**Soybean Lecithin High in Free Fatty Acids for Broiler Chicken Diets: Impact on Performance, Fatty Acid Digestibility and Saturation Degree of Adipose Tissue**

#### **Alberto Viñado, Lorena Castillejos \* and Ana Cristina Barroeta**

Animal Nutrition and Welfare Service, Department of Animal and Food Science, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain; Alberto.Vinado@uab.cat (A.V.); Ana.Barroeta@uab.cat (A.C.B.)

**\*** Correspondence: lorena.castillejos@uab.cat

Received: 4 September 2019; Accepted: 6 October 2019; Published: 14 October 2019

**Simple Summary:** The search of alternatives for soybean oil, as a dietary energy source, has generated a lot of interest in broiler feeding due to economic and supply reasons. Soybean lecithin, as a co-product derived from the soybean oil degumming process, and its blending with other by-products derived from the vegetable oil refining process such as acid oils, may represent an alternative energy source for broiler chicken diets formulation. The current study has demonstrated that soybean lecithin high in free fatty acids can be included in grower–finisher diets, as a partial replacer of soybean oil or in combination with an acid oil, without impairing performance or fatty acid digestibility and causing minor changes in the fatty acid composition of the abdominal fat pad.

**Abstract:** Two experiments were conducted to evaluate the inclusion of soybean lecithin with a high free fatty acid content (L) in starter and grower–finisher broiler diets, as well as its influence on performance, energy and fatty acid (FA) utilization and the FA profile of the abdominal fat pad (AFP). A basal diet was supplemented with soybean oil (S; Experiment 1) or acid oil (AO; Experiment 2) at 3%, and increasing amounts of L (1%, 2% and 3%) were included in replacement. The inclusion of L did not modify performance parameters (*p* > 0.05). The S replacement by L reduced energy and total FA utilization (*p* ≤ 0.05) in starter diets; however, in grower–finisher diets, a replacement up to 2% did not modify energy and FA utilization (*p* > 0.05). The AO substitution by L produced no modifications on energy and FA utilization (*p* > 0.05) during the starter phase, while the blend of 1% of AO and 2% of L resulted in the best combination in terms of the FA digestibility. The FA profile of the AFP reflected the FA composition of diets. The addition of L could replace, up to 2% or be blended with AO in broiler grower–finisher diets as an energy source.

**Keywords:** broiler chickens; alternative energy source; soybean lecithin; phospholipids; vegetable acid oil; digestibility balance; free fatty acids; triacylglycerols

#### **1. Introduction**

Co-products and by-products derived from the vegetable oil refining process may represent an interesting and economic alternative to conventional fat sources used in broiler feeding, such as soybean oil. During degumming, most phospholipids (PL) present in crude soybean oil are extracted, generating a co-product known as crude soybean lecithin. Lecithins are defined as a lipid mixture highly composed of PL, but they are also rich in glycolipids, carbohydrates and neutral lipids, such as triacylglycerols [1]. Soybean lecithin is an available low-cost energetic source [2] with a similar fatty acid (FA) profile to soybean oil [3,4]. In addition, its elevated surface-active PL content of soybean lecithin represents an added value as an emulsifier; hence, its dietary inclusion may improve fat

absorption [5,6]. However, soybean lecithin has a high viscosity that hampers its inclusion during feed manufacturing. For this reason, in order to facilitate its homogeneous blending in feed, mixing lecithin at different ratios with acid or crude oils is a common practice [7]. On the other hand, vegetable acid oils derived from the chemical refining process of crude oils are normally composed of a large quantity of free fatty acids (FFA; 40%–60%) and represent an important source of energy [8,9]. Nevertheless, it has been observed that a high dietary FFA concentration may reduce energy utilization by impairing dietary fat solubilization in the gastrointestinal tract [5].

We hypothesized that soybean lecithin could be considered as an alternative energy source for broiler chicken diets, in replacement or combined with other fats, with no negative effects on the performance, nutrient digestibility, and FA composition of adipose tissue. Therefore, a total of two experiments were conducted to assess the potential use of a soybean lecithin high in FFA (L) as an alternative energy source in broiler feeding when combined with soybean oil (S; Experiment 1) or a monounsaturated vegetable acid oil (AO; Experiment 2). The evaluation was based on the study of the influence of L inclusion on performance, feed energetic content, FA digestibility, and, thus, the effect on the FA profile of the abdominal fat pad (AFP) of the broiler carcass.

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

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

The experiments were performed at Servei de Granges i Camps Experimentals (Universitat Autònoma de Barcelona, Bellaterra, Spain), were in accordance with the European Union Guidelines (2010/63/EU), and were approved by the Animal Ethics Committee (CEEAH) of the same institution (number code: 4006). Two different trials of 38 days (d) each were performed with a feeding program in two phases: Starter (from 0 to 21 d) and grower–finisher (from 22 to 38 d). Experimental diets (Table 1) were based on wheat and soybean meal, presented in mash form, and were formulated to meet or exceed FEDNA (Fundación Española para el Desarrollo de la Nutrición Animal) requirements [10]. Furthermore, titanium dioxide (TiO2) was used as an inert marker at 5 g/kg in order to perform digestibility balances.


**Table 1.** Ingredient composition of the starter and grower–finisher broiler chicken diets on an as-fed basis (Experiments 1 and 2).

<sup>1</sup> Soybean oil and soybean lecithin high in free fatty acids and monounsaturated acid oil in different blending proportions. <sup>2</sup> Provides per kg feed: Vitamin A (from retinol), 13,500 IU; vitamin D3 (from cholecalciferol), 4800 IU; vitamin E (from alfa-tocopherol), 49.5 IU; vitamin B1, 3 mg; vitamin B2, 9 mg; vitamin B6, 4.5 mg; vitamin B12, 16.5 μg; vitamin K3, 3 mg; calcium pantothenate, 16.5 mg; nicotinic acid, 51 mg; folic acid, 1.8 mg; biotin, 30 μg; Fe (from FeSO4·7 H2O), 54 mg; I [from Ca(I2O3)2], 1.2 mg; Co (from 2 CoCO3·3 Co(OH)2·H2O), 0.6 mg; Cu (from CuSO4·5 H2O), 12 mg; Mn (from MnO), 90 mg; Zn (from ZnO), 66 mg; Se (from Na2SeO3), 0.18 mg; Mo [from (NH4)6Mo7O24], 1.2 mg; organic acids (starter diets at 4 g/kg; grower–finisher diets at 3 g/kg); β-glucanase 350 IU; xylanase 1125 IU.

Experiment 1: A total of 96 Ross 308 newly hatched female broiler chickens were randomly assigned to one of four experimental treatments (six replicates/treatment) and allocated in cages (four birds/cage). A control basal diet was supplemented with S at 3% (S3), and increasing amounts of L (soybean lecithin blended with soybean acid oil in a 5:1 proportion) were included in replacement of S as added fat: 1% (S2–L1), 2% (S1–L2) and 3% (L3).

Experiment 2: A total of 120 Ross 308 newly hatched female broiler chickens were randomly assigned to one of five experimental treatments (six replicates/treatment) and allocated in cages (four birds/cage). A control basal diet was supplemented with AO (a 1:1 blend of olive pomace acid oil and sunflower acid oil) at 3% (AO3), and increasing amounts of L were included in replacement of AO: 1% (AO2–L1), 2% (AO1–L2) and 3% (L3). The S3 diet was included as a reference treatment.

#### *2.2. Animal Husbandry and Controls*

The animals were obtained from a local hatchery (Pondex S.A.U., Juneda, Spain), weighed, wing-tagged and randomly distributed in cages with a grid floor and a tray for excreta collection. The temperature and light program used was consistent with the specifications in the Ross 308 lineage management handbook [11], and the animals were allowed to consume feed and water ad libitum. Broiler body weight (BW) was recorded individually at 21 and 38 d post-hatch, whereas feed intake was measured by cage at 21 and 38 d post-hatch. The data were used to calculate the average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) from both phases and the overall results of each experiment. Mortality was recorded daily to adjust ADG and ADFI. Two nutritional balances were performed for each experiment between d 9 and 11 (starter period) and d 36 and 37 (grower–finisher period), where excreta samples (free of contaminants) were taken on each day of the digestibility balance (once per d), homogenized, freeze-dried, ground, and kept at 4 ◦C until further analysis. At the end of each experiment, all the animals used in both experiments were slaughtered in a commercial abattoir, and carcasses were recovered.

Carcasses (total BW excluding blood and feathers) were weighed, and the AFP (from the proventriculus surrounding the gizzard down to the cloaca) of each bird was removed and weighed in order to calculate the AFP carcass percentage. Furthermore, representative sample of the AFP of each bird was taken, pooled by replicate, and frozen at −20 ◦C for further analysis.

#### *2.3. Laboratory Analyses*

Experimental oil samples (S, L and AO) were chemically characterized, as shown in Table 2.

The FA composition was analyzed by gas chromatography following the methodology described by Guardiola et al. [12]. The acid value was determined according to International Organization for Standardization (ISO) 660 [13], and the acidity was expressed as the FFA percentage of oleic acid. In the case of the soybean lecithin high in FFA, the acetone insoluble matter was analyzed using the Ja 4–46 method from the American Oil Chemists' Society (AOCS) [14], and the PL composition was determined by HPLC (D450 MT1, Kontron; Eching, Germany) according to the method described by Helmerich and Koehler [15].

Regarding the experimental feed samples, the proximate analysis was performed following AOAC methodology [16]: Ether extract (Method 920.39), crude protein (Method 968.06), ash (Method 942.05), dry matter (Method 934.01), and crude fiber (Method 962.09). The gross energy content was determined for oil, feed and excreta samples by an adiabatic bomb calorimeter (IKA-Kalorimeter system C4000; Staufen, Germany). Titanium dioxide was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES; Optima 3200 RL, Perkin Elmer; Waltham, MA, USA) in Experiment 1, while it was determined in Experiment 2 by the method described by Short et al. [17].

The FA profile of the feed and excreta was analyzed by adding nonadecanoic acid (C19:0, Sigma-Aldrich Chemical Co.; St. Louis, MO) as an internal standard and following the method described by Sukhija and Palmquist [18], whereas in the case of the AFP, the method described by Carrapiso et al. [19] was used. The final extract obtained was injected into a gas chromatograph

(HP6890, Agilent Technologies; Waldbronn, Germany) following the method conditions described by Cortinas et al. [20].


**Table 2.** Chemical analysis of the experimental added oils.

SFA: Saturated fatty acid; MUFA: Monounsaturated fatty acid; PUFA: Polyunsaturated fatty acid; UFA:SFA: Unsaturated-to-saturated fatty acid ratio; PUFA:SFA: Polyunsaturated-to-saturated fatty acid ratio; FFA: Free fatty acid; AI: Acetone insoluble matter; Total PL: Total phospholipids; PC: Phosphatidylcholine; PI: Phosphatidylinositol; PE: Phosphatidylethanolamine; AP: Phosphatidic acid; LPC: Lysophosphatidylcholine; GE: Gross energy; N.D.: Not determined. <sup>1</sup> S: Soybean oil; L: Soybean lecithin high in free fatty acids; AO: Monounsaturated acid oil. <sup>2</sup> Percentage of total product.

#### *2.4. Calculations and Statistical Analysis*

The apparent digestibility of FA (%) was calculated using the following equation:

The apparent digestibility of FA = 1 − {(TiO2 in feed/FA concentration in feed)/ (TiO2 concentration in excreta/FA concentration in excreta)}.

The apparent metabolizable energy (AME) of the diets was calculated multiplying the apparent absorption of the gross energy by its corresponding diet gross energy. Both calculation formulas were in accordance with Rodriguez-Sanchez et al. [21].

Cage means were used as the experimental unit (six replicates/treatment) in performance (except BW), FA digestibility, the FA profile of the AFP, and the AME values of the diets. A Shapiro–Wilk test indicated a normal distribution of the data. In Experiment 1, data were analyzed by a one-way ANOVA using R Statistics (Version 3.3.1; R Core Team, Vienna, Austria), with treatment as the main factor. In Experiment 2, soybean oil treatment (S3) was compared against the AO3 treatment separately with a one-way ANOVA (S3 vs. AO3), whereas diets containing co-products and by-products were compared with a one-way ANOVA (AO3 vs. AO2–L1 vs. AO1–L2 vs. L3). Tukey's multiple-range test was performed to determine whether means were significantly different (*p* ≤ 0.05). The linear model used was: Yij = μ + α<sup>i</sup> + εj, where μ is the global mean, α is the treatment effect, and ε is the residual error.

#### **3. Results**

#### *3.1. Experimental Fats and Diets Composition*

The FA profiles of S and L (Table 2) were similar regarding polyunsaturated FA (PUFA) content; nevertheless, L presented a higher content in saturated FA (SFA) and a lower content in monounsaturated FA (MUFA) than S. In the case of AO, oleic acid was the most abundant FA, followed by linoleic acid. Furthermore, the three added fats differed in their average unsaturated-to-saturated FA ratio (UFA:SFA), where S and AO presented higher average values (5.14 and 5.60, respectively) than L (3.74); the three fats also differed in their average polyunsaturated-to-saturated FA ratio (PUFA:SFA), where AO presented the lowest value (2.04), followed by L (2.82) and S (3.67). Concerning FFA content, AO presented the highest value, representing its main lipid molecular structure (52.9%), whereas L showed a medium average content (24.1%), and S showed the average lowest value (1.95%). Additionally, both S and AO presented higher average values of gross energy (39.8 and 39.5 MJ/kg, respectively) than L (34.4 MJ/kg).

The proximate analysis results and the FA profile of the experimental diets are shown in Table 3 (Experiment 1) and Table 4 (Experiment 2). The experimental treatments showed a similar macronutrient content, and their main differences were related to the FA profile and the energetic content. In Experiment 1, the replacement of S by L increased dietary SFA in starter (9.4%) and grower–finisher diets (11.9%), whereas a decrease in MUFA was observed (11.3% and 7.1% for starter and grower–finisher diets, respectively), causing a reduction in dietary the UFA:SFA. In Experiment 2, the replacement of AO by L increased dietary SFA (9.0% and 7.2%, for starter and grower–finisher diets, respectively) and dietary PUFA (28.4% and 36.8% for starter and grower–finisher diets, respectively). On the contrary, this replacement reduced the MUFA content (42.8% and 36.7% for starter and grower–finisher diets, respectively). The replacement of AO by L reduced the UFA:SFA, whereas it increased the PUFA:SFA.


**Table 3.** Analyzed gross energy, macronutrient content and fatty acid composition of starter and grower–finisher broiler chicken diets (Experiment 1).

SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids; UFA:SFA: Unsaturated-to-saturated fatty acid ratio; PUFA:SFA: Polyunsaturated-to-saturated fatty acid ratio; GE: Gross energy. <sup>1</sup> S3: Soybean oil (S) at 3.00%; S2–L1: S at 2.00% and soybean lecithin high in free fatty acids (L) at 1.00%; S1–L2: S at 1.00% and L at 2.00%; L3: L at 3.00%. <sup>2</sup> Samples were analyzed twice.


**Table 4.** Analyzed gross energy, macronutrient content, and fatty acid composition of starter and grower–finisher broiler chicken diets (Experiment 2).

SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids; UFA:SFA: Unsaturated-to-saturated fatty acid ratio; PUFA:SFA: Polyunsaturated-to-saturated fatty acid ratio; GE: Gross energy. <sup>1</sup> S3: Soybean (S) oil at 3.00%; AO3: Acid oil (AO) at 3.00%; AO2–L1: AO at 2.00% and soybean lecithin high in free fatty acids (L) at 1.00%; AO1–L2: AO at 1.00% and L at 2.00%; L3: L at 3.00%. <sup>2</sup> Samples were analyzed twice.

#### *3.2. Growth Performance and Abdominal Fat Deposition*

The growth performance and abdominal fat deposition parameters of Experiments 1 and 2 are shown in Tables 5 and 6, respectively.

**Table 5.** Growth performance and abdominal fat pad deposition of broiler chickens according to different dietary added fats (Experiment 1).


BW: Body weight; ADFI: Average daily feed intake; ADG: Average daily gain; FCR: Feed conversion ratio; RSE: Residual standard error. <sup>1</sup> S3: Soybean oil (S) at 3.00%; S2–L1: S at 2.00% and soybean lecithin high in free fatty acids (L) at 1.00%; S1–L2: S at 1.00% and L at 2.00%; L3: L at 3.00%.

In both experiments, performance parameters were not affected by the replacement of the added fats (S and AO) by L in any phase, nor were the overall parameters of the experiments (*p* > 0.05). Nevertheless, in Experiment 2, the S replacement by AO impaired the feed conversion ratio in the grower–finisher phase and the global period of the experiment (*p* ≤ 0.05); the AO replacement by L tended to improve the feed conversion ratio (*p* = 0.055). Concerning the effect of dietary added fats on abdominal fat deposition, no significant differences were observed between experimental treatments (*p* > 0.05).


**Table 6.** Growth performance and abdominal fat pad deposition of broiler chickens according to different dietary added fats (Experiment 2).

<sup>1</sup> S3: Soybean oil (S) at 3.00%; AO3: Acid oil (AO) at 3.00%; AO2–L1: AO at 2.00% and L at 1.00%; AO1–L2: AO at 1.00% and L at 2.00%. L3: L at 3.00%. <sup>2</sup> S3 was not included in the statistical analysis against diets containing co- and by-products. x,y ANOVA AO3 vs S3: Values within the same row with no common superscripts are significantly different, *p* ≤ 0.05. BW: Body weight; ADFI: Average daily feed intake; ADG: Average daily gain; FCR: Feed conversion ratio; RSE: Residual standard error.

#### *3.3. Digestibility Balances*

The influence of the added fats on the dietary feed AME and the FA digestibility in both feeding periods can be seen in Table 7 (Experiment 1) and Table 8 (Experiment 2).

The digestibility balance of Experiment 1 indicated, in starter diets, that the partial and total replacement of S by L (S2–L1, S1–L2, and L3) negatively affected the feed AME value (*p* < 0.001) and the FA digestibility. Animals fed diets with 2% and 3% of L (S1–L2 and L3) showed a lower total fatty acid (TFA; *p* = 0.017), MUFA (*p* = 0.026) and PUFA (*p* = 0.004) digestibility, and they tended to absorb SFA worse than animals fed S3 (*p* = 0.055). In the case of grower–finisher diets, animals fed L3 presented a lower feed AME (*p* < 0.001), and a lower TFA (*p* = 0.020), oleic acid (*p* < 0.001) and PUFA (*p* = 0.003) digestibility as compared to animals fed S3. However, no differences were observed between S3 and treatments with partial replacement by L (S2–L1 and S1–L2).

Results from Experiment 2 showed that the S3 treatment presented a higher dietary AME and TFA digestibility than AO3 in both periods (*p* ≤ 0.05). Regarding the use of co-products (AO and L) as added fats, in the starter period, replacing AO by L led to no observable differences in the feed AME and the digestibility of TFA, SFA and MUFA (*p* > 0.05). Nevertheless, L3 presented a higher digestibility of linolenic acid (*p* = 0.011) in contrast to AO3. On the other hand, grower–finisher diets showed differences between treatments in the SFA, MUFA and PUFA digestibility. The total replacement of AO by L (L3) did not modify the dietary AME or the digestibility of TFA and SFA, but it caused a lower MUFA (*p* < 0.001) and a higher linolenic acid (*p* = 0.006) digestibility. The lowest feed AME value was observed in AO2–L1 (*p* < 0.001), which was consistent with the FA digestibility. The AO2–L1 treatment presented a lower TFA and MUFA digestibility than AO3 (*p* ≤ 0.05), and it presented a lower SFA digestibility than AO1–L2 and L3 (*p* < 0.01). Nonetheless, animals fed AO1–L2 did not show differences with the AO3 treatment and presented a higher MUFA digestibility in comparison to L3 (*p* < 0.001).


**Table 7.** Feed apparent metabolizable energy value and fatty acid digestibility of starter and grower–finisher broiler chicken diets according to added fat sources (Experiment 1).

a–c Values within the same row with no common superscripts are significantly different, *p* ≤ 0.05. AME: Apparent metabolizable energy; TFA: Total fatty acid; SFA: Saturated fatty acid; MUFA: Monounsaturated fatty acid; PUFA: Polyunsaturated fatty acid; RSE: Residual standard error. **<sup>1</sup>** S3: Soybean oil (S) at 3.00%; S2–L1: S at 2.00% and soybean lecithin high in free fatty acids (L) at 1.00%; S1–L2: S at 1.00% and L at 2.00%; L3: L at 3.00%.


**Table 8.** Feed apparent metabolizable energy value and fatty acid digestibility of starter and grower–finisher broiler chicken diets according to added fat sources (Experiment 2).

<sup>1</sup> S3: Soybean oil at 3.00%; AO3: Acid oil (AO) at 3.00%; AO–L1: AO at 2.00% and soybean lecithin high in free fatty acids (L) at 1.00%; AO–L2: AO at 1.00% and L at 2.00%; L3: L at 3.00%. <sup>2</sup> S3 was not included in the statistical analysis against diets containing co-products. a–c Values within the same row with no common superscripts are significantly different, *<sup>p</sup>* ≤ 0.05; x,y ANOVA S3 vs. AO3: Values within the same row with no common superscripts are significantly different, *p* ≤ 0.05. AME: Apparent metabolizable energy; FA: Fatty acid; SFA: Saturated fatty acid; MUFA: Monounsaturated fatty acid; PUFA: Polyunsaturated fatty acid; RSE: Residual standard error.

#### *3.4. Fatty Acid Composition of Abdominal Fat Adipose Tissue*

The effect of dietary added fat on the FA composition of the AFP can be seen in Table 9. Total replacement of S by L increased SFA, in particular palmitic acid concentration (*p* < 0.01), whereas it reduced the UFA:SFA and the PUFA:SFA (*p* < 0.01). Furthermore, a tendency for a reduction of linoleic acid concentration (*p* = 0.069) was observed. In contrast to S3, animals feed AO3 presented an AFP with a higher MUFA concentration, concretely oleic acid (*p* ≤ 0.05), and a lower PUFA content, concretely linoleic and linolenic acid (*p* ≤ 0.05), thus reducing the PUFA:SFA (*p* ≤ 0.05). Finally, the use of L as a substitute for AO caused an increase in PUFA, specifically linoleic and linolenic acid (*p* < 0.01), and a reduction in the MUFA content (*p* < 0.01). In this case, the PUFA:SFA increased as long as L replaced AO (*p* = 0.014).


**Table 9.** Fatty acid composition of abdominal fat pad of broiler chickens according to different fat sources <sup>1</sup> in diet (Experiments 1 and 2).

<sup>1</sup> S3: Soybean oil (S) at 3.00%; S2–L1: S at 2.00% and soybean lecithin high in free fatty acids (L) at 1.00%; S1–L2: S at 1.00% and L at 2.00%; AO3: Acid oil (AO) at 3.00%; AO2–L1: AO at 2.00% and L at 1.00%; AO1–L2: AO at 1.00% and L at 2.00%; L3: L at 3.00%. <sup>2</sup> S3 was not included in the statistical analysis against diets containing co-products. a–c Values within the same row with no common superscripts are significantly different, *p* ≤ 0.05. x,y ANOVA S3 vs. AO3: Values within the same row with no common superscripts are significantly different, *p* ≤ 0.05. SFA: Saturated fatty acid; MUFA: Monounsaturated fatty acid; PUFA: Polyunsaturated fatty acid; UFA:SFA: Unsaturated-to-saturated fatty acid ratio; PUFA:SFA: Polyunsaturated-to-saturated fatty acid ratio; RSE: Residual standard error.

#### **4. Discussion**

#### *4.1. Chemical Composition of the Experimental Fats and Diets*

The gross energy content of the added fats indicated that L resulted in being less energetic than S and AO. This fact is a direct consequence of PL releasing less energy than triacylglycerol and FFA. Furthermore, the L included in both experiments contained high levels of FFA (24.1%) because it was blended with soybean acid oil. The standard FFA content of soybean lecithin products is normally established at between 1.0% and 3.0% [1,22]. It is important to mention that available literature regarding the use of a soybean lecithin high in FFA in monogastric nutrition is scarce, and the literature review was based on studies that used a regular soybean lecithin with a lower FFA content. The chemical composition of the experimental diets reflected the FA profile of the added fats. The dietary UFA:SFA was reduced, as L was included in the replacement of S, which was also reported by Soares and Lopez-Bote [3].

#### *4.2. Growth Performance and Abdominal Fat Deposition*

The inclusion of L as a substitute for S did not lead to any negative effect on growth efficiency. Results agree with Azman and Cifti [23], who observed that a partial replacement (50%) of soybean oil by a soybean lecithin (4% and 6% of total added fats for starter and grower–finisher diets, respectively) did not modify final the BW or the global feed conversion ratio. However, the replacement of S by AO reduced feed conversion efficiency in the grower–finisher phase and the global period of Experiment 2. Some authors have stated that acid oils present a lower nutritive value than native oils due to their main lipid molecular structure being FFA, negatively affecting FA absorption and energy utilization [6,24].

Regarding abdominal fat deposition, results indicated that the different added fats included had no influence. It was demonstrated by Ferrini et al. [25] that animals fed a diet high in SFA content (PUFA:SFA = 0.25) presented a higher AFP deposition than animals fed diets rich in PUFA (PUFA:SFA = 6.72). The lack of differences observed in fat deposition in the present studies could be related to the slight changes in saturation degree between treatments (grower–finisher S3 and L3 PUFA:SFA = 1.95 and 1.70, respectively).

#### *4.3. Digestibility Balances*

Results extracted from Experiment 1 showed that, in terms of FA and energy utilization, the substitution of S by L at any level in starter diets is not recommended. However, the results in adult broilers suggest that L can partially replace S up to 2%. In accordance with our results, Huang et al. [4] observed, in young broiler chickens, that the partial (1%) and total replacement (2%) of soybean oil by soybean lecithin reduced the feed AME content. In the case of adult broilers, they reported that the partial (0.5% and 1%) and total (2%), replacement of soybean oil by soybean lecithin did not affect the feed AME value or ether extract utilization. Tancharoenrat et al. [26] indicated that young chicks present a limited capacity to digest and absorb fats; however, this capacity is improved from two weeks of life. Our results are consistent with this fact due to the fact that grower–finisher broilers showed a better utilization of L than starter broilers.

In Experiment 2, the comparison between S3 and AO3 demonstrated the lowering effect of the high FFA content on the FA digestibility and the feed AME, as other authors have previously stated [27,28]. It has been established that the presence of monoacylglycerols is essential for a correct solubilization of the products derived from the lipolysis into mixed-micelles [24]. In addition, Sklan [24] also suggested a direct relationship between monoacylglycerol presence in the duodenum and bile secretion, justifying the lower FA absorption rate of acid oils in comparison to crude oil. These facts were confirmed by Rodriguez-Sanchez et al. [9], who observed that a high presence of FFA was related to an insufficient solubilization and absorption of lipolysis products, and, in particular, this fact was more pronounced with unsaturated diets than saturated ones. The blending of AO and L in starter diets did not modify the FA digestibility except for linolenic acid, which was enhanced by the L inclusion. However, in grower–finisher diets, the blending of 1% of AO and 2% of L resulted in the best option in terms of FA utilization. Some authors have suggested that soybean lecithin, as an emulsifier, may enhance lipid absorption—in particular, SFA and long-chain FA—by facilitating FA incorporation inside the micelles [5,6]. However, in accordance with Soares and Lopez-Bote results [3], no improvement of the SFA digestibility related to L inclusion was demonstrated in the present experiments. This lack of effect could be related to the highly unsaturated degree of the experimental diets used in the present study. On the other hand, in the grower–finisher phase, the AO1–L2 treatment resulted in the best option, thanks to an improvement in linolenic acid, along with a tendency for a growth of the PUFA digestibility (*p* = 0.071), which suggests an emulsifying effect. It is well known that blending fats with a complementary FA profile and different lipid molecular structures (triacylglycerols, FFA and PL) produces positive interactions in terms of the AME content and the FA digestibility [2,27,28]. The synergic effect observed between 1% of AO and 2% of L can be explained because it might have been an adequate proportion of PL capable of better solubilizing FFA in the mixed micelle, facilitating its absorption. On the other hand, it is important to comment on the grower–finisher results shown in the AO2–L1 treatment, which was also a blending treatment but showed the lowest feed AME value and the lowest TFA digestibility. Results may suggest that replacing an acidic oil by a less energetic oil with a high acidity, such as L (Table 2), caused an elevated proportion of the FFA:PL, thus leading to an insufficient presence of PL capable of solubilizing the FFA into the mixed micelles. As a consequence, a chemical characterization of the different fats and oils used as energy sources can provide important information about the possible interactions between different lipid molecular structures, as Roll et al. [28] have previously stated.

