU counts detected in cecal samples of broilers in treated (T) and control (C) groups. Table S1: Significantly different taxa according to *p*-value (≤0.05) shown by differential abundance analysis between the two experimental groups.

**Author Contributions:** Conceptualization, V.B. and M.C.; methodology, V.B., M.C., C.G. and R.R.; software, A.A., V.P. and M.C.; validation, V.B., M.C., A.A., V.P., G.S., C.G. and R.R.; formal analysis, M.C., C.G. and R.R.; investigation, M.C., C.G. and R.R.; resources, V.B.; data curation, M.C., V.P., A.A., C.G. and R.R.; writing—original draft preparation, V.B., M.C., V.P., A.A., G.S., C.G. and R.R.; writing—review and editing, V.B., M.C., V.P., A.A., G.S., C.G. and R.R.; visualization, V.P., A.A., M.C. and V.B.; supervision, V.B.; project administration, V.B.; funding acquisition, V.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by InQpharm Animal Health.

**Acknowledgments:** The authors would like to thank InQpharm Animal Health for the kind support in providing the test product.

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

#### **References**


© 2020 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/).

### **Associations Between** *IGF1, IGFBP2* **and** *TGFß3* **Genes Polymorphisms and Growth Performance of Broiler Chicken Lines**

**Bozena Hosnedlova 1,\*, Katerina Vernerova 2, Rene Kizek 1,3,4, Riccardo Bozzi 5, Jaromir Kadlec 6, Vladislav Curn 2, Frantisek Kouba 7, Carlos Fernandez 8, Vlastislav Machander <sup>9</sup> and Hana Horna <sup>9</sup>**


Received: 31 March 2020; Accepted: 23 April 2020; Published: 5 May 2020

**Simple Summary:** The main goal of breeding programs for broiler chickens is to increase growth rate and breast and thigh muscles weight. The candidate gene approach is a powerful technique for genetically improving performance traits in chickens. We studied the associations of the single nucleotide polymorphisms of three genes involved in protein synthesis, glucose metabolism and cell proliferation (*IGF1, IGFBP2, TGF*β*3*) with performance traits in the Hubbard F15 and Cobb E chicken lines. Based on our results, it can be concluded that the *TGF*β*3* gene could be used as a candidate gene marker for chicken growth traits.

**Abstract:** Marker-assisted selection based on fast and accurate molecular analysis of individual genes is considered an acceptable tool in the speed-up of the genetic improvement of production performance in chickens. The objective of this study was to detect the single nucleotide polymorphisms (SNPs) in the *IGF1, IGFBP2* and *TGFß3* genes, and to investigate their associations with growth performance (body weight (BW) and average daily gain (ADG) at 14, 21, 28, 35 and 42 days of age) and carcass traits in broilers. Performance (carcass) data (weight before slaughter; weights of the trunk, giblets, abdominal fat, breast muscle and thigh muscle; slaughter value and slaughter percentage), as well as blood samples for DNA extraction and SNP analysis, were obtained from 97 chickens belonging to two different lines (Hubbard F15 and Cobb E) equally divided between the two sexes. The genotypes were detected using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) methods with specific primers and restrictase for each gene. The statistical analysis discovered significant associations (*p* < 0.05) between the *TGF*β*3* SNP and the following parameters: BW at 21, 28 and 35 days, trunk weight and slaughter value. Association analysis of BWs (at 21, 28 and

35 days) and SNPs was always significant for codominant, dominant and overdominant genetic models, showing a possible path for genomic selection in these chicken lines. Slaughter value was significant for codominant, recessive and overdominant patterns, whereas other carcass traits were not influenced by SNPs. Based on the results of this study, we suggested that the *TGF*β*3* gene could be used as a candidate gene marker for chicken growth traits in the Hubbard F15 and Cobb E population selection programs, whereas for carcass traits further investigation is needed.

**Keywords:** chicken; SNP; *IGF1*; IGFBP2; TGFß3; Hubbard F15; Cobb E; growth; meat

#### **1. Introduction**

With the growth of the human population [1], the total amount of meat consumed increases at a global level worldwide [2]. Meat consumption rose worldwide from 23.08 kg per person per year in 1961 to 43.22 kg per person per year in 2013 [2]. Chicken meat is one of the most consumed types of meat worldwide [3].

Growth performance and carcass traits are the most important economic traits in broiler chicken production, and are controlled by a number of genes [4]. Growth is a complex process that involves the regulated coordination of a wide range of neuroendocrine pathways [5]. Therefore, it is very difficult to achieve rapid genetic improvement in these traits using only traditional selection methods. The growing knowledge of the structure and function of the chicken genome can be beneficial, and can lead to the recognition of causal genes and the development of new selectable molecular markers.

Although the *Gallus gallus* (chicken) genome was first sequenced as early as 2004 [6], it still required further improvements [7–9]. The newest version of the chicken genome assembly (Gallus\_gallus-5.0; GCA\_000002315.3), built from combined long single molecule sequencing technology, finished bacterial artificial chromosomes (BACs) and improved physical maps, was presented in 2017 [10]. Since the methodological approach has improved, the originally reported size of the chicken genome has increased from 1.05 [6] to 1.23 Gb, which has contributed to the increased number of genes observed [10]. Initial assemblies have been found insufficient for the more complete discovery of allelic contributions to complex traits [10], leading to ongoing efforts to improve the quality of the chicken reference genome [8,11].

However, the genetic improvement of polygenic traits, including growth performance and meat production, can be accomplished by marker-assisted selection that is more accurate in estimating the animal's genetic value [12]. The molecular markers linked to quantitative trait loci (QTLs) are not affected by environmental conditions. Therefore, they could increase the speed and effectiveness of animal breeding progress. As soon as the relationship between a DNA polymorphism and an important trait is revealed, the DNA marker may be used [13]. The candidate gene approach has become a powerful technique for the genetic improvement in chicken breeding programs, and can result in increased efficiency in detecting the required production performance traits [4].

The main objectives of the strategy in commercial broiler breeding programs include increasing growth rate and breast muscle weight, reducing abdominal fat content, improved feed efficiency and increased fitness. The relationships between these individual production traits are very complex and some of them are very difficult to measure. Therefore, the use of molecular marker-assisted selection (MMAS) is necessary. In case that the favorable allele is rare, a larger positive impact can be expected [14].

The purpose of the present study was to identify polymorphisms and evaluate the association between polymorphisms in three studied genes—*IGF1* (insulin-like growth factor 1), *IGFBP2* (insulin-like growth factor binding protein 2) and *TGFß3* (transforming growth factor β)—with growth performance and meat production in chickens from two broiler lines: Hubbard F15 and Cobb E. The biological functions and interdependence of these genes are shown in Figure 1.

**Figure 1.** Signaling cascade of insulin-like growth factor 1 (IGF-1) and its potential impacts in metabolism, its interactions with transforming growth factor β3 (TGF-β3) and the biological functions of the *IGF1*, *IGFBP2* and *TGFß3* genes. IGF-1 bioavailability is modulated by IGF binding proteins (IGFBPs) [15]. IGF-1 action is mediated by its binding to its receptor [16], the type 1 insulin-like growth factor receptor (IGF-1R). IGF-1R is a heterotetramer composed of two extracellular α subunits and two transmembrane β subunits, as shown in Figure 1. α subunits are cysteine-rich regions, whereas β subunits possess a tyrosine kinase domain, which constitutes the signal transduction mechanism [16]. Tyrosine phosphorylation activates a signaling cascade [17]. IGF-1 has autocrine, paracrine [18,19] and endocrine effects [18]. IGF-1 binds toits receptor (IGF-1R) in the cell membrane, resulting in autophosphorylation and the recruitment of the adaptor proteins–insulin receptor substrate IRS-1, IRS-2, and the proto-oncogene tyrosine-protein kinase (SRC) homology and collagen protein (SHC). The serine/threonine kinase (AKT) is activated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) and by the mammalian target of rapamycin (mTOR)-containing complex mTOR-C2, leading to the phosphorylation at threonine 308 and serine 473, respectively. Activated AKT regulates downstream signaling molecules such as tuberous sclerosis protein 1/2 (TSC-1/2), which inhibit mTOR-C1 complex and regulate the ribosomal protein S6 kinase 1/2 (S6K-1/2) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EB-P1) phosphorylation, FOXO transcription factors, glycogen synthase kinase-3β (GSK-3β), p27, BCL-2 antagonist of cell death (BAD), and BCL-2. These substances are involved in some cellular processes such as protein synthesis, glucose metabolism and cell survival. SHC activation induces the activation of the RAS/mitogen-activated protein (MAP) kinase pathway, resulting in enhanced cell proliferation [15]. Activation of IRS induces the activation of intracellular RAF/MEK/ERK/RAS and PI3K signaling pathway. The first mentioned pathway mediates mitosis, and the second one mediates metabolism and cell growth effect through AKT [20]. After the ligand (IGF-1) binds to its receptor (IGF-1R), PI3K is activated, cell proliferation is promoted by activating the mitogen-activated protein kinase (MAPK) cascade, and apoptosis is blocked by inducing the phosphorylation and the inhibition of proapoptotic proteins such as BAD [21]. The protein IGFBP-2 encoded by the gene of the same name is able to control the biological actions of IGFs [22] and TGFß [23] in vivo via the endocrine, autocrine or paracrine pathways. The protein TGFß-3 encoded by the *TGF*β*3*gene controls the growth, proliferation and differentiation of cells, cell motility and apoptosis. TGFß-3 plays an essential role in the development of skeletal muscles. It also can suppress the formation of tumors [24]. Adapted from [15,25–27] based on other works: [21,28–53].

The insulin-like growth factor 1 gene (*IGF1*) has been identified as a biological candidate gene for growth, body composition, metabolic and skeletal characteristics, and is also a positional candidate gene for growth and fat deposition in chicken [54]. This gene is involved in growth of various tissues such as muscle and bones [55]. The chicken *IGF1* gene was mapped to 165.95 cM on chromosome 1 (GGA1). In a broiler-layer F2 population used to map body weight (BW) QTL by a genome scan, a QTL affecting BW at 6 weeks of age has been detected at 160 cM (confidence interval (CI) 114 to 180 cM) on chicken GGA1 [56]. In the same F2 cross, a QTL at 150 cM (CI from 100 to 182 cM) on GGA1 affecting abdominal fat weight (AFW) has been ascertained [57].

The bioavailability of the insulin-like growth factors (IGFs) is regulated by a family of structurally conserved insulin-like growth factor binding proteins (IGF-binding proteins; IGFBPs) [58–60]. IGFBPs selectively bind to IGF-1 and IGF-2 proteins but do not bind to insulin [61]. More than 99% of IGF molecules circulate in blood serum as complexes with these specific and high affinity-binding proteins. Although IGF-binding protein 3 (IGFBP-3) is a main component and binds over 75% of the circulating IGF [62], IGFBP-2 is sensitive to dietary protein level, and may play a substantial role in the modulation of the growth-promoting effect of circulating IGF-1 by creating the IGF-1-IGFBP-2 complex in chickens [63]. IGFBP-2 is the predominant IGFBP in serum for different species [64]. IGFs, IGFBPs and IGFBP proteases are the major regulators of somatic growth and cell proliferation [65]. IGFBP-2 controls the biological actions of IGFs [22] and TGFs [23] in vivo via endocrine [22,23], autocrine [23] or paracrine mechanisms [22], and affects the growth and development of animals [66]. IGFBP-2 might indirectly affect adipocyte differentiation by controlling IGF [67] and TGF-β biological actions in fat tissue [68].

The transforming growth factor β (TGF-β) belongs to a large family of multifunctional growth factors [69], with important regulatory roles in embryonic and adult development [70], such as morphogenesis, development and differentiation [69]. Polypeptide growth factors of TGF-β family regulate a number of cellular processes such as cell proliferation, differentiation, migration, adhesion and apoptosis [70]. TGF-β plays a key role in maintaining both bone and articular cartilage homeostasis [71].

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

#### *2.1. Experimental Population—Animals*

The chicken hatching eggs were produced and the experiment was performed in the testing station of broilers (fattening test No. 1148) at the state-owned enterprise International Testing of Poultry, Ustrasice (Czech Republic). After hatching, 50 chickens from each of the two broiler lines Hubbard F15 and Cobb E were stalled in windowless air-conditioned hall with deep bedding and controlled light mode (Table 1). Stocking density was 6.1 chicks/m2. The hall was disinfected with Virkon before the chickens were stored. The chickens were watered by automatic dropper drinking basins and fed with three feed mixtures, differently for particular period of fattening, from tube feeders *ad libitum*. Hypermangan solution was applied to the water in the first days of age. The composition (contents of main nutrients) in individual complete feed mixtures BR1, BR2 and BR3 for fattening broiler chickens up to the 10th, 35th and 42nd day of age, respectively, are shown in Table 2.



**Table 2.** The content of nutrients in feeding mixtures for broilers (BR1, BR2, BR3) in different periods of the experiment.

\* The feeding mixtures were produced in ZZN Pelhrimov, a.s., according to given recipes.

#### *2.2. Phenotypic Data*

Body weight (BW) was measured at 14, 21, 28, 35 days and before slaughter at 42 days of age. The mortality during experiment was 3% (sudden death syndrome). Chickens were slaughtered at 42 days of age and the slaughter analysis was performed. The carcass traits, such as weight of trunk, giblets, abdominal fat, breast muscle (with and without skin) and thigh muscle (with and without skin), as well as slaughter value and slaughter percentage were investigated. The slaughter value was calculated as the ratio between the weight of the carcass (trunk weight) and the weight at 42 days of age before slaughter, and the slaughter percentage was calculated as the ratio between the sum of weight of the trunk and giblets and the weight at 42 days.

#### *2.3. SNP Genotyping*

#### 2.3.1. DNA Extraction

Genomic DNA for genotyping assays was extracted from whole blood samples, which were collected from 97 chickens at 42 days of age before slaughtering. Blood was taken from *vena ulnaris* into 1.5 mL EDTA-treated microtubes. For extraction of genomic DNA, chelex 100 was used, and the concentration and purity of genomic DNA were verified by spectrophotometer Shimadzu BioSpec-nano (Shimadzu Corporation, Kyoto, Japan).