#### *4.4. Fatty acid Composition of Abdominal Fat Adipose Tissue*

The FA profile of the AFP reflected the FA profile of the diets, in accordance with most of the published data [25,29]. Though some authors have reported that the presence of different dietary lipid molecular structures, such as randomized FA, influences the FA profile of the AFP [29,30], our results demonstrate that the saturation degree of the AFP is more influenced by the dietary saturation degree rather than by the lipid molecular structures (triacylglycerols, FFA and PL) present in the feed.

#### **5. Conclusions**

In summary, the inclusion of soybean lecithin high in FFA is suitable in grower–finisher diets as a partial replacer of soybean oil up to 2% without impairing performance, FA and energy utilization. Regarding to the use of a combination of co-products as an energy source, the best strategy in grower–finisher diets is a blend of 2% of high FFA soybean lecithin and 1% of monounsaturated vegetable acid oil; this is due to synergistic interactions on FA and energy utilization. Finally, the FA profile of the diets has a stronger impact on the FA profile of the AFP rather than the different lipid molecular structures.

**Author Contributions:** Conceptualization: A.V., L.C., and A.C.B.; methodology: A.V., L.C., and A.C.B.; formal analysis: A.V., L.C., and A.C.B.; investigation: A.V., L.C., and A.C.B.; data curation: A.V., L.C., and A.C.B.; writing—original draft preparation: A.V., L.C., and A.C.B.; writing—review and editing: A.V., L.C., and A.C.B.

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

**Acknowledgments:** The English of this manuscript was proofread by Chuck Simmons, a native English-speaking university instructor of English.

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

#### **References**


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

### **E**ff**ects of Mannanoligosaccharide Supplementation on the Growth Performance, Immunity, and Oxidative Status of Partridge Shank Chickens**

**Minyu Zhou 1, Yuheng Tao 1, Chenhuan Lai 1, Caoxing Huang 1,2, Yanmin Zhou <sup>3</sup> and Qiang Yong 1,2,\***


Received: 25 September 2019; Accepted: 14 October 2019; Published: 16 October 2019

**Simple Summary:** To keep animals healthy and maintain sustainability, modern poultry production industry uses functional feed additives such as mannanoligosaccharides to minimize the potential threat of disease and protect the intestinal mucosa against invading microorganisms. However, most of them are obtained by chemical synthesis that may cause environmental pollution. Thus, we found a way to produce mannanooligosaccharides by an enzyme called β-mannanase to avoid pollution. This enzyme is produced by the fungus species *Aspergillus niger*. In the present study, we evaluated such enzymatic mannanooligosaccharide and found it can improve oxidative status and immunity in broiler chickens.

**Abstract:** Mannanoligosaccharides (MOS) can be used in poultry production to modulate immunity and improve growth performance. So, we hypothesized that our enzymatic MOS could achieve the same effects in broilers. To investigate this, a total of 192 one-day-old Partridge Shank chickens were allocated to four dietary treatments consisting of six replicates with eight chicks per replicate, and they were fed a basal diet supplemented with 0, 0.5, 1 and 1.5 g MOS per kg of diet(g/kg) for42 days. Treatments did not affect the growth performance of chickens. Dietary MOS linearly increased the relative weight of the bursa of Fabricius and jejunal immunoglobulin M (IgM) and immunoglobulin G (IgG) content, whereas it linearly decreased cecal *Salmonella* colonies at 21 days (*p* < 0.05). The concentration of jejunal secretory immunoglobulin A (sIgA) and IgG at 42 days as well as ileal sIgA, IgG, and IgM at 21 and 42 days were quadratically enhanced by MOS supplementation (*p* < 0.05). Also, chickens fed MOS exhibited linear and quadratic reduction in jejunal malondialdehyde (MDA) accumulation (*p* < 0.05). In conclusion, this enzymatic MOS can improve the immune function and intestinal oxidative status of Partridge Shank chickens.

**Keywords:** mannanoligosaccharide; growth performance; immunity; oxidative status; Partridge Shank chickens

#### **1. Introduction**

Oligosaccharides, such as mannanoligosaccharides (MOS) are now widely used as functional feed additives in modern poultry production. MOS are indigestible to monogastric animals and can inhibit colonization of pathogenic microorganisms in the intestinal tract by binding pathogenic bacteria that possess mannose-specific type-I fimbriae and by its prebiotic activity. At the other hand, MOS have been found to enhance the growth of some probiotics such as cecal *Lactobacillus* species and *Bifidobacterium* species.

Extensive reports have proved that dietary MOS supplementation can enhance immunity and intestinal health, resulting in better growth performance of animals under both normal and adverse conditions [1–6]. Additionally, some exciting findings on MOS research have currently been observed by Bozkurt et al. [7], Attia et al. [8] and Zheng et al. [9], who have shown that dietary MOS addition can act as a free radical scavenger to improve the body's antioxidant capacity through inhibiting lipid peroxidation and/or elevating antioxidant enzymes activities in laying hens, broilers, and sheep. Furthermore, Liu et al. [10] have reported that the inclusion of dietary MOS can relieve hepatic oxidative damage of fish under adverse conditions. It has been demonstrated that dietary MOS supplementation increases water-holding capacity and tenderness [8,11], whereas it decreases the fat content of muscle in animals [8,12]. In a published paper, Zhang et al. [13] illustrated that dietary yeast cell wall inclusion, a widely used MOS product, reduced the concentration of malondialdehyde (MDA), an end-product of lipid peroxidation, in raw and boiled muscles in broilers.

MOS originates from different sources, and it has been repeatedly reported that various mannanases from bacteria, fungi, and plants can hydrolyze different mannan-containing polysaccharides to yield MOS [14–22]; however, the supply of MOS is not adequate to meet the demand. So, an economically viable technique for producing MOS has yet to be identified and developed. *Amorphophallus konjac* K. Koch is an underutilized agricultural material with low commercial value in China where it is typically used as animal feed and as a gelling and thickening ingredient for human foods [23]. It has been recognized as a safe material according to the FDA (Food and Drug Administration) [24]. Almost 60% of konjac is glucomannan, a previously noted precursor to MOS. The glucomannan from *Amorphophallus konjac* (KGM) and MOS from glucomannan consist of a linear chain of β-1,4-d-glucose and d-mannose. Structural studies of MOS from KGM revealed that it contains only glucose and mannose at a molar ratio of 1:1.6 [23]. In addition, it was found that branching occurs at β-1,6- glucoses approximately three times for every 32 sugar residues [25]. Finally, it has been found that most MOS has a degree of polymerization (DP) between 2 and 6. Little is known about the effect of this MOS on broilers, especially Partridge Shank chickens, an important local chicken breed. We hypothesized that the MOS would exhibit a high bioavailability in vivo. The current study was therefore conducted to evaluate the effects of enzymatic MOS from KGM on the growth performance, immunity, and antioxidant status of Partridge Shank chickens.

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

The experimental procedures used in this study were approved by the Nanjing Agricultural University Institutional Animal Care and Use Committee. The ethical code is NJAU20171104.

#### *2.1. Mannanoligosaccharide*

Mannanoligosaccharide (MOS) was prepared from KGM produced by the laboratory using enzymatic hydrolysis. The KGM used in this experiment was prepared from *Amorphophallus konjac* bought from the local market of Yunnan Province of China. The enzyme used was β-mannanase produced from *Aspergillus niger* by the laboratory. Hydrolysis was performed for 2 h at pH 5.0 with an environmental temperature of 50 ◦C. Post hydrolysis, enzymatic hydrolysate was free flowing. The enzyme activity was inactivated by putting enzymatic hydrolysate in a beaker into boiling water for 10 min, then ultrafiltration was used to separate the impurities to get MOS. Finally, spray drying (BUCHI, Flawil, Switzerland) was used to prepare solid MOS.

#### *2.2. Husbandry, Diets and Experimental Design*

A total of one hundred and ninety-two one-day-old Partridge Shank chicks with similar initial weight obtained from a commercial hatchery were randomly allocated into four dietary treatments. Each treatment included 48 chicks that consisted of six replicates (one cage per replicate). Birds in the four treatments were fed a basal diet supplemented with 0, 0.5, 1 and 1.5 g MOS per kg of diet for 42 days. Ingredient composition and nutrient content of the basal diets are presented in Table 1. Birds had free access to mash feed and water in three-level cages (120 cm <sup>×</sup> 60 cm <sup>×</sup> 50 cm; 0.09 m2 per chick) in a temperature-controlled room with continuous lighting. The temperature of the room was maintained at 32 to 34 ◦C for the first 3 days and then reduced by 2–3 ◦C per week to a final temperature of 26 ◦C. At 21 days and 42 days of age, birds were weighed after feed deprivation for 12 h and feed intake was recorded by replicate (cage) to calculate average daily feed intake (ADFI), and average daily gain (ADG). Birds that died during the experiment were weighed, and the data were included in the calculation of feed conversion ratio (FCR).


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

<sup>1</sup> Premix provided per kilogram of diet: vitamin A (transretinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 3000 IU; vitamin E (all-rac-α-tocopherol), 30 IU; menadione, 1.3 mg; thiamin, 2.2 mg; riboflavin, 8 mg; nicotinamide, 40 mg; choline chloride, 600 mg; calcium pantothenate, 10 mg; pyridoxine·HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; vitamin B12 (cobalamin), 0.013 mg; Fe (from ferrous sulphate), 80 mg; Cu (from copper sulphate), 8.0 mg; Mn (from manganese sulphate), 110 mg; Zn (from zinc oxide), 60 mg; I (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.3 mg; <sup>2</sup> the nutrient levels were as fed basis; <sup>3</sup> Values based on analysis of triplicate samples of diets.

#### *2.3. Sample Collection*

At 21 and 42 days, one bird (close to the average body weight of birds in each cage) from each replicate (48 birds in total) was selected and weighed after feed deprivation for 12 h. After that, blood samples (around 5 mL each) were taken from the wing vein and centrifuged at 4450× *g*, 15 min at 4 ◦C to separate serum, which was frozen at −20 ◦C until analysis. After blood collection, the chickens were euthanized by cervical dislocation and immediately necropsied. Following necropsy, the whole gastrointestinal tracts were quickly removed. Bursa of Fabricius, thymus, and spleen were then collected and weighed to calculate the relative organ weights using the following formula: relative weight of immune organ (g/kg) = immune organ weight (g)/body weight (kg). Jejunum (from the end of the pancreatic loop to the Meckel's diverticulum) and ileum (from Meckel's diverticulum to the ileocecal junction) were then excised free of the mesentery and placed on a chilled stainless-steel tray. The jejunal, and ileal mucosa were scratched carefully using a sterile glass microscope slide, which were then rapidly frozen in liquid nitrogen and stored at −80 ◦C for further analysis. Then cecum samples were quickly removed aseptically, and cecal contents were cultured to determine the population of *Lactobacillus*, *Salmonella* and *Escherichia coli*.

#### *2.4. Microflora Population Measurement*

Approximately 0.2 g of aseptically removed cecal contents were diluted in 2 mL of sterilized saline (154 mmol/L), and then three 10-fold serial dilutions were made from the diluted cecal contents (10<sup>−</sup>3, 10−<sup>4</sup> and 10−<sup>5</sup> for *Salmonella*; 10<sup>−</sup>4, 10−<sup>5</sup> and 10−<sup>6</sup> for *Escherichia coli* and *Lactobacillus*). A 100 μL portion of the last three dilutions were then spread evenly onto plates. *Escherichia coli* colonies were enumerated on MacConkey agar (Qingdao Hope Bio-Technology Co. Ltd., Qingdao, Shandong, China) at 37 ◦C for 24 h. *Lactobacillus* were enumerated on MRS agar (Qingdao Hope Bio-Technology Co. Ltd., Qingdao, Shandong, China) medium at 37 ◦C for 48 h. *Salmonella* colonies were determined on Bismuth sulfite agar (Qingdao Hope Bio-Technology Co. Ltd., Qingdao, Shandong, China) and incubated at 37 ◦C for 24 h. All plates with countable colonies were enumerated and averaged to express log CFU (Colony-Forming Units) per gram of cecal content.

#### *2.5. Determination of Mucosal Immune and Antioxidant Parameters*

Approximately 0.3 g mucosal samples from jejunum and ileum were homogenized (1:9, wt/vol) with ice-cold 154 mmol/L sodium chloride solution using an Ultra-Turrax homogenizer (Tekmar Co., Cincinatti, OH, USA) and then centrifuged at 4450× *g* for 15 min at 4 ◦C. The supernatant was then collected and stored at −20 ◦C for subsequent analysis.

Total superoxide dismutase (T-SOD) activity, and malondialdehyde (MDA) content were analyzed using commercial diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer's instructions. The activity of T-SOD was analyzed by the hydroxylamine method [26], and one unit of T-SOD was defined as the amount of enzyme per milliliter of mucosa required to produce 50% inhibition of the rate of nitrite production at 37◦C. MDA concentration was measured by barbiturate thiosulfate assay [27], and was expressed as nanomole per milliliter of mucosa.

Concentrations of immunoglobulin M (IgM), immunoglobulin G (IgG), and secretory immunoglobulin A (sIgA) were measured in appropriately diluted mucosal samples by enzyme-linked immunosorbent assay (ELISA) using microtiter plates and chicken-specific IgM, IgG, sIgA ELISA quantitation kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). All results were normalized against total protein concentration in each sample for inter-sample comparison. Finally, total protein concentration was determined by using a total protein quantitation kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

#### *2.6. Statistical Analysis*

Data was analyzed by one-way analysis of variance (ANOVA) using SPSS statistical software (Ver. 19.0 for windows, SPSS Inc., Chicago, IL, USA). The replicate (cage) was defined as the experimental unit. Polynomial contrasts were used to test the linear and quadratic effects of MOS levels. The level of significance was *p* < 0.05 in all analyses. Results are presented as means alongside their pooled standard errors of means.

#### **3. Results**

#### *3.1. Growth Performance*

Chickens given basal diets supplemented (Table 2) with MOS exhibited similar growth performance compared with the control group during the 42-day study (*p* > 0.05).


**Table 2.** Growth performance of Partridge Shank chickens fed diets supplemented with or without mannanoligosaccharide (MOS).

MOS = mannanoligosaccharide; ADG = average daily gain; ADFI = average daily feed intake; FCR = feed conversion ratio; SEM = standard error of means (each treatment included 48 chickens and consisted of 6 replicates); L = linear; Q = quadratic.

#### *3.2. Realtive Immune Organ Weights*

As shown in Table 3, the inclusion of MOS quadratically increased the relative weight of bursa of Fabricius at 21 days (*p* < 0.05), but this effect was not observed at 42 days (*p* > 0.05). Also, the relative weights of the thymus and spleen were not altered by the MOS diet (*p* > 0.05).

**Table 3.** Immune organ weights from Partridge Shank chickens fed diets supplemented with or without MOS (g/kg).


MOS = mannanoligosaccharide; relative immune organ weight that was expressed relative to body weight; SEM = standard error of means (each treatment included 48 chickens and consisted of 6 replicates); L = linear; Q = quadratic.

#### *3.3. Cecal Microflora Population*

In Table 4, it can be seen that MOS had a linear effect on *Salmonella* colonies (*p* < 0.05) in the cecal content at 21 days. However, cecal *Escherichia coli* and *Lactobacillus* colonies were not affected by MOS supplementation during the whole experiment (*p* > 0.05).


**Table 4.** Microflora population in the cecal content of Partridge Shank chickens fed diets supplemented with or without MOS (log CFU/g content).

MOS = mannooligosaccharide; SEM = standard error of means (each treatment included 48 chickens and consisted of 6 replicates); L = linear; Q = quadratic.

#### *3.4. Intestinal Immunoglobulins Contents*

Chickens exhibited similar content of sIgA in the jejunal mucosa among groups at 21 days (Table 5, *p* > 0.05). MOS linearly increased jejunal IgM and IgG contents (*p* < 0.05) at 21 days and quadratically increased jejunal sIgA and IgG levels at 42 days *(p* < 0.05). Simultaneously, ileal sIgA, IgM and IgG contents were quadratically increased in 42 days (*p* < 0.05).

**Table 5.** Intestinal immunoglobulins contents of Partridge Shank chickens fed diets supplemented with or without MOS (μg/mg protein).


MOS = mannooligosaccharide; sIgA = secretory immunoglobulin A; IgM = immunoglobulin M; IgG = immunoglobulin G; SEM = standard error of means (each treatment included 48 chickens and consisted of 6 replicates); L = linear; Q = quadratic.

#### *3.5. Intestinal Oxidative Status*

As shown in Table 6, chickens fed MOS exhibited linear and quadratic reduction in jejunal MDA accumulation at 21 days (*p* < 0.05), and quadratic effect on ileal MDA content at 42 days (*p* < 0.05). However, intestinal SOD activity was similar among treatments (*p* > 0.05).

**Table 6.** Intestinal antioxidant status of Partridge Shank chickens fed diets supplemented with or without MOS.


MOS = mannooligosaccharide; MDA = malondialdehyde; T-SOD = total superoxide dismutase; SEM = standard error of means (each treatment included 48 chickens and consisted of 6 replicates); L = linear; Q = quadratic.

#### **4. Discussion**

#### *4.1. Growth Performance*

Sims et al. [28] and Attia et al. [29] demonstrated that dietary MOS supplementation can improve the growth performance of poultry under normal conditions. In broilers, Geier et al. [30] found that when broiler feed contained MOS, the growth performance of broilers was unchanged. This study demonstrated that MOS supplementation exerted no significant effect on the growth performance of broilers, and this was consistent with the findings of Munyaka et al. [31], who reported that dietary supplementation with yeast-derived MOS preparation did not alter growth performance and mortality in broilers. In contrast, Churchil et al. [32] observed that yeast-derived MOS inclusion increased the body weight of broilers. In addition, Gao et al. [33] demonstrated that the growth performance of broilers was optimized by adding the yeast-derived MOS. Therefore, the unchanged growth performance observed in this study may be associated with the source of MOS used as the dietary supplement; that is, the broilers may digest less nutrients from our MOS. Based on this result, further studies are needed to evaluate the influences of different sources of MOS on the growth performance of chickens, and to evaluate how to further process our MOS so that it can increase nutrient digestibility of chickens.

#### *4.2. Relative Immune Organ Weights*

Relative immune organ weights could partially reflect the development and growth of immune organs. The current study showed that MOS quadratically increased the relative weight of bursa of Fabricius at 21 days, which plays a vital role in development and maturation of B-lymphocytes and the diversification of specific antibodies [34]. Thus, MOS supplementation may increase the weight of bursa by stimulating the proliferation of bursal lymphocytes. Also, digestive microbial antigen stimulation plays a vital role in the development of lymphoid organ tissue [35]. Li et al. [36] reported that the increased weight of bursa may be associated with possible changes to the intestinal microorganism population induced by yeast derived MOS supplementation. Dietary MOS supplementation, therefore, represents a nutritional strategy that could favor intestinal colonization of beneficial bacteria, thereby conferring intestinal health benefits to the host. Further study is required to verify this conjecture.

#### *4.3. Cecal Microflora Population*

MOS in this experiment is a plant-derived oligosaccharide, which can promote the growth of *Bifdobacteria*, which decreases colonization by enteric pathobionts like *Salmonella* and *Escherichia coli*, regulates immune signaling, and improves mucosal integrity [37,38]. It is well documented that MOS competitively adsorbs to the mannosespecific type 1 fimbriae of *Escherichia coli* and other pathogens, thereby limiting their colonization of the intestinal epithelium. This phenomenon results in the pathogens ultimately being excreted from the intestine [39,40]. Muthusamy et al. [41] reported that dietary MOS lowered *Salmonella* spp. and *Escherichia coli* number in the small intestine (duodenum, jejunum and ileum) of broilers with poor health or *Salmonella* challenged. In this study, MOS had a linear decreasing effect on *Salmonella* colony in the cecal content at 21 days, indicating that the prepared MOS can decrease colonization by enteric pathobionts. Different results were found by Li et al. [36] whereby MOS supplementation did not alter *Escherichia coli* and *Salmonella* colonies in the cecal content (only a decreased tendency was noted). Thus, oligosaccharides from different sources and different chain lengths may have different results on different intestinal microorganisms. This hypothesis requires further research to prove it.

#### *4.4. Intestinal Immunoglobulins*

The immune system guards the body against foreign substances and protects it from invasion by pathogenic organisms. In chickens, three classes of immunoglobulins participate in immune system maintenance. These immunoglobulins have been identified as IgM, IgG and IgA [42]. sIgA plays an important role in the protection and homeostatic regulation of intestinal mucosal epithelia separating the outside environment from the inside of the body. The primary function of sIgA is referred to as immune exclusion, a process that limits the access of numerous microorganisms and mucosal antigens to the thin and vulnerable mucosal barriers [43]. Savage et al. [44] reported that when feeding MOS to broilers, the concentration of IgA in the bile increased 14.2%, and that the MOS may have a mechanism that directly protects the mucosa. The present study showed that MOS linearly increased jejunal IgM and IgG contents at 21 days, while it quadratically increased sIgA and IgG contents at 42 days. Simultaneously, ileal sIgA, IgM and IgG contents were quadratically increased at 42 days. Similar results were also observed by Li et al. [36] and Gao et al. [33]. We assumed that the main target of the prepared MOS is located in the intestine, and it may simulate the development of intestinal cells in the jejunum and ileum to secrete more immunoglobulins. This result indicates that the prepared MOS can improve intestinal immune status.

#### *4.5. Intestinal Oxidative Status*

Reactive oxygen species (ROS) are produced during normal metabolism in cells, but concentration of ROS exceeding the antioxidant protection levels of cells can cause widespread damage to DNA, proteins and endogenous lipids [45]. SOD is generally regarded as one of the main antioxidant enzymes in scavenging the oxygen free radical [46]. The MDA is the main end product of lipid peroxidation by ROS, and increased MDA accumulation is an important indication of lipid peroxidation [47]. MOS from konjac has been reported to display relatively good antioxidative properties [48]. In poultry, enhanced SOD activity in the serum of broilers fed dietary MOS has recently been found by Attia et al. [49]. Bozkurt et al. [7] reported that dietary MOS supplementation could decrease MDA concentration in both eggs and liver, and increase SOD activity in the liver in laying hens. In this study, MOS

linearly and quadratically decreased jejunal MDA accumulation in 42 days and it had quadratic effect on ileal MDA accumulation at 42 days. This was in agreement with the results of Liu et al. [10], who demonstrated that dietary MOS inclusion decreased MDA accumulation in fish under adverse conditions. According to the literature, dietary MOS supplementation can accelerate gastrointestinal maturation and increase nutrient absorption for better growth performance in organisms [50–52], which may simultaneously and indirectly contribute to improving the adsorption and utilization of small molecules related to the synthesis of antioxidants. Thus, in the current study, elevated oxidative status in the intestinal mucosa by MOS supplementation might also be related to the promotion of MOS addition on the gut ecology and digestive function in animals [51,52].

#### **5. Conclusions**

In this study, MOS did not affect growth performance whereas it improved immune function (enhanced relative weight of bursa of Fabricius, enhanced jejunal sIgA and IgG contents and ileal sIgA and IgG levels), intestinal oxidative status (decreased jejunal MDA content), and regulated the cecal microflora population (reduced cecal *Salmonella* population) in Partridge Shank chickens.

**Author Contributions:** Conceptualization, M.Z., Y.Z., Q.Y.; Methodology, M.Z., Y.Z.; Investigation, M.Z., Y.T.; Project administration, C.L.; Writing—original draft preparation, M.Z.; Writing—review and editing, C.H.; Supervision, Q.Y.

**Funding:** This research was funded by the Governmental Public Industry Research Special Funds for Projects (Grant No. 201404615).

**Acknowledgments:** This study was completed at the College of Animal Science and Technology of Nanjing Agricultural University (Nanjing, China). The technical assistance of graduate students in this study is gratefully acknowledged.

**Conflicts of Interest:** The funder had no role in the design, analysis or writing of this article. All authors approved the submission of this manuscript and declare no conflict of interest. The manuscript has not been published previously, and is not under consideration for publication elsewhere.

#### **References**


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

*Article*

### **E**ff**ects of Chromium-Loaded Chitosan Nanoparticles on the Intestinal Electrophysiological Indices and Glucose Transporters in Broilers**

**Sajid Khan Tahir 1, Muhammad Shahbaz Yousaf 1,\*, Sohrab Ahmad 1, Muhammad Khurram Shahzad 1, Ather Farooq Khan 2, Mohsin Raza 1, Khalid Abdul Majeed 1, Abia Khalid 1, Hafsa Zaneb 3, Imtiaz Rabbani <sup>1</sup> and Habib Rehman <sup>1</sup>**


Received: 27 September 2019; Accepted: 14 October 2019; Published: 17 October 2019

**Simple Summary:** Chromium is an important trace element responsible for the metabolism of glucose by enhancing insulin activity. This study was planned to evaluate the effects of chromium-loaded chitosan nanoparticles on the transport of glucose or amino acid across jejunum, gene expression of glucose transporters, and glycogen contents of liver and muscle. The results revealed that an increase in the supplemented dose of chromium-loaded chitosan nanoparticles decreased liver glycogen content and glucose transport across jejunum, while the muscle glycogen, gene expression of glucose transporters, and amino acid transport remained unaffected.

**Abstract:** The present study aimed to evaluate the effect of chromium-loaded chitosan nanoparticles (Cr-CNPs) on the electrophysiological indices, gene expression of glucose transporters, and tissue glycogen in broilers. A total of 200 one-day-old broilers were randomly divided into five groups, with each having five replicates (n = 8). Group A was fed a corn–soybean meal diet, while the diets of groups B, C, D, and E were supplemented with 200, 400, 800, and 1200 μg/kg of Cr as Cr-CNPs, respectively. On day 35, the jejunum was collected for electrophysiological study, gene expression of glucose transporters, and tissues glycogen determination. The basal short-circuit current and tissue conductance before the addition of glucose was the same in all groups. Following the addition of glucose, the change in short-circuit current decreased (*p* < 0.05) in the jejunal tissues of birds supplemented with 400 and 1200 μg Cr-CNPs compared with the control group. Gene expression of SGLT-1 and GLUT-2 remained unaffected with supplementation. The serum glucose and liver glycogen concentration decreased (*p* < 0.05) linearly with supplementation, while no effect was observed on muscle glycogen. In conclusion, Cr-CNPs supplementation decreases the glucose absorption and liver glycogen content, without affecting the gene expression of glucose transporters.

**Keywords:** nanoparticles; chromium; supplementation; electrophysiology; Ussing chamber; poultry

#### **1. Introduction**

Glucose metabolism of avian species differs from that of mammals; i.e., the birds have higher blood glucose concentration and lower insulin levels [1]. Furthermore, avian species are considered less sensitive to insulin than mammals [2]. Chromium (Cr), a trace element, is known to increase insulin sensitivity in mammals [3] whereas, in broilers, Cr supplementation has reduced the serum glucose concentration [4]. The Cr level in poultry feed is yet to be appropriately recommended [5]. It is believed that poultry diets containing Cr can meet the requirements of the birds reared under the standard management conditions specific to broilers. However, several studies reported a positive effect of Cr supplementation on the production performance and carcass traits of broilers [6–8].

Intestinal absorption of carbohydrates occurs via glucose transporters [9]. In birds, most of the glucose or amino acid transport occurs in the jejunum [10]. The sodium-dependent glucose co-transporter transports glucose, along with sodium, from the intestinal lumen, while the sodium-independent glucose transporter is responsible for the transport of glucose along the basolateral side [11]. The transport of Glucose and amino acid through the intestine can be evaluated by studying the electrical variables with the help of an Ussing chamber. The effect of chromium-loaded chitosan nanoparticles (Cr-CNPs) on the electrophysiological indices and gene expression of glucose transporters is yet to be reported in poultry. Limited studies are available regarding the effect of organic chromium picolinate and chromium histidinate on the glucose transporters in layers exposed to heat stress [9]. The current research is aimed to explore the effect of Cr-CNPs supplementation on the electrophysiological parameters, gene expression of glucose transporters, and tissue glycogen content in broilers reared under standard management conditions.

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

All the experimental procedures used were approved by the Ethical Review Committee of the University of Veterinary and Animal Sciences, Lahore, Pakistan, vide letter No. DR/498.

#### *2.1. Preparation of Chromium-Loaded Chitosan Nanoparticles*

The Cr-CNPs were prepared and characterized at the Interdisciplinary Research Center in Biomedical Research, COMSATS University Islamabad (Lahore Campus), Pakistan, according to the method described by Wang et al. [12]. Briefly, 1% (*w*/*v*) solution of chitosan was prepared by dissolving chitosan into a 0.5% acetic acid solution with the pH adjusted to 3.5. The chitosan solution was then stirred for one hour. During stirring, 200 mg/L of chromium chloride solution was added to the chitosan solution to get a suspension of chitosan and chromium chloride. The pH of the suspension was adjusted to 6.5, and stirring was continued for five hours. Subsequently, the precipitate was centrifuged at 12,000 g for 15 min at room temperature and washed with distilled water to get Cr-CNPs.

#### *2.2. Experimental Animals, Grouping, Diet, and Management*

Two hundred male broiler chicks (Hubbard), were randomly divided into five groups, with each having five replicates (n = 8). Birds in group A (control) were given the non-supplemented basal diet, as shown in Table 1 [13], while the birds in the groups B, C, D, and E were fed the same diet but supplemented with graded levels of Cr-CNPs i.e., 200, 400, 800, and 1200 μg/kg of Cr as Cr-CNPs, respectively, for 35 days. The feed and water were provided ad libitum. Temperature and relative humidity on day 1 was kept at 35 ± 1.1 ◦C and 65 ± 5%, respectively. The temperature was decreased by 3 ◦C per week until it reached 26 ◦C on day 21.


**Table 1.** Composition of the diet (g/kg).

<sup>1</sup> Provided vitamins per kg of the feed: vitamin A (retinol), 11000 IU; vitamin B-12 (cyanocobalamin), 0.0132 mg; vitamin D3 (cholecalciferol), 2200 IU; vitamin E (alpha-tocopherol), 22 IU; choline chloride, 440 mg; riboflavin, 8.8 mg; pantothenic acid, 22 mg; ethoxyquin, 250 mg; menadione, 2.2 mg; pyridoxine, 4.4 mg; folic acid, 1.1 mg; biotin, 0.22; thiamin, 4.4 mg. <sup>2</sup> Supplied minerals per kg of the feed: Cu (CuSO4), 20 mg; Zn (ZnO), 200 mg; Mn (MnSO4), 240 mg; Fe (FeSO4), 120 mg; I (KI), 0.92 mg; Ca, 150–180 mg. Measured Chromium 4.05 mg/kg. \* Calculated according to NRC 1994.

#### *2.3. Sample Collection and Processing of Tissue*

On day 35, eight birds per group were randomly selected and their jejunal segments were prepared as described earlier by Rehman et al. [14]. Briefly, after exsanguination, a segment of jejunum was removed, washed thoroughly with an ice-cold Ringer's buffer solution, and transferred to the laboratory in buffer. The serosal layer was stripped off the jejunum. The jejunum was subsequently opened longitudinally along the mesenteric border, rinsed with Ringer's solution to remove the luminal contents, and then gassed with carbogen (95% O2 and 5% CO2) till mounting on the Ussing chamber.

Blood samples were taken to determine the glucose level. One hundred milligrams of tissue from each liver and pectoral muscle was collected for quantification of glycogen contents. For mRNA quantification of glucose transporters, the collected jejunal segments were washed with ice-cold normal saline. All the samples were stored at −80 ◦C for further analyses.

#### *2.4. Measurement of Electrophysiological Indices*

The jejunal mucosa (with stripped-off serosal layer) was cut into four pieces of 1 cm2 and mounted in between two compartments of the Ussing chamber. The exposed area of the chamber was 0.95 cm2 [15]. Damage to the edge of the tissue was minimized by silicon rubber rings on both sides of the tissue. Buffer solution (16 mL) was added to the chambers on each side. The buffer solution contained (in mM) 1.2 CaCl2, 115 NaCl, 25 NaHCO3, 20 Mannitol, 5 KCl, 2.4 Na2HPO4, 1.2 MgCl2, and 0.4 NaH2PO4, with the pH adjusted to 7.4. The buffer solution was continuously aerated with carbogen (95% O2 and 5% CO2) and maintained at 37 ◦C. Buffer osmolarity was measured (Osmomat 030, Gonotec GmbH, Berlin, Germany) and adjusted to 300 mOsmol/L, using mannitol. Tissues were allowed to equilibrate for 20 min under open circuit and then short-circuited by clamping the voltage at 0 mV for 5 min. Following equilibrium, 10.0 mM glucose or L-glutamine was added to the mucosal side, and the peak electrical response was measured. Electrical measurements like short-circuit current (Isc) and transmural tissue conductance (Gt) were observed with an automatic

computer-controlled voltage-clamp device (Mussler, Aachen, Germany) to assess the electrogenic transport of glucose or L-glutamine linked to sodium across the jejunal mucosa.

#### *2.5. Extraction of RNA and Quantification of Glucose Transporters*

The mRNA expression of glucose transporters (sodium-dependent glucose transporter-SGLT-1 and sodium-independent glucose transporter-GLUT-2) were determined by real-time PCR (Router gene 5 plex real-time PCR, Qiagen, Hilden, Germany). The oligonucleotide primers sequence for SGLT-1 [16], GLUT-2 [17], and ß-actin [17] (housekeeping gene) used for PCR amplification are shown in Table 2. The RNA extraction of the jejunal mucosa was done by using Trizol (Invitrogen, Karlsruhe, Germany), according to the manufacturer's instructions. The total RNA was quantified by using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). A total of 5 μg RNA was reverse transcribed to complementary DNA (cDNA), using First-Strand cDNA synthesis kit (Thermo Scientific™, Waltham, MA, USA). Real-time PCR was performed using the SYBER green maxima PCR kit (Thermo Scientific™, Waltham, MA, USA), as per manufacturer's instructions. The following PCR program was set on the machine to amplify the target mRNA in tissue extracts: 95 ◦C for 3 min, followed by 40 cycles of 95 ◦C for the 30 s, 60 ◦C for 30 s, and 72 ◦C for 30 s. To determine the melting points of the amplified cDNA and to confirm the production of a single product, a dissociation curve was generated after 40 cycles. Relative mRNA expression levels of SGLT-1 and GLUT-2 were determined using the 2−ΔΔCT method [18].


**Table 2.** Primer sequences used during real-time PCR.

SGLT-1 (Na+-dependent glucose and galactose transporter); GLUT-2 (Na+-independent glucose, galactose and fructose transporter).

#### *2.6. Quantification of Tissues Glycogen and Serm Glucose*

Liver and muscle glycogen contents were quantified by using iodine assay described by Bennet et al. [19] and Dreiling et al. [20], with some modifications. Briefly, 100 mg liver and muscle samples were homogenized using chilled perchloric acid to solubilize the glycogen. The homogenate was then centrifuged at 2500 rpm for 10 min at 4 ◦C. The supernatant was collected, and the pellet was re-homogenized with chilled perchloric acid. After another round of centrifugation, the supernatant thus collected was added to the previously collected supernatant and subjected to iodine assay. The absorbance of samples or glycogen standards was measured at 460 nm, using an EPOCHTM microplate spectrophotometer (Biotek Instruments Inc., Winooski, VT, USA). The serum glucose concentration was estimated by the commercially available kit (DiaSys, Germany Germany), according to manufacturer's instruction using the same EPOCHTM microplate spectrophotometer.

#### *2.7. Statistical Analysis*

Data were statistically analyzed using Statistical Package for Social Sciences (SPSS for windows version 20.0, IBM). Data were presented as means ±SEM and were analyzed using one-way analysis of variance (ANOVA). For group differences, Tukey's post hoc test was used. Polynomial contrasts were used to determine the linear, quadratic, and cubic effects of Cr-CNPs supplementation at *p* < 0.05.

#### **3. Results**

The basal short-circuit current (Isc) and transmural tissue conductance (Gt) before the addition of glucose did not vary between the supplemented groups and the control group (Table 3). After the addition of glucose to the mucosal side, the change in short-circuit current decreased linearly (*p* < 0.05) in the groups C and E compared with the control group (Figure 1). However, no effect on the change in transmural tissue conductance was observed with Cr-CNPs supplementation after addition of glucose (Figure 1).

**Table 3.** Effect of Cr-CNPs supplementation on initial short-circuit current and tissue conductance before the addition of glucose.


Data are presented as Mean ± SEM. Group A = control—without Cr-CNPs supplementation. Group B = offered 200 μg Cr-CNPs/kg of feed. Group C = offered 400 μg Cr-CNPs/kg of feed. Group D = offered 800 μg Cr-CNPs/kg of feed. Group E = offered 1200 μg Cr-CNPs/kg of feed. ISci = initial short circuit current. Gti = initial tissue conductance.

**Figure 1.** Effect of Cr-CNPs supplementation on (**A**) change in short-circuit current (ΔISc) and (**B**) change in tissue conductance (ΔGt) after addition of glucose to the jejunum in broilers. Labeled bars without a common letter differ significantly, *p* < 0.05. Data are presented as Mean ± SEM. Group A = control—without Cr-CNPs supplementation. Group B = offered 200 μg Cr-CNPs/kg of feed. Group C = offered 400 μg Cr-CNPs/kg of feed. Group D = offered 800 μg Cr-CNPs/kg of feed. Group E = offered 1200 μg Cr-CNPs/kg of feed.

Prior to the addition of glutamine addition, the basal Isc and Gt were similar in all supplemented groups compared to the control group (Table 4). After the addition of glutamine to the mucosal side, no effect of Cr-CNPs supplementation was observed on the change in short-circuit current and change in transmural tissue conductance, as shown in Figure 2.


**Table 4.** Effect of Cr-CNPs supplementation on initial short-circuit current and tissue conductance before the addition of L-glutamine.

Data are presented as Mean ± SEM. Group A = control—without Cr-CNPs supplementation. Group B = offered 200 μg Cr-CNPs/kg of feed. Group C = offered 400 μg Cr-CNPs/kg of feed. Group D = offered 800 μg Cr-CNPs/kg of feed. Group E = offered 1200 μg Cr-CNPs/kg of feed. ISci = initial short circuit current. Gti = initial tissue conductance.

**Figure 2.** Effect of Cr-CNPs supplementation on (**A**) change in short circuit current (ΔISc) and (**B**) change in tissue conductance (ΔGt) after addition of L-glutamine to the jejunum in broilers. Mean values with different small letters on the same bar differ significantly at *p* < 0.05. Data are presented as Mean ± SEM. Group A = control—without Cr-CNPs supplementation. Group B = offered 200 μg Cr-CNPs/kg of feed. Group C = offered 400 μg Cr-CNPs/kg of feed. Group D = offered 800 μg Cr-CNPs/kg of feed. Group E = offered 1200 μg Cr-CNPs/kg of feed.

The mRNA expression of glucose transporters, i.e., SGLT-1 and GLUT-2, remained unaffected by the Cr-CNPs supplementation compared to the control group, as shown in Figure 3.

**Figure 3.** Effect of Cr-CNPs supplementation on gene expression of glucose transporters, i.e., (left side) SGLT-1, and (right side) GLUT-2 in broilers. Data are presented as mean ± SEM. Group A = control—without Cr-CNPs supplementation. Group B = offered 200 μg Cr-CNPs/kg of feed. Group C = offered 400 μg Cr-CNPs/kg of feed. Group D = offered 800 μg Cr-CNPs/kg of feed. Group E = offered 1200 μg Cr-CNPs/kg of feed.

The liver glycogen concentration in groups D and E decreased linearly (*p* < 0.05) with the Cr-CNPs supplementation compared with the control group. No significant effect of Cr-CNPs supplementation was found on muscle glycogen concentration (Table 5). The serum glucose level decreased linearly (*p* < 0.05) with the Cr-CNPs supplementation (Table 5).

**Table 5.** Effect of Cr-CNPs supplementation on liver and muscle glycogen concentration (mg/100g) and blood glucose concentration (mg/dL) in broilers.


Data are presented as mean ± SEM. a–b Within the same row, different superscript indicates significantly different means at *p* < 0.05. Group A = control—without Cr-CNPs supplementation. Group B = offered 200 μg Cr-CNPs/kg of feed. Group C = offered 400 μg Cr-CNPs/kg of feed. Group D = offered 800 μg Cr-CNPs/kg of feed. Group E = offered 1200 μg Cr-CNPs/kg of feed.

#### **4. Discussion**

Chromium (Cr) is a biologically active trace element that plays a key role in metabolic activities in the body. Upon absorption, Cr is found in the blood either in free form or in bound-to-globulin proteins, transferrin, or complexes like glucose tolerance factor [21]. The bioavailability of organic chromium is higher than the inorganic form due to its increased absorption rate [22]. The inorganic form irreversibly binds with the undigested content in the intestine, and, hence, its absorption and bioavailability are limited [23]. The absorption of Cr can be enhanced by chelation, as it prevents the precipitation of chromium at alkaline pH in the poultry intestine [24].

The intestine of birds is highly absorptive for water and electrolytes. The electrical current across the epithelium is due to the net movement of ions. The transport of glucose and amino acids across the membrane occurs by either the paracellular or transcellular pathway [14]. Paracellular transport occurs without the expense of energy, while transcellular transport utilizes energy to transport glucose via sodium-dependent glucose transport (SGLT-1) and amino acids by carrier proteins in the luminal and basolateral membranes [25]. Transcellular transport of glucose and amino acids in poultry occurs in the small intestine and colon [26,27]. Most of the sodium-dependent uptake of glucose and amino acids via carrier proteins takes place in the jejunum [10]. The addition of glucose and amino acids to the luminal side of the intestine potentiates carrier-mediated transport, along with the enhanced uptake of luminal sodium. In response, the intestinal membrane depolarizes, and increased cytoplasmic sodium stimulates the sodium–potassium ATPase pump in the basolateral membrane, which, in turn, increases the net movement of sodium from the mucosal to the serosal side. These events bring changes in the electrical variable of the intestine and increase the short-circuit current [14,28,29]. In the present study, the basal short-circuit current and tissue conductance remained unaffected with Cr-CNPs supplementation before the addition of glucose or glutamine, which indicates good preservation and preparation of the tissues [30]. After the addition of glucose to the mucosal side, the change in short-circuit current (ΔISc) decreased linearly in jejunal tissues of Cr-CNPs-supplemented birds, but no effects were observed in ΔISc upon the addition of glutamine to the mucosal side. The decline in ΔISc after glucose addition reflects a decrease in sodium transport across the intestinal membrane. The change in tissue conductance did not vary in tissues of Cr-CNPs-supplemented birds after the addition of glucose or glutamine. To the best of our knowledge, no data are available regarding the effects of the Cr-CNPs on the electrophysiological indices in poultry. Gammelgaard et al. [31] conducted in vitro permeation studies with an Ussing chamber by using pig intestine to compare the absorption of organic and inorganic chromium. They found no response due to adsorption of chromium to the chambers.

The uptake of carbohydrates at the level of the intestine is facilitated by glucose transporters. The GLUT-2 is responsible for the exit of monosaccharides from the enterocytes by facilitated diffusion, while the SGLT-1 mediates the uptake of monosaccharides [32]. The SGLT-1 is expressed in the intestine and kidney [33]. It was reported that glucose absorption was decreased in SGLT-1-deficient mice, which depicts the role of SGLT-1 in maintaining the sodium–glucose homeostasis [15]. In our study, no effect was observed on the expression of GLUT-2 and SGLT-1 with the Cr-CNPs supplementation. Contrary to our study, Orhan et al. [9] reported an increase in the expression of SGLT-1 and GLUT-2 with chromium picolinate and chromium histidinate in layers subjected to heat-stress conditions. The possible reasons could be difference in chromium sources or environmental conditions. In our study, the ΔISc on mucosal addition of glucose decreased linearly with Cr-CNPs supplementation, but gene expression of SGLT-1 remained unaffected. The expression of GLUT-2 is, however, upregulated (*p* > 0.05) with the increase in Cr-CNPs concentration, which might have facilitated transportation of other monosaccharides, including fructose or galactose. However, translation of mRNA to GLUT-2 protein is still debatable and calls for further investigations into the role of Cr-CNPs. It may be due to the lack of consistency between mRNA and protein concentration data [34].

Chromium is a cofactor of glucose tolerance factor and enhances insulin function to increase the cellular uptake of glucose [35]. In our study, the liver glycogen concentration decreased linearly with Cr-CNPs supplementation, but no effect of Cr-CNPs supplementation was found on muscle glycogen concentration. Brooks et al. [1] reported no effects on muscle glycogen or liver glycogen in broilers supplemented with 200, 400, or 800 μg/kg of Cr as chromium propionate. Also, Cr supplementation at 1 mg/kg diet to a low-Cr diet increased liver glycogen synthetase activity but did not affect liver glycogen concentrations in rats [36]. Chromium supplementation also did not affect muscle glycogen in broilers [37], rats [36], humans [38], and sheep [39]. In birds, the liver is the major site of glycogen storage, and liver glycogen is a readily available source of glucose for homeostasis [19]. In our study, the linear decrease in liver glycogen may be the result of glycogenolysis in order to maintain homeostasis that was affected by the linear decrease in glucose absorption from the intestine following Cr-CNPs supplementation.

#### **5. Conclusions**

In conclusion, Cr-CNPs supplementation decreases the absorption of glucose across the jejunal mucosa, with a concomitant decrease in liver glycogen concentration. However, it does not affect the expression of glucose transporters. Further insights are required to explore the effect of Cr-CNPs on the pathways involved in glucose metabolism and transportation.

**Author Contributions:** Conceptualization, S.K.T., M.S.Y., and H.R.; data curation, S.K.T., M.S.Y., and M.R.; formal analysis, S.K.T., M.S.Y., S.A., M.K.S., A.F.K., A.K., and I.R.; funding acquisition, H.R.; methodology, S.K.T., M.S.Y., K.A.M., H.Z., and H.R.; supervision, M.S.Y. and H.R.; writing—original draft, S.K.T.; writing—review and editing, M.S.Y. and H.R.

**Funding:** This study is funded by the Pakistan Agricultural Research Council (PARC), Islamabad, Pakistan, under Agricultural Linkages Program with Project No. AS-091.

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

#### **References**


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

## **E**ff**ects of Probiotics as Antibiotics Substitutes on Growth Performance, Serum Biochemical Parameters, Intestinal Morphology, and Barrier Function of Broilers**

#### **Tengfei He, Shenfei Long, Shad Mahfuz, Di Wu, Xi Wang, Xiaoman Wei and Xiangshu Piao \***

State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; hetengfei@cau.edu.cn (T.H.); longshenfei@cau.edu.cn (S.L.); shadmahfuz@yahoo.com (S.M.); superwudee@163.com (D.W.); wang\_xi\_1998@163.com (X.W.); 13031901582@163.com (X.W.)

**\*** Correspondence: piaoxsh@cau.edu.cn; Tel.: +86-10-6273-3588; Fax: +86-10-6273-3688

Received: 21 October 2019; Accepted: 13 November 2019; Published: 18 November 2019

**Simple Summary:** The abuse of antibiotics in animals feed may cause antibiotic-resistant microbes and antibiotic residue in animal products. Probiotics (PB) have been used in the feed industry for several decades due to their beneficial effects on immunity and the growth of livestock and poultry. However, the efficiency of PB on animals varies due to the types and dose of PB. Therefore, investigating the effects of PB (*Bacillus subtilis*, *Bacillus licheniformis*, and *Saccharomyces cerevisiae*) as an antibiotic substitute on growth performance and intestinal health status in broilers is valuable and meaningful.

**Abstract:** The aim of this study was to investigate the effects of the combination of probiotics replacing antibiotics on growth performance, serum biochemical parameters, intestinal morphology, and expression of tight junction proteins in intestinal mucosa of broilers. A total of 168 Arbor Acres broilers (45.04 ± 0.92 g) were randomly divided into three treatments, with seven replicates per treatment, and eight broilers per replicate. The experiment included phases 1 (d 0 to 21) and 2 (d 21 to 42). The dietary treatments contained a corn soybean meal-based diet (control group; CON); an antibiotic group (basal diet + 75 mg/kg chlortetracycline; CTC), and a probiotics group (basal diet + probiotics (500 mg/kg in phase 1 and 300 mg/kg in phase 2; *Bacillus subtilis* 5 <sup>×</sup> 109 CFU/g, *Bacillus licheniformis* 2.5 <sup>×</sup> 1010 CFU/g and *Saccharomyces cerevisiae* 1 <sup>×</sup> 109 CFU/g; PB). The results showed broilers fed PB had improved (*p* < 0.05) feed conversion ratio (FCR) in phase 1 and increased (*p* < 0.05) average daily gain (ADG) in phase 2, as well as improved (*p* < 0.05) ADG and FCR overall (d 0 to 42). The apparent total tract digestibility (ATTD) of dry matter, organic matter, gross energy, and crude protein was increased (*p* < 0.05) in broilers fed PB, while the ATTD of dry matter and organic matter was enhanced in broilers fed CTC compared with CON. Broilers fed PB showed increased (*p* < 0.05) serum total antioxidant capacity concentrations and tended to have higher (*p* = 0.06) level of serum immunoglobulin M in phase 1 compared with CON. These broilers also had increased (*p* < 0.05) level of serum immunoglobulin A in phase 2 in comparison with CON and CTC. Moreover, broilers fed CTC and PB showed increased (*p* = 0.05) villus height to crypt depth ratio in duodenum, as well as higher (*p* < 0.05) mRNA expression of zonula occludens-1 in jejunum compared with CON. In conclusion, dietary supplementation with PB as chlortetracycline substitute could improve the growth performance, nutrient digestibility, serum antioxidant capacity, jejunal mucosal barrier function, and intestinal morphology of broilers.

**Keywords:** antibiotics; broiler; growth performance; intestinal health; probiotics

#### **1. Introduction**

The wide application of antibiotics has greatly improved the growth performance of livestock and poultry, whereas the abuse of antibiotics in animal feeds may cause antibiotic residue in animal products and the direct selection of antibiotic-resistant microbes, which may cause harm in humans [1]. Broilers, which are one of the fastest growing applications of animal husbandry, face significant problems that impact their growth performance and intestinal health [2]. Therefore, seeking alternatives for in-feed antibiotics for broilers has gained enormous interest currently.

Studies show that herb extracts [3], essential oils [4], and probiotics (PB) [5] could be used as antibiotic substitutes in animals. Among these, PB have been used in feed processing for decades due to their beneficial effects on immune function and growth rate, as well as their low production cost [2]. *Bacillus licheniformis*, which is generally recognized as safe, has been extensively used for a long time in the poultry industry and has demonstrated a positive effect in aiding nutrient digestion and absorption in the host's body [6,7]. In addition, research has proved that *Bacillus subtilis* improves broiler growth and performance equally as well as antibiotics such as bacitracin methylene disalicylate and avilamycin, and supplementation of *Bacillus subtilis* not only improves broiler performance but also positively impacts villi histomorphometry [8]. These bacteria can also produce digestive enzymes, such as protease, amylase, and lipase, and promote the digestion and absorption of nutrients. Bacterial components, such as cell wall sugar and peptidoglycan, can also promote the growth and development of immune organs in poultry [2]. *Saccharomyces cerevisiae* is a type of anaerobic bacteria, which is rich in protein, nucleic acid, vitamins, polysaccharides, and other nutrients, and its cell wall has a special spatial structure, which can reduce the toxicity of mycotoxins in animals [2]. However, less is known about the effect of the mixture of these three probiotics (*Bacillus subtilis*, *Bacillus licheniformis*, and *Saccharomyces cerevisiae*) on ameliorating impairment of growth performance and intestinal health in broilers.

Therefore, the aim of this study was to explore the effect of dietary inclusion of *Bacillus subtilis, Bacillus licheniformis*, and *Saccharomyces cerevisiae* in broiler diets, on growth performance, nutrient digestibility, serum immunoglobulin, antioxidant function, intestinal barrier function, and intestinal morphology.

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

The experimental protocols used in this experiment were approved by the Institutional Animal Care and Use Committee of China Agricultural University (Beijing, China) (No. AW09089102-1). The experiment was carried out at the National Feed Engineering Technology Research Center of the Ministry of Agriculture Feed Industry Center Animal Farm (Hebei, China).

#### *2.1. Experimental Products*

The main components of the PB were *Bacillus subtilis* 5 <sup>×</sup> 109 CFU/g, *Bacillus licheniformis* 2.5 <sup>×</sup> <sup>10</sup><sup>10</sup> CFU/g, and *Saccharomyces cerevisiae* <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>9</sup> CFU/g, which were provided by Beijing Smistyle Sci. and Tech. Development Co., Ltd.

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

A total of 168 one-day-old as-hatched Arbor Acres chicks (weighing 45.04 ± 0.92 g) were purchased from Arbor Acres Poultry Breeding Company (Beijing, China). All the broilers were randomly divided into 3 treatments, 7 replicates per treatment, and 8 chickens per replicate. The trial was divided into two phases: phase 1 (day 0 to 21) and 2 (day 21 to 42). The test period was 42 days. The dietary treatments contained a corn soybean meal-based diet (control group, CON); an antibiotic group (basal diet + 75 mg/kg chlortetracycline, CTC), and a probiotics group (basal diet + probiotics (500 mg/kg in phase 1 and 300 mg/kg in phase 2; PB). The feed formulation was based on National Research Council (NRC, 1994) [9] and the formulation is shown in Table 1.


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

1. Vitamin A, 11,000 IU; vitamin D, 3025 IU; vitamin E, 22 mg; vitamin K3, 2.2 mg; thiamine, 1.65 mg; riboflavin, 6.6 mg; pyridoxine, 3.3 mg; cobalamin, 17.6 μg; nicotinic acid, 22 mg; pantothenic acid, 13.2 mg; folic acid, 0.33 mg; biotin, 88 μg; choline chloride, 500 mg; iron, 48 mg; zinc, 96.6 mg; manganese, 101.76 mg; copper, 10 mg; selenium, 0.05 mg; iodine, 0.96 mg; cobalt, 0.3 mg. 2. Crude protein was the analyzed value. Other values were calculated.