#### 2.3.2. Optimalization of PCR-RFLP Assay

Polymerase chain reaction (PCR) was performed for all assays in total volume 25 μL mixture containing 1 μL genomic DNA, 10 pmol of each primer and 12.5 μL PPP Master Mix (Top-Bio, s.r.o., Vestec, Czech Republic). Sequences of sets of primer pairs for all three gene polymorphisms used in PCR assays are shown in Table 3.The primers for the *IGF1* genotyping were designed according to Moody et al. [72]. The thermal profile included pre-denaturation at 94 ◦C for 2 min followed by 30 cycles 94 ◦C for 30 s, 67 ◦C for 30 s and 72 ◦C for 50 s, with a final extension of 72 ◦C for 7 min. Thermocycler BIOER Life ECO (Hangzhou Bioer Technology Co,. Ltd., Bin An Rd, Hi-tech (Binjiang) District, Hangzhou, China) was used for DNA amplification. SNP of *IGF1* gene was detected after digesting PCR product with *Hinf*I restriction endonuclease (Fermentas, Vilnius, Lithuania) at 37 ◦C overnight. For detection of *IGFBP2* genotypes PCR amplification was done using primer set by Li et al. [73]. Amplification was performed under following conditions: pre-denaturation at 94 ◦C for 2 min followed by 30 cycles 94 ◦C for 30 s, 54 ◦C for 30 s and 72 ◦C for 30 s, with a final extension of 72 ◦C for 7 min. PCR products were digested (restriction fragment length polymorphism–RFLP) with *Eco72*I restriction endonuclease (New England Biolabs, Ipswich, MA, USA) at 37 ◦C overnight. The PCR primers designed by Li et al. [69] were applied for *TGF*β*3.* The PCR reaction conditions were the same as for *IGFBP2*, except the annealing temperature which was 58 ◦C. Gene fragments

were subjected to digestion by *Bsl*I restriction enzyme (New England Biolabs, Ipswich, MA, USA) at 37 ◦C overnight.


**Table 3.** Primers used in polymerase chain reaction (PCR) assay.

<sup>a</sup> Moody et al. (2003) [72], Zhou et al. (2005) [54]. <sup>b</sup> Li et al. (2006) [73]. <sup>c</sup> Li et al. (2003) [69]. \* All possibilities of fragments.

#### 2.3.3. Electrophoresis

The PCR products were visualized by 2% and restriction patterns by 3% agarose gel electrophoresis and ethidium bromide staining. The ENDURO™ 250 V power supply (Labnet International, Inc., New York, USA) and the HU13 midi horizontal gel electrophoresis unit (Scie-Plas Ltd., Cambourne, Cambridge, UK) were used for DNA electrophoresis. Syngene™ Ingenius 3 Manual Gel Documentation System (Syngene) was used for photo-documentation.

#### *2.4. Statistical Analysis*

Associations of three different polymorphisms of *IGF1*, *IGFBP2* and *TGF*β*3* genes with growth characteristics and carcass data in 97 chicken belonging to two different lines (Hubbard F15 and Cobb E), equally divided between two sexes, were studied. Genotypes were tested for Hardy-Weinberg equilibrium (HWE) using a chi-square (χ2) test in R/SNPassoc Package (R Development Core Team). Whole-Genome association analyses were performed assuming five different genetic models (inheritance patterns) using R/SNPassoc Package (R Development Core Team): codominant, dominant, recessive, overdominant and log-additive effect. The level of significance was tested at the nominal 5% significance level after correcting for the number of tests performed (Bonferroni correction). Phenotypes were represented by the carcass data collected on poultry, whereas line and sex were included in the model as fixed effects. Hardy-Weinberg equilibrium (HWE) was calculated and tested using χ<sup>2</sup> test at the 0.05 level of statistical significance.

Another statistical analysis was performed using box and forest plots. Average slopes of growth curve and total integrals for the weight sum of the trunk, giblets, abdominal fat, breast and thigh muscles at 42 days of age (at the slaughter of chickens) in both chicken lines, for both sexes and for all genotypes observed, were evaluated using the laboratory information system Qinslab (Prevention Medicals, Studenka, Czech Republic).

#### **3. Results**

The *IGF1*/*Hinf*I PCR-RFLP analysis of 97 DNA samples obtained from chicken belonging to broiler lines Hubbard F15 and Cobb E showed only two from three genotypes, namely *AA* (378 + 244 + 191 bp), and *AC* (622 + 378 + 244 + 191 bp), as shown in Figure 2. The *AA* homozygotes (73.20%) predominated over heterozygotes (26.80%)–Table 4, Figure 3. No *CC* homozygous individuals were detected on either of the two broiler lines. Correspondingly, the frequency of allele *A* is much higher (86.60%) than allele *C* (13.40%) in the investigated chicken population, as is evident from Table 4.

In contrast, in *IGFBP2*/*Eco72*I polymorphism, all three genotypes (*AA, AB*, and *BB*) were found, however, *BB* (265 + 102 bp) homozygotes showed very low frequency (4.12%). The most represented genotype was *AB* (367 + 265 + 102 bp) with a frequency of 56.70%.

Also, in *TGFß3*/*Bsl*I SNP, all three genotypes were detected, with the highest observed genotypic frequency in heterozygotes (42.27%) followed by 36.08% and 21.65% in *AA* (145 + 75 + 74 bp) and *BB (*125 + 75 + 74 + 20 bp), respectively.

Only for IGFBP2 frequencies in total population, Hardy-Weinberg equilibrium (HWE) was identified: *p* < 0.01.

**Figure 2.** The restriction fragment length polymorphism (RFLP) patterns for *IGF1* (*AA:* 378 + 244 + 191 bp; *AC:* 622 + 378 + 244 + 191 bp; *CC*–it was not detected), *IGFBP2* (*AA*: 367 bp; *AB*: 367 + 265 + 102 bp; *BB:* 265 + 102 bp) and *TGF*β*3* (*AA*: 145 + 75 + 74 bp, *AB*: 145 + 125 + 75 + 74 bp; *BB*: 125 + 75 + 74 + 20 bp). Agarose 2%, 120 V, 60 min, Tris-borate-ethylenediaminetetraacetic acid (EDTA) (TBE) buffer. M–marker.


**Table 4.** Genotype and allele frequencies of *IGF1, IGFBP2* and *TGF*β*3* genes in the chicken population.

**<sup>a</sup>** The numbers in brackets are percentage frequencies (relative frequencies). **<sup>b</sup>** HWE–Hardy-Weinberg equilibrium;

\* statistically significant (*p* < 0.05)*;* nf–not found.

**Figure 3.** Genotype distribution of individual genes in both chicken lines.

Tables 5–7 show average values of growth performance and carcass traits in both chicken lines, according to individual genotypes. The highest average BW at 42 days was achieved in the Cobb E line, with the *AC* genotype of *IGF1* (2967.50 g), *BB* genotype of *IGFBP2* (3170.00 g) and the *AB* genotype of *TGF*β*3* (3104.29 g). The highest average breast muscle (without skin) was found in a Cobb E chicken with an *AC* genotype of *IGF1* (629.25 g), a *BB* genotype of *IGFBP2* (753.00 g) and an *AB* genotype of *TGF*β*3* (653.62 g). The Cobb E chicken with an *AC* genotype of *IGF1* (52.50 g) and a *BB* genotype of *IGFBP2* (63.00 g) also had the highest average abdominal fat weight (AFW); whereas for *TGF*β*3*, the highest AFW was found in the *AA* genotype (54.95 g). The highest average thigh muscle (with skin) were measured in a Cobb E line chicken with an *AC* genotype of *IGF1* (519.75 g), a *BB* genotype of *IGFBP2* (552.00 g) and an *AB* genotype of *TGF*β*3* (538.72 g). On the contrary, the lowest average values of BW at 42 days and AFW showed in the Hubbard F15 line chicken with an *AA* genotype of *IGF1* (2585.00 and 35.32 g, respectively), a *BB* genotype of *IGFBP2* (2413.33 g and 30.33 g, respectively) and an *AA* genotype of *TGF*β*3* (2541.33 g and 31.53 g). The lowest breast muscle (without skin) showed the Hubbard F15 line chicken with an *AA* genotype of *IGF1* (501.75 g), an *AA* genotype of *IGFBP2* (498.75 g) and an *AA* genotype of *TGF*β*3* (494.33 g). The lowest thigh muscle (with skin) was observed in a Hubbard F15 line chicken with an *AA* genotype of *IGF1 (*470.68 g) and a *BB* genotype of *IGFBP2* (445.00 g) and in a Cobb E line chicken with a *BB* genotype of *TGF*β*3* (458.67 g).


**Table 5.** The average growth performance and carcass traits in the chicken population (according to *IGF1* genotypes).


**Table 5.** *Cont.*

BW–average body weight; SD–standard deviation; CI–confidence interval (95%); \* (g); \*\* slaughter value = weight of trunk/BW at 42 days \* 100 (%); \*\*\* slaughter percentage = weight of trunk+ weight of giblets/BW at 42 days \* 100 (%).



BW–average body weight; SD–standard deviation; CI–confidence interval (95%); \* (g); \*\* slaughter value = weight of trunk/BW at 42 days \* 100 (%); \*\*\* slaughter percentage = weight of trunk + weight of giblets/BW at 42 days \* 100 (%).


**Table 7.** The average growth performance and carcass traits in the chicken population (according to *TGF*β*3* genotypes).

BW–average body weight; SD–standard deviation; CI–confidence interval (95%); \* (g); \*\* slaughter value = weight of trunk/BW at 42 days \* 100 (%); \*\*\* slaughter percentage = weight of trunk + weight of giblets/BW at 42 days \* 100 (%).

By means of statistical software analysis, the relationships between SNPs and individual traits were identified. The fixed effects included in the "whole" model were sex and line, and the *p*-values obtained were adjusted by the number of tests using Bonferroni correction.

Only the *TGF*β*3* SNP (Table 8) resulted in statistical significance for the following parameters: body weight at 21, 28 and 35 days, trunk weight and slaughter value. The *p* values were significant for codominant, dominant and overdominant genetic models, with the exception of the slaughter value, which was not significant for the dominant inheritance pattern.

Figure 4 shows average slopes of growth curve (14–42 days of age) in individual genotypes of both chicken line and sexes.

Figure 5 represents total integrals for the sum of the trunk, giblets, abdominal fat, breast and thigh muscles at 42 days of age in all genotypes observed.


**Table 8.** Results of statistical analysis for testing association between *TGF*β*3* polymorphism and growth performance and carcass traits in the chicken population.

BW–body weight; ns–no significant SNP after Bonferroni correction. Statistical significances at significance level 0.05 are highlighted in bold.

**Figure 4.** Forest plot of body weight (g) of chicken (at 14–42 days of age) of both lines in individual genotypes. (**A**) Males; (**B**) females; (**C**) total. Statistical characteristics of individual variants: average (n), median, standard deviation (SD), *p*-value (compared to average value), minimum (min) and maximum (max) value. Symbol H is Hubbard F15; C is Cobb E. The line segments represent confidence interval–CI (95%).

**Figure 5.** The weight sum of the trunk, giblets, abdominal fat, breast muscle with and without skin and thigh muscle with and without skin. *IGF1* gene with genotype *AA* and *AC* (**A**), *IGFBP2* with genotype *AA* and *AB* (**B**), *TGF*β*3* with genotype *AA, AB* and *BB* (**C**). The pink symbol—median, the grey symbol—mean, square indicates the weight (number of samples). A comparison of individual genotypes (**D**). Statistical characteristics of individual variants: number (n), mean and median. The line segments represent confidence interval–CI (95%); m—males, f—females.

#### **4. Discussion**

The study of candidate genes is one of the primary methods to determine whether specific genes are related to economically important traits in farm animals [69]. We performed genotyping of the SNP of three genes linked to consumer-priced characteristics in chicken meat.

One of the main hormones required to normal growth process and muscle development is insulin-like growth factor 1 (IGF-1) [74]. The chicken *IGF1* gene consists of four exons and three introns, spanning more than 50 kb on chromosome 1 [75].

*IGF1* encodes the same-name protein (IGF-1), which has a similar molecular structure to insulin [76] and induces insulin-like metabolic effects in muscle and adipose tissues [65]. This protein plays an important role in the proliferation, differentiation and metabolism of myogenic cell lines in chickens [76]. IGF-1 is one of the three ligands (insulin, IGF-1, IGF-2) belonging to the IGF system, which also includes three cell surface binding receptors (InsR, IGF-1R, IGF-2R), and insulin-like growth factor binding proteins (IGF binding proteins, IGFBPs) and IGFBP protease [77]. In addition, the IGF-1 protein is a potent mitogen and an essential stimulus for the differentiation of adipocytes [78]. The production and secretion of IGF-1 is affected by age, nutritional status, and several hormones [79]. The predominant source of IGF-1 is the liver and some other tissues, including muscle, brain and kidney [80].

IGF-1 binds to the type 1 insulin-like growth factor receptor (IGF-1R), which plays a critical role in signaling cell survival and proliferation [21]. However, IGF-1 can also bind, albeit with lower affinity, to the insulin receptor [16], regulating some metabolic functions [25].

Insulin-like growth factors (IGFs) provide essential signals for the control of embryonic, as well as postnatal development in vertebrates [81]. In addition to the growth hormone (GH), IGF-1 is one of the two main hormones required to support normal growth in chicken. Optimal growth requires a "set-point" concentration of both IGF-1 and triiodothyronine (T3) in blood circulation. Pituitary GH plays a role in controlling the circulating levels of both IGF-1 and T3 [74]. IGFs stimulate hepatic glycogen, increase DNA synthesis and promote tissue growth in chicken [82]. The highest level of *IGF1 mRNA* expression was detected in the chicken liver. High levels of *IGF1 mRNA* (10%–30% of the value in the liver) were expressed in spleen, lung and brain of chickens. *IGF1 mRNA* expression was

also observed in other extrahepatic tissues such as the kidney, heart, intestine, thymus and muscle of chickens, but these expression levels were less than 4% of that in the liver [83].

The abundant expression of *IGF1* gene was detected in the liver of normal chicken, but no *IGF1 mRNA* expression was found in that organ of dwarf chicken [83]. The expression of hepatic *IGF1 mRNA* level and circulating IGF-1 concentration were significantly higher in chicken with a high growth rate, compared to the line with low growth rate, supporting the hypothesis of its stimulatory effect during post-hatching growth of chickens as stated by Beccavin et al. [84]. The liver is the main site of IGF-1 production during post-hatch growing stages of chicken as described by Kita et al. [85].

IGF-1 is significantly altered by the genotype, suggesting a pivotal role in the control of growth rate in broiler chickens [84].

The SNPs within the chicken *IGF1* promoter were reported by numerous previous studies [54,86–92] but according to the author's best knowledge, no research work on gene constitution of *IGF1* SNP in the Hubbard F15 and Cobb E lines has been reported until now.

Genotype frequency analysis indicated that the *AA* genotype (73.20%) was of higher frequency than the *AC* (26.80%) and *CC* (0%) genotypes in both chicken lines, which is consistent with another study [92]. For the other two genes (*IGFBP2, TGF*β*3*), the predominance of heterozygous (*AB*) genotypes was detected. Interestingly, in Hubbard F15, a distribution of both allele in *TGF*β*3* was identical (Table 4).