#### *2.3. Detection Index and Measuring Method*

#### 2.3.1. Growth Performance

The body weight and feed intake of the broilers were registered on day 0, 21, and 42, and the average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated.

#### 2.3.2. Nutrient Retention

At day 39–42, 200 g of the excreta sample was collected for each replicate, feathers and the like in the excreta were removed, and the sample was then oven dried at 65 ◦C for 72 h. All samples were ground to pass through a 1 mm screen (40 mesh) before analysis. Feed or fecal samples were analyzed for dry matter (DM), crude protein (CP), crude fat (EE), and ash according to Association of Official Agricultural Chemists (AOAC, 2012) [10]. The gross energy (GE) in feed and fecal samples were determined by an automatic isoperibol oxygen bomb calorimeter (Parr 1281, Automatic Energy Analyzer; Moline, IL, USA). Organic matter (OM) was calculated as 1 − ash content (DM-base). Nutrient retention was determined by the equation as follows: Apparent total tract digestibilitynutrient (ATTD) = 1 − (Crdiet × Nutrientfeces)/(Crfeces × Nutrientdiet).

#### 2.3.3. Serum Antioxidant and Immune Function

At day 21 and 42, one broiler chicken with a body weight close to the average was selected for each replicate. A quantity of 4 mL of blood was collected from the wing vein and centrifuged at 3000 r/min for 10 min, and the supernatant was dispensed into a 0.5 mL Eppendorf tube and stored at −80 ◦C. The contents of serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were determined by spectrophotometric methods using a spectrophotometer (Leng Guang SFZ1606017568, Shanghai, China) following the instructions provided by manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The contents of serum malondialdehyde (MDA) were determined using kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The contents of serum immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) were measured by an ELISA kit (IgG, IgM, and IgA quantitation kit; Bethyl Laboratories, Inc., Montgomery, TX, USA).

#### 2.3.4. Intestinal Morphology

On day 42 of this experiment, two broilers were slaughtered from each replicate. The abdominal cavity was dissected and the intestine was separated. Segments of the mid-duodenum, mid-jejunum, and mid-ileum were taken and rinsed with cold physiological saline (0.9% saline), then immediately stored in 10% buffered formalin. Conventional paraffin embedding, sectioning, HE staining, and six straight and complete fluffs were selected for each section, and the height of the villi and the depth of the crypt corresponding to the villi were determined. The height of random orientated villi and their adjoined crypts were determined with a light microscope using a calibrated eyepiece graticule [11].

#### 2.3.5. The Level of Claudin-1, Occludin and ZO-1 Gene Expression in Jejunal Mucosa

On day 42 of this experiment, the jejunal mucosa was taken from the broilers and then stored in liquid nitrogen. Total RNA extraction was done using Trizol Reagent (TaKaRa, Dalian, China), and the purity and concentration of total RNA were measured by ultraviolet spectrophotometer. Total RNA (1μg) was reverse-transcribed into cDNA using Prime Script RT Reagent Kit (TaKaRa, Dalian, China) according to the direction of the manufacturer's protocol. The primers were synthesized by TaKaRa Biotechnology (TaKaRa, Dalian, China), which were obtained from the published works of Shao et al and Li et al [12,13], and are shown in Table 2. Real-time PCR was conducted according to Li et al [14].


**Table 2.** Sequences of the primers used for the determination of gene expression levels.

ZO-1: zona occludens-1.

#### *2.4. Statistical Methods*

Data was subjected to Analysis of variance (ANOVA) using the GLM procedure of SAS (SAS Institute, 2008) [15]. The replicate was the experimental unit. Significantly different means were separated by Duncan's multiple range test. Results were expressed as least squares means and SEM. Significance was designated at *p* ≤ 0.05, while a tendency for significance was designated at 0.05 < *p* ≤ 0.10.

#### **3. Results**

#### *3.1. Growth Performance*

As can be seen from Table 3, dietary supplementation with CTC and PB had no significant effect on the ADFI of broilers compared with CON. In phase 1, broilers fed PB showed improved FCR compared with CON and CTC (*p* < 0.05). In phase 2, broilers fed PB showed improved ADG in comparison with CON (*p* < 0.05) and had no significant difference with CTC. Overall (day 0 to 42), broilers fed PB had improved ADG and FCR compared with CON (*p* < 0.05) and enhanced ADG compared with CTC (*p* < 0.05).


**Table 3.** Effects of probiotics on growth performance of broilers 1.

<sup>1</sup> CON, control; CTC, chlorotetracycline (75 mg/kg); PB, probiotic (500 mg/kg in phase 1, 300 mg/kg in phase 2); SEM, standard error of the mean. <sup>2</sup> Treatment, the specific *p* value of the diet effect in the ANOVAs analysis. a,b values in the same row with different letters are significantly different at *p* < 0.05.

#### *3.2. The ATTD of Nutrients*

The effects of PB on the ATTD of nutrients in broilers are shown in Table 4. Compared with CON, the DM and OM were increased (*p* < 0.05) in broilers fed PB and CTC. In addition, broilers fed PB also showed enhanced (*p* < 0.05) GE and CP compared with CON.


**Table 4.** Effects of probiotics on the nutrient retention of broilers (%, day 42) 1.

<sup>1</sup> CON, control; CTC, chlorotetracycline (75 mg/kg); PB, probiotic (500 mg/kg in phase 1, 300 mg/kg in phase 2); SEM, standard error of the mean. <sup>2</sup> Treatment, the specific *p* value of the diet effect in the ANOVAs analysis. a,b values in the same row with different letters are significantly different at *p* < 0.05.

#### *3.3. Serum Antioxidant Status*

The effects of PB on the antioxidant status of broilers are shown in Table 5. Compared with CON, broilers fed CTC and PB showed increased serum T-AOC concentration in phase 1 (*p* < 0.05). There was a tendency of enhancing concentration of SOD (*p* = 0.06), GSH-Px (*p* = 0.06), and reducing (*p* = 0.07) level of MDA in serum of broilers fed PB compared with CON in phase 1. In phase 2, broilers fed PB had higher (*p* < 0.05) concentration of GSH-Px and lower (*p* < 0.05) level of MDA in serum in comparison with CON.


**Table 5.** Effects of probiotics on the serum antioxidant function of broilers 1.

<sup>1</sup> CON, control; CTC, chlorotetracycline (75 mg/kg); PB, probiotic (500 mg/kg in phase 1, 300 mg/kg in phase 2); SEM, standard error of the mean. <sup>2</sup> Treatment, the specific *p* value of the diet effect in the ANOVAs analysis. a,b values in the same row with different letters are significantly different at *p* < 0.05.

#### *3.4. Serum Immunoglobulins*

The effects of PB on serum immunoglobulins of broilers are shown in Table 6. In phase 1, broilers fed PB (*p* = 0.07) and CON (*p* = 0.06) tended to show enhanced level of IgM compared with CTC, while broilers fed PB increased (*p* < 0.05) level of IgA in phase 2 in comparison with CTC and CON.


**Table 6.** Effects of probiotics on the serum immunoglobulins function of broilers (ug/mL) 1.

<sup>1</sup> CON, control; CTC, chlorotetracycline (75 mg/kg); PB, probiotic (500 mg/kg in phase 1, 300 mg/kg in phase 2); SEM, standard error of the mean. <sup>2</sup> Treatment, the specific *p* value of the diet effect in the ANOVAs analysis. a,b values in the same row with different letters are significantly different at *p* < 0.05.

#### *3.5. Intestinal Morphology*

The effects of PB on the intestinal morphology of broilers are shown in Table 7. Compared with CON, the duodenal villus height to crypt depth ratio was significantly increased (*p* < 0.05) in broilers fed CTC and PB. In addition, these broilers tended to showed lower crypt depth in duodenum, as well as higher villus height to crypt depth ratio in ileum compared with CON.


**Table 7.** Effects of probiotics on intestinal morphology of broilers (day 42) 1.

<sup>1</sup> CON, control; CTC, chlorotetracycline (75 mg/kg); PB, probiotic (500 mg/kg in phase 1, 300 mg/kg in phase 2); SEM, standard error of the mean. <sup>2</sup> Treatment, the specific *p* value of the diet effect in the ANOVAs analysis. a,b values in the same row with different letters are significantly different at *p* < 0.05.

#### *3.6. Jejunal Mucosal Barrier Functions*

The effects of PB on jejunal mucosal barrier function of broilers are shown in Table 8. Compared with CON, broilers fed CTC and PB showed higher gene expression of zonula occludens-1 (ZO-1) in jejunum (*p* < 0.01), and had no significant effect on the gene expression of claudin-1 and occludin in jejunum.

**Table 8.** Effects of probiotics on gene expression levels of claudin-1, occludin and ZO-1 genes in the jejunum barrier function of broilers (day 42) 1.


<sup>1</sup> CON, control; CTC, chlorotetracycline (75 mg/kg); PB, probiotic (500 mg/kg in phase 1, 300 mg/kg in phase 2); SEM, standard error of the mean. <sup>2</sup> Treatment, the specific *p* value of the diet effect in the ANOVAs analysis. a,b values in the same row with different letters are significantly different at *p* < 0.05.

#### **4. Discussion**

The current study showed that broilers fed PB improved ADG in phase 2, and ADG and FCR overall. Our results are consistent with the study of Kalia et al. [16], who reported that a diet supplemented with mixed PB could improve the body weight gain and feed efficiency, and decrease mortality in broilers. However, studies conducted by Ahmad et al. [17] and Fathi et al. [18] showed PB had no significant effects on improving FCR. This difference might be due to the variation of survivability of PB in the intestine of broilers and the dose rate of PB used for broilers. The possible reason for the current positive effect on performance could be explained by the *Bacillus subtilis* in PB improving the immune response [19] and the positive effect of PB on modulating the microbiota structure (such as reducing the content of *Salmonella Enteritidis*) [20]. The improvement of performance might also be due to PB increasing nutrient retention (GE, CP, DM, and OM). Research has shown that PB is able to improve the activity of digestive enzymes of animals [21]. Moreover, dietary PB supplementation could produce some metabolites, including organic acids, to enhance the nutrient retention in broilers [22]. The current study showed dietary inclusion of PB, namely, *Bacillus subtilis*, *Bacillus licheniformis*, and *Saccharomyces cerevisiae*, has the same effects as CTC in improving growth performance, which indicates that PB could be a potential antibiotics substitute.

Current research indicates that addition of PB had a positive role on antioxidant functions in broilers. In agreement with our results, Capcarova et al. and Wen et al. [23,24] found that some probiotics could be beneficial in oxidation resistance, scavenging reactive oxygen species, and promoting antioxidant capability. With regard to antioxidant capacity, the endogenous antioxidant defense system in animals also relies on other external sources, such as probiotics, which are the natural source for prevention of the oxidative stress induced by reactive oxygen species [25]. Collectively, this study suggested that PB can possess antioxidant capacity in broilers.

The current study also showed dietary PB supplementation had a positive effect on serum immunoglobulin, which is in agreement with Fathi et al. [18], who reported improving effects of PB on IgM and cell-mediated immunity. The reason may be that *Bacillus subtilis* had a positive effect on enhancing antibodies against the Newcastle disease of broiler chicks [19]. PB *Bacillus subtilis* could also enhance humoral immune responses and stimulate the host's mucosal immune system by interacting with intestinal epithelial cells in broilers [26]. The mechanism of PB on the immunity of broilers may also result because PB can protect animals from pathogen colonization by competing for epithelial binding sites and nutrients, strengthening the intestinal immune response, and producing antimicrobial bacteriocins. [22]

The current study showed that dietary PB supplementation can increase the ratio of villus height to crypt depth, which indicates that PB can promote the development of the absorptive surface of duodenum and ileum in broilers. This might be due to the beneficial bacteria in PB, which may improve crypt cell proliferation in the small intestine, and thus help increase the growth rate in broilers [17]. In addition, the *Bacillus licheniformis* in PB can colonize and form niches in the small intestine, which positively protects the villi from pathogens and improves the growth of villi [27]. However, Sohail et al. [28] found that PB had no effect on stress-induced injury in the intestinal morphology of 42-day-old chickens, which might be due to the variation of types and amounts of PB used in different studies. Moreover, the improvement of intestinal morphology and integrated intestinal barrier are important for epithelial cell function, which might be the reason for the improved ATTD of nutrients [29].

The function of the intestinal barrier and the absorption of nutrients can be directly affected by the damage of the mucosal epithelium, and PB can regulate intestinal immunity and tight junction protein mRNA expression of broilers [30]. Current research indicates that the addition of PB to diets can promote the gene expression of ZO-1 in jejunal mucosa of broilers and improve the jejunal mucosal barrier function of broilers. PB in diets can decrease the feed weight gain ratio and intestinal coliform, and can also increase the duodenal villus height to crypt depth ratio. These results suggest that the supplementation of a PB mixture in the diet can effectively improve part of the intestinal barrier function. PB has been shown to be adherent to the intestinal epithelium, resistant to acidic conditions, and capable of antagonizing and competitively eliminating certain pathogens in vivo [31]. In contrast, the PB mixture used in this study consisted of *Bacillus licheniformis*, *Bacillus subtilis*, and *Saccharomyces cerevisiae*. *Bacillus licheniformis* and *Bacillus subtilis* are aerobic bacteria that use oxygen in the intestine to provide an anaerobic environment for the colonization of anaerobic bacteria, such as *Lactobacilli* and *Bifidobacteria*. Therefore, these lactic acid-producing bacteria produce a more acidic environment, which impairs the growth of opportunistic pathogens [32].

#### **5. Conclusions**

The results of this experiment showed that the addition of probiotics (500 mg/kg in phase 1, 300 mg/kg in phase 2) could improve broilers' growth performance, nutrient retention, and serum antioxidant capacity, and improve their intestinal health via improving jejunal mucosal barrier function and intestinal morphology. The results indicated that the current probiotics could be used as a chlortetracycline substitute in the diet of broiler chickens.

**Author Contributions:** Conceptualization, X.P.; Data curation, T.H.; Formal analysis, S.L. and X.W. (Xiaoman Wei); Investigation, X.P.; Project administration, S.M. and D.W.; Software, X.W. (Xi Wang); Writing—original draft, T.H. and S.L.; Writing—review & editing, T.H.

**Funding:** This research is funded by the National Natural Science Foundation of China (31772612) and CARS 35.

**Acknowledgments:** We want to thank the support by the National Natural Science Foundation of China (31772612), CARS 35 as well as Beijing Smistyle Sci. and Tech. Development Co., Ltd.

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

#### **References**


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

### *Review* **Use of Medicinal Mushrooms in Layer Ration**

#### **Shad Mahfuz and Xiangshu Piao \***

State Key laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China; shadmahfuz@yahoo.com

**\*** Correspondence: piaoxsh@cau.edu.cn; Tel.: +86-10-6273-3588; Fax: +86-10-6273-3688

Received: 17 October 2019; Accepted: 12 November 2019; Published: 21 November 2019

**Simple Summary:** The extensive use of antibiotics in the poultry industry to increase production performance has led to human health hazards. The use of natural herbs as antibiotic substitutes has been reported in the poultry feed industry. Therefore, the objective of this review was to determine the effect of different levels of mushrooms and their extract in diet on laying performance and health status. On the basis of previous findings, dietary supplementation using mushrooms as a natural feed supplement sustained laying performance and improved immunity in laying hens.

**Abstract:** Application of different medicinal mushrooms intended to enhance production performance and health status has created an importance demand in poultry production. One goal of using medicinal mushrooms is to get rid of antibiotics in poultry feed without affecting the optimum performance. Increasing concerns about this issue have led to more attention on antibiotic substitutes and a significant demand for them for organic egg production. Thus, supplementation with medicinal mushrooms is a new concept for research in layer production, however, there is still a great deal of confusion about inclusion levels and the mode of action of medicinal mushrooms on production performance and health status in laying hens. Taking this into account, this review outlines the experimental uses of medicinal fungi on the growth performance, laying performance, egg quality, and health status of layer birds based on previous findings to date. Finally, we highlight that supplementation with medicinal fungi can play a role on the immunity, health, and production performance in laying hens.

**Keywords:** medicinal mushrooms; laying hens; health status; performance

#### **1. Introduction**

Traditionally, mushrooms have been used for highly valued food and pharmaceutical purposes because of their role as a tonic and their benefit to health [1]. Cultivated edible mushrooms are good sources of protein, have low-fat content, and are cholesterol free [2]. Mushrooms are also very popular as a quality protein containing essential amino acids, adequate vitamins, minerals, and are rich source of different unsaturated fatty acids [3]. Different bioactive components have been extracted from the fruiting body and mycelium part of mushroom and tested in invitro studies. Polysaccharides are considered to be the most activate component in mushrooms which have immune stimulating activities [3]. In addition, the polysaccharides in mushroom have been found to produce different cytokines and increase the weight of immune stimulating organs in laboratory animals [4,5]. Presently, researchers have become interested in the role of medicinal mushrooms in poultry production systems.

Antibiotics as feed additives have been used as growth and health promoters in poultry production [6], however, because of the appearance of microorganisms that are resistant to specific antibiotics, the application of antibiotics in poultry ration has been forbidden or restricted in the developed countries [7,8]. As a result, exploring new growth-promoting alternatives to antibiotics has become a hot topic of research for several years [9]. Chickens are very sensitive to immunosuppressive

stressors and infectious diseases [10]. Different infections are responsible for reduction in growth rates, poor egg production, and mortality, which have resulted in huge economic losses in the poultry industry. There is a direct relationship between feeding and the immune system of the host [11]. Various attempts, through genetic manipulation, dietary alterations, various medicinal supplements, etc. have been tried to reduce the cholesterol content in meat and eggs, and therefore improve their health status [12].

At present, there are several scientific works about the health promoting benefits of involving mushrooms in farm animals. Currently, poultry researchers are committed to using unconventional natural feed supplement as a substitute for antibiotics that have been proven as possible ways to enhance the health and to improve the production in poultry. Although it is known that mushrooms are medicinally importance for chickens health, unfortunately, the inclusion level of mushrooms in poultry diets is still under consideration. Findings from past reports have highlighted that their inclusion may enhance production performance and health in laying hens [13]. Taking this into consideration, this review is focused on the importance of the medicinal mushrooms as an alternative for antibiotics that can improve the performance and the immunity in laying hens.

#### **2. Common Medicinal Mushrooms Used in Layer Study**

A group of mushrooms have been identified as medicinal mushrooms, in recent years, due to their biological properties both invivo and invitro studies. The phylum Basidiomycota is the most predominant among the mushrooms species that has been proven to be a medicinal mushroom [14]. On the basis of some previous studies, we have identified some common medicinal mushrooms that can be used as a source of active substances for optimum performance and health status in layer chickens. A list of major medicinal mushrooms that were used in poultry ration during the previous years is presented in Table 1 and Figure 1.

**Figure 1.** Photographs of different medicinal mushrooms: (**a**) Flammulina velutipes, (**b**) *Hericium erinaceus*, (**c**) Agaricus brasiliensis, (**d**) Pleurotus ostreatus, (**e**) *Lentinula edodes*, (**f**) Ganoderma lucidum, (**g**) *Cordyceps sinensis*, and (**h**) Tremella fuciformis.


**Table 1.** Botanical classification and distribution of medicinal mushrooms used in poultry ration 1.

<sup>1</sup> K, kingdom; P, phylum; C, class; O, order; F, family; G, genus; and Sp, species.

#### **3. Biological Role of Medicinal Mushrooms**

Mushrooms have been reported to have many useful functions including antitumor, anticancer, antihypertensive, cholesterol lowering effect, antioxidant properties, anti-inflammatory, immune-modulatory function, as well as anti-bacterial, antiviral, and antifungal activities on human and animal health [15,16].

#### *3.1. Antitumor Activities*

The shiitake mushroom (*Lentinus edodes*) is rich in antitumor agents which play a role inhibiting cancer cell growth [17]. Aqueous extracts from the vegetative submerged mycelia of cultivated *Ganoderma lucidu*, and *Lentinus edodes* have been reported to have antitumor activities [18]. *F. velutipes* mushrooms have been reported to hold bioactive compound having antitumor functions [19]. The extract of *F. velutipes* mushroom has been used to oppose breast cancer cells [20]. Recently, significant novel components with anticancer function were discovered in *F. velutipes* by Chinese researchers. These researchers discovered a sesquiterpene, which is known as flammulinol A, along with other flammulinolides A–G derived from *F. velutipes* mushroom that were effective against several cancer cell lines [21]. A recent study by Dong et al. [22] reported that polysaccharide, purified from *Ganoderma applanatum* mushroom, was effective against human breast cancer in an invitro study.

#### *3.2. Antioxidant Activities*

Today, the antioxidant properties of different medicinal mushrooms are well-known. Some previous studies have reported that the polysaccharides and oligosaccharide present in medicinal mushrooms show antioxidant functions [23]. Conventional uses of butylated hydroxyanisole and butylated hydroxytoluene as synthetic antioxidants can be hazardous to humans, and therefore there is a need to discover natural antioxidant products [24]. Tang et al. [3] stated that the phenolic ingredients present in mushrooms may have the capacity to withdraw the oxidation of the LDL for their anti-inflammatory activities. A fibrinolytic enzyme that was successfully purified and derived from the culture supernatant of needle mushroom was reported by Park et al. [25]. The antioxidant activities depend on different parts and varieties of mushrooms. Zeng et al. [26] stated that *F. velutipes* mushroom hold a higher phenolic amount with the highest antioxidant activities. Different mushrooms were found to exhibit vitaminC and selenium that can play a role in antioxidant functions [14]. A recent study by Lin et al. [27] found that the *Cordyceps sobolifera* (Ascomycetes) mushroom exhibits antioxidant properties as a functional food and dietary supplement. In addition, *Agaricus brasiliensis* are considered potential auxiliaries for the treatment of patients with rheumatoid arthritis due to their capacity to reduce oxidative stress [28]. The anti-inflammatory and antioxidant properties of *A. bisporus* biomass extracts from an in vitro culture were reported by Muszynska et al. [29]. In their studies, incubation of Caco-2 cells with *A. bisporus* extracts resulted in decreased expression of cyclooxygenase-2 and prostaglandin F2α receptor as compared with the lipopolysaccharide (LPS) or TNF-α-activated cells. The antioxidant activity of *Pleurotus ostreatoroseus* (Agaricomycetes) mushroom was also noted by Brugnari et al. [16].

#### *3.3. Lipid Metabolism Activities*

The positive role of golden needle mushroom on lipid metabolism in male hamsters was reported by Yeh et al. [30]. Their study showed that both the extract and the powder originating from needle mushroom were capable of reducing serum and liver tissue cholesterol level in hamsters. Another study by Yang et al. [31] found a lower level of plasma triglyceride, total cholesterol (TC), and low-density lipoprotein cholesterol in diet-induced hyperlipidemic rats fed *Hericiumerinaceus* mushroom exo-polymer. Lovastatin, as well as γ-aminobutyric acid (GABA), were identified from *F. velutipes* fruiting bodies [32]. Lovastatin is used to reduce cholesterol production that can diminish risks of heart diseases [32,33]. Another study by Harada et al. [34] reported very effective results

by decreasing the systolic pressure in rats using GABA-mediated *F. velutipes* mushroom powder. *ß*-D-glucan and its derivatives present in medicinal mushrooms ensured their cholesterol lowering effects by reducing the absorption or increasing the faecal excretion [35]. The oyster mushroom is also famous for its cholesterol reducing functions [36].

#### *3.4. Antimicrobial Activities*

The antimicrobial properties of medicinal mushrooms are well established. The extracts derived from medicinal *Pleurotus* species mushroom have been reported to have potential antibacterial and antifungal functions [37,38]. An invitro experiment was conducted by Sknepnek et al. [39] with reishi mushroom (*Ganoderma lucidum*) on antimicrobial functions. Their studies concluded that the liquid *Ganoderma lucidum* mushroom beverage at a 0.04 mg/mL concentration was very useful against *Staphylococcus epidermidis* and *Rhodococcus equi*. In addition, it was very useful against *Bacillus spizizenii, B. cereus,* and *R. equi* at a 0.16 mg/mL concentration. Nedelkoska et al. [40] reported that the mushroom fruiting body was very effective against different bacteria. Kashina et al. [41] stated that the mushroom, *F. velutipes,* exhibited inhibitory activities in opposition to two different harmful fungi (*Sporothrix schenckii* and *Candida albicans*). Enokipodins have been found in the needle mushroom that has antimicrobial functions [42].

#### *3.5. Immune Functions*

The immune functions of mushrooms are well known. Different protein and various peptides present in mushrooms are able to modify the immune response positively [14]. Invitro immune-modulatory studies with *F velutipes* showed that raw 264.7 cells were stimulated to secret nitric oxide upon administration of 200 to 500 μg/mL *F.velutipes* polysaccharide (FVPA2). The FVPA2 also encouraged the proliferation of the spleen lymphocytes and B lymphocytes in experimental mice [43]. Manayi et al. [44] used the extract of *Ganoderma applanatum* mushroom at a concentration of 1000 mg/kg diet on the defense mechanisms in rainbow trout. This study found the potential ability of *G. applanatum* mushroom extract to activate immunologic parameters in rainbow trout. Lee et al. [45] found that the mushroom could increase the concentration of IFNγ that has a toxic function against lymphoma cell. The polysaccharides of needle mushroom were found to produce different cytokines and increase the weight of immune stimulating organs in laboratory animals [4,5]. The mushroom polysaccharides increased the body weight of experimental mice and the weight ratio of the thymus and spleen, as well as it could modulate the T cell subpopulation of thymocytes and splenocytes [30]. Moreover, the polysaccharides of mushroom increased NO (nitric oxide), TNF-a, IL-1b, and IL-6 production, and lymphocyte proliferation in mice model [46].

#### *3.6. Nutritional Roles*

Mushrooms are very popular for their nutritional values. Mushrooms have been reported as a good source of six major nutrients which include carbohydrates, especially dietary fiber, proteins, vitamins, minerals, lipids, and water. Rich in proteins, carbohydrates, and fiber with low fat are the unique features of the medicinal mushroom. In addition, different types of essential amino acids (AA) have been found in mushroom [47–49]. The nutritional component of different mushroom showed dry matter (DM) 74% to 89.6%; crude protein (CP) 8.9% to 14.8%; carbohydrate 43.33% to 69.40%; total detergent fiber (TDF) 1.9% to 7.40%; crude fat (EE) 1.75% to 3.91%; ash (total mineral) 4.91% to 8.40%; calcium (Ca) 2.21% to 3.05%, and phosphorus (P) 1.68% to 1.88% [3,50,51].

#### **4. Medicinal Mushrooms in Layer Chicken Ration**

The data regarding the role of medicinal mushrooms in layer chicken performance are summarized in Table 2. Hence, there has been considerable debate regarding current findings on laying hens' performance, as well as many variables that have been associated with the current findings such as mushroom species, use dosage, method of application (either non-fermented or fermented with

beneficial organisms), part of the mushroom (either fruiting bodies or stem base), and the treatment period. However, collectively, many scientists agreed that mushrooms could have a positive role by improving the laying percent, table egg quality, egg yolk cholesterol level, as well as immunity in laying hens. Further studies are needed to detect the actual dose for optimum performance in layer chickens.


<sup>1</sup> ND, Newcastle disease; IB, infectious bronchitis (IB); AI, Avian influenza; Ig, immunoglobulin; IL, interleukin; n-6, omega-6 fatty acid; and FCR, feed conversion ratio.

#### *4.1. Application of Medicinal Mushrooms on Performance and Egg Quality*

There have been limited studies conducted, in previous years, to evaluate the effects of medicinal mushrooms in laying hens. Mahfuz et al. [52] conducted a study to examine the role of *Fammulina velutipes* mushroom stem wastes (FVW) on growth performance, and immunity in pullet birds on basic of different levels (2%, 4%, and 6%). They found that the final live weight was greater (*p* < 0.05) in mushroom fed groups at all levels (2%, 4%, and 6%) than that of the control and antibiotics diets. No differences (*p* > 0.05) were found for the average daily feed intake, average daily weight gain, and feed conversion ratio (FCR) among treatments. Dry matter (DM), crude protein (CP), and ether extract (EE) retention were higher (*p* < 0.05) in FVW diets than the control and antibiotic diets. Excreta DM was higher (*p* < 0.05) and pH was lower (*p* < 0.05) in FVW diets than the control and antibiotic

diets. The higher body weight, in this study, must be related to higher nutrient retention in mushroom supplemented groups. In addition, the Excreta DM was higher in the mushroom supplemented groups which suggests that incorporated FVW reduced excreta moisture, which can prevent the wet litter in poultry house, as well as increase the absorption of nutrients and reduce the ammonia gas production from excreta in chicken house. Teye et al. [58] stated that high moisture content, high temperatures, and high pH can facilitate the production of ammonia from excreta. Mahfuz et al. [53], consequently, assessed the role of *Flammulina velutipes* in laying hens ration with different levels of mushrooms (2%, 4%, and 6%) in experimental diets and did not find any differences (*p* > 0.05) in laying performance parameters such as average daily egg production percentage, egg mass, FCR, etc., in laying hens. The number of unmarketable table eggs was fewer (*p* < 0.05) in mushroom fed diets as compared with the control diets. This study also found suitability for calcium retention in eggshells with FVW diets as compared with the control and the antibiotic diets. It was hypothesized that the higher calcium retention could be related to a higher number of marketable eggs in mushroom fed groups. No effects on egg production percentage, egg mass, and FCR ensured the fact that feeding mushroom did not have any adverse effects on laying performance. Lee et al. [13] found that feeding *F. velutipes* mycelium had no adverse effects on egg production percentage, feed intake, and FCR, in laying hens, but the egg weight was found greater (*p* < 0.05) in the 1% and 3% mushroom feeding groups than the control diets. Furthermore, feeding mushroom at the 4% level resulted in significantly higher (*p* < 0.05) egg albumen height, haugh unit, eggshell weight, and shell thickness. It was thought that mushrooms contain higher level of CP that leads to increased egg albumin and might have an effect on shell gland for continuous eggshell formation. On the contrary, Na et al. [59] found that dietary inclusion of mushroom had no effect on eggshell weight, shell thickness, and haugh unit. Finally, the authors suggested that mushroom, as a natural resource of feed for laying hens, can be used at the 5% level without affecting normal performance.

*Lentinula edodes* are commonly known as Shiitake mushroom which has long been considered to be a medicinal mushroom. An experiment was conducted with the shiitake mushroom on laying performance and egg quality by Hwang et al. [54]. Higher (*p* < 0.05) egg production and higher (*p* < 0.05) haugh unit in eggs were reported by feeding shiitake mushroom than that of the control group. However, the other laying parameters including egg weight, shape index, shell thickness, albumen height, yolk color, and the egg sensory evaluation (e.g., appearance, color, flavor, oily nature) were not affected (*p* > 0.05). Egg yolk fatty acids, especially linoleic acid, total omega-6 fatty acid (n-6), and the polyunsaturated fatty acid were found to be higher (*p* < 0.05) in 0.5% mushroom feeding groups than the control group, however, palmitoleic acid and α-linolenic acid were lower (*p* < 0.05) in the 0.5% mushroom feeding group than the control fed group. In addition, the cholesterol concentration of egg yolk was lower (*p* < 0.05) in the 0.5% mushroom fed diet than the control group [54]. Foods rich in total omega-3 fatty acid (n-3), total omega-6 fatty acid (n-6), as well as polyunsaturated fatty acid (PUFA) are very helpful for health. A diet enriched withn-3 PUFA is considered to have preventative functions for people with vascular diseases [60]. Willis et al. [61] reported that adding *Lentinula edodes* mushroom mycelium extract to layer diets had no significant effects on laying performance. In a subsequent study, Willis et al. [62] further investigated that birds fed with fungus myceliated grain could successfully induce molting and allowed egg production earlier than the control group. This study concluded that fungus myceliated meal can be an effective alternative to conventional feed withdrawal methods, for the successful initiation of molt and maintenance of post-molt performance.

The mushroom, *Cordyceps militaris,* was used in layer diets to evaluate the performance and egg yolk cholesterol level by Wang et al. [57]. The results showed significantly lower (*p* < 0.05) egg cholesterol in mushroom fed groups with 1% and 2% levels than the control fed groups. In addition, improved (*p* < 0.05) FCR with greater (*p* < 0.05) egg weight were found at the 2% level mushroom fed group than in the control group. However, no significant differences were observed on the eggshell weight, egg yolk weight, shell thickness, and egg yolk color, among the treatment groups.

Lee et al. [13] used the spent mushroom (*Hypsizygus marmoreus*) substrates in layer ration to evaluate the feeding effects on egg production and table egg quality. None of the production performance parameters were affected by feeding mushroom *Hypsizygus marmoreus* during the entire study period. However, the egg yolk color scores were higher (*p* < 0.05) in mushroom fed groups than the control group. They concluded that fermented spent mushroom can be used, up to15% in layer ration, without affecting normal laying percent and table egg quality.

A wild medicinal mushroom, *Ganoderma lucidum,* was used for pullet performance in a study by Ogbe et al. [55]. No significant effects were observed for the feed intake by feeding *Ganoderma lucidum* mushroom in pullets. However, FCR was improved (*p* < 0.05) in mushroom groups than the non supplemented control group. Lee et al. [56] used *Pleurotus eryngii* mushroom to evaluate the performance in laying hens. Egg cholesterol level was lower (*p* < 0.05) in the mushroom groups than the control diets, however they did not observe any significant differences on egg production performance by feeding mushroom base diets in laying hens. The haugh unit was greater (*p* < 0.05) in the 1% and 2% experimental diet groups. The authors finally concluded that laying hens fed with the residue of *Pleurotus eryngii* mushroom could produce lower cholesterol in eggs.

#### *4.2. Application of Medicinal Mushrooms on Health Status in Layer Chickens*

Body immunity and inner organ weight are good indicator of health status in chickens. No significant difference was found for inner relative organ (liver, gizzard, spleen, and abdominal fat) weights between control and antibiotic fed diets [52], however, the bursa weight was higher (*p* < 0.05) in the mushroom fed diets than the control and antibiotic fed diets [52]. No effects on inner organ weights ensured that feeding mushroom did not have any toxic effects on pullet chickens, whereas the higher bursa weight ensured the improved immune status in experimental chickens. Higher bursa weight is an indicator of better health status and a sound physiological response to body immune system [63]. The appropriate level of immune sub-parameters such as immunoglobulin, cytokines, protein, and some biochemical index are important to maintaining the immune response in host. Antibody titers against Newcastle disease (ND), infectious bronchitis (IB), and Avian influenza (AI) virus vaccines were found to be higher (*p* < 0.05) in mushroom stem waste fed diets in pullet [52]. In addition, serum immunoglobulin parameters (IgA, IgG, and IgM) were found to be higher (*p* < 0.05) in mushroom fed diets than the control and antibiotic fed diets in the experimental pullet. Bai et al. [64] stated that serum immunoglobulin concentrations can generate humoral immune response in animals due to their important roles on immune function fighting against various infections. In addition, supplementation of β-glucan from edible mushroom had a significant immune stimulatory effect in chickens [65]. Similarly, the antibody titers against infectious bursal diseases virus were greater in mushroom *Ganoderma lucidum* fed groups than the control diets, in pullet [55]. In another study by Mahfuz et al. [66] antibody response on ND was greater (*p* < 0.05) in the 6% mushroom stem fed group and IB were greater (*p* < 0.05) in all levels of mushroom fed groups than both the positive and negative control diets, in laying hens. This study further demonstrated that the serum cytokines concentrations (IL-2, IL-6, IL-4, and TNF-α) were higher (*p* < 0.05) in mushroom feed groups than the control and antibiotic fed groups, in laying hens. The polysaccharides in medicinal mushrooms have strong immune modulatory activity and possess antioxidant activity that could enhance nonspecific and specific immune responses invitro [46]. In addition, cytokines are known to be regulators of the immune status. The function of IL-2 relies on the commencement of B and T lymphocytes cells. However, the activation of Th1 depends on secretion of IL-2, TNF-α, along with other cytokines that create the cellular immunity [67]. Similarly, Jarosz et al. [68] reported that the function of Th2 depends on IL-4, with other cytokines secretion that stimulates humoral immunity.

Lee et al. [13] found that the number of pathogenic bacteria, especially *Salmonella* spp., *E coli,* and *Clostridium* spp. were lower (*p* < 0.05) in mushroom (*Flammulina velutipes*) fed groups than the control diets, in laying hens. Similarly, Willis et al. [61] reported that adding *Lentinula edodes* mushroom mycelium extract in layer diets, could decrease the number of pathogenic bacteria *Salmonella* spp. in the

caecum and crop of birds fed with mushroom extracts. Lee et al. [56] used mushroom *Pleurotus eryngii* in layer ration. This study found that both the serum triglyceride and the serum cholesterol were lowered (*p* < 0.05) in mushroom fed groups than the control. Moreover, the dietary inclusion of dried mushroom at the 1% and 2% level showed greater (*p* < 0.05) serum antioxidant enzyme activities, in laying hens. This is due to a higher content of phenolic substance and different minerals, especially selenium, in mushrooms. Dietary supplementing selenium could enhance the body weight gain and antioxidant enzyme activities in chickens [69]. The author finally concluded that the residue of *Pleurotus eryngii* mushroom could improve the antioxidant status in layer chickens. Sun et al. [70] reported that edible mushrooms have a hypo-cholesterolemic effect on health and suggested its use as an oral medicine. The improved antioxidant status of chickens fed with different medicinal mushrooms was due to the presence of phenolic compounds, especially phenolic acid, which is the major naturally occurring antioxidant components found in mushrooms.

#### **5. Conclusions and Future Perspectives**

This review highlights that medicinal mushrooms could be fruitfully used as an effective natural growth promoter, as well as an immune boosting agent, in layer birds. In spite of the brood uses of medicinal mushrooms in layer diets, further studies by various researchers are recommended regarding the dosages of medicinal mushrooms on optimum performance and immune response, in laying hens. Therefore, future study should examine the use of medicinal mushroom in reaction to a pathogen challenge, as well as dosages. We suggest future research on medicinal mushrooms as alternates for antibiotics in laying hens so that it can be an effective strategy for organic egg production and encourage future researchers to discover the aspects of medicinal mushrooms that are important to immunity and health status that previous studies were not able to explore.

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

**Acknowledgments:** The authors would like to express their sincere gratitude to Mr. Austin Faust, English Language Teacher, the Ministry of Agriculture Feed Industry Centre (MAFIC), the College of Animal Science and Technology, the China Agricultural University (CAU), China for the English language and grammar revision of the manuscript.

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

#### **References**


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

### *Review* **The Genus** *Allium* **as Poultry Feed Additive: A Review**

#### **Damini Kothari 1, Woo-Do Lee 1, Kai-Min Niu <sup>2</sup> and Soo-Ki Kim 1,\***


Received: 4 October 2019; Accepted: 20 November 2019; Published: 26 November 2019

**Simple Summary:** The routine and unregulated use of in-feed antibiotics as growth promoters in poultry have been linked to the development of antimicrobial resistance, a serious global threat to the human, animal, and environment health. Growing public health concerns about food and environmental safety intensified the search for effective antibiotic alternatives in poultry production. The aim of this review is to present the current state of knowledge on the use of alliums as effective poultry feed additives in relation to their effects on growth performance, disease infections, gut and immune modulation, and product quality.

**Abstract:** The genus *Allium*, belonging to the family Amaryllidaceae has been known since ancient times for their therapeutic potentials. As the number of multi-drug resistant infections has increased due to in-feed antibiotic usage in poultry, the relevance of alliums as feed additives has been critically assessed. Garlic and the other *Allium* species, such as onions, leek, shallot, scallion, and chives, have been characterized to contain a plethora of bioactive compounds such as organosulfur compounds, polyphenols, saponins, fructans, and fructo-oligosaccharides. Consequently, alliums have been validated to confer antioxidant, antibacterial, antiviral, immunostimulatory, gut homeostasis, and lipid- as well as cholesterol-lowering properties in poultry. This review intends to summarize recent progress on the use of edible alliums as poultry feed additives, their beneficial effects, and the underlying mechanisms of their involvement in poultry nutrition. Perspectives for future research and limitations are also briefly discussed.

**Keywords:** *Allium*; feed additive; beneficial effects; organosulfur compounds; polyphenols; poultry

#### **1. Introduction**

The prolonged and unregulated use of antibiotics driven by a growing demand for animal products lead to the emergence of antibiotic resistance, a global threat to the animal and human health [1–3]. Poultry is the world's primary source of animal protein and it represents one of the highest consumers of antibiotics as growth promoters [3]. The European ban on sub-therapeutic use of antibiotics (1831/2003/EC, 2006) and the growing awareness among the consumers of the fatalistic effects of antibiotic resistance as well as residues in animal products intensified the hunt for effective in-feed antibiotic surrogates without affecting animal productivity or product quality [4–6]. However, the major challenges associated with antibiotic-free poultry production are poor growth performance, lower productivity, and increased morbidity as well as mortality in birds [7,8]. Many reviews shedding light on efficient and cost-effective antibiotic alternatives in poultry have been published in recent times [2,6,9–11]. Recently, plant-derived feed additives have gained considerable interest as sustainable substitutes in poultry diets [12,13]. An effective plant-derived additive in poultry (broilers, layers, and quails) is expected to stimulate feed intake, improve digestive enzyme secretions, activate

immune system, modulate gut microbiota, as well as have antibacterial, coccidiostatical, antiviral, antioxidant and/or anti-inflammatory activities [12–14]. In this context, *Allium* holds immense promise due to a variety of bioactive compounds including organosulfur compounds (OSCs), polyphenols, saponins, fructans, fructo-oligosaccharides (FOS), among many others. The genus *Allium* of the Amaryllidaceae family consists of ca. 850 species and represents one of the most studied plants of medicinal importance [15]. Extensive literature is available on the therapeutic properties of *Allium* spp. in humans, however, there is poor evidence in the poultry counterpart.

In the last three decades, alliums, in particular onion (*A. cepa*) and garlic (*A. sativum*), as well as garlic chives (*A. hookeri*) more recently have been reported to be incorporated into poultry diets to investigate their effects. However, the published literature on the effects of allium feeding in poultry have generated great inconsistency, making it impossible to draw a generalized conclusion on the efficacy of such feed additives. The discrepancies may be due to the heterogeneity of the composition of allium preparations, subject recruitment (broiler, layers, quails, etc.), dosage, duration of study, and so forth. This review combs the existing literature and gleans information to present an updated relevance of *Allium* spp. as effective poultry feed additives. We discuss the vast array of allium compounds in relation to their bio-functionalities. Emphasis was given to the dietary effect of *Allium* spp. on growth performance, infectious diseases, immunomodulatory properties, gut microbiota as well as gut morphology, and product quality in poultry. Moreover, this review discusses the lacunae to be surmounted for optimal application of alliums in poultry.

#### **2. Overview of Major Bioactive Compounds in** *Allium*

#### *2.1. Organosulfur Compounds*

The genus *Allium* is a rich source of organosulfur compounds (OSCs), which are one the main bioactive compounds of the plants [16,17]. The major OSCs in *Allium* spp. include allyl cysteines, S-alk(en)yl-L-cysteine sulfoxides (ACSOs), thiosulfinates, and sulfides in varying amounts [18]. The characteristic aroma in different *Allium* spp. are mainly associated with the different levels of ACSO precursor, namely alliin (S-allyl-L-cysteine sulfoxide; garlic and elephant garlic), methiin (S-methyl-L-cysteine sulfoxide; garlic, onions, leeks, and shallots), propiin (S-propyl-L-cysteine sulfoxide; shallots), and isoalliin (S-1-propenyl-L-cysteine sulfoxide; onions and shallots) [19,20]. The synthesis of the OSCs is depicted in Figure 1 and starts with the transformation of γ-glutamyl peptides into ACSOs by the action of γ-glutamyl transpeptidase and oxidase in the cytoplasm of plant cells. When the bulbs are cut or crushed alliin is transformed into the allicin (alkenyl alkene thiosulfinate) by the action of a vacuolar lyase, alliinase. Allicin immediately decomposes into diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), diallyl tetrasulfide (DATTS), dipropyl disulfide (DPDS), ajoenes, and vinyldithiins depending on their manufacturing process [21,22]. The direct catabolism of γ-glutamyl cysteine leads to the formation of water-soluble S-allylcysteine (SAC) and S-allylmercaptocysteine (SAMC) [23]. The OSCs and their transformation products are well-studied antimicrobial agents [24]. Several antimicrobial compounds have been extracted and identified from many spp. of *Allium* including garlic (*A. sativum* L.), onion (*A. cepa* L.), shallot (*A. ascalonicum* L.), elephant garlic (*A. ampeloprasum* L. var. *ampeloprasum* auct.), rosy garlic (*A. roseum*) [24], garlic chives (*A. hookeri*) [25], and wild garlic (*A. ursinum*) [26]. Although the antimicrobial mechanism of these compounds has not been well defined, it seems that it is associated with the inhibition of important thiol-dependent enzymatic systems (alcohol dehydrogenase, thioredoxin reductase, trypsin, other proteases, RNA and DNA polymerases) and antioxidant activity, which have a multiple inhibitory effect on the microbial cell [27–29]. The potent antimicrobial activities of OSCs is also related to the number of disulfide bounds, i.e., DATTS > DATS > DADS > DAS [30].

**Figure 1.** Major organosulfur compounds (OSCs) of *Allium* spp.

#### *2.2. Polyphenolic Compounds*

Another important class of bioactive compounds in alliums includes polyphenols [31,32]. The health-promoting activity of dietary polyphenols seems to be related to their antioxidant and anti-inflammatory activities [33]. Allium vegetables contain high levels of polyphenolic compounds, particularly phenolic acids, flavonoids, and their derivatives. *Allium* spp. are amongst the richest sources of dietary flavonoids [34]. Leighton et al. [35] found that flavonoid levels in the edible portion of allium vegetables (leeks, shallots, green onions, garlic, and onions) range from > 0.03 to 1 g/kg of vegetables. Flavonoids identified in onions were quercetin di-glucosides, quercetin 4'-glucoside, quercetin aglycone, and in some cases, isorhamnetin monoglucosides or kaempferol monoglucosides [36]. Quercetin glucosides of onion are more bioavailable than other quercetin-rich foods such as tea and apples [37]. The main phenolic acids found in alliums include *p*-Coumaric acid, ferulic acid, sinapic acid, gallic acid, and protocatechuic acid [38]. However, very few studies used allium flavonoids as feed additives to promote growth, immune, and antioxidant response for animals [39,40].

#### *2.3. Saponins*

Saponins are surface-active glycosides with triterpenoid or steroidal aglycone. Allium plants contain steroidal saponins, which are mainly divided into three groups based on their structure: spirostanols, furostanols, and open-chain (cholestane-type) saponins [41]. Saponin accumulation in the root organs is reported to be higher than in the aerial parts (stem and leaves) of alliums [42]. Until now, as many as 290 steroidal saponins (130 spirostanols, 140 furostanol, and 18 cholestane-type) have been identified in more than 40 different *Allium* species [41]. Allium saponins are not pungent and have many biological properties including antispasmodic, antifungal, haemolytic, anti-inflammatory, cholesterol-lowering, and cytotoxic activities. Moreover, saponins have the advantage of being more stable to food processing and cooking than the relatively unstable OSCs [43].

#### *2.4. Fructans and Fructo-Oligosaccharides*

Water-soluble fructans and fructo-oligosaccharides (FOS) together with glucose, fructose, and sucrose constitute the main non-structural carbohydrates in *Allium* species [44]. Fructans from various spp. of *Allium* including *A. cepa* (onion), *A. cepa* L. var. *ascalonicum* (shallot), *A. ampeloprasum* L. var. *porrum* (leek, 3 cvs.), *A. schoenoprasum* L. (chives), *A. sativum* L. (garlic), *A. fistulosum* L. (Japanese bunching onion/Welsh onion), *A. tuberosum* Rottl. ex. spr. (Chinese chives) have been characterized [44]. Several in vitro and in vivo studies witnessed the immunomodulatory [45–47], prebiotic [48], antiviral [49], and gastroprotective [50] effects of allium poly- and oligosaccharides. Lee et al. [46] reported the influenza A virus inhibitory activity of the fructan from *A. fistulosum* in an animal model and it was suggested to be mediated by host immune functions since the polysaccharide did not show any direct inhibitory effect on the virus replication in vitro. The immunomodulatory effect was attributed to promotion of phagocytosis, release of NO, and expressions of several immune-related cytokines [interleukin (IL), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ)] [47,48,51].

#### **3.** *Allium* **spp. as Poultry Feed Additives**

Literature search was conducted to accumulate the latest findings in implication of alliums as poultry feed additives and their role in growth performance, lipid metabolism, poultry infectious diseases, immunomodulation, gut modulation, and product quality (Table 1). The sections below outline the key components and mechanisms responsible for these functions.

#### *3.1. E*ff*ects on Growth Performance*

Several studies have documented the benefits of *Allium* spp. (in particular onion and garlic) on growth performance in poultry by improving weight gain, feed intake, and/or feed efficiency [52–54]. Farhani et al. [52] found that onion extract (1%) in drinking water improved growth performance and blood biochemical characteristics. They attributed the effect to the onion FOS, which might help in maintaining beneficial gut microorganisms and improve nutrient absorption. Goodarzi et al. [54] speculated that the OSCs of onion have increased nutrient absorption and thereby improved growth performance in broilers. In addition, onion in diet can reduce blood glucose stimulating the nervous system for higher feed intake, which can lead to increased weight gain [52,54].

The precise mechanisms behind the improved growth performance in poultry fed alliums remain unclear. However, some researchers have linked this improvement to the increased feed intake of allium supplemented diets [55]. Generally, garlic is used as seasonings to improve the flavor and hence it might improve the palatability of feed, thus increasing voluntary feed intake. Brzóska et al. [55] reported that garlic extract (2.25 mL/kg of feed) stimulated the appetite of chickens, which resulted in significantly greater feed intake and thereby higher body weight gains. Sheoran et al. [56] and Kirubakaran et al. [57] hypothesized that the improvement in weight gain of the birds using garlic in their rations may probably be due to allicin. Kirubakaran et al. [57] postulated that garlic in broiler diet may increase salivary flow rate and gastric juice secretion, resulting in improved digestibility and higher body weight. Negative effects on growth performance in broilers were also observed with the supplementation of 1 g of garlic powder/kg feed and 15 g of garlic bulb/kg feed [58,59]. The inclusion of alliums may reduce diet palatability due to their pungency and as a result the feed intake and body weight of animals decrease [58,60,61]. While Aji et al. [62] reported ineffectiveness of low doses (0.025 and 0.05 g of onion and garlic/kg feed) to produce any observable effects and suggested that the dosage of alliums as an important factor. However, several studies reported no significant effects on growth performance parameters such as feed intake, body weight gain, or feed efficiency in broilers by the dietary supplementation of alliums [14,60,62–64]. In the case of layers, most of the studies found no significant changes in performance when layer diets were supplemented with alliums [65–68]. Some researchers suggested that well-nourished healthy poultry reared under clean and ideal conditions, often do not respond to growth-promoting supplements, while the stressed or challenged birds may give better results with the same supplements [49,66,67,69–72]. Intriguingly, Ao et al. [66] indicated garlic supplementation could increase growth performance in broilers by reducing the concentration of cortisol, the stress hormone. The variability in the efficacy of alliums on animal performance could also be attributed to the variation in the product fed, dosage, duration, and subjects used among the studies.


**Table 1.** Effect of dietary supplementation of *Allium* spp. in poultry.


**Table 1.** *Cont.*

ND: Not determined; BWG: Body weight gain; FCR: Feed-conversion ratio; HDL-C: High-density lipoprotein-cholesterol; LDL-C: Low-density lipoprotein-cholesterol; VLDL-C: Very low-density lipoprotein-cholesterol; LPS: Lipopolysaccharide; α-1-AGP: Alpha-1-acid glycoprotein; SOD: Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde; NE: Necrotic enteritis; WBC: White blood cells; IgG: Immunoglobulin G; TBARS: Thiobarbituric acid reactive substances; ATTR: Apparent total tract retention of nutrients; PUFA: Polyunsaturated fatty acid; SFA: Saturated fatty acid; EPEF: European production efficacy factor.

#### *3.2. Hypolipidemic and Hypocholesterolemic E*ff*ects*

Alliums (especially garlic) have a traditional place in folk medicine as hypolipidemic and hypocholesterolemic agents in many cultures. Elevated blood cholesterol and triacylglycerides in animal proteins are associated with the increased risk of cardiovascular disease in humans. Several researchers have opined that garlic exhibited hypocholesterolemic effects in poultry including broilers, layers, and quails through the inhibition of key enzymes such as malic enzyme, fatty acid synthase, glucose-6-phosphate dehydrogenase, and 3-hyydroxy-methyl-glutaryl-CoA (HMG-CoA) reductase involved in cholesterol and lipid synthesis [81,83,85–87]. Allicin is thought to be the potentially active component [81,87]. However, Lanzotti et al. [43] reported that allicin is very unstable and not present in intact garlic or in any garlic products. Moreover, the acidity of the stomach prevents the formation of allicin [88]. Some researchers attributed the cholesterol-lowering effect of garlic to the steroidal saponins possibly by inhibiting cholesterol absorption in the intestine or a direct effect on cholesterol metabolism [41]. The exact mechanism by which garlic reduces plasma cholesterol concentration is remaining elusive.

#### *3.3. E*ff*ects on Infectious Diseases*

Recently, allium derived feed additives have been given useful results against several infectious diseases in broilers such as the infection of *Escherichia coli*, *Salmonella*, *Clostridium perfringens*, and *Eimeria* [27,71,72,89–91]. Elmowalid et al. [89] reported that garlic dietary supplementation for three weeks provided in vivo protection against multi-drug resistant *E. coli* O78 challenge in broilers by reducing the mortality rates to >10% from 60% (control, non-supplemented birds). The authors suggested that the bioactive phenolic and non-phenolic compounds in garlic are responsible for this effect. Lee et al. [71] reported less loss of body weight gain, decreased lesion score, and oocyst shedding by *A. hookeri* dietary supplementation in necrotic enteritis (NE) challenged (*Clostridium*/*Eimeria* co-infection) commercial broilers. Another in vivo study with the dietary supplementation of two garlic metabolites (10 ppm) namely propyl thiosulfinate (PTS) and propyl thiosulfinate oxide (PTSO) revealed increased body weight gain, decreased fecal oocyst excretion, a higher profilin antibody response, and greater spleen cell proliferation in *E. acervulina*-infected chickens as compared with the infected birds fed a non-supplemented control diet [73]. Ali et al. [91] also reported that the dietary supplementation of garlic powder (15g/kg of feed) reduced oocysts shedding and lesion score as well as lowering mortality, and improved histopathology of the small intestines in the supplemented group. They ascribed these effects to the presence of allicin and phenolic compounds in garlic. The allicin has antioxidant and antiparasitic activity which directly kill the sporozoites [73]. The phenolic compounds in garlic act on the cytoplasmic membrane of *Eimeria* and make changes in their cation permeability, leading to the death of pathogens [91].