With regards to the genotype frequencies of *IGF1*/*Hinf*I gene polymorphism, from the three known restriction patterns, only two genotypes were detected: *AA* and *AC*, with an almost three times higher prevalence of *AA* homozygotes over heterozygotes. The *CC* homozygous genotype was not found in either chicken line, which is consistent with the finding of Moe et al. [90], which reported an absence of the *CC* genotype in two commercial broiler strains (Chunky and Cobb). The noticeable predominance of allele *A* (86.60%) over allele *C* (13.40%) (Table 4) observed in our study is in conspicuous accordance with previous studies. As Moe et al. [90] have shown, allele *C* occurs especially in native chickens, for example in nine Japanese native chicken breeds (Chabo, Ehime-jidori, Gifu-jidori, Koeyoshi, Koshamo, Mikawa, Satsuma-dori, Engie and Tokuchijidori), the frequency of this allele to be 1.0. Our finding of low incidence of *C* allele in broiler chicken also corresponds with the results of genotyping performed by Anh et al. [4], who observed the *CC* genotype of *IGF1* gene with very low frequencies (0.13 to 0.15) in all populations of crossbreds from commercial parent stock broilers with four Thai synthetic chicken lines (the Kaen Thong, Khai Mook Esarn, Soi Nin, and Soi Pet). The *IGF1* SNP gene constitution of these four Thai synthetic chicken lines was studied by Promwatee et al. [92], who found that the *AA* genotype had a considerably lower frequency than the *AC* and *CC* genotypes in all chicken lines except Soi Noi, in which the *AA* and *CC* genotypes were similar. With the exception of Soi Nin, there was the predominance of the *C* allele—the frequency of the *A* allele was lower than that of C in all lines except Soi Noi, where both allelic frequencies were the same. This indicates that allele *C* is evidently typical for native chicken breeds. A higher frequency of allele *A* than that of allele *C* in commercial broiler stocks compared to native chicken can be explained as a result of selection effect on growth traits [90].

It can be concluded that the incidence of a higher *A* allele frequency over *C* allele in the *IGF1* locus observed in our study could be a result of a long-term selection strategy applied in the populations of chosen broiler lines that are the subject of this study.

Various studies reported associations between *IGF1* polymorphism and growth traits in chickens. Zhou et al. [54] and Amills et al. [86] reported that polymorphism of the *IGF1* gene in the promoter and 5 -untranslated region (5 -UTR) was directly associated with chicken growth rate. Bian et al. [89] found that haplotypes based on three *IGF1* polymorphisms (c.-366A > C, c.528G > A and c.\*1024C > T–in 5 -flanking, exon 3 and 3 -flanking regions of *IGF1*) were associated with BW traits.

In our study, *AC* genotype of *IGF1* evinced the highest average BW at 42 days in both chicken lines. This genotype also corresponded with a higher average AFW, breast muscle weight (with or without skin), thigh muscle (with or without skin), slaughter value and slaughter percentage in both lines. On the contrary, the *AA* genotype of *IGF1* was associated with the average lowest BW at 42 days, trunk weight, AFW, breast and thigh muscles, slaughter value and slaughter percentage in both lines. However, no significant difference was identified.

These results are inconsistent with the study of Zhou et al. [54], which observed that broiler line with fragment sizes of 378, 244 and 191 bp (*AA* genotype) showed greater improvement of marketable BW. Additionally, in Thai native chickens, the *AA* genotype resulted in a higher BW compared to the *AC* and *CC* genotypes [93]. Promwatee et al. [92] found, in two synthetic lines (Khai Mook Esarn, Soi Pet), the association between the *AA* genotype and BW at 8 and 12 weeks of age and average daily gain (ADG) at 0–12 and 0–14 weeks. On the contrary, in the Soi Nin synthetic line, BW at 8 and 12 weeks and ADG at 0–12 weeks were associated with the *AC* genotype. In the fourth synthetic line (Kaen Thong), no significant association was found [92]. In Thai native chickens (Chee), the *IGF1* gene was significantly associated with BW at 12 and 16 weeks of age, and ADG during 0–12 and 0–16 weeks of age [94].

IGFBP-2 binds to insulin-like growth factors [64]. IGFBP-2 is the predominant binding protein produced during adipogenesis of white preadipocytes [95]. IGFBP-2 is secreted by white adipocytes and contributes to the prevention of diet-induced obesity [96]. The circulating IGFBP-2 level was significantly and negatively correlated with fasting plasma glucose, triglycerides, low-density lipoprotein (LDL) cholesterol, IGF-1, IGF-2 and insulin C-peptide [97].

IGFBP-2 regulates a broad spectrum of physiological processes involved in growth, development, and differentiation [73]. Both inhibitory and stimulatory effects of IGFBP-2 on cell proliferation have been reported [98]. IGFBP-2 plays an important role in growth and fat metabolism [64]. IGFBP-2 is the predominant IGF binding protein produced during adipogenesis, and is known to increase the insulin-stimulated glucose uptake in myotubes [99]. IGFBP-2 stimulates glucose uptake in a phosphatidylinositol-3-OH kinase (PI3K)-dependent manner. Adipocytes treated with insulin and IGF-1 for 30 min exhibited a significant (*p* < 0.001) increase in PI3K phosphorylation when compared with the control cells. Similarly, IGFBP-2 induced a significant increase in PI3K phosphorylation in 3T3-L1 adipocytes treated for either 30 min (*p* < 0.01) or 24 h (*p* < 0.001). Similarly, IGFBP-2 induced a noticeable increase in AKT phosphorylation in 3T3-L1 adipocytes treated for either 30 min (*p* < 0.05) or 24h (*p* < 0.01) [99]. IGF-1 significantly (*p* < 0.001) increased, whereas insulin failed to induce (*p* > 0.05) AMP-activated protein kinase (AMPK) phosphorylation in 3T3-L1 adipocytes. Similarly, the treatment of adipocytes with IGFBP-2 for either 30 min or 24 h induced a significant (*p* < 0.001) increase in AMPK phosphorylation [99].

Among the seven IGFBPs, IGFBP-2 is the main binding protein secreted by differentiating white preadipocytes, indicating a potential role in the development of obesity. Overexpression of IGFBP-2 was associated with decreased susceptibility to obesity and improved insulin sensitivity [78]. IGFBP-2 expression was associated with fat mass percentage (*p* < 0.02). It was demonstrated that IGFBP-2 is expressed by subcutaneous abdominal adipocytes of obese individuals and that the expression elevated with increasing adiposity and reducing insulin sensitivity [100].

The main functions of IGFBPs are: (1) acting as carrier proteins for circulating IGF-1 and controller of its flow from the vascular space to tissues; (2) increasing IGF-1 half-life and regulating its metabolic clearance [101]; (3) modulating the interaction between IGF-1 and its receptor, and thus indirectly controlling IGF-1 biological activity [102]; (4) modulating IGF-1 in target tissues, inhibiting or activating its specific actions: cell proliferation, differentiation, survival and migration [62,103–105]; and (5) providing a specific localization pool of IGF-1, because IGFBPs can associate with cell membranes or the extracellular matrix (ECM) [106]. Moreover, some IGFBPs can possess some biological effects outside the IGF-1 signaling pathways, such as apoptosis induction and proliferation/inhibition in some tumors [105].

The *IGFBP2* gene has a total length of 32 kb and it is composed by four exons, 2.0 kb (rat) and 1.6 kb (human) *mRNAs* are generated, and the mature protein is approximately 31 kDa and 36 kDa in rats and humans, respectively [107]. The chicken *IGFBP2* gene spans to more than 38 kb on chromosome 7 (GGA7), consists of four exons, and presents similar organization compared with rats and humans. The chicken *IGFBP2* gene is expressed in a majority of tissues, such as liver, muscle, kidney, heart, ovary, brain, intestine and other tissues [108]. *IGFBP2* gene expression was downregulated in the visceral white adipose tissue of mice, and its circulating levels were reduced in obese mice [96]. Eckstein et al. [109] reported that IGFBP-2 level negatively affected bone size and mineral content in mice, suggesting it was an important regulator of bone biology in vivo.

As for another gene necessary for growth and development processes, the analysis of *IGFBP2*/*Eco72*I gene polymorphism in the present study showed all three known genotypes (*AA, AB*, and *BB*), with an obvious predominance of the heterozygous genotype (56.70%). However, *BB* (265 + 102 bp) homozygotes showed very low frequency (4.12%). The most represented genotype *AB* (367 + 265 + 102 bp) had a similar frequency to the heterozygotes (53.21%) detected by Li et al. [73], who found almost identical frequencies of both homozygotes (*AA* 22.96%, *BB* 23.83%).

The predominance of *A* allele over *B* allele in *IGFBP2* locus in our study may be, similarly to *IGF1* locus, a long-term selection strategy employed in these chicken populations.

The study of Li et al. [73] indicated that chicken *IGFBP2* gene intron 2 C1032T (accession number AY 326194) polymorphism was associated with growth and body composition traits in an F2 population. Moreover, the *IGFBP2* gene was found to be highly expressed in abdominal fat [73]. QTL for fat deposition was mapped between the marker brackets *LEI0064* and *ROS0019* (75 kb to 27 Mb) on *GGA7* in the chicken linkage map [57], which covers the chicken *IGFBP2* gene (23 to 24 Mb). In Thai native chickens (Chee), the *IGFBP2* gene was significantly associated with body weight at 4 weeks of age, ADG during 0–4 weeks of age and breast width at 16 weeks of age [94].

An excessive abdominal fat in chickens is undesirable and is therefore sought to be reduced, in order to improve the quality of the final product. The IGFBP-2 could inhibit the biological actions of IGF in vivo via endocrine or paracrine mechanisms [22] and indirectly control adipocyte differentiation by regulating the actions of IGF [67]. The structure and function of the *IGFBP2* gene has been analyzed in detail, however, the association of this gene with growth features in chickens has been little studied [66].

In our study, heterozygous genotype *AB* of *IGFBP2* resulted—in both chicken lines—in a higher average BW at 42 days, trunk weight, AFW, breast and thigh muscles, slaughter value and slaughter percentage compared with *AA* genotype (Table 6). On average, chickens with the *IGFBP2-BB* genotype grew slower and simultaneously deposited less fat in the body. These differences, however, were not statistically significant. The lowest breast muscle (without skin) was observed in the Hubbard F15 line chicken with an *AA* genotype of *IGFBP2* (498.75 g).

The findings of higher BW and AFW in heterozygotes in our research are not consistent with the findings of the study of Li et al. [73], which found that F2 chicken homozygous for the *B* allele (*IGFBP2-BB*) had a higher AFW than birds of the other two genotypes.

The results point to the potential identification of *IGFBP2* as a candidate gene for altering the growth rate and abdominal fat [73]. Reduced growth was associated with increased hepatic *IGFBP2 mRNA* expression and elevated serum IGFBP-2 levels [22], further suggesting IGFBP-2 as a negative growth regulator in vivo [73].

TGF-βs are represented in birds and mammals by three isoforms of secreted cytokines TGF-β1, TGF-β2 and TGF-β3 [70]. The research of Li et al. [69] supported the broad effects of *TGF*β genes on the growth and development of chickens. Recently, eight from 17 polymorphic sites of the *TGF*β*3* gene [53 (T → C), 1653 (C → T), 1755 (A → G), 3343 (C → T), 3540 (C → T), 4786 (C → T), 7263 (C → T) and 7471 (G → A)] have been significantly related to reproduction traits, indicating these polymorphic sites as potential assistant selection markers for improvement of reproductive capacity of Liboyaoshan chicken [110].

The *TGF*β*3* gene could be a marker for genetically improving duration fertility in hens. In the recent study performed by Gu et al. [111], four SNPs were identified in intron 1 of *TGF*β*3*, and were significantly associated with the duration of fertility in hens (*p* < 0.05). In addition, they identified multi-copy and copy number variants (CNVs) in chicken *TGF*β*3*, and later determined significant associations between *TGF*β*3* CNVs and duration fertility in hens. Specifically, the *TGF*β*3* copy number exhibited a significant positive correlation with its expression (*p* < 0.05).

A significant association between the *TGF*β*3*-*Bsr*I polymorphism and mortality between 14 and 42 days in broiler chickens was reported by Ye et al. [112].

In addition, significant effects of *TGF*β*3-Bsr*I polymorphism on the cecum content *Salmonella enteritidis* bacterial load were found [113], which could have been of great importance, especially in commercial broiler chicken farms. A moderate association (*p* < 0.17) was found between the *TGF*β*3*-*Bsr*I sire allele and antibody response to the *S. enteritidis* vaccine [113]. Polymorphism in the restriction site of *TGF*β*3*-*Bsr*I was associated with *S. enteritidis* burden. The heterozygote *A*/*C* had the highest *S. enteritidis* burden in the cecum, spleen and liver compared with the other two genotypes (*p* < 0.01). The *C*/*C* genotype of *TGF*β*3*showed the lowest bacterial burden for Village Chickens, whereas in Red Junglefowl, the *A*/*A* genotype exhibited the lowest *S. enteritidis* colonization [114]. *Salmonella enterica* serovar Enteritidis infection is a common concern in poultry production for its negative effects on growth, as well as food safety for humans [114].

In the study of Li et al. [69], the *TGF*β*3* polymorphism in broilers crossed with Leghorn was associated with traits of growth and body composition, such as BW, ADG, breast muscle weight, abdominal fat and spleen weight. In our study, for the *TGF*β*3* gene, *AB* genotype was the most common in both chicken lines. The allele A was identified as a dominant allele in Cobb E (64.89%), whereas in Hubbard F15, the frequency of both alleles was identical. This finding is different from another study analyzing *TGF*β*3* genotypes in breeder hens [115], where the allele *B* was a dominant allele at *TGF*β*3* locus, due to it having the highest frequency (0.81).

For the *TGF*β*3* gene, different tendencies were observed in the association of the *A* and *B* alleles with the traits observed within both chicken lines. In Cobb E, the *AB* genotype of *TGF*β*3* resulted in the highest average BW at 42 days (3104.29 g). The highest AFW had Cobb E chicken with *AA* genotype of *TGF*β*3* (54.95 g). On the contrary, in Hubbard F15, the highest average BW at 42 days and AFW were observed in chickens with the *BB* genotype (2618.00 g, 37.33 g, respectively).

The highest average breast muscle (without skin) was found in the *AB* genotype of *TGF*β*3* in both lines *(*in Cobb E chicken: 653.62 g and in Hubbard F15: 515.55 g). The highest average thigh muscle (with skin) was also found in the *AB* genotype of *TGF*β*3* in both lines, with the highest average value in Cobb E (Cobb E: 538.72 g, Hubbard F15: 476.75 g).

On the contrary, the lowest average value of BW at 42 days was observed in the *AA* genotype of *TGF*β*3* in both lines (in the Hubbard F15 line chicken with the *AA* genotype: 2541.33 g and in Cobb E in individuals with the *AA* genotype: 2778.50 g). The lowest AFW was found in the chicken with the *AA* genotype in the Hubbard F15 line (31.53 g), and in Cobb E in birds with the heterozygous genotype *AB* (48.90 g). The lowest breast muscle (without skin) was in the Hubbard F15 line chicken with the *AA* genotype of *TGF*β*3* (494.33 g) and in Cobb E in the birds with the *BB* genotype (551.00 g). The lowest thigh muscle (with skin) was observed in the Hubbard F15 line chicken with the *AA* genotype (469.00 g), and in the Cobb E line chicken with the *BB* genotype of *TGF*β*3* (458.67 g).