Salem et al. [27] assessed the efficacy of garlic extract (40 mg/mL) in experimentally *S. typhimurium* induced salmonellosis in Cobb broiler chicks. The garlic extract used in the study contained allicin, alliin, allylsulfide, E-ajoene, and vinyldithiin. The mortality rate was decreased from 53.3% to 13.3% after treatment with garlic extract. The body weight of the infected chickens was significantly improved with the treatment of garlic extract when compared with infected non-treated groups. The post-mortem lesions were less severe in the garlic-treated infected chicks as compared with control infected chicks. The authors suggested the efficacy of garlic against multidrug resistant *Salmonella* by reducing its invasion, resistance to antimicrobial agents, and biofilm formation ability. Jimoh et al. [90] reported that garlic at the various supplementation levels reduced the caecal load of *C. perfringens* as compared with the control group and attributed to the OSCs.

Kavindra and Shalini [92] reported in vitro anthelminthic potential of garlic oil (2%, 4%, and 6%) against *Ascaridia galli* diseases in poultry birds. Mechanistically, the garlic oil reduced significantly the glucose uptake, glycogen content, oxygen consumption, and relative activity of acid and alkaline phosphomonoesterases in the parasite. However, an in vivo study by Velkers et al. [28] failed to observe efficacy of allicin from garlic against experimentally induced *A. galli* infection in chickens with no significant effect on worm load. Shojai et al. [93] observed an inhibitory effect of garlic extract against infectious bronchitis virus in specific pathogen-free (SPF) embryonic eggs and they suggested that the garlic extract could have an effect on the virus in replication phase. From the above discussion, it can be inferred that allium compounds at a certain inclusion rate can alleviate the negative effects of infections in chickens and mediate multiple disease-related signaling pathways.

#### *3.4. E*ff*ects on Intestinal Microbiota and Morphology*

The gastrointestinal tract of poultry harbors complex assemblages of microorganisms (microbiome) mainly dominated by the phyla Firmicutes (*Lactobacillus, Streptococcus*, *Bacillus*, *Enterococcus*), Bacteroidetes (*Bacteroides*, *Bifidobacterium*), Proteobacteria (*Escherichia*, *Salmonella, Campylobacter, Shigella)* and Actinobacteria [94]. This gut microbiome is recognized as a key player in governing host growth performance and health by providing nutrients from indigestible dietary substrates, competitive exclusion of pathogens, detoxification, strengthening the gut barrier, and modulation of immune system [95,96]. Pan and Yu [97] suggested an intertwined relationship of the gut microbiome with poultry host and diet. Therefore, any perturbation in the taxonomic composition of gut

microbiota (called dysbiosis) may underlie its contribution to symptoms of a disease condition like that in humans. Recently, few studies strengthened the applicability of alliums (mainly garlic and onion) as poultry feed additive in the gut microbiota modulation in regard to diversity and composition [53,54,74,75,80,98]. Supplementation of onion showed a significant reduction in the population of *E. coli* and increased significantly *Lactobacillus* and *Streptococcus* species. Similarly, Goodarzi et al. [99] and Shargh et al. [100] also reported higher *Lactobacilli* spp. and reduced *E. coli* load in ileum of onion fed broilers. Shin et al. [101] hypothesized that the phylum Proteobacteria may potentially serve as biomarker for gut dysbiosis in humans. Intriguingly, Kim et al. [102] demonstrated that lower numbers of certain gut pathogens such as *E. coli* may improve broiler performance. Sheoran et al. [56] and Kirubakaran et al. [57] also indicated that the lower *Staphylococcus aureus* and *E. coli* as well as aflatoxins producing fungi in the intestine fostered nutrient digestibility which in turn improve weight gain of the birds.

Allicin has also been reported to improve and regenerate the physiological structure of the intestinal epithelium layer, and enhance crypt depth and villus height, which ultimately support its digestive capacity through increased absorption of nutrients and assimilation [103]. However, the instability and poor bioavailability of allicin question its effects in vivo [23]. Ur Rahman et al. [53] observed that onion supplementation significantly increased dimensions (villus height, width, crypt depth, and surface area) of duodenum, jejunum, and ileum. The authors hypothesized that larger intestinal villi are associated with higher absorption of the nutrients and reduction of *E. coli* in the intestine. Mehmood et al. [104] also reported that supplementation of onion in the feed significantly increased villus height, crypt depth, and surface area of the jejunum in broilers.

Karangiya et al. [80] indicated that garlic supplementation (10g/kg feed) increased the absorptive surface area of the intestine (villus height, width, and crypt depth) and correlated with the higher body weight gain in broilers. Diets containing garlic-derived propyl propane thiosulfonate (PTS-O) (0.045 and 0.090 g/kg feed) has also been shown to improve absorption surface at the ileal level in broilers [74]. In an extended study, Peinado et al. [75] observed a decrease in the numbers of enterobacteria, in particular lactobacilli and an increase in bacteroides in the broiler intestine with the dietary supplementation of PTS-O (0.045 and 0.090 g/kg feed). Although generally regarded as a beneficial group, the higher number of lactobacilli is linked to the impairment in fat digestion or absorption in poultry due to their bile-deconjugation activity [105,106]. Thomas et al. [107] suggested that the higher bacteroidetes was responsible for the improved performance in chickens since bacteroidetes are involved in fermentation of high molecular weight carbohydrates, activation of T-cell mediated immune responses, prevention of potential pathogens, bile acid metabolism, and transformation of toxic and/or mutagenic compounds. Likewise, Ruiz et al. [98] observed lower diversity indices of ileal mucosa-associated microbiota in chickens fed the PTS-O–supplemented diet, which was ascribed to the bactericidal effect of PTS-O against enterobacteria, coliforms, *E. coli*, *C. jejuni*, and *Salmonella* spp., as also observed by Peinado et al. [75]. In addition, PTS-O was able to significantly increase and modulate the composition of bifidobacteria in growing broilers; which are considered as excellent candidates of probiotics in broilers [108]. Another study involving PTS-O supplementation indicated negative correlations between relative abundances of *Escherichia*–*Shigella* or enterobacteria (crop, ileum and caeca) and growth performance as well as fat digestibility in PTS-O fed broilers [109]. When garlic extract (0.04 or 0.06 g/kg feed) was gavaged to broilers reduced number of *E. coli* and *Staphylococcus aureus* in the ileo-caecal digesta and improved nutrient digestibility were observed [110]. Kırkpınar et al. [111] reported that garlic oil alone or in combination with oregano, reduced *Clostridium* counts in the ileum of broilers. However, total organism, *Streptococcus*, *Lactobacillus* spp., and coliform counts were not affected by the dietary treatments. The lower *Clostridium* counts were ascribed to the antibacterial effects of essential oils.

Notwithstanding the fact that specific mechanistic studies how dietary alliums affect chicken gut health and physiology are limited; it is clear from the above-cited findings that alliums participate in gut homeostasis to foster an intestinal environment conducive to commensals by reducing the expansion of pathogenic microorganisms. However, a better understanding of the gut/microbe interactions and gut microbial diversity using next generation sequencing will provide new opportunities for the improvement of poultry health and production.

#### *3.5. E*ff*ects on Immune Response*

Poultry diet and nutrition are critical determinants of birds' immune response. Several studies have advocated disease prevention or immune enhancing effects of alliums in poultry, however, very few studies investigated the underpinning mechanisms for their specific immunomodulatory effects. For instance, Kim et al. [73] investigated the effects of two garlic secondary metabolites (10 ppm) namely PTS and PTSO on the in vitro and in vivo parameters of chicken gut immunity during experimental *E. acervulina* infection. In vitro, PTSO/PTS treatment dose-dependently killed invasive *E. acervulina* sporozoites and stimulated splenocyte proliferation. In vivo feeding of PTSO/PTS provided increased protective immunity following live *E. acervulina* challenge infection, as indicated by improved bodyweight gains, reduced fecal oocyst shedding, and higher anti-profilin serum antibody titers, compared with the non-supplemented controls. In PTSO/PTS-fed birds, microarray hybridization identified 1227 transcripts, whose levels were significantly altered (552 up-regulated and 675 down-regulated) in the intestinal intraepithelial lymphocytes (IEL) involving immuneand cardiovascular-related gene pathways and networks. The authors observed a simultaneous and interactive effects of PTSO/PTS dietary supplementation on adaptive (increased splenocyte proliferation and anti-prolifin titers) and innate immunity [downregulation of toll-like receptors (TLR) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)] in chickens against coccidiosis.

Hanieh et al. [112] reported that the dietary alliums (garlic and onion) have a potential to enhance the humoral immune functions in White Leghorn chickens following immunization with Newcastle disease virus (NDV), sheep red blood cells (SRBC) and *Brucella abortus* (BA). The authors observed that alliums (10 g/kg feed) enhanced anti-NDV, anti-SRBC, and anti-BA antibody production, which might be due to increased CD4/CD8 cells, following immunization. Moreover, the relative weight of spleen and thymus were increased in case of garlic supplementation, which was ascribed to the enhanced lymphocyte proliferation and the increase in WBC counts. The mechanism of improved humoral immune functions by the dietary alliums against three antigens was further delineated by a subsequent in vitro study on the lymphocytes and peritoneal macrophages from white Leghorn chickens (male) [113]. The authors observed that garlic and onion extract augmented concanavalin A (ConA)-induced splenocyte and thymocyte proliferations, and gene expression of IL-2 and IFN-γ as well as higher microbicidal activity and reactive oxygen species production in macrophages. They speculated different mechanisms of immune modulation by garlic and onion. Garlic had a direct stimulatory effect on the immune cells, whereas onion had an indirect stimulatory effect, the antioxidant activity of high flavonoids of onion may be a plausible explanation [112,113]. In contrast, Jafari et al. [114] and Goodarzi et al. [54] failed to report any significant effect on antibody titers against NDV in garlic and onion fed broilers, respectively.

Another study investigated the effect of dietary supplementation of *A. hookeri* on the inflammatory immune activities in the jejunum during the immunological stress induced by *Clostridium*/*Eimeria* co-infected commercial broilers [71]. The authors observed downregulated expression of pro-inflammatory cytokines such as IL-1β, IL-8, IL-17A, inducible nitric oxide synthase (iNOS), and lipopolysaccharide induced TNF-α (LITAF) in jejunum of NE challenged group by the dietary supplementation of *A. hookeri* as compared with control. In addition, the dietary supplementation of *A. hookeri* significantly increased expressions of tight junction (TJ) proteins [junctional adhesion molecule (JAM), occludin, and zonula occludens 1 (ZO1)] and intestinal mucin 2 (MUC2). These proteins play crucial roles in the regulation of intestinal permeability and barrier function [49].

Garlic dietary supplementation modulated chicks' innate immune response via various mechanisms including phagocytosis augmentation, bactericidal activity enhancement and nitric oxide (NO) production reduction, together with triggering the IL-1β, IL-6 and IFN-γ cytokines expression levels in comparison with the non-supplemented chicks against multi-drug resistant *E. coli* O78 challenge [89].

Lee et al. [49] reported that the dietary supplementation of *A. hookeri* promoted gut integrity and enhanced innate immunity during an immunological stress induced by lipopolysaccharide (LPS) in young broiler chicken. They observed decreased levels of alpha-1-acid glycoprotein (α-1-AGP), a marker for systemic non-specific inflammation or gut barrier health. In LPS-challenged groups, chickens fed diets supplemented with *A. hookeri* (1 or 3g/kg feed) exhibited lower transcript levels of pro-inflammatory cytokines (IL-1b, IL-8, TNF superfamily member 15, and LITAF) as compared with the non-supplemented fed chickens. Furthermore, the dietary supplementation of *A. hookeri* significantly upregulated the expression levels of TJ proteins (JAM, occludin, and ZO1) and MUC2.

Garlic powder and holy basil leaf powder either alone or in combination in the broiler's diet have potent immune modulating activity by showing a stimulatory effect on relative mRNA expression of TLR 2, TLR 4, and TLR 7 in the commercial broilers [56]. However, Toghyani et al. [14] reported no influence of dietary garlic (4g/kg feed) on immune-related parameters such as antibody titers, lymphoid organs' weight, albumin to globulin ratio, and heterophil to lymphocyte ratio in broilers and speculated that a higher dose is required to elicit any immune response. Indeed, the aforementioned data related to the immune effects of dietary alliums in poultry form the basis of further studies involving the mechanisms of molecular signaling and immune response initiation. Therefore, in a more long-term perspective, the assessment of variation among immune system components of poultry in response to allium supplementation will offer a better understanding of nutritional immunomodulation to reduce risk and manage field infections.

#### *3.6. E*ff*ects on Product Quality*

Dietary strategies are valuable options to improve nutritional value as well as oxidative stability and sensory properties of poultry meats and eggs. The antibacterial, anticoccidial, antifungal, antiviral, and antioxidant activity, as well as the immune-enhancing activity of allium-derived compounds have garnered attention in improving the poultry product quality. The plasticity and extraordinary responsiveness of poultry eggs to dietary factors make them the most attractive targets for nutrition modulation [115]. Several studies reported the use of alliums toward the improvement of egg quality [67,69,81–83,85]. Ao et al. [69] found a better fatty acid profile in egg yolk with higher poly-unsaturated fatty acid and lower saturated fatty acid by the dietary garlic (30 g/kg feed). Damaziak et al. [116] indicated that administration of the dietary onion extract to hens resulted in heavier eggs, with a higher content of egg yolk and better quality of albumen. The genus *Allium* has an exceptional ability to absorb, metabolize, and store selenium as organoselenium compounds such as selenomethionine and selenocysteine [117]. Olobatoke and Mulugeta [67] had given the possible explanation to the increased egg weight in laying hens is the absorption of garlic compounds (selenomethionine and selenocysteine) and their subsequent deposition in the egg yolk. Additionally, alliums are rich source of polyphenols (gallic acid, ferulic acid, quecetin, kaempferol, and flavonoid glycosides), potent antioxidants. The Haugh unit, albumen height, and pH are the indicators of the freshness of eggs, which tends to decrease during storage. Lim et al. [76] reported that with the dietary garlic in layers, the Haugh unit was improved during storage possibly due to the antioxidant effect from allicin and organoseleniums. Allicin inhibits the formation of superoxide by the xanthine/xanthine oxidase system, probably via a thiol exchange mechanism [118]. Wakebe [119] reported that the inclusion of selenomethionine in the layer diet (0.3 ppm/kg feed) resulted in higher Haugh units, which was ascribed to increased glutathione peroxidase activity in the egg yolk and white. Mahmoud et al. [85] proposed another explanation in that the garlic enhances the egg's antioxidant status by upgrading the glutathione peroxidase activity in yolk and albumen; this thereby increased egg quality during storage with better albumen height, Haugh unit, and pH probably because of less lipid and protein oxidation. In an organoleptic assessment, Motozono et al. [120] reported an off flavor in eggs with garlic dietary

supplementation (20 g crushed garlic/kg feed) in layers, while Birrenkott et al. [121] and Olobatoke and Mulugeta [67] reported no differences in color and flavor in eggs from hens consuming up to 30 g dietary garlic powder per kg feed. Damaziak et al. [116] indicated that the effect of dietary allium on the taste of eggs may be determined by both supplementation level and duration.

The inclusion of alliums in poultry diets has also been reported to improve color stability, fatty acid composition [63], sensory properties [14,111], and the anti-oxidative ability of meat [64,66,79]. Choi et al. [63] reported the color stability of meat (highest redness and yellowness) by the incremental levels of garlic powder (1–5%) dietary supplementation and the effect was ascribed to the reduced metmyoglobin formation and oxidation in thigh muscle of chicks. The same study observed better fatty acid profile in garlic supplemented groups by protecting the oxidation of unsaturated fatty acid. However, Abdullah et al. [122] reported no effects of garlic supplementation on the meat quality such as cooking loss percentage, shear force, color coordinates). The improved anti-oxidative capability of chicken meat by the dietary allium supplementation was attributed to the accumulation of antioxidant compounds such as flavonoids and OSCs [66,79]. Indeed, the utilization of dietary alliums to improve the quality of poultry products should be done carefully because high doses of the allium may reduce overall acceptability with altered taste and odor.

#### **4. Factors Determining the E**ff**ectiveness of Alliums in Poultry Feed**

Several variables need to be considered while recognizing the efficacy and safety of alliums in poultry (Table 2). From the above-cited studies, it is evident that six different kinds of allium formulations were mainly used in poultry, i.e., powder (sun- or air-dried), juice, purified extract, oil, aged extract, and paste. These processed alliums contain a variety of OSCs (major bioactive constituents) which differ greatly from their intact forms, depending on their manufacturing process. Most of these preparations were not chemically characterized and thus cannot be generalized under a single umbrella to have a biological response in poultry. Thiosulfinates, the most bioactive OSCs, are volatile and can evaporate rapidly, leading to largely varied final concentrations in the feed [123]. The pungent smell of thiosulfinates [21] might also affect the feed palatability, depending on the applied dosage. In the published literature, the inclusion rates of alliums in poultry have been reported to be very wide ranging from 0.001% to 10% (Table 1). For instance, Aji et al. [62] reported ineffectiveness of a low dose (25 and 50 mg of allium/kg feed) of onion and garlic supplementation while Varmaghany et al. [58] indicated negative effects of a high dose of garlic supplementation. Therefore, identification of an optimal dosage of alliums will also determine its effectiveness in poultry. Fujisawa et al. [124] reported that thiosulfinates might lose antimicrobial activity by reacting with sulfhydryl (SH) compounds of other feed components (proteins). The thermally unstable nature of allium bioactive constituents [16,20] also affects their application in feed production, since thermal processing is an important step to decontaminate the harmful microorganisms of feed. The OSCs have poor water solubility [125], which further limits their application in feed. While considering these factors, the higher cost of allium-based feed cannot be overlooked. Apart from these, the poultry responses might also be affected by various factors such as the feed type (pellet or mash) and quality, duration of study, hygiene, subject recruitment (broiler, layers, quails, etc.), age, health status (healthy or challenged) and environmental factors among many others [126]. Indeed, without proper standardized formulation, in practice the choice of an economically feasible allium-based feed additive is compromised in poultry diets.


**Table 2.** Factors determining the effectiveness of alliums in poultry.

#### **5. Fortification**/**Preservation of** *Allium* **Bioactivity**

The instability and volatility of allium bioactive compounds prompted animal nutritionists to devote intensive efforts in the search for new stabilization techniques that could ensure feed safety and quality as well as enhance modern preservation methods in the feed industry.

#### *5.1. Fermentation*

Fermentation has significantly improved bioactivities and organoleptic properties of alliums (Table 3). Allium fermentation resulted in higher polyphenolic content via the deglycosylation of complex phenolic glycosides to their simpler derivatives by the action of glucosidases, thereby increasing their antioxidant activity as well as bioavailability [127–129]. Fermentation could also reduce the pungent smell of alliums and hence expected to improve the palatability of feed [128–130]. Furthermore, fermented alliums can act as a viable source of probiotics and provide host health benefits [131]. Bernaert et al. [132] hypothesized that fermentation can be used as a stabilization technique for the preservation of antioxidant activity in *A. ampeloprasum* var. *porrum*. Hossain et al. [133] reported an increase in feed intake with the fermented garlic supplementation as compared with the control diet in broilers. Thus, the allium 'probiotication' may offer a cost-effective approach in the manufacture and storage processes of a feed additive by extending shelf-life and maintaining desired sensory properties in addition to the host health benefits.


**Table 3.** Fermentation of *Allium* spp. with respect to compositional changes and bioactivities.


**Table 3.** *Cont.*

#### *5.2. Microencapsulation*/*Nanotechnology*

Microencapsulation is one of the most effective approaches for protecting bioactive compounds against oxidation, heat and evaporation, controlled delivery, uniform distribution, storage stability, masking off-flavours, and extending the shelf life without affecting their physical, chemical or functional properties [143]. Milea et al. [143] and Akdeniz et al. [144] successfully encapsulated phenolic compounds extracted from onion skin. Piletti et al. [145] reported that β-cyclodextrin encapsulation of garlic oil increased thermal stability and water solubility, as well as preserved antimicrobial activity. However, the use of microencapsulation is based on several factors, including feasibility, practicability, and cost [146].

Nanoparticles can be used as possible feed supplements for poultry to improve overall health and feed conversion ratio [147]. The formulation of plant-derived bioactive compounds using nanotechnology may result in their improved activity at low dosage [148]. Sundari et al. [149] reported turmeric extract nanoparticle as a feed additive which improved meat quality at low dosage without affecting performance in broilers. Xu et al. [125] converted natural organosulfur compounds into nanometer-sized iron sulfides (nFeS) with improved antibacterial activity and antibiofilm efficacy in vitro. Jini and Sharmila [150] synthesized silver nanoparticles from *A. cepa*, which have higher in vitro antidiabetic and antioxidant activities. However, the toxicity of nanoparticles due to nano size and the high cost hinder their practical application in poultry.

#### **6. Future Perspectives**

The aforementioned findings are testimony to the fact that the appreciation of alliums as poultry feed additives exhibits tremendous opportunities as well as hurdles. Therefore, the scientists, veterinarians, and commercial partners must work together to thwart the limitations for optimal efficacy of alliums, from poultry health and economic perspectives. Future research in this field will help us to better understand their mechanism of action and optimal dosage as well as efficient delivery methods (fermentation, microencapsulation and/or nanotechnology). Since alliums are a hub of bioactive compounds which might affect poultry production synergistically, the dietary supplementation of dried alliums or their extracts poses an advantage over the single extracted compound. Moreover, the synergistic effect of alliums with other antibiotic alternatives such as prebiotic, probiotic, organic

acid, etc. together with good management and farming practices will be the key to achieve sustainable poultry production. Moreover, researchers in this field should be encouraged to publish even the negative or no effects of alliums in poultry.

#### **7. Conclusions**

It is evident from our discussion that alliums harbor a variety of bioactive compounds such as organosulfur compounds, flavonoids, fructans, fructo-oligosaccharides, saponins, etc., and thereby justifying their usefulness as feed additives for poultry production. Recently, several studies have established that alliums in poultry diets have a significant modulatory effect on their growth performance indices, lipid metabolism, gut ecosystem as well as immune responses, especially when poultry are experiencing stress and disease challenge conditions. In addition, the alliums also improve the nutritional quality of poultry products via their enrichment in antioxidant (flavonoids) and organoselenium compounds. However, their application in poultry production has been largely circumvented due to inconsistent efficacy among studies, lack of a clear understanding of the mechanisms of action, non-availability of a standard as well as chemically characterized formulation, and higher cost. The processing methods, such as extraction, encapsulation, fermentation, and heating strongly influence the chemical composition, ergo, the biological activity of alliums. Therefore, poultry nutritionists must understand the inherent differences among the allium products used in various studies, along with their potential role in providing desired potential effects when added to poultry feed. A standardized procedure should be developed for an allium-based feed additive retaining its bioactive components. The OSCs and polyphenol contents of allium products may serve as proxy for their strategic application in poultry nutrition feeding programs. In addition, to preserve the effectiveness of alliums as a poultry feed additive, the optimization of dosage regimens that encompasses bioavailability could also be a suitable strategy. This review is expected to inspire investigations on alliums as feed additives for poultry health and disease management.

**Author Contributions:** Conceptualization, D.K.; search of literature, D.K.; W.-D.L. and K.-M.N.; writing—review and editing, D.K.; supervision, S.-K.K.; project administration, S.-K.K.; funding acquisition, S.-K.K.

**Funding:** This study was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (118051-03).

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

#### **References**


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

#### *Article*

### **Growth Performance of Broilers as Influenced by Di**ff**erent Levels and Sources of Methionine Plus Cysteine**

**Abd Ur Rehman 1, Muhammad Arif 1, Muhammad M. Husnain 1, Mahmoud Alagawany 2, Mohamed E. Abd El-Hack 2,\*, Ayman E. Taha 3, Shaaban S. Elnesr 4, Mervat A. Abdel-Latif 5, Sarah I. Othman <sup>6</sup> and Ahmed A. Allam <sup>7</sup>**


Received: 3 November 2019; Accepted: 27 November 2019; Published: 1 December 2019

**Simple Summary:** The current work evaluated the utilization of different sources of methionine either from DL-methionine (DL-Met) or L-methionine (L-Met) using different concentrations of dietary methionine plus cystine (Met + Cyst) in broiler chickens. Results showed that a better edible meat yield could be obtained by supplementing Met + Cyst at the rate of 80% of the digestible lysine.

**Abstract:** The objective of this work was to evaluate the utilization of methionine from DL-methionine (DL-Met) and L-methionine (L-Met) with different levels of dietary methionine plus cystine (Met+ Cyst) in broilers. The experimental diets were formulated by using three levels of Met + Cyst, i.e., 74%, 77% and 80% of digestible lysine. Met + Cyst was provided either from DL-Met or L-Met. A total of 450 day-old broilers were divided into six groups (five replicates of 15 birds each) in a 3 × 2 factorial arrangement under completely randomized design. Weight gain (WG), feed intake (FI) and feed conversion ratio (FCR) was determined. At the end of the experiment (35 days), two birds from each replicate were slaughtered to determine carcass characteristics and serum homocysteine. Results indicate that the combined effect of L-Met and DL-Met significantly affected (*p* < 0.05) the WG in the starter period and FI in the finisher period. Neither source nor level of methionine influenced (*p* > 0.05) the FI, WG and FCR of broilers during the starter, finisher or overall phase of growth. The interaction between sources and levels of methionine did not influence (*p* > 0.05) the feed intake, weight gain and FCR during the overall phase of growth. Source of methionine had no (*p* > 0.05) effect on carcass characteristics. Methionine levels had a significant effect (*p* < 0.05) on carcass weight, chest weight and thigh weight. The interaction between sources and levels of methionine had a significant (*p* < 0.05) effect on the liver weight. The sources of methionine had significant (*p* < 0.05) effects on the liver and heart weight, while methionine levels significantly influenced (*p* < 0.05) the liver and gizzard weight. Finally, it was concluded that if DL-Met and L-Met are included in feed at a standard level, they are equally effective as a source of methionine for broilers.

**Keywords:** methionine; levels; sources; growth; carcass; broiler

#### **1. Introduction**

Methionine has a vital role in the metabolic functioning of animals and humans, which is why it is also known as functional amino acid. Methionine is considered as the first limiting amino acid in broilers and its deficiency may cause reduced growth performance, metabolic disorder and impaired immune system [1,2]. It plays a vital role in the production of energy through the synthesis of protein; it also enhances the broilers' livability, efficiency of feed and growth performance [3–5]. Also, a methyl group that is provided by sulfur-adenosyl methionine is required for many metabolic reactions such as epinephrine, carnitine, choline and creatine synthesis [5,6]. Synthetic sources of methionine (L-methionine (L-Met), DL-methionine (DL-Met) and DL-2 hydroxy–4-(methyl) butanoic acid (LMA)) are included in commercial broiler feed to optimize the dietary level of methionine. However, synthetic methionine is very expensive and the availability of methionine from different synthetic sources is controversial [7]. The availability of methionine from L-Met, DL-Met and LMA is 100%, 99% and 88%, respectively. L-Met is directly used by the animal as a precursor for protein synthesis and metabolized through the trans-sulfuration pathway to produce cysteine and glutathione [8,9]. Methionine hydroxy analog free acid (MHA-FA) is chemically different from DL-Met because it has a hydroxyl group at the asymmetric carbon atom, whereas DL-Met has an amino group. This chemical difference lowers the bio-availability of MHA compared to DL-Met [10,11].

Different levels of methionine in the diet of poultry have been reported by researchers, ranging from 0.3–1.2% during the initial period and 0.3–0.9% during the growth period of poultry. It has been suggested that commercial poultry production does not need more than 0.38% and 0.50% methionine in grower and starter diets, respectively, for the optimum feed efficiency and growth of broilers, although high rates of methionine are necessary to boost the immune system [12]. Reports regarding the dietary level of methionine are controversial. Kalinowski [13] studied the effect of DL-Met levels (0.32%, 0.38%, 0.44%, and 0.50%) with a constant level of L-cystine (0.40%) on slow and fast growing broilers from 3 to 6 weeks of age, and observed that weight gain was not affected and the feed conversion ratio (FCR) was improved with the highest level of methionine. However, Xie et al. [14] reported that increasing levels of DL-methionine (0.285%, 0.385%, 0.485%, 0.585% or 0.685%) resulted in decreased feed intake and weight gain because of higher plasma homocysteine concentration. This might be related to differences in the source of methionine used. Because of deamination of other amino acids during conversion of D-Met to L-Met, different sources of methionine may perform differently. Ribeiro et al. [8] observed that L-Met addition in broiler diet provided better FCR as compared to DL-Met, and MHA. Lui et al. [15] observed that the bioavailability of MHA-FA was greater than DL-Met. Data regarding the use of different methionine sources with varying dietary Met + Cyst levels are scarce, thus the main objective of this study was to evaluate the utilization of methionine from DL-Met and L-Met with different levels of dietary Met + Cyst in broilers.

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

The animal experiment was conducted in accordance with the recommendations and guidelines of the Committee on the Ethics of Animal Experiments of Sargodha University, Sargodha, Pakistan.

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

The experiment was conducted at the poultry research center at the College of Agriculture, University of Sargodha (Sargodha, Pakistan). A total of 450 day-old broiler chickens (Ross 308-mixed sex) with similar body weight were randomly divided into 6 groups in a 3 × 2 factorial arrangement under completely randomized design (CRD). Each group had five replicates (pens) of 15 birds. Six experimental diets were formulated (Tables 1 and 2) by using 3 levels of Met + Cyst (74%, 77% and 80% of digestible lysine) and two sources (DL-Met and L-Met) of methionine. Chickens were reared in suitable pens, under the same managerial, hygienic and environmental conditions. Each diet was randomly allotted to each group for five consecutive successive weeks.


**Table 1.** Ingredients and nutrients of starter diets (1–21 days).

<sup>1</sup> LM 74, 77 and 80 and DLM 74, 77 and 80 indicate inclusion of L-methionine and DL-methionine at the rate of 74%, 77% and 80% of digestible lysine, respectively. ME = Metabolizable energy. \* Provides per kg of diet: 20 MIU Vitamin A; 5 MIU Vitamin D3; 60 g Vitamin E 50;2gVitamin K3;6gVitamin B2; 45 g Vitamin B3; 12 g Vitamin B5; 5 g Vitamin B6; 12.5 g Vitamin B9; 12.5 g Vitamin B12; 275 g Manganese (MnSO4); 150 g Ferrous (FeSO4); 200 g Zn (ZnSO4); 75 g Cu (CuSO4); 75 g Selenium; 4 g Potassium iodide.

#### *2.2. Housing and Management*

The housing area was cleaned and fumigated before the arrival of the chicks. Fumigation was done by using KMnO4 and formalin. Similar management conditions (floor space, temperature, relative humidity, light and ventilation) were provided to all replicates. Feed and water were provided ad libtium.


**Table 2.** Ingredients and nutrients of finisher diets (22–35 days).

<sup>1</sup> LM 74, 77 and 80 and DLM 74, 77 and 80 indicate inclusion of L-methionine and DL-methionine at the rate of 74%, 77% and 80% of digestible lysine, respectively. \* Provides per kg of diet: 20 MIU Vitamin A; 5 MIU Vitamin D3; 60 g Vitamin E 50; 2 g Vitamin K3;6gVitamin B2; 45 g Vitamin B3; 12 g Vitamin B5;5gVitamin B6; 12.5 g Vitamin B9; 12.5 g Vitamin B12; 275 g Manganese (MnSO4); 150 g Ferrous (FeSO4); 200 g Zn (ZnSO4); 75 g Cu (CuSO4); 75 g Selenium; 4 g Potassium Iodide.

#### *2.3. Growth Performance*

Feed intake and weight gain were recorded through the test periods (the starter period corresponds to 1–21 days of age, the finisher period to 22–35 days of age, and the overall period to 1–35 days of age). The feed intake was calculated by the difference between feed supplied and refusal at each period. The feed conversion ratio (FCR) was calculated by dividing feed intake by weight gain [16].

#### *2.4. Carcass Evaluation and Serum Homocysteine*

At the end of the experiment, two birds of average body weight from each replicate were randomly selected and slaughtered to determine the carcass characteristics (live weight, carcass weight, eviscerated weight, chest weight and thigh weight) and weight of visceral organs (liver, heart and gizzard).

#### *2.5. Blood Sampling*

Blood samples (*n* = 5) were collected from the wing vein at 35 days of age without anticoagulant for serum separation. Samples were centrifuged at 1435× *g* for 5 min at 4 ◦C to obtain clear sera, which was collected for homocysteine analysis using chromatographic assay [17].

#### *2.6. Statistical Analysis*

Data collected were analyzed by using the analysis of variance technique in a 3 × 2 factorial arrangement under CRD. Means of all parameters were separated by using Tukey's test with the assistance of software (SAS® 9.3 Software).

#### **3. Results**

#### *3.1. Growth Performance*

The combined effect of L-Met and DL-Met significantly affected (*p* < 0.05) the weight gain of broilers in the starter period. Neither the source nor levels of methionine influenced (*p* > 0.05) the feed intake, weight gain and FCR of broilers during the starter, finisher or the whole period (Table 3).

**Treatments Feed Intake (g) Weight Gain (g) FCR (g Feed**/**g Gain) Source** × **Level 1–21 days 22–35 days 1–21 days 22–35 days 1–21 days 22–35 days** LM 74 1321.5 2105.0 <sup>b</sup> 934.8 <sup>b</sup> 1282.4 1.4191 1.9386 LM 77 1342.8 2149.1 a,b 1011.8 <sup>a</sup> 1209.1 1.3291 1.7873 LM 80 1338.2 2154.9 <sup>a</sup> 1023.4 <sup>a</sup> 1282.5 1.3076 1.8108 DLM 74 1335.1 2077.3 <sup>b</sup> 914.1 <sup>b</sup> 1129.0 1.3293 1.6395 DLM 77 1320.8 2097.3 <sup>b</sup> 1005.7 <sup>a</sup> 1188.9 1.4054 1.8183 DLM 80 1331.5 2150.7 <sup>a</sup> 1008.1 <sup>a</sup> 1214.9 1.3225 1.6195 SEM 5.857 15.630 18.002 68.759 0.0262 0.0142 Source LM 1334.2 2134.9 990.00 1258.0 1.3524 1.8455 DLM 1329.1 2109.8 984.97 1177.6 1.3519 1.6925 SEM 3.382 9.0243 10.393 39.698 0.0151 0.0601 Level 74 1328.3 2123.2 970.3 1205.7 1.3742 1.7891 77 1331.8 2129.9 976.4 1199.0 1.3672 1.8028 80 1334.8 2114.0 1015.8 1248.7 1.3150 1.7151 SEM 4.142 11.052 12.72 48.620 0.0185 0.0737 *p*-Values Source × Level NS \* \* NS NS NS Source NS NS NS NS NS NS Level NS NS NS NS NS NS

**Table 3.** Effect of different sources and levels of methionine plus cystine on growth performance of broilers during the starter and finisher phases.

LM 74, 77 and 80 and DLM 74, 77 and 80 indicate inclusion of L-methionine and DL-methionine at the rate of 74%, 77% and 80% of digestible lysine, respectively. a,b Means sharing different superscripts differ significantly (*p* < 0.05). NS = Non-significant (*p* > 0.05). \* = Significant (*p* < 0.05).

Data presented in Table 3 indicate that the interaction between source and levels of methionine had a significant effect (*p* < 0.05) on the feed intake and weight gain during the finisher and starter period, respectively. The results regarding feed intake in the finisher period revealed that the best values (*p* < 0.05) were achieved at an 80% ratio of L-Met. However, no significant differences in feed intake were observed between LM80 and DLM80. During the starter period, the highest values of weight gain (*p* < 0.05) were achieved with a 77% or 80% ratio of L-Met and DLM in comparison with a ratio of 74%.

As shown in Table 4, the interaction between sources and levels of methionine did not influence (*p* > 0.05) the feed intake, weight gain and FCR during the overall phase of growth.


**Table 4.** Effect of different sources and levels of methionine plus cystine on growth performance of broilers during overall experimental period (1–35 days).

LM 74, 77 and 80 and DLM 74, 77 and 80 indicate inclusion of L-methionine and DL-methionine at the rate of 74%, 77% and 80% of digestible lysine, respectively. NS = Non-significant (*p* > 0.05).

#### *3.2. Carcass Characteristics*

The source × level of methionine had a significant (*p* < 0.05) effect on thigh weight and nonsignificant (*p* > 0.05) effect on live weight, carcass weight, after skin removal, eviscerated weight and chest weight (Table 5). The source of methionine had a non-significant effect (*p* > 0.05) on the carcass characteristics of broilers. Level of methionine had a significant (*p* < 0.05) effect on carcass weight, chest weight and thigh weight and a non-significant (*p* > 0.05) effect on live weight, after skin removal and eviscerated weight.

#### *3.3. Weight of the Visceral Organs*

As presented in Table 6, the interaction between sources and levels of methionine had a significant (*p* < 0.05) effect on liver weight, while the effect on heart and gizzard weight was non-significant (*p* > 0.05). On the other hand, with regard to liver weight, there was no significant difference between LM80 and DLM80. The sources of methionine had a significant (*p* < 0.05) effect on the liver and heart weight while the effect on the gizzard weight was non-significant (*p* > 0.05), since LM increases liver and heart weight when compared to DLM. Liver and gizzard weights were gradually increased as the levels of methionine increased from 74% to 77% to 80% (*p* < 0.05).



LM 74, 77 and 80 and DLM 74, 77 and 80 indicate inclusion of L-methionine and DL-methionine at the rate of 74%, 77% and 80% of digestible lysine, respectively. a,b Means sharing different superscripts differ significantly (*p* < 0.05). NS = Non-significant (*p* > 0.05). \* = Significant (*p* < 0.05).



LM 74, 77 and 80 and DLM 74, 77 and 80 indicate inclusion of L-methionine and DL-methionine at the rate of 74%, 77% and 80% of digestible lysine, respectively. a,b Means sharing different superscripts differ significantly (*p* < 0.05). NS = Non-significant (*p* > 0.05). \* = Significant (*p* < 0.05).

#### *3.4. Serum Homocysteine*

The combined effect of L-Met and DL-Met had no (*p* > 0.05) effect on serum homocysteine level (Table 6). Neither source nor level of methionine had a significant (*p* > 0.05) influence on serum homocysteine level.

#### **4. Discussion**

The body weight gain of broilers was significantly increased (*p* < 0.05) in the starter period due to the combined effect of L-Met and DL-Met. These findings of growth performance confirmed the reports of earlier researchers, Ahmed and Abbas [18] who studied the effect of dietary methionine levels above the nutrient requirements of poultry (NRC) [19] recommendation on performance and carcass traits in broiler birds. Four dietary levels of methionine, 0%, 100%, 120% and 130% of the NRC recommendation were used. Weight gain was significantly higher by 110% and 130% of NRC methionine than that of the control diet. Better weight gain with L-Met than DL-Met is also supported by the findings of Katz and Baker [20] who observed that L-Met provided better and more efficient weight gain than D-Met or DL-Met. At a level of supplementation near the requirement, equal efficiency was attained because L-Met is 100% absorbed in the body as compared to DL-Met.

The feed intake and FCR in the starter period remained unchanged by the combined effect of L-Met and DL-Met; this is supported by other researchers [21,22] who have also observed no significant difference in feed intake and FCR due to supplementation of L-Met and DL-Met because when the diet was supplemented with methionine alone, some methionine was converted to cysteine. The presence of small excess amounts of cysteine depressed the feed intake without a proportional reduction in weight gain because the presence of cysteine reduces the metabolic damage. The combined expression of methionine and cystine as sulfur amino acids restricts the efficient use of feedstuff, and also results in inconsistency in requirements. The conversion of methionine to cysteine was nonequivalence [23] and cystine oxidation occurs when it increases beyond the limit, resulting in inefficiency in the accurate estimate of the requirement of individuals. The replacement value for cysteine in broilers that are 3–6 weeks old is 52% [24], however, Wheeler and Latshaw [25] reported 43% and NRC [18] reported 47% as the recommended value. It has been estimated that about 10% of dietary protein is diverted by the broiler in the first 6 weeks to the formation of feathers [26]; this process is high in cysteine [27]. Engler et al. [28] reported that less cysteine is required by male broiler genotypes that are low feathering (L/k+) after the age of 3 weeks, and this results in a 15% advantage in the feather weight of the k+/k<sup>+</sup> bird at the age of 48 days [29]. If the nutrients are stored in the feathers then it will not be available for other purposes; while the muscle of breast nourishment rate is reduced by the continuous production of keratin, which limits the supply of nutrients [30]. Therefore, the cystine deficiency results in the reduction in the recovery of breast meat and also decreases the extent of feathering. Our finding of no effects of the sources of methionine on the starter phase of broilers is supported by other researchers [22,31] who observed that L-Met and DL-Met did not affect growth performance due to conversion of DL-Met into L-Met. It seems that lower metabolization of D–amino acid due to the lower amount of D–amino acid oxidase in young broilers may reduce the utilization of higher amounts of DL-Met, which leads to metabolic stress and inhibition of body weight gain in the starter phase. Our findings regarding unaltered feed intake, weight gain and FCR of broilers in the starter period due to different levels of methionine are similar to those of other researchers [32,33], which might be attributed to the satisfaction of methionine requirements at the lower standard level.

The results regarding feed intake of broilers in the finisher period indicated significant differences (*p* < 0.05) between L-Met and DL-Met. This finding confirmed the reports of earlier researchers [34] who observed better feed efficiency of chicks fed an L-Met diet as compared to DL-Met because the supplementation of either L-Met or DL-Met have beneficial effects on villus development in association with increased glutathione production and levels of total antioxidant capacity, and reduced protein oxidation in the duodenum. Supplementation of L-Met has a better effect on redox status and development of the gut of young chicks as compared with DL-Met.

Our finding of no changes in the weight gain of broilers in the finisher period by the combined effect of L-Met and DL-Met is the similar to other researchers [33,35,36] who observed that L-Met or DL-Met did not significantly influence the weight gain of broilers in the finisher period. This was because when large quantities of methionine are added in the feed, excess methionine is converted into homocysteine and higher amounts of homocysteine in the body reduce the body weight of broilers [37]. No significant differences (*p* > 0.05) were observed by different type of methionine on feed intake, weight gain, and FCR of broilers in the finisher period. This finding confirms the reports of earlier researchers [32,33,38]. Because d-amino acid oxidase, the key enzyme that converts D-Met to L-Met, exists only in the liver and kidney, D-Met is not utilized directly by the cells of the gastrointestinal tract until it is converted to L-Met either in the liver or kidneys. Research has also shown that the expression of this enzyme is very low for young animals. Therefore, L-Met is the only biologically functional form of methionine that is readily utilized by the intestinal cells of young animals. The quantity of methionine had no effect (*p* > 0.05) on the performance of broilers in the finisher period. This finding confirms the reports of earlier researchers [21,22] who observed that levels of methionine had no effect on feed intake, average daily gain, feed efficiency and FCR of broilers because DL-Met is readily converted into the L-isomer by the animal. Also, our finding that L-Met and DL-Met had no combined effect on feed intake, weight gain and FCR during the overall phase of broiler growth is supported by other researchers [33,35,36] who also observed that L-Met or DL-Met did not influence the feed intake, weight gain and overall FCR of broilers. Zhang [7] studied the effect of different dietary methionine source supplementation including L-Met, DL-Met and DL-2-hydroxy-4-(methylthio) butanoic acid (DL-HMTBA) on growth performance. He observed no differences among L-Met, DL-Met and DL-HMTBA for weight gain and feed efficiency. No effect of methionine sources on overall growth of broiler has also been found by other researchers [21,22,31] who observed that L-Met and DL-Met had no effect on the overall phase of broilers.

The results regarding thigh weight indicated that the highest value (*p* < 0.05) was achieved at the 80% ratio of L-Met, while the values achieved at the 80% ratio of DL-Met were lower than L-Met. The significant effect of L-Met and DL-Met on the thigh weight of broilers is supported by other researchers [39,40] who also observed that methionine sources improved the thigh weight of broilers because methionine has a role in the synthesis of creatinine in thigh muscles. No differences (*p* > 0.05) were observed for different types of amino acid on the carcass characteristics of broilers. This finding confirmed the reports of earlier researchers [31,41] who observed that the type of methionine had no effect on live weight, carcass weight, after skin removal, eviscerated weight and chest weight because L-Met is directly absorbed in the body and DL-Met, MHA is first converted into L-Met and then absorbed in the body. Also, Drazbo et al. [42] found that the source of dietary methionine had no effect on carcass yield or breast muscle quality.

The levels of methionine had a significant (*p* < 0.05) effect on carcass weight, chest weight and thigh weight. This finding confirms previous studies [43,44], where levels of methionine had a significant influence on the thigh weight, chest weight and carcass weight. This is because D-Met is oxidatively converted to α-ketoanalogues of L-Met, 2-keto-4(methylthio) butanoic acid (KMB) by the enzyme D-amino acid oxidase, which is a proximal oxidase containing flavin adenine dinucleotide (FAD) as a cofactor. Then, KMB is converted into L-methionine by the transfer of nitrogen from the donor amino acid, which is catalyzed by ubiquitous transaminases. In chickens, many amino acids like glutamic acid, arginine, isoleucine and alanine are used for transamination of KMB [7,11].

Results regarding liver weight indicated that the highest value (*p* < 0.05) was observed at the 80% ratio of L-Met while the values achieved at the 80% ratio of DL-Met were lower than L-Met. The combined effect of L-Met and DL-Met had a significant (*p* < 0.05) effect on the liver weight of broilers. Our finding of unaltered heart and gizzard weight due to the combined effect of L-Met and DL-Met is supported by other researchers [45,46] who observed that DL-Met and herbal methionine had no significant effect on the carcass yield, breast meat and eviscerated weight of broilers. The significant differences observed between the heart and liver weight of birds fed different types of

amino acid are corroborated by Ahmed and Abbas [45] who observed that dietary supplementation of methionine significantly affected the liver and heart weight. Ribeiro et al. [35] observed that DL-Met had a significant effect on the gizzard weight in heat stress conditions, which is similar to our findings of differences in gizzard weight due to the type of amino acid.

Unaltered homocysteine due to the combined effect of L-Met and DL-Met in the diet of broilers was supported by the findings of Pillai et al. [47]. They observed that dietary methionine had no effect on hepatic homocysteine remethylation. No effect of the source of methionine on serum homocysteine level of broilers confirms the findings of Harter and Baker [48] who observed that methionine was stored in the plasma of birds fed excess methionine, but plasma levels of homocysteine, cystathionine, and cystine remained unchanged. Haulrik et al. [49] found that high methionine and high protein diet did not significantly increase homocysteine concentration as compared to low methionine and low protein diet, which confirms our findings that there was no change in serum homocysteine level due to different amino acid sources.

On the basis of these results, it may be concluded that if DL-Met and L-Met are included at a standard level in feed, they are equally effective as a source of methionine for broilers. However, better carcass traits may be achieved if Met + Cyst is added at the rate of 80% of digestible lysine.

**Author Contributions:** Data curation, A.U.R. and M.E.A.E.-H.; Formal analysis, M.A.A.-L.; Investigation, M.A. (Muhammad Arif); Methodology, M.M.H.; Resources, S.I.O.; Software, A.E.T.; Visualization, M.A. (Mahmoud Alagawany) and A.A.A.; Writing—review & editing, M.E.A.E.-H. and S.S.E.

**Acknowledgments:** This research was funded by the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University through the Fast-Track Research Funding Program.

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

#### **References**


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

*Article*

### **Dietary Betaine Improves Intestinal Barrier Function and Ameliorates the Impact of Heat Stress in Multiple Vital Organs as Measured by Evans Blue Dye in Broiler Chickens**

**Majid Shakeri 1, Jeremy James Cottrell 1, Stuart Wilkinson 2, Weicheng Zhao 1, Hieu Huu Le 1, Rachel McQuade 3, John Barton Furness <sup>4</sup> and Frank Rowland Dunshea 1,\***


Received: 27 November 2019; Accepted: 21 December 2019; Published: 23 December 2019

**Simple Summary:** Heat stress alters the normal physiological status, compromising the function of organs such as the small intestine. However, evidence exists of a wider distribution of organ dysfunction, stemming from factors such as a reduction in blood flow due to redistribution to the skin for increased radiant heat loss to the environment. Simultaneously, assessing organ dysfunction at multiple locations presents technical difficulties, and hence studies are lacking. Therefore, the aim of this experiment was to determine the pattern of Evans Blue Dye distribution as a cost-effective indicator of organ dysfunction in HS chickens supplemented with betaine. The results showed that Evans Blue Dye concentration increased in the kidney and muscle during heat stress, while such concentration was reduced with betaine. Therefore, betaine could improve the broiler's tolerance to heat stress, and Evans Blue Dye may be a useful tool for investigating the effects of heat stress on broiler organ dysfunction.

**Abstract:** In a 2 × 2 factorial design, 60 male Ross-308 broilers were fed either a control or 1 g/kg betaine diet and housed under thermoneutral (TN) or heat stress (HS) conditions. Broilers were acclimated to diets for 1 week under TN (25 ◦C), then either kept at TN or HS, where the temperature increased 8 h/day at 33 ◦C and 16 h/day at 25 ◦C for up to 10 days. Respiration rate (RR) was measured at four time points, and on each of 1, 2, 3, 7 and 10 days of HS, 12 broilers were injected with 0.5 mg/kg of Evans Blue Dye (EBD) solution to quantify regional changes in tissue damage. Betaine was quantified in tissues, and ileal damage was assessed via morphometry and transepithelial resistance (TER). Heat stress elevated RR (*p* < 0.001) and resulted in reduced villous height (*p* = 0.009) and TER (*p* < 0.001), while dietary betaine lowered RR during HS (*p* < 0.001), increased betaine distribution into tissues, and improved ileal villous height (*p* < 0.001) and TER (*p* = 0.006). Heat stress increased EBD in the muscle and kidney of chickens fed the control diet but not in those receiving betaine. Overall, these data indicate that supplemented betaine is distributed to vital organs and the gastrointestinal tract, where it is associated with improved tolerance of HS. Furthermore, EBD markers help reveal the effects of HS on organs dysfunction.

**Keywords:** heat stress; betaine; Evans blue dye; physiological responses; broiler chickens

#### **1. Introduction**

Due to the impacts of a changing climate, heat stress (HS) is of increasing concern for animal production. Broilers are sensitive to HS due to the presence of feather coverage, increased selection for muscling, and because they lack sweat glands. Heat stress compromises efficient broiler production in part by reducing voluntary feed intake. However, some studies have shown that the reduction in feed intake does not fully explain the reduction in growth rate [1]. The reasons for this include factors such as altered endocrine status [2,3]. Furthermore, increased evaporative heat loss by panting and radiant heat loss by blood flow redistribution also compromise efficient growth. As the distribution of blood flow to the skin to facilitate radiant heat loss relies on a commensurate reduction in blood flow elsewhere, continued HS can precipitate dysfunction in affected organs due to reduced nutrient delivery and removal of metabolic by-products. Heat stress compromises intestinal barrier integrity in broiler chickens and other species [4–6], presumably by splanchnic blood restriction, and that can lead to bacterial translocation and a systemic inflammatory response [7,8].

While investigations into the etiology of heat stroke have confirmed the central role of gastrointestinal tract (GIT) damage [9], it is apparent that heat stroke has a wider pattern of organ damage than the GIT alone, with increased incidences of renal and liver injury [10,11]. It is likely that compromised hepatic and renal function also contribute to reduced growth efficiency in broilers and other production species during heat exposure. While quantifying systemic changes in organ damage is technically difficult, this may in part be overcome by using blood-borne markers such as Evans Blue Dye (EBD). Within the blood, EBD binds tightly to plasma albumin and is used as an exogenous marker of plasma volume [12]. Following inflammation or tissue injury, EBD extravasates into the surrounding tissue where it may be quantified as a marker of tissue damage [13,14]. Identifying localised sites of stress is a useful strategy for the development of amelioration strategies, as has been demonstrated with the supplementation of antioxidants in mitigating the impact of HS on intestinal permeability [4]. Therefore, the aims of this experiment were to investigate changes in EBD extravasation in the broiler during HS. Furthermore, the organic osmolyte betaine has been demonstrated to be an effective supplement for ameliorating the effects of HS. It protects cells against osmotic inactivation, improves water retention of cells [15], reduces core body temperature by reducing the activity of the ion pumps required for osmoregulation, allowing more energy for growth [16–18], and acts as a methyl donor for homocysteine remethylation [19]. Therefore, the secondary aim of the experiment was to determine whether supplementation of betaine ameliorated the effects of HS and altered the pattern of EBD extravasation.

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

#### *2.1. Animal Ethics*

The experiment was approved by The University of Melbourne, Australia (Protocol no. 1814704.1).

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

Four week-old male Ross-308 chickens (*n* = 60) were obtained from a local commercial farm (Turosi, Bannockburn, Victoria, Australia) located within 2 h driving distance from The University of Melbourne. Chickens were randomly allocated to 4 equally sized pens (1.9 × 3.4 m) in two environmentally controlled rooms. All pens were covered with wood shavings (8–10 cm deep) with 4 drinkers and 4 feeders for each pen. The chickens were allowed to acclimate to the pens and facility for 7 days at a constant 25 ◦C (thermoneutral, TN). From the arrival in the facility, chickens were given either a standard finisher control diet (CON, *n* = 30), which was formulated as a commercial finisher diet (Feedworks, BESTMIX, CP 21.3% and 12.65 MJ ME/kg) and exceeded nutrient requirements [20], or a CON plus 1 g/kg betaine (Betafin S1, DuPont, Marlborough, UK) diet (BET, *n* = 30). After 7 days acclimation, the temperature in one room increased to 33 ◦C for 8 h/day (9 a.m.–5 p.m., 16 h/day 25 ◦C) to induce heat stress (HS) for 10 days while the alternate room was maintained under TN conditions. The relative humidity for both rooms was between 40–55% during the experiment. Light was provided 20 h/day, and chickens had ad libitum access to feed and water during the period of experiment. On each of days 1, 2, 3, 7 and 10 of environmental treatment, 3 chickens from each pen (each diet × temperature group) were assessed for EBD extravasation.

#### *2.3. Physiological Responses*

Respiration rate (RR) was measured at 11:00 a.m. after chickens had been exposed to 1, 3, 7 and 10 days HS (corresponding to 8, 10, 14 and 17 days in the facility and consuming the experimental diets). Chickens were filmed with a cell phone (iPhone 7, Apple Inc., Cupertino, CA, USA) and then the number of breaths taken over a 20-s period was quantified and then expressed as breaths per min.