Association analysis showed that *Bsl*I genotypes of *TGF*β*3* are related to some performance traits (Table 8). The statistical analysis revealed a significant association of *TGF*β*3* with BW at 21, 28 and 35 days and trunk weight in the codominant (negative value), dominant and overdominant (positive values) genetic model, and with slaughter value in codominant (negative value), recessive and overdominant (positive values) genetic model.

The average slopes of the growth curve (14–42 days of age) constructed according to the line, genotype and sex (Figure 4) was confirmed to be a result of linear growth, as well as lineage and sex differences in body weight. Constructing the graphs of total integrals for the weight sum of the trunk, giblets, abdominal fat, breast and thigh muscles at 42 days of age (at the slaughter of chickens) in both chicken lines (with a separate evaluation of both sexes) and all genotypes observed (Figure 5) showed interesting tendencies. However, no statistically significant dependence was observed, despite apparent differences.

#### **5. Conclusions**

The presented study demonstrated that the point mutation can affect chicken growth, and confirmed some significant associations between SNP and growth traits. Based on these findings, it can be concluded that the *TGF*β*3* gene could be applied as a candidate gene marker for chicken growth traits in the Hubbard F15 and Cobb E broiler line population selection program. However, further association analysis will be required to clarify the effects of this marker on growth and production traits in the broiler chicken population.

**Author Contributions:** All authors participated in the manuscript. B.H. performed genetic analyses and wrote manuscript, K.V. optimized laboratory methodology, performed genetic analyses and participated in writing manuscript, R.K. evaluated experimental data and critically revised the manuscript, R.B. evaluated genetic analyses, J.K. collected samples and revised manuscript, V.C. expertly revised the manuscript, F.K. and C.F. revised manuscript, V.M. and H.H. were responsible for running and conducting the experiment. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the project of the Ministry of Education, Youth and Sports of the Czech Republic No. MSM 6007665806.

**Acknowledgments:** The authors would like to thank a state-owned enterprise International Testing of Poultry Ustrasice (Czech Republic) for performing production trait analysis, Vaclav Rehout for support for research, and Ing. Jana Karlickova and Irena Vankova Nestavalova for their technical assistance. The authors would also like to thank Josef Ruzicka for the development of the laboratory information system Qinslab (Prevention Medicals, Studenka, Czech Republic). CF would like to express his gratitude to RGU for its support.

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

#### **Abbreviations**


*Animals* **2020**, *10*, 800


#### **References**


© 2020 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 Inclusion of Bilberry and Walnut Leaves Powder on the Digestive Performances and Health of Tetra SL Laying Hens**

**Roua Gabriela Popescu 1,**†**, Sorina Nicoleta Voicu 1,2,**†**, \*, Gratiela Gradisteanu Pircalabioru 3, Alina Ciceu 1,4, Sami Gharbia 1,4, Anca Hermenean 4, Sergiu Emil Georgescu 1, Tatiana Dumitra Panaite <sup>5</sup> and Anca Dinischiotu <sup>1</sup>**


Received: 16 April 2020; Accepted: 5 May 2020; Published: 9 May 2020

**Simple Summary:** In poultry, diet composition influences growth performance, egg production, as well as digestion. In this study, the effects of dietary additives obtained from bilberry and walnut leaves powder on the digestive performances of Tetra SL hens were evaluated by histologic and morphometric analyses of the intestinal mucosa as well as by the enzymatic activity measurements of alpha-amylase, invertase, maltase, and trypsin correlated with cecum microbiota.

**Abstract:** The purpose of this study was to examine the effects of dietary inclusion of two additives at the final concentration of 0.5% bilberry (E1) and 1% walnut (E2) leaves powder in the basal diet on digestive health of hens. A total number of 90 Tetra SL hens were divided into two experimental groups (E1 and E2) and one control group (C) consisting of 30 hens each. After four weeks, 10 hens of each group were sacrificed and tissue samples and intestinal content were taken from the duodenum, jejunum, and cecum in order to perform histological, enzymatic, and microbiota analyses. In groups E1 and E2, the histological analysis showed a significant increase of villus height, resulting probably in increased absorption of nutrients in duodenum and jejunum. A decrease in the specific activity of alpha-amylase and trypsin in E1 and E2 for both duodenum and jejunum compared to the control one was also recorded. In addition, the maltase and invertase specific activity in duodenum increased, a tendency that was kept for maltase but not for invertase in jejunum. The cecal microbiota of E1 and E2 individuals was characterized by an increase of *Firmicutes* and *Lactobacilli* and a decrease of *Enterobacteriaceae*. In conclusion, our results indicate that bilberry and walnut leaves additives in feed may improve the health status of the poultry gastrointestinal tract.

**Keywords:** bilberry leaves; walnut leaves; laying hens; digestive enzyme activities; nutrition

#### **1. Introduction**

Tetra SL is a brown egg-laying hybrid widely used for its excellent internal and external egg quality as well as for efficient and long-term egg production. Animal performance improvement is the most important detail from an economic point of view, especially in the livestock industry. Composition of diets given to hens [1] influences growth performance, egg production, as well as digestion. In poultry nutrition, energy suppliers and proteins are the most important feed constituents after water. Cereals provide about 70% of energy, whereas other sources supply the rest. Recently, phytogenic feed additives received attention as alternatives to prebiotics, probiotics, and antibiotics in laying hen nutrition. Some herbal feed inclusions were shown to improve performance, immunity, and antioxidant status in laying hens [2]. Other ones did not affect body weight but improved egg production; weight; and quality regarding yolk color, cholesterol, and malondialdehyde compared to the control [3]. Furthermore, bilberry and walnut leaves powder included in the diet of laying hens increased the antioxidant properties of egg yolks [4] but did not affect other performance indicators.

The avian gastrointestinal tract is divided into nine discrete segments: the oral cavity, esophagus, crop, proventriculus, gizzard, small intestine, ceca, large intestine, and the cloaca. The small intestine is the major site for digestion and absorption of nutrients and influences the rates of energy intake, feeding behavior, and energy allocation [5]. The electrolytes together with digestive enzymes secreted by pancreas and intestinal glands and those produced by mucosal cells are responsible for the hydrolysis of macronutrients. The initial digestion of feed is carried out by pepsin and pancreatic proteases, peptidases, lipase, and amylase [6,7].

Currently, there are limited scientific data regarding the relationship between the nutritional quality of feed used in poultry farming and digestive enzymes [8,9]. Bird [10] determined the distribution of trypsin and amylase in different segments of duodenum in Leghorn chickens. Thus, the first three quarters of the duodenum have a content of 45% trypsin, with 55% in the last part, and for amylase, the content is 23% in the first part, most of the activity of amylase in duodenum being in the last third, almost from the point where the pancreatic ducts communicate with the duodenum [11].

Functional anatomical and histological characteristics of the avian gastrointestinal tract are critical to their feed conversion efficiency. To facilitate maximal absorption of dietary components, the intestinal mucosa is highly convoluted and specialized. The epithelium is folded into villi and the epithelial cells have apical microvilli, forming a brush border observed by optical microscopy. These infoldings increase the small intestinal surface area for absorption by about 600-fold, resulting in a higher capacity for nutrient absorption [12]. Intestinal morphology (villus height and crypt depth) changes in response to exogenous agents. Deeper crypts indicate faster tissue turnover as they contain progenitor cells. Intestinal mucins are high molecular weight glycoproteins secreted by goblet cells. In chickens, mucins are observed to be extensively expressed by goblet cells in the colon and small intestine [13].

Also, the gastrointestinal tract of poultry has a diverse and complex microbiota that plays a significant role in the digestive process and the absorption of nutrients, maintaining immune system development and pathogen exclusion, which are vital for improvement of digestion, health, and growth performance [14]. Poultry microbiota composition depends on many factors including the exposure to growth environmental, intestinal segment, and diet [15]. The esophagus, crop, and cloaca are considered semi-aerobic environments, allowing mixed communities of aerobes, micro-aerobes and facultative anaerobes, including members of the α, β, and γ-Proteobacteria. The internal sections of the gastrointestinal tract located between the crop and cloaca are dominated by obligate or facultative anaerobes, including members of the *Firmicutes* and *Proteobacteria* [16].

Obviously, the improvement of poultry growth performance depends on intestinal health and consequently on microbiome composition. An appropriate microbiota is favored by different vegetal additives. In this context, a method to improve animal performance is the use of vegetal feed additives, which have beneficial effects in livestock production as well as in health and nutrition of animals, which might arise from activation of feed intake and digestive secretions. These have also antimicrobial, antiviral, antioxidant, and immunomodulatory properties [17,18].

Starting from the fact that plants with high phenolic content have strong antioxidant power [19], and due to the few data regarding the use of plant leaves as additives in the diets of laying hens and their nutritional assessment, in this study, we have chosen to use bilberry and walnut leaves powder. *Vaccinium myrtillus* is a species of the genus *Vaccinium* from the family *Ericaceae* [20]. Fruit and aerial parts of plant are known as a natural source of food and drink due to their richness in nutritional and antioxidant compounds and can also be integrated into food supplements and pharmaceuticals for preventing urinary tract infections [21] and cerebral vascular accidents [22]. Bilberry has several effects such as prevention or even reversal in a considerable degree of age-related object memory decline of rats [23] and antioxidant [24,25], anti-inflammatory, anticancer, anti-neurodegenerative, and cardioprotective effects [26,27] due to their phenolic compounds, including proanthocyanidins, flavonoids, stilbenoids, phenolcarboxylic acid derivatives, and flavonol glycosides [20].

*Juglans regia L*. belonging to the *Juglandaceae* family is the most well-known member, representing an important species of deciduous trees. Walnut leaves have been considered as a beneficial source of health, with important amounts of phenolic compounds [28], and have been intensively used in traditional medicine [29] for the treatment of venous insufficiency and hemorrhoids and for their antidiarrheal, anthelmintic, astringent, keratolytic, antifungal, hypoglycemic, hypotensive, and sedative effects [29]. Also, they have high content in flavonoids, alkaloids, saponins, steroids, and tannins [30].

Previously, it has been shown that walnut leaves administration reduced the proliferation of *Clostridium perfringens* in chickens [31]. Also, Mousavi et al. [32] showed that supplementation of broiler chicken diet with a green husk of walnut powder improved the function of the immune system. The goal of this study was to examine the consequences of the dietary inclusion effects of additives obtained from bilberry and walnut leaves powder on the microbiota from cecum correlated with digestive performances of Tetra SL hens by histologic and morphometric analyses of the intestinal mucosa as well as the enzymatic activities measurements. As far as we know, this is one of the first studies regarding the correlation between dietary inclusion of some plant leaves and the health of poultry gastrointestinal tract.

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

#### *2.1. Plant Material and Antioxidant Capacity*

Phytochemical characterization of plant material and leaf samples of bilberry and walnut were obtained from local pharmacies (S.C. Stefmar productie S.R.L, Râmnicu Vâlcea, Romania). The leaf extracts of bilberry and walnut were prepared according to the method described by Cos, arcă et al. [33]. A mass of 1 g of dried vegetable material was mixed with 40 mL distilled water and heated at 90 ◦C for 45 min with shaking. The suspension was then centrifuged at 2370× *g*, and the supernatant was stored at −20 ◦C until analysis was done [34].

The total polyphenol content in extracts was quantified according to the Folin–Ciocalteu method as described previously [35]. A sample of 50 μL was homogenized with 250 μL of 1/10 diluted Folin–Ciocalteu reagent and incubated for 1 min at room temperature. Then, a volume of 750 μL of 7.5% (w/v) Na2CO3 solution was added. The mixture was brought to a final volume of 5 mL and then incubated in the dark for 2 h at 25 ◦C. At the end, the optical density was measured at 760 nm using distilled water as a blank. The concentration of polyphenols was calculated using a gallic acid calibration curve. The results were expressed in milligrams of gallic acid (GAE) per gram of dried weight (d.w.) (mg GAE/g dw).

The antioxidant capacity of leaf extracts of bilberry and walnut was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical according to the method of Burits and Bucar [36]. Different leaf extract concentrations were mixed with 0.04% DPPH at a ratio of 1:100. After 30 min of incubation in the dark at room temperature, the absorbance of samples was measured at 517 nm using a FlexStation 3 multi-mode microplate reader (Molecular Devices LLC, San Jose, CA, USA) [37].

Oxygen radical absorbance capacity (ORAC) assay was performed according to the method of Davalos et al. [38]. A volume of 20 μL of extract or phosphate buffer (for blank) was incubated with 120 μL of 70 nM fluorescein for 15 min in darkness at 37 ◦C. The peroxyl radical was generated by adding 60 μL of 12 mM 2,2 -azobis (2-amidino-propane) dihydrochloride (AAPH), which was freshly prepared before each test. After 80 min of incubation in darkness at room temperature, the fluorescence intensity (FL) was recorded (excitation wavelength at 485 nm; emission wavelength at 520 nm) for 80 min at intervals of one minute using the FlexStation 3 multi-mode microplate reader (Molecular Devices LLC, San Jose, CA, USA). In parallel, a standard curve was prepared with Trolox (6-hydroxy-2,5,7,8-tetramethylcroman-2-carboxylic acid) at concentrations ranging 0–100 μM (0, 12.5, 25, 50, 75, and 100 μM) [37].

#### *2.2. Hens and Experimental Treatments*

A total of 90 Tetra SL laying hens (aged 32 weeks) was assigned into two experimental groups (E1 and E2) and one control group (C) with 30 birds each (ten replicates each; 3 birds/replicates; and a total of 30 cages) and housed in an experimental hall under controlled environmental conditions (temperature, humidity, and ventilation) in 3-tier batteries and 16 h/24 h light regimen. Feed and water were offered ad libitum during the experiment. The corn–soybean meal basal diet (2800 kcal/kg metabolizable energy (ME) and 17.8% crude protein (CP) was the same for all groups as described by Untea et al. [4], which contained 30% corn; 31.46% wheat; 4% gluten, 21.2% soybean meal; and 1.46% vegetable oil and other components per kg. Unlike the diet formulation for group C, the experimental diets included two different herbal feed additives as follows: 0.5% bilberry leaves (E1) and 1% walnut leaves (E2). Diet formulations were calculated in agreement with the feeding requirements of laying hens as given by National Research Council [39]. After four weeks, 10 hens of each group (randomly selected from each cage) were slaughtered with the approval (case no. 5148/10.08.2018) of the Ethical Committee of the National Research-Development Institute for Animal Nutrition and Biology, Balotes,ti, Romania (Ethical Committee no. 52/30.07.2014).

Performance parameters regarding feed intake, feed conversion ratio, egg production, egg weight, and laying percentage were monitored on the experimental period. Herbal feed additives used did not influence the production performances of the birds except for the weight of the eggs, the results being presented previously by Untea et al. [4] as a part of this study. Also, the antioxidant stability of egg yolk was increased due to higher concentrations of lutein and zeaxanthin, that is very important for human nutrition [4].

#### *2.3. Sample Collection for Analyses*

The small intestine from each individual was collected, and intestinal content and tissue samples were taken from the duodenum and jejunum in order to perform enzymatic and histologic analyses. The intestinal contents from duodenum jejunum and cecum were also collected. The total protein extracts were obtained by homogenization of 1 g tissue in 10 mL phosphate buffer, pH 7.4. The suspensions were kept one hour at 4 ◦C and centrifuged at 9600× *g* for 10 min. The resulting supernatants were used for the determination of maltase and invertase activities. Also, the activities of alpha-amylase and trypsin were measured in the intestinal content, whereas the microbiological analyses were performed on the cecal content.

#### *2.4. Light Microscopy Examination*

The intestinal segments were immersed in 4% paraformaldehyde in phosphate buffered saline (PBS) solution and dehydrated in a graded series of ethanol. Finally, each specimen was embedded in paraffin and cut into 4-μm sections using a microtome. Hematoxylin and Eosin (H&E) (Merck, Darmstadt, Germany) stain was performed. Ten villi and crypts of Lieberkühn from duodenum and jejunum segments of each bird were measured using an optical microscope (Olympus BX43, Tokyo, Japan), a camera (30 XC Olympus, Tokyo, Japan), and image analysis software (Olympus Cell

Sense Dimension, Tokyo, Japan). For measurement of villus height and widths of crypt, mucosa segments were randomly selected from each cross section. A total of 10 villus heights (measured from the tip of the villus to the villus–crypt junction) and the depth of 10 crypts (measured from the crypt–villus junction to the base of the crypt) from cross sections of each individual were analyzed.

#### *2.5. Enzymatic Analysis*

#### 2.5.1. Maltase Activity Assay

Maltase (EC 3.2.1.20) activity was determined using the Maltase assay kit instructions from My BioSource (San Diego, CA, USA). For each sample, an appropriate control was prepared. A volume of 25 μL from sample was mixed with 50 μL substrate (maltose) and incubated for 20 min at 37 ◦C. Then, the reaction was stopped by 25 μL stop solution and the mixture was centrifuged at 4000× *g* for 10 min. The supernatant was mixed with 200 μL of chromogenic agent, homogenized, and incubated for 15 min at 37 ◦C. The absorbance was measured using the Flex Station 3 Multi-Mode Microplate Reader (Molecular Devices LLC, San Jose, CA, USA) automatic plate reader at 505 nm. The enzymatic activity was calculated using a calibration curve with glucose. The results obtained were expressed in U/mg of protein.

#### 2.5.2. Invertase Activity Assay

The enzymatic activity of invertase (EC 3.2.1.26) was evaluated according to the Invertase assay kit instruction (code MAK 118; Sigma-Aldrich, Darmstadt, Germany). Briefly, over a volume of 40 μL from each sample was added 5 μL of sucrose solution in each well. Then, the mixture was incubated for 20 min at room temperature. After this interval, a volume of 90 μL reaction mixture containing enzyme and dye reagent was added and incubated for 20 min in dark. The absorbance was measured at the FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices LLC, San Jose, CA, USA) automatic plate reader at 570 nm. As a standard, a 100 μM glucose solution was used. The results obtained were expressed in U/mg of protein.

#### 2.5.3. Alpha-Amylase Assay

Alpha-Amylase (EC 3.2.1.1) activity was determined according to Bernfeld [40]. The amount of reducing sugars released from soluble starch was measured using an alkaline 3, 5-dinitrosalicylic acid (DNS) reagent. Therefore, 100 μL of 20 mM Na2HPO4–NaH2PO4 (pH 6.9) buffer and 100 μL of 1% soluble starch solution were mixed with 5 μL of sample and incubated for 10 min at 25 ◦C. The reaction was stopped by addition of 200 μL of DNS reagent. The reaction mixture was heated at 100 ◦C for 4 min. The reducing groups were quantified at 546 nm with a FlexStation 3 Multi-Mode microplate reader. One unit of activity represented the amount of enzyme that released one μmole of maltose in one minute at 25 ◦C. Enzyme activity was expressed as specific activity (units per gram of protein).

#### 2.5.4. Trypsin Assay

Trypsin (EC 3.4.21.4) activity was determined according to the method described by Hummel [41] using *N-p*-Tosyl-L-arginine methyl ester hydrochloride (TAME) as substrate. The change in absorbance at 247 nm was measured for 10 min at 25 ◦C on a FlexStation 3 Multi-Mode microplate reader (Molecular Devices LLC, San Jose, CA, USA). The reaction mixture contained 216.6 μL of 50 mM Tris-HCl (pH 8.0) buffer, 30 μL of 10 mM TAME, and 8.66 μL of sample. One unit of trypsin activity was defined as the amount of enzyme hydrolyzing one micromole of TAME in one minute at 25 ◦C. Enzyme activity was expressed as specific activity (units per gram of protein).

#### *2.6. Protein Determination*

Protein concentration was determined by Bradford method [42] using bovine serum albumin as standard.

#### *2.7. Microbiota Characterization*

The Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) technique was used to analyze the changes induced by the different diets at the microbiota level. Microbial DNA extraction was performed using a commercial kit (AllPrep PowerViral DNA/RNA Kit, Qiagen, Hilden, Germany). Briefly, 250 mg of cecal content was subjected to treatment with cell lysis matrix (PowerBead Tubes, Glass 0.1 mm, Qiagen, Hilden, Germany) as well as enzymatic digestion that resulted in nucleic acid isolation. Nucleic acids were subsequently subjected to purification (based on MB Spin Columns, according to the manufacturer instructions). Microbial DNA concentration and purity were spectrophotometrically quantified. The concentration of all DNA samples was adjusted to 3 ng/μL in DNAse and RNAse free water. The relative abundance of microorganisms in cecal DNA was measured by qRT-PCR on Applied Biosystem ViiA7. The total number of bacteria in the samples was quantified using universal primers for 16S rRNA. qRT-PCR reactions were performed using SYBR Green Master mix (Applied Biosystems) and specific primers for different taxa (all primers were selected from literature, and their sequences are shown in Table 1) [43–46]. Each PCR reaction included 200 nM forward and reverse primer, 9 ng DNA, and 2xSYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). The samples were incubated at 95 ◦C for 5 min and then amplified by 40 cycles of 95 ◦C for 10 s, 60 ◦C for 30 s, and 72 ◦C for 1 s.


**Table 1.** Primer sequences used for microbiota characterization.

#### *2.8. Statistical Analysis*

Statistical analysis of data was performed with GraphPad Prism software (Version 6, GraphPad, San Diego, CA, USA) using one-way ANOVA, followed by Bonferroni's post hoc test. For histology, enzymology, and microbiota experiments, the number of replicates was n = 10. All values are expressed as mean ± standard deviation (SD) of three replicates, and the data were considered statistically significant only if the *p*-values were less than 0.05.

#### **3. Results**

#### *3.1. Composition Analysis of Vegetable Additives*

The antioxidant potential of each leaf extract was estimated using three methods: total polyphenols content measurement, DPPH free-radical scavenging activity, and ORAC assay (Table 2).


**Table 2.** Phytochemical characterization of plant material.

TPC: Total polyphenolic content; GAE: Gallic Acid Equivalents; d.w: dry weight; DPPH: the antioxidant capacity using the 2,2-diphenyl-1-picrylhydrazyl radical; ORAC: Oxygen radical absorbance capacity value. All data are reported as mean plus or minus standard deviation (SD), (n = 3).

According to our data, the total polyphenol content is almost double in the bilberry leaves compared with the walnut leaves (Table 2).

Also, the extract from bilberry leaves exhibited an 84.8% DPPH scavenging activity, confirming that this feed additive acts as a free radical scavenger. At the same time, it was an effective scavenger of the 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical, according to the ORAC test. The antioxidant activity of walnut leaves extract was lower, presenting a 57.59% DPPH scavenging activity and almost half capacity to counteract the ABTS radical in comparison with bilberry leaves.

#### *3.2. Histology of the Duodenum*

The duodenal villi were lined by a simple columnar epithelium, followed by a *lamina propria* made by connective tissue rich in vascular network, which absorbs the digestive products, and a *muscularis mucosa*, which underlies the base of crypts. The epithelium has a normal aspect, consisting of absorptive cells, called enterocytes, and individual goblet cells, which secrete mucin, for protection and lubrication of the intestinal contents. Brunner's glands were not present in duodenum (Figure 1).

**Figure 1.** Effect of bilberry and walnut leaves supplementation diet on duodenum morphology of laying hens: The basal diet served as the control, and different levels of herbal feed additives were supplemented to the basal diet as follows: 0.5% cranberry leaves (E1) and 1% walnut leaves (E2). Sce: simple columnar epithelium; lp: lamina propria; cr: crypt; mm: muscularis mucosa. bb: brush border; H&E (Hematoxylin and Eosin) stain; (n = 10); n: number of replicates.

#### *3.3. Histology of the Jejunum*

Histology of the jejunum has a normal aspect (Figure 2).

The average lengths of villi of duodenum were 891.55 μm (control), 1158.84 μm (E1), and 1263.44 μm (E2), whereas the average widths of crypts of duodenum were 189.94 μm (control), 189.49 μm (E1), and 189.46 μm (E2) (Table 3).

Also, the average lengths of villi of jejunum were 905.98 μm (control), 1258.04 μm (bilberry leaves E1), and 1248.7 μm (walnut leaves E2). Moreover, the average widths of the crypts of jejunum were 209.51 μm (control), 210.26 μm (E1), and 211.39 μm (E2) (Table 3). Villus height (Table 3) was significantly higher for the experimental groups compared to control (*p* < 0.001).

**Figure 2.** Effect of bilberry and walnut leaves supplementation diet on jejunum morphology of laying hens: The basal diet served as a control, and different levels of herbal feed additives were supplemented to the basal diet as follows: 0.5% cranberry leaves (E1) and 1% walnut leaves (E2). Sce: simple columnar epithelium; lp: lamina propria; Cr: crypt; mm: muscularis mucosa. bb: brush border; H&E (Hematoxylin and Eosin) stain; (n = 10); n: number of replicates.

**Table 3.** Measurements of the villi length and widths of crypt for the control and experimental groups.


C group: basal diet/control group; E1 group: basal diet with 0.5% bilberry leaves and E2 group: basal diet with 1% walnut leaves. All data are reported as mean values ± standard deviation (SD) and statistical significance, where ns *p* > 0.05, \*\*\* *p* ≤ 0.001; (n = 10); n: number of replicates.

#### *3.4. Intestinal Enzymes Activities*

In duodenum, the activity of maltase (Table 4) increased insignificantly by 157% for E1 by 264% for E2 compared to control. In jejunum, the same enzymatic activity decreased by 86% in the case of the E1 group, while for E2, it was increased by 330% (Table 4) compared to control. Regarding the specific activity of invertase in duodenum, it was unmodified in group E1 whereas, in group E2, it increased significantly by almost 11.61% compared to the control (Table 4). In contrast, the administration of basal diet enriched with 1% walnut leaves decreased significantly by 8.89% in jejunum. The basal diet supplemented with 0.5% bilberry leaves (E1) determined an increase of invertase specific activity at the jejunum level compared to the control level.

**Table 4.** Influence of dietary source on enzymatic specific activity (U/mg protein) of maltase, invertase, alpha-amylase, and trypsin of duodenum and jejunum of laying hens.


The basal diet served as a control (C), and different levels of herbal feed additives were supplemented to the basal diet as follows: 0.5% bilberry leaves (E1) and 1% walnut leaves (E2). All data are reported as mean values ± standard deviation (SD) and statistical significance, where ns: *p* > 0.05; \* *p* ≤ 0.05; \*\* *p* ≤ 0.01; \*\*\* *p* ≤ 0.001; (n = 10); n: number of replicates.

Our experimental data revealed a decrease in the specific activity of alpha-amylase in experimental groups for both duodenum and jejunum compared to the control. Furthermore, the enzymatic activity of trypsin from both intestinal segments was insignificantly decreased and below the control group level in all experimental groups.

#### *3.5. Intestinal Microbiota*

The modification of the diet recipe caused the appearance of changes in the intestinal microbiota. These were evident at the phylum level as well as at the bacterial population level. In general, *Firmicutes* and *Bacteroidetes* are the most abundant phyla, followed by *Proteobacteria*, *Actinobacteria*, *Verrucomicrobia*, and *Fusobacteria* [47]. The major phyla that make up the gut microbiota (*Bacteroidetes* and *Firmicutes*) have been analyzed. The administration of the commercial recipe enriched with bilberry leaves (E1) or walnut leaves (E2) led to a moderate decrease in the level of *Bacteroidetes* and an increase in the abundance of *Firmicutes* (Table 5).

**Table 5.** The relative abundance of *Firmicutes, Bacteroidetes, Lactobacillus* sp, and *Enterobacteriaceae* phyla as determined by The Real-Time Quantitative Reverse Transcription PCR (qRT-PCR): Eubacteria 16S rRNA was used for normalization.


C: control group; E1 group: basal diet with 0.5% bilberry leaves and E2 group: basal diet with 1% walnut leaves. All data are reported as mean values ± standard deviation (SD) and statistical significance, where ns: *p* > 0.05, \*\* *p* ≤ 0.01; (n = 10); n: number of replicates.

Generally, the personalization of diet formulas aims to induce beneficial modifications of the microbiota by increasing the number of bacteria with probiotic potential (e.g., lactobacilli) and by decreasing the level of potentially pathogenic bacteria (pathobionts) such as *Enterobacteriaceae*. In this

context, we quantified the abundance of lactobacilli and members of the *Enterobacteriaceae* family in isolated cecal samples from hens fed with different dietary formulas. For both analyzed groups, a significant decrease of the level of *Enterobacteriaceae* was observed as well as a statistically significant increase of the level of lactobacilli (Table 5).

#### **4. Discussion**

The poultry industry is one of the most dynamic animal industries. Feed efficiency and high performance of birds as well as the quality of eggs are the crucial goals in poultry production. In this context, the quality of diet along with environmental conditions and health of birds need to be considered to achieve these goals [48].

Nowadays, different herbal additives have been investigated for antioxidant, antimicrobial, and anti-inflammatory activities; growth-promoting effects; and egg quality. The beneficial impact of herbal additives could be due to the polyphenolic composition, which influences sugar digestion and absorption of nutrients in the small intestine [49]. Higher antioxidant protection of bilberry leaves compared to walnut ones could be conferred by the raised polyphenol content that can neutralize free radicals and can inhibit the propagation of free-radical reactions [50,51].

In the current study, histological evaluation showed that bilberry and walnut leaves powder supplementation of diets have exerted beneficial effects in the duodenum and the jejunum morphology, materialized in significant increases of villus height, resulting probably in increased adsorption of nutrients.

This hypertrophy of villi and, by default, of their epithelial cells resulted in an increased surface area and capacity of absorption [52], corresponding to raised activities of intestinal enzymes [53,54] probably due to feed supplementation with bilberry and walnut leaves powder. Previously, it has been stated that villi height and crypt depth ratio in the small intestine has a direct influence on the absorptive function [55]. These have been observed in chickens after the administration of zeolite [56], L-glutamine [57], and clinoptilolite [55].

In poultry, the activity of digestive enzymes located in the brush border membrane of enterocytes plays a significant role in the physiological processes occurring in the digestive tract that depend on nutritional feeding and characteristics of diet [5]. Previously, it has been shown that activities of digestive enzymes are affected by the amount, composition, and regime of food intake during the growing phase [11]. Several studies have shown that the activities of proteases in the intestinal juice are modified according to the amount of protein in the diet, while the activities of amylase and lipase depend on the content of food in carbohydrates and lipids as substrates for their activity [7].

According to our data, a strong negative correlation between the total polyphenol content and a decreased alpha-amylase and trypsin activities both in duodenum and jejunum was observed (Table 2).

The alpha-amylase catalyzes hydrolysis of the internal α-1, 4-glycosidic linkages in starch, generating glucose and maltose. In our study, the activity of alpha-amylase is decreased in both experimental groups E1 and E2 for duodenum and jejunum compared to control. Considering that amylase activity is diminished, probably oligosaccharides escaped digestion in the small intestine and reached the cecum, where they were fermented by microbiota producing short chain fatty acids [58] and other aminated compounds with beneficial impact on hens. There are three main short chain fatty acids, namely acetate, propionate, and butyrate, that represent signaling molecules with epigenetic impact [59].

Trypsin is responsible for protein and peptides degradation into amino acids. The peptides hydrolysis stage is important in protein absorption because, although these are smaller in size compared to proteins, they are still too large to be absorbed by the small intestine mucosa [60]. Our results show that trypsin activities from duodenum and jejunum were insignificantly decreased and below the control level in all experimental groups E1 and E2, probably due to covalent attachment of the phenolic compounds from feed additives to reactive nucleophilic sites of the enzyme, affecting its

three-dimensional conformation and the active site. As a result, the rate of the catalyzed reaction decreased significantly [61].

Maltase and invertase are key enzymes of carbohydrate digestion; therefore, the relationship between cecum microbiota and small intestine enzyme activity could be due to glucose and fructose presence in the gastrointestinal tract, which could favor the *Lactobacilli* strains' presence as previously has been proved [62,63].

It has been shown that partial digests of protein in the culture medium was essential for growth of some lactic bacteria [64]. Also, peptides resulting in the small intestine due to trypsin activity could favor the relative abundance of *Firmicutes* [65].

An interesting observation is that, in hens from the E1 and E2 groups, duodenal invertase activity increased compared to control, whereas in the case of jejunum, this activity was higher for E1 and decreased for E2. Invertase is an enzyme with two active centers [66], one that catalyzes the hydrolysis of sucrose and the other involved in the hydrolysis of both maltose and other alpha-glucosides.

Probably due to the increased activity of invertase in the E2 group in duodenum, a lower amount of sucrose was present in jejunum, and this could explain the decreased invertase activity (Table 4) and the increased maltose hydrolysis (Table 4).

For *Aves* species, the digestion starts at the oral cavity and ends at the cloacal level, crossing several "stations" represented by a series of organs involved in the process [67]. The cecum is presented in the form of two extensions at the intersection of the small intestine with the large intestine [68] and is specialized in fiber digestion, nitrogen recycling through urine and osmotic regulation, and water resorption [69]. At the same time, certain types of B vitamins are released (thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folic acid, and vitamin B12). Dietary fiber components are not digested by endogenous digestive enzymes and consequently are substrates for bacterial fermentation in the distal part of the gut [70].

Our results indicate that group E2 (commercial recipe + 1% walnut leaves) has the highest probiotic potential, with this type of diet leading to a 5-fold increase in the level of lactobacilli and a significant decrease in *Enterobacteriaceae* compared to the control group. Moreover, probably higher concentrations of short chain fatty acids produced in the small intestine decreased the *Enterobacteriaceae* population [71], which is negatively correlated with the *lactobacilli* population (Table 5). Similarly, Leusnik et al. [72] showed that dietary supplementation with bilberry extract (80 mg/kg of feed) significantly decreased the size of *Enterococcus* spp. populations in broilers on day 28 of the experiment.

#### **5. Conclusions**

Our results indicate that basal diets enriched with bilberry and walnut leaves powder might change positively the microbiota of hens by modulating several digestive enzymes that favor the development of lactobacilli and decrease Enterobacteriaceae. As a result, we could conclude that supplementation of basal feed with herbal additives might increase the health status of poultry.

**Author Contributions:** Conceptualization, A.D. and A.H.; methodology, R.G.P., S.N.V., A.C., S.G., and G.G.P.; software, R.G.P.; validation, R.G.P., S.N.V., and G.G.P.; formal analysis, R.G.P. and S.N.V.; investigation, R.G.P., S.N.V., A.C., S.G., and A.H.; writing—original draft preparation, R.G.P., S.N.V., A.H., and G.G.P.; writing—review and editing, A.H. and A.D.; supervision, S.N.V. and A.D.; project administration T.D.P. and S.E.G.; funding acquisition, S.N.V. and A.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a grant of the Romanian Ministery of Research and Innovation, CCCDI—UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0473/"From classical animal nutrition to precision animal nutrition, scientific foundation for food security", within PNCDI III.

**Acknowledgments:** This work was supported by a grant of the Romanian Ministery of Research and Innovation, CCCDI—UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017-0473/"From classical animal nutrition to precision animal nutrition, scientific foundation for food security", within PNCDI III "Poultry feeding—a natural way of maintaining gut health, poultry performance, and food quality under conditions imposed by the ban of antibiotics".

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

#### **References**


© 2020 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 Inclusion of Di**ff**erent Doses of** *Persicaria odorata* **Leaf Meal (POLM) in Broiler Chicken Feed on Biochemical and Haematological Blood Indicators and Liver Histomorphological Changes**

**Muhammad Abdul Basit 1,2,\*, Arifah Abdul Kadir 1,\*, Teck Chwen Loh 3, Saleha Abdul Aziz 4, Annas Salleh 5, Ubedullah Kaka <sup>6</sup> and Sherifat Banke Idris 1,7**


Received: 5 May 2020; Accepted: 17 June 2020; Published: 16 July 2020

**Simple Summary:** The frequent use of antimicrobial growth promoters (AGPs) in poultry feed leads to antimicrobial resistance, resulting in a ban on their subtherapeutic use in food-producing animals. In this context, there is a dire need to find safe and potential alternatives. Recently, phytobiotics, especially herbs, have gained attention and have been studied extensively for their possible use as alternative poultry feed additives. *Persicaria odorata* is a herb (phytobiotic) that is reported to possess antioxidant, antimicrobial, immunomodulatory, and hepatoprotective properties. This study is the first of its kind to assess the effects of different doses of supplementation of *Persicaria odorata* leaf meal (POLM) on haematological blood indicators, serum biochemistry, organ parameters, and histomorphology of the liver in broiler chickens. The results revealed that the dietary supplementation of POLM enhanced the growth performance, positively improved the haematological indices and serum biochemistry profile with no deleterious effects on internal organs, and ameliorated the histomorphology of the liver, even at dietary supplementation of 8 g/kg. Thus, POLM would be safe at an inclusion rate of 8 g/kg as an alternative phytogenic feed additive in broiler chickens.

**Abstract:** This research was conducted to estimate the effects of *Persicaria odorata* leaf meal (POLM) on haematological indices, serum biochemical attributes, and internal organs parameters, including histomorphological features of the liver, in broiler chickens. A total of 120 one-day-old male broiler chicks (Cobb-500) were randomly allocated into four experimental groups. The dietary treatments were basal diet (BD), which served as the control (C), along with BD + 2 g/kg POLM (Po2), BD + 4 g/kg POLM (Po4), BD + 8 g/kg POLM (Po8), which were the supplemented groups. The body weight gain (BWG) showed a linear increase and feed conversion ratio (FCR) showed a linear decrease with increasing POLM dosage at day 42 (*p* < 0.05) and for the overall growth performance period

(*p* < 0.01). On day 21 and day 42, the values of red blood cells (RBCs), white blood cells (WBCs), haemoglobin (Hb), and packed cell volume (PCV) showed linear increases (*p* <0.05) as the dosage of POLM increased in the diet. On day 21, dietary supplementation of POLM linearly decreased (*p* < 0.05) the serum activity of alkaline phosphatase (ALP), aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), and serum levels of urea and creatinine. On the other hand, serum levels of total protein (TP), albumin, and globulin showed a linear increase (*p* < 0.05) as the POLM dosage increased. On day 42, the serum activity of AST and ALT and serum levels of glucose, cholesterol, triglycerides, urea, and creatinine showed linear decreases (*p* < 0.05) with increased levels of POLM in the diet. However, POLM supplementation linearly increased (*p* < 0.05) the serum levels of TP and globulin. Dietary inclusion of POLM did not influence the organ parameters and showed no adverse effects on the liver histomorphology. In conclusion, supplementation of POLM increased the growth performance, improving haematological indices and serum biochemistry profiles of broiler chickens without any deleterious effects on the liver histomorphology. The results of the present study provide evidence that POLM can be safely used at a dose rate of 8 g/kg of feed as an alternative to conventional antimicrobial growth promoters (AGPs).

**Keywords:** broiler chicken; feed additive; blood haematology; phytobiotics; serum biochemistry

#### **1. Introduction**

The ban against in-feed inclusion of antimicrobial growth promoters (AGPs) has increased the momentum in research to find potential alternatives [1,2]. An increasing interest has been seen in the study of phytobiotics as alternatives to AGPs. Among phytobiotics, herbs are of particular significance because of their secondary bioactive metabolites, such as flavonoids, which are potent antioxidants, thus helping to prevent oxidative stress and reduce the risk of developing chronic diseases [3,4]. Additionally, they are anti-inflammatory, immunomodulatory [5,6], antimicrobial, anthelmintic, [3,4,7], detoxifying, and digestion-stimulating substances [8].

Herbs have shown positive effects on the performance and biological health of broiler chickens [9,10], can improve haematological blood indicators and serum biochemical attributes [11–13], and have also been reported to regulate the kidney and liver functions [14,15]. Among such herbs, *Persicaria odorata*, of the family Polygonaceae, is of important significance. *P. odorata* is a perennial herb that can grow up to 1.0 m in lowlands and 1.5 m in hilly areas. This plant possesses long and lanceolate leaves measuring 0.5–2.0 cm in width and 5–7 cm in length [16,17], and is used traditionally and regularly in Southeast Asian cuisine. *P. odorata* has many common names, such as Vietnamese cilantro and Vietnamese mint. In Malaysia, Indonesia, Singapore, and Brunei, it is called "*daun laksa*" or "*daun kesum*" [16]. The dried grounded leaves of *P. odorata* contain 3.5% crude protein, 0.83% crude fat, 10.66% crude fiber, and 1.83% ash [18]. It is a powerful antioxidant [17] that contains essential oils [19] and flavonoids [20]. Among these flavonoids, myricetin, quercetin, gallic acid, and coumaric acid are essential bioactive compounds [21]. Its high polyphenolic content, quercetin and myricetin, has been suggested to be responsible for its antioxidant activity [22,23], which can inhibit lipid peroxidation [24]. Moreover, previous studies have shown that *P. odorata* was non-toxic in murine model [25,26].

Phytobiotics are assumed to be natural, safe, and residue-free substances, which may have mildly toxic effects compared to commonly used synthetic AGPs [27]. Literature is scarce about the safe use of herbal plants and their optimal dosage; thus, there is a dire need to estimate the appropriate dosage and the possible side effects of natural feed additives, which might be used as safe alternative growth promoters in poultry production. The present study aims to estimate the effects of different doses of supplementation of POLM on the growth performance, haematological blood indicators, serum biochemical attributes, histomorphology of the liver, and internal organ parameters in broiler chickens.

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

#### *2.1. Source and Preparation Method for Diets*

Fresh samples of *P. odorata* were obtained from the Universiti Putra Malaysia herbal farm. The obtained samples were authenticated from the Biodiversity Unit, Institute of Biosciences, Universiti Putra Malaysia, and deposited with voucher no. SK3296/18 for future reference. For sample preparation, fresh leaves of *P. odorata* were dried using an oven set to 50 ◦C for 72 h and milled to a fine powder. The obtained sample (fine powder) was stored at 4 ◦C until further use.

#### *2.2. Experimental Birds and Diets*

The experimental procedures and animal handling were approved by the institutional animal care and use committee, Universiti Putra Malaysia (UPM/IACUC/AUP-R033/2018). A total of 120 one-day-old male broiler chicks (Cobb500) were procured from a local hatchery. Upon arrival, the birds were wing-tagged and allocated randomly into 4 treatment groups with 5 replicates of 6 birds each. The birds were raised in cages with wire meshed floor (length 120 cm × width 120 cm × height 45 cm), which were placed in a conventional open-sided shed. The birds in all replicates were reared under the same environmental and management conditions. The cyclic temperature in the house ranged between a maximum of 34 ◦C and a minimum of 24 ◦C, while the humidity ranged between a maximum of 91% and a minimum of 65%. Commencing from day one, birds had free access to water and feed, while the lighting was continuous. The basal diet without any premixing of anticoccidial, antimicrobial, and antioxidant drugs or feed enzymes was procured from the feed supplier and processed into experimental diets at a feed mixing facility at the Universiti Putra Malaysia. The current study consisted of four dietary treatments, which were given for 42 days (starter period = 1 to 21days, finisher period = 22 to 42 days). The dietary treatments were the basal diet (BD), which served as the control (C); and BD + 2 g/kg POLM (Po2), BD + 4 g/kg POLM (Po4), and BD + 8 g/kg POLM (Po8), which were the treatment groups. The experimental diets were formulated in the composition that meets or exceed NRC [28] recommendations (Table 1). The experimental broiler chickens were vaccinated with Newcastle disease (ND) and infectious bronchitis (IB) vaccines at day 4 and day 21, respectively; and vaccinated with the infectious bursal disease (IBD) vaccine on day 7 via an intra-ocular route.


**Table 1.** Ingredients (% as feed) and nutritional analysis of the basal diet.

<sup>†</sup> Premixed administered vitamins per (kg) of dietary feed: Vitamin K (menadione) 1.33 (mg); Vitamin A (retinol), 1950 (μg); Vitamin D3 30 (μg); Vitamin E, 0.02 (mg); riboflavin, 2.0 (mg); Biotin, 0.03 (mg); Vitamin B12, 0.03 (mg); VitaminB1, 0.83 (mg); Vitamin B3 24 (mg); Vitamin B6, 1.37 (mg); Folic acid, 0.33 (mg); Calcium D-Panthothenate, 3.69 (mg). †† Premixed administered minerals per kg of dietary feed: Zinc, 100.01 (mg); iron, 120.0 (mg); Mg, 16.0 (mg); I, 0.8 (mg); Co, 0.6 (mg); Cu, 19.99 (mg). Diet C = Control (0 g/kg medicinal herb; *P. odorata*); Diet Po2 = 2 g/kg *P. odorata*; Diet Po4 = 4 g/kg *P. odorata*; Diet Po8 = 8 g/kg *P. odorata*; \* Calculated according to NRC [28].

#### *2.3. Growth Performance Measurement*

The initial body weights of the birds were recorded on day 1, followed by weekly recording of live body weight (BW) and feed intake (FI) throughout the entire experiment. The body weight gain (BWG) and feed conversion ratio (FCR) were calculated. Mortality was recorded whenever it occurred.

#### *2.4. Sample Collection*

On day 21 and day 42, two broiler chickens were randomly selected per replicate (*n* = 10) per experimental group. From each bird, the blood samples were collected via brachial (wing) vein to check haematological blood indicators and serum biochemical indices.

#### *2.5. Analysis of Haematological Blood Indicators*

For haematological blood indicator analyses, the blood samples were collected in the K3 EDTA tubes (BD Vacutainer®, Franklin Lakes, NJ, USA). Blood haematology parameters, including red blood cell (RBCs) and white blood cell (WBCs) counts, haemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentrations (MCHC) were measured within 2 h post blood collection using a haematology analyser (ABC Vet®, ABX Diagnostics, Montpellier, France).

#### *2.6. Serum Biochemical Indices*

For serum biochemical analyses, blood samples were collected in a plain tube (BD Vacutainer®, Franklin Lakes, NJ, USA) and subjected to centrifuge at 3000×*g* for 15 min to obtain serum, which was stored at −20 ◦C until further analyses. The serum biochemical indices, including aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), alkaline phosphatase (ALP), urea, creatinine, triglycerides, cholesterol, glucose, total protein (TP), albumin, and globulin; and serum electrolytes, including sodium (Na), chloride (Cl), and potassium (K), were measured with commercial kits (Roche "Basal" Diagnostica, Rotkreuz, Switzerland) using an autochemistry analyser (Bio Lis 24i Chemistry Analyser, Tokyo, Japan).

#### *2.7. Determination of Relative Internal Organ Weights*

On day 42, two birds per replicate (*n* = 10) from each experimental group were selected randomly and slaughtered according to the procedure designated in Malaysian Standard (MS) 1500: 2009 [29]. The following equation was used to calculate the dressing percentage (DP):

$$\text{LPP} = \text{(eviscerated carcases} \text{weight/live weight)} \times 100 \tag{1}$$

The weights of the gizzard, heart, liver, kidney, spleen, pancreas, and bursa of Fabricius were taken and expressed as percentages (%) of the live body weight.

#### *2.8. Liver Histomorphology Assessment*

The liver samples were collected from birds slaughtered to determine internal organ parameters. For histomorphological studies, the liver tissues were kept for 48 h in 10% buffered formalin and subjected to a series of dehydration cycles in an automated tissue processor (Leica ASP 3000, Wetzlar, Germany). The liver tissues were embedded using a paraffin embedding system (Leica RM 2155, Wetzlar, Germany). Tissue sections up to 4–5 μm in size were obtained using a microtome (Leica Jung Multicut 2045, Wetzlar, Germany) and stained using haematoxylin and eosin staining. For the histomorphology, the tissues were examined under a light microscope (Leica DM LB2, Wetzlar, Germany).

#### *2.9. Data Analyses*

The data analyses were carried out with SAS 9.4 [30] (SAS Institute Inc., Cary, NC, USA) by one-way ANOVA using the general linear model procedure. Group differences were compared by Duncan's multiple range test. The effects of POLM supplementation at different doses were measured using an orthogonal polynomial contrast test for linear and quadratic effects. The differences were considered as significant at *p* < 0.05.

#### **3. Results**

#### *3.1. Growth Performance*

The growth performance of broiler chickens is shown in Table 2. On day 21, compared to the control BWG was significantly increased (*p* < 0.05) in the Po8 group; however, FI and FCR were not affected (*p* > 0.05) by dietary supplementation of POLM. On day 42, compared to the control, BWG was significantly increased (*p* < 0.05) in POLM-supplemented groups (Po2, Po4, and Po8). Furthermore, FCR was decreased (*p* < 0.05) in POLM-supplemented diets compared to the control group. Additionally, FI was not affected (*p* > 0.05) by dietary supplementation of POLM. Regarding the overall growth performance of broiler chickens (days 1–42), the maximal increase (*p* < 0.05) for the BWG and the lowest (*p* < 0.05) FCR were seen in dietary group Po8 compared to the control group. In addition, the BWG showed a linear increase (*p* < 0.05) and FCR showed a linear decrease (*p* < 0.05) with increasing POLM dosage on day 42 (*p* < 0.05) and for the overall growth performance period.


**Table 2.** Effects of different doses of supplementation of *Persicaria odorata* leaf meal (POLM) on the growth performance of broiler chickens.

a–c indicate that values in the same row with different superscripts are significantly different (*p* < 0.05). FI: feed intake; BWG: body weight gain; FCR: feed conversion ratio; C: control; basal diet alone; Po2: basal diet+ POLM 2 g/kg; Po4: basal diet+ POLM 4 g/kg; Po8: basal diet+ POLM 8 g/kg. SEM: standard error of mean.

#### *3.2. Haematological Blood Indicators*

Data in Tables 3 and 4 illustrate the impacts of different doses of POLM on the haematological blood indicators of broilers on day 21 and day 42, respectively. On day 21, compared to the control, RBC counts were improved (*p* < 0.05) in the Po8 group. On the other hand, PCV was significantly increased in the POLM-supplemented groups (Po2, Po4, and Po8) compared with the control group. The Hb concentration was higher (*p* < 0.05) in the Po4 group and the maximum value (*p* < 0.05) was recorded in the Po8 group compared to the control group. Furthermore, the WBC counts were increased (*p* < 0.05) in dietary group Po8 relative to the control group. However, MCV, MCH, and MCHC were not affected

(*p* > 0.05) by the dietary supplementation of POLM. In addition, POLM supplementation linearly increased RBC (*p* = 0.048), Hb (*p* = 0.048), and WBC counts (*p* = 0.0001) with increasing POLM dosage.

**Table 3.** Haematological blood indicators of broilers fed with different supplementation doses of POLM at day 21.


a,b indicate that values in the same row with different superscripts are significantly different (*p* < 0.05). RBCs: red blood cells; PCV: packed cell volume; Hb: haemoglobin; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; WBCs: white blood cells; C: control; basal diet alone; Po2: basal diet+ POLM 2 g/kg; Po4: basal diet+ POLM 4 g/kg; Po8: basal diet+ POLM 8 g/kg.

**Table 4.** Haematological blood indicators of broilers fed on different supplementation doses of POLM at day 42.


a,b indicate that values in the same row with different superscripts are significantly different (*p* < 0.05). RBCs: red blood cells; PCV: packed cell volume; Hb: haemoglobin; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; WBCs: white blood cells; C: control; basal diet alone; Po2: basal diet+ POLM 2 g/kg; Po4: basal diet+ POLM 4 g/kg; Po8: basal diet+ POLM 8 g/kg.

On day 42, compared with the control group, RBC, WBC, PVC, and Hb counts increased (*p* < 0.05) in the Po8 group. On the other hand, POLM supplementation did not influence (*p* > 0.05) MCV, MCH, or MCHC values in experimental broiler chickens. In addition, POLM supplementation linearly increased RBC (*p* = 0.046), Hb (*p* = 0.047), MCV (*p* = 0.016), MCHC (*p* = 0.039), and WBC counts (*p* = 0.0001) with increasing POLM dosage.

#### *3.3. Serum Biochemistry*

Data in Tables 5 and 6 show the influences of different doses of POLM on the serum biochemistry of broilers on day 21 and day 42, respectively. On day 21, compared to the control group, the AST activity in the serum was significantly decreased (*p* < 0.05) with increasing levels of POLM supplementation. However, the serum activity of ALT was decreased in Po8 group compared with the control group. On the other hand, the activity of ALP in the serum was not affected (*p* > 0.05) by dietary supplementation of POLM. The serum TP level was increased (*p* < 0.05) in dietary group Po8 compared to the control group. Furthermore, the serum levels of albumin and globulin (except Po2) were increased (*p* < 0.05) in POLM-supplemented groups compared to the control group. In contrast with the control group, except for Po2, the serum levels of urea and creatinine were decreased (*p* < 0.05) in POLM-supplemented groups. On the other hand, supplementation of POLM did not influence (*p* > 0.05) serum levels of glucose, cholesterol, triglycerides, K, or Cl in experimental broiler chickens. In addition, there was a linear decrease in the serum activity levels of ALP (*p* = 0.048) and AST (*p* = 0.000) and the serum levels

of urea (*p* = 0.044) and creatinine (*p* = 0.000) as the POLM dosage increased. However, a linear and quadratic decrease in the serum activity of ALT (*p* = 0.000) was observed with increasing POLM dosage. The POLM supplementation linearly increased the serum levels of TP (*p* = 0.000), albumin (*p* = 0.022), and globulin (*p* = 0.000) with increasing supplementation levels in the diet.

**Table 5.** Biochemical indicators of chicken blood from birds fed different supplementation doses of POLM at day 21.


a–d indicate that values in the same row with different superscripts are significantly different (*p* < 0.05). ALP: alkaline phosphatase; AST: aspartate aminotransferase; ALT: alanine aminotransferase; C: control; basal diet alone; Po2: basal diet+ POLM 2 g/kg; Po4: basal diet+ POLM 4 g/kg; Po8: basal diet+ POLM 8 g/kg.

**Table 6.** Biochemical indicators of chicken blood from birds fed different supplementation doses of POLM at day 42.


a–c indicate that values in the same row with different superscripts are significantly different (*p* < 0.05). ALP: alkaline phosphatase; AST: aspartate aminotransferase; ALT: alanine aminotransferase; C: control; basal diet alone; Po2: basal diet+ POLM 2 g/kg; Po4: basal diet+ POLM 4 g/kg; Po8: basal diet+ POLM 8 g/kg.

On day 42, the activity of AST and ALT in the serum was decreased (*p* < 0.05) with increasing POLM supplementation. However, the serum activity of ALP was not influenced by (*p* > 0.05) POLM supplementation in experimental broilers. The serum level of TP was increased (*p* < 0.05) in POLM supplementation groups compared to the control group. The serum levels of albumin in the Po8 group and globulin in Po4 and Po8 groups were significantly increased (*p* < 0.05) compared with the control group. Serum levels of cholesterol, triglycerides, and urea were decreased (*p* < 0.05) with increasing POLM supplementation levels, in comparison with the control group. Birds in Po8 and Po4 groups showed decreased serum creatinine levels compared with the Po2 and control groups. However, POLM supplementation did not vary (*p* > 0.05) the serum level of Na, Cl, or glucose in the experimental birds. In addition, there was a linear decrease in serum levels of AST (*p* = 0.003), glucose (*p* = 0.002), cholesterol (*p* = 0.000), and triglycerides (*p* = 0.000) as the POLM supplementation increased. Furthermore, linear and quadratic decreases in serum levels of ALT (*p* = 0.000; 0.004), urea (*p* = 0.000; 0.03), and creatinine (0.000; 0.022) were observed as the POLM supplementation levels increased. Moreover, the serum levels of TP (*p* = 0.001) and globulin (*p* = 0.006) showed linear increases with increased supplementation of POLM.

#### *3.4. Dressing Percentage and Relative Internal Organs Weights*

The dietary supplementation of POLM did not influence (*p* > 0.05) the dressing percentages or relative internal organs weights across experimental broiler chickens (Table 7). Moreover, there were linear increases in the dressing percentage (*p* = 0.040) and relative spleen weight (*p* = 0.000) with increasing levels of POLM.

**Table 7.** Dressing percentages and relative internal organs weights of broilers fed different supplementation doses of POLM at day 42.


C: control; basal diet alone; Po2: basal diet+ POLM 2 g/kg; Po4: basal diet+ POLM 4 g/kg; Po8: basal diet+ POLM 8 g/kg.

#### *3.5. Morphological Analyses of Liver*

Figure 1a–d show the histomorphologies of the liver sections of the experimental birds. The histomorphological examination of the liver section of the control group showed congestion of the central vein (CV), with loosening of the endothelium and vacuolar degeneration of the hepatocytes (Figure 1a). The liver lobule sections of the POLM-supplemented groups (Po2, Po4, and Po8) showed normal architecture of the hepatic lobules and central veins with intact endothelia and hepatic sinusoids. Furthermore, multifocal areas of the RBCs were seen in sinusoidal capillaries, without any infiltrative evidence of inflammatory cells within the liver parenchyma (Figure 1b–d). A gradual histomorphological improvement in the normalcy level of the hepatocytes was noticed with increasing POLM supplementation levels. The hepatocyte architecture was clearer and showed no vacuolation or degenerative changes in the Po8 group compared to Po2 and Po4, where low levels of vacuolation and fatty changes were noticed within hepatocytes in a centrally magnified area of the hepatic lobule sections.

**Figure 1.** Photomicrograph image. (**a**) Liver lobule section of the control group, showing partial congestion (black arrow) in the central vein (CV) and vacuolar degeneration of the hepatocytes (black arrowhead). (**b–d**) Liver lobule sections of the Po2, Po4, and Po8 groups, showing central veins with intact endothelia, RBCs within sinusoids (white arrow), and radiating sinusoidal spaces (white arrowhead); there is no evidence of the infiltration of inflammatory cells in the liver parenchyma. (**c**) Hepatocytes showing normal architecture (asterisk)**.** The magnified area in the center of the image shows histomorphological features of hepatocytes, where the hepatocytes in the Po8 group (**d**) have the clearest and healthiest architecture compared to the other groups (H&E:Haematoxylin and Eosin; 400X).

#### **4. Discussion**

#### *4.1. Growth Performance*

Supplementation of POLM enhanced the growth performance of broiler chickens. In this study, compared to the control group, BWG was significantly increased (*p* < 0.05) in the Po8 group on day 21; however, FI and FCR were not affected (*p* > 0.05) by dietary supplementation of POLM. Aroche et al. [31] reported that the dietary inclusion of phytobiotics in the form of 0.5 % mixed powder of *M. citrifolia*, *P. guajava*, and *A. occidentale* improved the feed efficiency, which resulted in increased BWG. The *P. guajava and M. citrifolia* contain flavonoids and possess antioxidant and antimicrobial properties [32]. The flavonoid contents of these phytobiotics are assumed to increase the growth performance in supplemented broiler chickens [33,34]. The secondary metabolites of herbs such as alkaloids, tannins, and flavonoids positively influence the birds' health, as they possess antimicrobial, anti-inflammatory, and antioxidant properties [35]. Thus, the inclusion of phytobiotics as a dietary supplement help to improve the growth in chickens [36–38].

On day 42, compared to the control group, BWG was increased (*p* < 0.05) and FCR was significantly decreased in POLM-supplemented groups (Po2, Po4, and Po8). Additionally, The BWG showed a linear increase and FCR showed a linear decrease (*p* < 0.05) with increasing POLM dosage. Supplementation of POLM resulted in enhanced BWG in broiler chickens, which may have been due to the flavonoids and the secondary bioactive compound quercetin, which has a primary role and was successfully quantified from POLM. Quercetin is a flavone that should improve growth in birds by upregulating growth hormone, triggering the hepatic growth hormone receptor; this stimulus increases the concentration of insulin-like growth factor-1 [39]. Kim et al. [40] reported increased broiler growth by supplementing quercetin in broilers. Quercetin can limit the effects of oxidative stress [41] and pro-inflammatory cytokines such as TNF-α, interleukin, and cyclooxygenase-2 [42], thus modulating the gut environment to better utilise the nutrients, improving the growth in birds.

The overall growth performance (1–42 days) of broiler chickens showed that compared to the control group, BWG was maximumly increased (*p* < 0.05) and FCR was significantly decreased (*p* < 0.05) in the Po8 dietary group. In addition, the BWG showed a linear increase (*p* < 0.01) and FCR showed a linear decrease (*p* < 0.01) with increasing POLM dosage. Salami et al. [43] identified that the incorporation of medicinal herbs as feed additives in the broiler chickens' diets improved the FCR in the last growth phase of the birds. Other studies also suggested the roles of flavonoids in the growth performance of broiler chickens [34,44]. The present study results are in agreement with Mpofu et al. [45], where inclusion of *L. javanica* at the rate of 5 g/kg in broiler chickens' diets had a positive impact on overall growth. In another study, Paraskeuas et al. [36] reported that inclusion of phytobiotics such as eugenol, menthol, and anethol at the rate of 100 to 150 mg/kg in feed improved the nutrient digestibility, thus enhancing the growth measures in broiler chickens.

Conclusively, the supplementation of POLM in broiler chickens showed a positive effect on growth performance, hence effectively increasing the BWG and feed efficiency with decreased FCR. These findings are in agreement with the positive results of the previous studies, where in-feed phytobiotics were tested in broiler chickens [31,45,46]. Thus, the present results show that POLM effectively enhanced the growth performance of broiler chickens, even at the supplementation rate of 8 g/kg.

#### *4.2. Haematological Blood Indicators*

Haematology blood tests of experimental animals are very significant when evaluating the toxic effects of a supplemented compound or plant extract. Haematology blood tests are also tools that can be used to determine the physiological and pathological statuses of the organisms [47]. The haematological blood indicators in this study were found to be within normal ranges [48]. The normal blood haematology values in this study indicated the adequacy of nutrients and better immune status of the broiler chickens supplemented with POLM.

The current study findings indicated significant increases in RBC and WBC counts, and in Hb and PCV values. These outcomes are comparable with the results of Reis et al. [49], who indicated that inclusion of phytobiotics such as cinnamic aldehyde, thymol, and carvacrol in broiler chickens significantly increased erythrocyte counts and haemoglobin in comparison with the control. Similar findings in another study were reported by Krauze et al. [50], who studied the dietary effects of probiotic *Bacillus subtilis* (0.25 g/L) *Enterococcus faecium* (0.25 g/L), and phytobiotics containing cinnamon oil (0.25 mL/L) in broiler chickens and found improvements in the immune system and parameters such as RBCs and Hb. In another experiment, Gilani et al. [11] examined the efficacy of organic acids and phytobiotics (possessing flavonoids) in poultry feed as alternatives to AGPs, observing significant increases in RBC and WBC counts, as well as an increase in PCV in broiler chickens. Similarly, broiler chickens fed Garden cress (*Lepidium satvium*) seed powder [51], cayenne pepper (*Capsicum frutescens*) and turmeric (*Curcuma longa*) powders [52], and pawpaw leaf and seed meal [53] showed increased values of Hb, PCV, and RBCs.

The present study results were not significant (*p* > 0.05) for MCV, MCH, or MCHC in experimental broiler chickens. These results affirm the findings of Oghenebrorhie and Oghenesuvwe [54], who reported no significant results for MCV, MCH, or MCHC among broilers supplemented with *Moringa oleifera* leaf meal (MOLM)**.**

In conclusion, the dietary supplementation of POLM improved the RBCs, WBCs, PCV, and Hb, suggesting better utilisation of the dietary nutrients.

#### *4.3. Serum Biochemistry*

Serum biochemical parameters show the metabolism of nutrients in the body and highlight the possible changes resulting from intrinsic and extrinsic factors [55,56]. The liver is one of the largest and most vital organs of living organisms, and it has a pivotal role in detoxification, metabolism, and elimination of endogenous and exogenous substances [57]. The activity levels of ALP, AST, and ALT are considered as diagnostic tools that may be used to evaluate hepatotoxicity [58]. Any pathological manifestation or toxicity results in enhanced activity levels of AST and ALT [59]. Moreover, their activity levels are considered as specific indicators of liver injury or impairment [60]. The current study results showed decreased (*p* < 0.05) serum activity of AST and ALT by increasing the POLM supplementation dosage. However, the serum activity of ALP was not influenced by (*p* > 0.05) POLM supplementation in experimental broiler chickens. The decreased activity of ALT and AST indicated the hepatoprotective nature of the POLM. The POLM possesses a significant concentration of flavonoids and secondary metabolites, including quercetin, which is believed to be responsible for hepatoprotective activity [17,61]. Farag and El-Rayes [62] revealed the hepatoprotective effect of quercetin from bee pollen in broilers, which has an ability to restrict oxidative damage to the liver. Another study by Odetola et al. [12] showed that the graded supplementation of *Petiveria alliacea* root meal in broiler chickens significantly decreased the activity of AST. In a previous study, Oloruntola et al. [63] found that the dietary inclusion of pawpaw and bamboo leaf meal significantly decreased the activity of ALT in broiler chickens.

Serum proteins are primarily synthesised in the liver and their concentrations reflect the functional status of hepatocytes. Any decline in the levels of serum proteins (TP, albumin, and globulin) may be the result of hepatic insufficiency, malnutrition, and active inflammation, which may be due to the recurrent infections and immune deficiency [64]. Furthermore, the serum protein levels of birds are considered important indicators for the determination of their health status. The fattening period of broiler chickens is very short, and there is a rapid accumulation of building proteins in the body tissues, which may significantly influence the concentrations of proteins in the blood, as well as their composition [65]. This rapid growth trend requires intensive erythropoiesis and haemoglobin synthesis, which can result in increased globulin production, potentially affecting the concentrations of serum protein levels in growing chickens [66,67]. The current study results showed that the inclusion of POLM significantly increased the levels of TP, albumin, and globulin compared to the control group. In addition, TP, albumin, and globulin showed linear increases with increasing supplementation of POLM. The present study results are in accordance with the results of Goerge et al. [68], who noted higher serum TP levels in broilers fed a ginger-powder-supplemented diet at starting and finishing phases. Abudabos et al. [69] reported trends for serum TP and globulin for broilers fed anise and thyme essential oils that were in agreement with the current study.

In birds, the normal reference range of serum glucose is 200 to 500 mg/dL [48]. The present study showed that the serum glucose concentrations were not influenced by POLM in the experimental chickens; however, numerically high values were recorded in the control group compared to supplemented groups. The current findings are in line with the study by Abudabos et al. [69], where serum glucose did not differ significantly in experimental broilers supplemented with phytogenic feed additives.

The serum concentrations of cholesterol and triglycerides are considered to be indicators of lipid metabolism [70]. The current study findings showed that the serum levels of triglycerides and cholesterol were not affected by POLM supplementation at day 21, however significant decreases in the levels of triglyceride and cholesterol were noted in POLM-supplemented groups relative to control on day 42. Furthermore, it was observed that the increasing dosages of POLM linearly decreased the serum levels of triglycerides and cholesterol. The current study results are endorsed by Vispute et al. [13], who reported that dill and hemp seed (possessing flavonoids) significantly decreased the serum levels of triglycerides in the final growth phase. Similarly, our results are in agreement with Zhang et al. [71], who specified that the supplementation of *Chinese bayberry* leaves in chickens' diets significantly decreased the serum concentrations of triglycerides and cholesterol. Our outcomes are affirmed by Zhou et al. [72] and Niu et al. [73], who reported that dietary supplementation of broilers with fermented *Ginkgo biloba* rations and fermented *Ginkgo biloba* leaves can significantly decrease the serum levels of triglycerides and cholesterol. Similar results were shared by Gilani et al. [11], who revealed that phytobiotics, organic acids, and their combinations resulted in significantly reduced serum levels of cholesterol and triglycerides in broiler chickens.

Electrolyte balance plays an important role in acid–base balance and ultimately modifies the performance of broiler birds. Any alteration in the acid–base balance results in malfunction of the biochemical and metabolic pathways, which results in an inability to maintain the physiological status of the birds. The minerals Na, K, and Cl are essential for acid–base and osmotic balance, as well as transport of substances across the cell membranes. Thus, they play vital roles in the metabolisms of living organisms. Any imbalance in these minerals can directly alter the acid–base balance, metabolic functions, and ultimately the performance of broiler chickens [74]. In the present study, the Na, K, and Cl values were within normal ranges. These results are in accordance with Malahubban and Ab Aziz, [75], who reported the graded supplementation of Misai Kucing (*Orthosiphon stamineus*) in broiler chicken.

The kidneys are considered the second target organs that may be injured due to metabolic dysfunctions. Kidney function plays a key role in measuring the possible toxicity of any compound. The status of kidney function can be measured via the increase or decrease in serum levels of urea and creatinine. Higher creatinine levels result from reduced glomerular filtration, which reflects kidney impairment [76], while an elevated serum urea level indicates cardiac and renal tissue injuries. The current study findings showed that serum levels of creatinine and urea were significantly decreased as the POLM dosage increased. These findings indicated that POLM had no deleterious effects on kidney function. Various studies using phytobiotics supplementation in broiler chickens have supported our present study results, including the work by Rubio et al. [77], Ahmad et al. [78], and Adegoke et al. [52].

In conclusion, our findings showed decreased activity of AST and ALT with reduced serum levels of urea and creatinine. These results highlighted that POLM supplementation was useful in terms of liver and kidney function, and was safe even at 8 g/kg in broiler chickens. Additionally, the increased blood protein levels (TP, albumin, and globulin) in this study might be due to the antioxidant and immunomodulatory properties of the POLM supplementation [5,6].

#### *4.4. Relative Internal Organs Weights*

The relative internal organs weights served as an indicator for the responses of animals towards any in-feed toxic substance that may result in an increase or decrease in internal organs weights [79]. In the current study, no macroscopic alterations, such as hypertrophy or atrophy, injury, and swelling, were noticed in any internal organ. Furthermore, dietary inclusion of POLM did not influence the relative organs weights in experimental broiler chickens. The outcomes of our present study are in agreement with Oloruntola [53], who observed that the relative internal organs weights of the broilers were not influenced by dietary inclusion of seed meal and pawpaw leaf meal. Similar observations were described in the work by Rubio et al. [77] and Vispute et al. [13], where dietary addition of phytobiotics did not influence the relative organs weights in broiler chickens.

In conclusion, the constant relative internal organs weights of the broilers across experimental groups suggested that, POLM supplementation had no adverse effect on internal organs of the broiler chickens.

#### *4.5. Histomorphological Analysis of the Liver*

The histomorphological study of the liver revealed that POLM supplementation did not show deleterious effects on liver tissues. The vacuolar degeneration was more frequent in the control group compared to the hepatic tissue samples of all the POLM-supplemented groups. The microscopic characteristics of the hepatic tissues showed positive impacts on the histomorphologies of the livers, as seen in Figure 1b–d compared to Figure 1a (control group). The histomorphological changes in the present study were comparable to the previous study by Quereshi et al. [80], in which the hepatoprotective effects of *fenugreek* seeds and *dandelion* leaves in broiler chickens resulted in normal architecture of the hepatic parenchyma. Additionally, the hepatoprotective effects of these phytobiotics were suggested to be due to the presence of flavonoids in *dandelion* leaves and *fenugreek* seeds. Klaric et al.'s [15] results also supported the results of the present study, where supplementation of phytobiotics such as propolis and bee pollen, which possess flavonoids, ameliorated the liver morphology compared to the control group. Additionally, normal hepatocytes without regressive lesions were noticed in the supplemented groups compared to the control. The control group showed extensive regressive lesions in liver tissue sections. In another study, Farag and El-Rayes [62] observed that the dietary supplementation of bee pollen in broiler chickens' diets ameliorated the hepatic parenchyma and reduced tissue injury. Furthermore, flavonoids such as quercetin might have a protective effect against oxidative damage of the liver.

Inflammatory responses and oxidative stress are key factors that can damage the liver. Any substance that can diminish the oxidative stress and inflammation can produce hepatoprotective effects and reduce hepatic injury. In the present study, the POLM supplementation produced protective effects on the hepatocytes. These hepatoprotective effects were primarily due to quercetin, which produce effects by limiting oxidative stress [41]; pro-inflammatory cytokines such as TNF-α, IL-6, and COX-2 [42]; and nuclear factor NF-κB, probably via interference of the signalling of the toll-like receptor TLR4 [81]. Furthermore, quercetin increases the non-enzymatic and enzymatic antioxidants by stimulating the Nrf2–ARE signalling pathway in cells, which might positively influence the liver status and function [82]. Conclusively, increasing the POLM supplementation levels has a gradual ameliorating histomorphological effect on hepatocytes. A predominantly healthy architecture of the liver parenchyma was noticed in POLM-supplemented group Po8 compared to the Po2 and control groups.

#### **5. Conclusions**

The current study results showed that the dietary inclusion of POLM supplementation in broiler chickens enhanced the growth performance and positively improved haematological blood indicators and serum biochemistry attributes, with no deleterious effects on the internal organs. Additionally, broilers chicken fed a diet supplemented with POLM at a rate of 8 g/kg showed the most promising results in terms of growth performance, as well as for the tested blood and serum biochemistry parameters, and retained relatively normal hepatic parenchyma. Thus, POLM supplementation at 8 g/kg would be the appropriate dose as an alternative feed additive for broiler chickens.

**Funding:** This research project was supported by a grant (Grant Code: GP/2018/9616700), Geran Putra, Universiti Putra Malaysia.

**Conflicts of Interest:** The authors declare that they have no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Author Contributions:** All authors have read and agreed to the published version of the manuscript. Conceptualization, M.A.B., A.A.K., T.C.L., S.A.A., and A.S.; methodology, M.A.B., A.A.K., T.C.L., S.A.A., and A.S.; sample collection M.A.B., A.A.K., U.K., and S.B.I.; formal analysis, M.A.B., A.A.K., T.C.L., S.A.A., A.S., and U.K.; investigation, M.A.B., A.A.K., T.C.L., A.S., U.K., and S.B.I.; resources, A.A.K. and T.C.L.; writing—original draft preparation, M.A.B., A.A.K., T.C.L., S.A.A., and A.S.; writing—review and editing, M.A.B., A.A.K., T.C.L., S.A.A., A.S., U.K., and S.B.I.; supervision, A.A.K., L.T.C., S.A.A., and A.S.; project administration, A.A.K.; funding acquisition, A.A.K.

#### **References**


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