#### *2.4. Evans Blue Dye Injection, Slaughter and Tissue Collection*

Chickens were injected with 0.5 mg/kg of an EBD solution (1.5% *w*/*w* EBD, Sigma, Aldrich, MO, USA in 0.9% saline solution) into the brachial vein on days 1, 2, 3, 7 and 10 of the environmental treatment. Three chickens were briefly removed from each pen for the injection, then returned to their designated rooms for 2 h. Chickens were then removed from the rooms, electrically stunned (Mitchell Engineering Food Equipment Pty Ltd., Queensland, Australia), placed in an inverted restraining funnel, slaughtered by severing the major blood vessels in the neck and then exsanguinated. Tissue samples were collected from the ileum, jejunum, muscle, liver, spleen and kidney for measuring EBD concentration. Furthermore, about 5 cm of ileum tissue and a piece of *psoas major* (breast muscle) were collected for morphometric analysis.

#### *2.5. Evans Blue Dye Extraction and Qualification*

Evans Blue Dye concentration was measured in the collected tissues according to a published method [13]. Tissue samples were dried in an oven at 70 ◦C for 48 h, then EBD extracted from 100 mg of dried and pulverised tissue with 500 μL formamide (Sigma, Aldrich, St Louis, MO, USA) and incubated at 55 ◦C for 24 h. Samples were then centrifuged for 15 min at 14,000× *g* and 4 ◦C, and the A610 of 200 μL of supernatant quantified in duplicate against standards using Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific Inc, Waltham, MA, USA). The obtained results were expressed as ng EBD per mg tissue dry weight.

#### *2.6. Intestinal Transepithelial Electrical Resistance*

Intestinal transepithelial electrical resistance (TER) was measured according to a previously published method [4]. Sections of ileum were collected immediately after euthanasia on days 3, 7 and 10 of the environmental challenge. After collection, sections were placed in chilled phosphate buffered saline, then transferred to Krebs solution (pH 7.4). The ileal sample was then opened along the mesenteric border and the external muscle was removed. The remaining layers were mounted onto a round slider (0.71 cm2) and placed into a two-part Ussing chamber (EasyMount Diffusion Chambers, Physiologic Instruments) and 5 mL Krebs' solution was added to each side. On the mucosal side, the 11.1 mM glucose was replaced with mannitol. Voltage and I*sc* readings were acquired using a PowerLab amplifier and recorded using LabChart®5 (AdInstruments Pty Ltd., Lexington, New South Wale, Australia). Tissue was left to equilibrate for 20 min before clamping the voltage to 0 V, and epithelial resistance was determined by administering five 2-s pulses of 2 mV. The TER was calculated by Ohm's law and multiplied by the exposed area.

#### *2.7. High-Performance Liquid Chromatography Analysis*

Betaine was quantified in ileum, kidney and spleen following derivatisation with bromophenacyl bromide catalysed with 18-crown-6 and the bromophenacyl esters quantified by High-Performance Liquid Chromatography [21]. Briefly, 100 mg of pulverised tissue samples were homogenised with a bead beater (AnytimeLabTrader LLC, Fallbrook, CA, USA) in 1 mL tris buffer (1 M, pH 7) then centrifuged at 10,000× *g* for 15 min. to obtain supernatant. The obtained supernatant was added to monopotassium phosphate (100 mmol/L) and derivatisation solution containing 4-bromophenacyl bromide (50 mmol/L) and 18-crown-6 (2.5 mmol/L) in acetonitrile and vortex mixed. The samples were heated at 80 ◦C in a block heater for 1 h, cooled to room temperature before filtering into a glass High-Performance Liquid Chromatography. The A254 of the bromophenacyl esters of betaine were then quantified versus standards using a High-Performance Liquid Chromatography following a 10 μL injection (Model 2998, Waters, Milford, MA, USA).

#### *2.8. Morphometric Analysis*

Tissue samples were collected on days 1, 2, 3, 7 and 10 of the environmental challenge. The midpoint of the ileal section, and *psoas major* were excised and transferred in 10% formalin (Sigma, Aldrich, St Louis, MO, USA) and fixed in paraffin wax [22]. Slides were prepared using 8 μm sections, stained by hematoxylin and eosin, and the villous height, crypt depth, ileum seromuscular layer and *psoas major* fibre diameter (width) were quantified using a light microscope equipped with a camera (Leica, ICC50 W, Wetzlar, Germany), and analysed with ImageJ software [23]. The distance from the tip of the villous to the villous crypt junction represents the villous height, crypt depth was defined as the depth of the invagination between adjacent villous, and seromuscular layer was the smooth muscular layer located under the crypt. A total of 10 samples per section were quantified.

#### *2.9. Statistics Analysis*

All data were analysed using ANOVA for the main and interactive effects of temperature and diet (CON vs. BET) and time (1, 2, 3, 7 and 10 days) using Genstat version 18 (VSNi Ltd., Hemel Hempstead, UK). Statistical significance was considered at *p* ≤ 0.05, and when achieved, a Duncan's multiple range post-hoc test was performed to differentiate between treatment groups, which were then labelled with differing alphabetic superscripts. Where skewed data occurred, normality was restored following a Log10 transformation and analysed as above. The predicted means were then back-transformed (10×) and presented in tables in parentheses. The replication for the main effects of temperature and diet were 30 chickens, respectively. The replication for the interaction between temperature and diet was 20 chickens per group and for temperature × diet × time was 3 chickens per treatment/time.

#### **3. Results**

#### *3.1. Respiration Rate*

Heat stress increased RR at each time point measured (*p* < 0.001, Figure 1). Dietary BET supplementation reduced RR during HS (*p* < 0.001) at each time point (Figure 1), but there was no effect of dietary BET under TN conditions. No main or interactive effects of time on RR were observed.

**Figure 1.** Respiration rate in broilers fed either a control diet (CON, round symbols) or betaine supplemented diet (BET, triangle symbols) after 1, 3, 7 and 10 days of being exposed to either thermoneutral (TN, filled symbols) or heat stress (HS, open symbols) conditions. The standard error of the difference for Temperature × Diet × Day is displayed on the data for the chickens fed the CON diet under TN conditions.

#### *3.2. Evans Blue Dye Distribution*

Evans Blue Dye concentration was quantified in muscle, liver, ileum, jejunum, spleen and kidney (Table 1). Heat stress increased EBD concentrations in the kidney (74 vs. 99 ng/mg, *p* = 0.007) but reduced concentrations in the spleen (213 vs. 162 ng/mg, *p* = 0.024). Dietary BET decreased EBD concentrations in the jejunum (94 vs. 76 ng/mg, *p* = 0.043), ileum (100 vs. 76 ng/mg, *p* = 0.028) and kidney (113 vs. 61 ng/mg, *p* < 0.001). There were significant interactions between diet and temperature in muscle and kidney such that HS increased EBD concentrations in chickens consuming the CON diet but not in those on the BET supplemented diet (Figure 2A,B). There was also an interaction between temperature and diet in the spleen where BET increased EBD concentrations under TN conditions, but not during HS (Figure 2C). There were no main or interactive effects of temperature, diet or time for EBD concentration in the liver, while interactions were observed for diet, temperature and time in the kidney, jejunum and ileum (Table 1). These interactions typically reflected that BET under HS conditions decreased tissue EBD concentrations. In the kidney, EBD concentration was higher under HS CON than all other groups after 1 day of HS. In the jejunum, this was observed after 7 days and in the ileum at day 10.


**Table 1.** Effects of a control diet (CON) or dietary betaine (BET) on Evans Blue Dye distribution in broilers housed under thermoneutral (TN) or heat stress (HS) conditions for 1, 2, 3, 7 and 10 days.


**Table 1.** *Cont*.

<sup>1</sup> <sup>+</sup> *p* < 0.10; \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001. Other main and interactive effects *p* > 0.10. Differing superscripts within a column denotes significant (*<sup>p</sup>* <sup>&</sup>lt; 0.05) differences for D × <sup>T</sup> × Day on a single day of the experiment. <sup>2</sup> Due to skewed data the values were Log10 transformed before statistical analysis. Back transformed means are presented in parentheses.

**Figure 2.** Changes in the distribution of Evans Blue Dye in (**A**) psoas major, (**B**) kidney, and (**C**) spleen in broilers during a thermoneutral (TN) vs. heat stress (HS) environmental challenge. Broilers were fed either a control diet (CON) or betaine diet (BET), and the mean represents the main effect of 5 time points (1, 2, 3, 7 and 10 day challenge). Means with differing superscripts denote *p* < 0.05. Refer to Table 1 for full interactive effects.

#### *3.3. Transepithelial Electrical Resistance*

Ileal TER was quantified after 3, 7 and 10 days of environmental challenge (Table 2; Figure 3). Overall, TER increased by BET (182 vs. 235 Ω.cm2, *p* = 0.006) and reduced by HS (256 vs. 161 Ω.cm2, *p* < 0.001). Ileal TER declined with time over the course of the experiment (*p* < 0.001). An interaction between HS and time occurred such that the TER of TN chickens at day 3 was almost double that of HS chickens (346 vs. 186 Ω.cm2, *p* = 0.049). An interaction between diet, temperature and day was observed, such than a reduction in ileal TER had taken place by day 10. Alternatively, the TER from HS BET chickens was nearly double than HS CON at this time (93 vs. 161 Ω.cm2) (Table 2).

**Table 2.** Effects of a control diet (CON) or dietary betaine (BET) on ileal transepithelial electrical resistance and ileal morphology in broilers under thermoneutral (TN) or heat stress (HS) conditions for 1, 2, 3, 7 and 10 days.


<sup>1</sup> \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001. Other main and interactive effects *p* > 0.10. Differing superscripts within a column denotes significant (*p* < 0.05) differences for D × T × Day on a single day of the experiment. <sup>2</sup> Due to logistical constraints, ileal transepithelial electrical resistance was only measured on days 3, 7 and 10. <sup>3</sup> Due to skewed data, the values were Log10 transformed before statistical analysis. Back transformed means are presented in parentheses.

**Figure 3.** Transepithelial electrical resistance (TER) changes in broilers during a thermoneutral (TN) vs. heat stress (HS) environmental challenge. Broilers were fed either a control diet (CON) or betaine diet (BET), and the mean represents the main effect of 3 time points (3, 7 and 10 day challenge). Refer to Table 2 for full interactive effects.

#### *3.4. Morphometric Analysis*

Ileal villous height decreased by HS (826 vs.779 μm, *p* = 0.009) and increased by dietary BET (741 vs.864 μm, *p* < 0.001) (Table 2; Figure 4). There was no main effect of HS on villous area, whereas dietary BET increased villous surface area (97 vs. 136 μm2, *p* < 0.001) (Table 2; Figure 5A). However, there were interactions such that villous surface area reduced over time particularly in those chickens exposed to HS. There was no main effect of HS on crypt depth, whereas it increased by dietary BET (152 vs. 192 μm, *p* < 0.001) (Table 2). However, there were interactions such that crypt depth decreased over time particularly in those chickens exposed to HS. The seromuscular layer depth decreased by HS (227 vs. 174 μm, *p* < 0.001) and increased by dietary BET (161 vs. 240 μm, *p* < 0.001) (Table 2; Figure 5B). However, there were interactions such that seromuscular layer depth increased over time in those chickens that were consuming the BET diet and housed under TN conditions, whereas it declined in those chickens exposed to HS (Table 2). Heat stress reduced psoas major fibre diameter (227 vs. 174 μm, *p* < 0.001), whereas it increased by dietary BET (161 vs. 240 μm, *p* < 0.001) (Figure 6). However, there were interactions such that psoas major fibre diameter was initially higher in chickens fed dietary BET and then increased over time in those chickens consuming the BET diet and housed under TN conditions, whereas it declined in those chickens exposed to HS. Psoas major fibre diameter remained constant in those chickens consuming the CON diet.

**Figure 4.** Representative photomicrographs of the ileum after 10 days of the experiment from broilers fed a control diet (CON, **A** and **C**) and betaine (BET, **B** and **D**) on villous height under thermoneutral (TN, **A** and **B**) or after 10 days being exposed to heat stress (HS, **C** and **D**).

**Figure 5.** Ileal morphology: (**A**) villous surface area and (**B**) seromuscular layer in broilers during a thermoneutral (TN) vs. heat stress (HS) environmental challenge. Broilers were fed either a control diet (CON) or betaine diet (BET) and the mean represents the main effect at 5 time points (1, 2, 3, 7 and 10 day challenge). Means with differing superscripts denote *p* < 0.05. Refer to Table 2 for full interactive effects.

**Figure 6.** Psoas major fibre diameter in broilers fed either a control diet (CON, round symbols) or betaine supplemented diet (BET, triangle symbols) after 1, 3, 7 and 10 days of being exposed to either thermoneutral (TN, filled symbols) or heat stress (HS, open symbols) conditions. Psoas major fibre diameter increased over the course of experiment in TN BET (*p* < 0.001) while HS groups reduced HS CON. The standard error of the difference for Temperature × Diet × Day is displayed on the data for the chickens fed the CON diet under TN conditions.

#### *3.5. Betaine Distribution*

Heat stress decreased betaine concentrations in the ileum (117.1 vs.84.7 μmol/g, *p* < 0.001), whereas they increased in the kidney (59.6 vs.74.2 μmol/g, *p* < 0.001) and spleen (59.9 vs. 64.2 μmol/g, *p* = 0.02) (Table 3; Figure 7). Overall, chickens supplemented with BET had higher betaine concentrations in the ileum (83.2 vs. 118.6 μmol/g, *p* < 0.001), kidney (60.5 vs. 73.4 μmol/g, *p* < 0.001) and spleen (57.7 vs. 66.4 μmol/g, *p* < 0.001) than their control counterparts. However, there were interactions such that ileal betaine concentrations increased over time under TN conditions particularly in those chickens consuming the BET diet. Conversely, for the kidney and spleen, the increase in tissue betaine concentrations in response to dietary BET were greater during HS than under TN conditions (Figure 7).


**Table 3.** Effects of a control diet (CON) or dietary betaine (BET) on tissue betaine concentration in broilers housed under thermoneutral (TN) or heat stress (HS) conditions for 1, 3, 7 and 10 days.

<sup>1</sup> \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001. Other main and interactive effects *p* > 0.10. Differing superscripts within a column denotes significant (*p* < 0.05) differences for D × T × Day on a single day of the experiment.

**Figure 7.** Changes in the distribution of betaine: (**A**) ileum, (**B**) kidney, and (**C**) spleen in broilers during thermoneutral (TN) vs. heat stress (HS) environmental challenge. Broilers were fed either a control diet (CON) or betaine diet (BET), and the mean represents the main effect of 5 time points (1, 2, 3, 7 and 10 day challenge). Means with differing superscripts denote *p* < 0.05. Refer to Table 3 for full interactive effects.

#### **4. Discussion**

Consistent with other experiments in this area [24], HS compromised the small intestinal mucosa, as evidenced by reduced villous height and crypt depth. Furthermore, as in other experiments HS reduced the TER of the small intestinal mucosa [6]. The current results indicated that HS did not influence EBD concentration in the jejunum or ileum suggesting there no discernible extravasation in the small intestine during HS. Taken together, these data support that the reduction in villous height observed was not due to an ablation of the villous, as observed in some other investigations into the effect of HS on the intestinal mucosa of pigs [25]. Alternatively, the reduction in crypt depth and absence of increases in EBD indicates that the reduction in villous height was likely due to reduced crypt cell proliferation and would be consistent with reductions in splanchnic blood flow observed in HS layers and other species.

Blood flow redistribution between organs in HS animals were quantified using radioactive microspheres to determine localised changes in capillary blood flow (CBF). In layer hens, it was found that that HS increased CBF to the skin, comb, wattles and upper respiratory tract while CBF in the digestive and reproductive tracts reduced by approximately one half [26]. In baboons, HS increased skin CBF by approximately 10% and was compensated by reductions in splanchnic and renal CBF of 35% and 27%, respectively [27]. The splanchnic bed and kidneys receive approximately 25% and 30% of cardiac output in resting animals. In addition, it has been postulated that this makes them sensitive to disruptions in reductions in blood flow, which in turn can precipitate oxidative stress and hypoxic damage [28], and is a prelude to loss of intestinal barrier function, inflammatory damage and bacterial translocation [29,30]. However, there are exceptions, and under milder forms of HS the consequences are likely reduced protein synthesis and crypt cell proliferation, resulting in a gradual decline in intestinal barrier function [6], which appears to be supported by the present findings.

The organs where differences in EBD distribution due to HS were observed were the kidney and spleen. The results from these organs were quite different, with HS increasing kidney EBD concentration, but alternatively reducing EBD concentration content in the spleen. That the kidney is a site of impairment during HS has been indicated in clinical studies [10,11]. Compared to the GIT, less is known about the etiology of HS mediated kidney damage; parallels may exist, as the kidneys also receive a large proportion of cardiac output, which can be disrupted by HS [27,31]. The interruption in blood flow may be more closely linked to decompensation and heat stroke and may not be applicable under HS [32]. Elsewhere, increased incidences of nephropathy have been recorded in rural communities in rural tropical communities, and this has been attributed to a warming climate, but it has also been postulated to be in part due to dehydration [33]. Although not quantified in this experiment, we previously quantified reductions in haematocrit in broilers [2,5] and in pigs, and this was accompanied by an apparent reduction in plasma volume, even with ad libitum water intake in all experiments [34]. There are fewer reports into the effects of HS on the spleen, and the result from this experiment was primarily driven by an increase in EBD concentration in the TN BET group, and TN and HS CON groups were not significantly different. Recently, it was observed in ducks that HS reduced spleen size, complementing production [35]. Additionally, a study observed that HS altered spleen lymphocyte populations [36]; collectively, these results might indicate that HS may compromise immune function.

Consistent with earlier experiments by our research group and others [2,5,37], betaine supplementation reduced respiration rate and rectal temperature, indicating partial amelioration of the effects of HS. Furthermore, betaine improved ileal villous height, area, crypt depth and seromuscular thickness. This result is in agreement with the experiments in young broilers up to 3 weeks of age [38,39], but differs to the results of [40], who also investigated the effects of betaine on finisher broilers. The experiment by [38] observed that the improved morphology was associated with improved resistance to coccidiosis infection, while morphology was not quantified. A study by [41] showed that dietary betaine reduced coccidiosis intestinal damage scores. As per our earlier work, betaine was shown to improve growth digestive function in HS broilers [5]. Still, it should be noted that in our earlier experiment, no improvement in ileal TER was observed. In summary, betaine was observed to improve productivity in broilers and other species, in part by improving intestinal morphology.

Although the fractional oral bioavailability of betaine has not been quantified, it has been reported to be readily available [42], and in broilers has been reported to be absorbed in the jejunum [38]. In this experiment, betaine concentrations increased with supplementation in the ileum, kidney and spleen, and previously we observed that supplementation increases plasma, liver and muscle concentrations [2]. Elevated ileal betaine concentrations may be an indication of betaine absorption across a wider area of the GIT than previously thought, while the contribution of arterial second-pass betaine metabolism cannot be excluded. Regardless, localised increases in betaine support a direct role for betaine in the ileum, as the ileum is not a site of betaine homocysteine methyltransferase expression [43], it is unlikely that the ileum is utilising betaine. Despite lower concentrations of betaine being quantified in the HS

ileum, as the ileum is not recognised as a site of betaine utilisation, the most likely role for betaine in the HS broiler ileum is as an osmolyte.

The effects of betaine on EBD distribution were that increases in EBD concentration in the HS CON group were not evident with BET, possibly indicating reduced extravasation and muscle damage. This may support the role of betaine in improving growth rates and feed conversion ratio in HS broilers [5], improved meat tenderness and reduced drip loss [2]. Likewise, increased EBD concentrations observed in the kidney of the HS CON were not evident in the HS BET group, also indicating amelioration of HS-mediated damage. As the kidney is a site of betaine homocysteine methyltransferase expression, it is possible that betaine protects the kidney through roles as a methyl donor or as an osmolyte. However, the effect of BET in the spleen was perplexing, increasing under TN but not HS conditions. Furthermore, overall reductions (independent of HS) in EBD were observed in the jejunum and ileum, which is consistent with the improvements in intestinal morphometry. Perhaps surprisingly, no effect of HS or betaine was observed on liver EBD concentration. Elsewhere, HS was observed to induce oxidative stress in the liver [28] and has been reported to be a major site of betaine distribution [38].

#### **5. Conclusions**

Supplementation of betaine partially ameliorated the physical symptoms of HS in finishing Ross-308 broilers and when supplemented betaine was widely distributed. In particular, betaine benefitted the small intestine, improving ileal resistance and villous height while reducing EBD concentrations, indicating an improvement in intestinal barrier function and gut health. It has been widely reported that HS compromises intestinal barrier function, a result that was supported by this experiment. However, by using EBD as a marker of organ dysfunction, it was apparent that a wider pattern of compromised function exists. This was evidenced in the muscle, kidney, jejunum, ileum and spleen having elevated EBD concentrations, likely reflecting underlying inflammation or damage. Importantly beneficial interactive effects with betaine were observed in muscle, jejunum, ileum and the kidneys, where elevated EBD concentrations were ameliorated by betaine. This indicates that the wide tissue distribution of betaine enables it to have multiple protective effects against HS, contributing to improved productivity and meat quality.

**Author Contributions:** Conceptualization, J.J.C., S.W., M.S., and F.R.D.; data curation, M.S. and F.R.D.; formal analysis, F.R.D., M.S.; funding acquisition, F.R.D., and J.J.C.; investigation, M.S., H.H.L., and W.Z.; methodology, M.S., H.H.L., J.B.F., and R.M.; project administration, F.R.D., J.J.C., and M.S.; resources, J.J.C. and S.W.; supervision, F.R.D., J.J.C., and S.W.; writing—original draft, M.S.; writing—review and editing, F.R.D., M.S., and J.J.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by MIRS and MIFRS Scholarships.

**Acknowledgments:** The authors are thankful to staff of the research facility for their support throughout the experiment. We would like to thank Tri Foods Pty. Ltd. for providing and transporting the chickens' feed. We would also like to thank the histology laboratory The University of Melbourne for processing the samples.

**Conflicts of Interest:** The authors have no conflict of interest. S.W. is employed by Feedworks Australia who provided the betaine and manufactured the diets used in these experiments.

#### **References**


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

### **E**ff**ect of Feed Additives as Alternatives to In-feed Antimicrobials on Production Performance and Intestinal** *Clostridium perfringens* **Counts in Broiler Chickens**

**Silje Granstad 1,\*, Anja B. Kristo**ff**ersen 1, Sylvie L. Benestad 1, Siri K. Sjurseth 1, Bruce David 2, Line Sørensen 3, Arnulf Fjermedal 4, Dag H. Edvardsen 5, Gorm Sanson 3, Atle Løvland <sup>2</sup> and Magne Kaldhusdal <sup>1</sup>**


Received: 6 January 2020; Accepted: 29 January 2020; Published: 3 February 2020

**Simple Summary:** For many years, antibiotics were added to chicken feed to prevent disease and promote growth. This practice has been banned or voluntarily abolished in many countries. However, most countries still allow the use of in-feed ionophorous coccidiostats, which are drugs that possess both antiparasitic and antibacterial properties. Concerns related to antimicrobial resistance have led to increased focus on broiler chickens raised without the use of any antimicrobial agents, and the interest in non-antibiotic feed additives with beneficial effects on gastrointestinal health and productivity is growing. In this study, feed additives with active components belonging to the product classes probiotics, prebiotics, phytogenics and/or organic acids were assessed for their effect on intestinal health and production performance in broiler chickens. Collectively, the group of non-antibiotic feed additives improved gut health and performance, but not to the same extent as the ionophorous coccidiostat narasin. Probiotics and prebiotics had the overall best performances during coccidia challenge, phytogenics improved overall feed conversion and reduced counts of the intestinal bacterium *Clostridium perfringens*, and organic acids increased weight gain independent of age. This study provides comparable and unbiased results from testing of alternatives to antibiotics in a uniform experimental model highly relevant to commercial conditions.

**Abstract:** Numerous non-antibiotic feed additives (alternatives to antibiotics, ATAs) have been marketed, but few have been evaluated under uniform testing conditions modelling commercial flocks. We compared 24 ATA treatments and the ionophorous coccidiostat narasin against a diet without any feed additives. Feed conversion ratio and body weight gain were registered from day 0 to 28 in Ross 308 chickens housed on litter floor. The chickens were challenged with *Eimeria* spp., and cecal *Clostridium perfringens* (CP) counts were investigated. Active components from all ATA classes had a positive impact on intestinal health or production performance. Whereas narasin had a strong CP-reducing effect in combination with performance-promoting impact, only two ATA treatments achieved significantly beneficial effects on CP counts as well as feed conversion during the time span following *Eimeria* challenge. Active components present in these two treatments include a *Bacillus subtilis* probiotic strain, short- and medium-chain fatty acids and *Saccharomyces cerevisiae* components. Different ATA classes had beneficial impact during distinct rearing phases and on

specific performance targets, suggesting that optimizing combinations and use of active components can make ATAs even more useful tools in broiler rearing without the use of in-feed antimicrobials. Further studies of promising ATAs and ATA combinations are required.

**Keywords:** broilers; feed additives; probiotics; prebiotics; phytogenics; organic acids; anticoccidials; necrotic enteritis; *Clostridium perfringens*; production performance

#### **1. Introduction**

The use of antimicrobial growth promoters (AGPs) was abolished in Sweden, Norway and Denmark in 1986, 1995 and 1998–1999, respectively [1]. As a response to this development, the use of ionophorous coccidiostats (e.g., narasin) in broiler feeds increased and became more important than before [2]. In 2006, the European Union implemented a total ban of AGPs, meaning that antimicrobials other than coccidiostats and histomonostats were no longer allowed as feed additives in the poultry industry [3,4]. Coccidiostats like narasin and other ionophores are still approved in the European Union for control of coccidiosis caused by the parasitic protozoans *Eimeria* spp. in poultry.

Ionophores are primarily approved for control of coccidiosis but may also have antibacterial and antiviral properties [5]. Narasin has a well-known inhibitory effect on the potential pathogen *Clostridium perfringens* (CP), which is associated with the intestinal disease necrotic enteritis (NE) in broiler chickens [6,7]. Selected ionophores have been suggested as novel antimicrobial agents to control infectious diseases in animals as alternatives to antimicrobial classes used to treat human disease [8]. Furthermore, concerns have been raised regarding the possibility that the use of narasin and other ionophores could be associated with bacterial resistance against antimicrobials used in human medicine, and that resistant bacteria could spread to humans both by direct contact with animals and through food supply [2,9]. These considerations have led to increased focus on conventional broilers raised without the use of any in-feed antimicrobial agents, including AGPs as well as ionophores and other coccidiostats. In 2015/2016, the Norwegian broiler industry abolished the routine use of in-feed coccidiostats, including narasin [10].

The former widespread practice of supplementing broiler feeds with AGPs was mainly based on the favorable influence of these compounds on production performance [2]. Impaired production performance leading to increased production costs is a main concern associated with rearing broilers without in-feed antimicrobials. The traditionally most commonly used AGPs are predominantly active against gram-positive bacteria [11], and many of these antimicrobials have been shown to suppress the proliferation of CP in vivo [12,13] and in vitro [14–16]. Several studies report an association between increased numbers of intestinal CP and growth depression in chickens [12,17,18], and collectively these findings suggest that antibacterial activity against CP may be involved in the 'antibiotic growth effect'. Development of NE and a subclinical form of this disease is associated with impaired production performance, cholangiohepatitis and high numbers of intestinal and fecal CP [19–21]. Infection with *Eimeria* spp. is considered an important predisposing factor for CP proliferation and development of NE in chickens [22,23].

The interest in non-antibiotic feed additives (hereafter: alternatives to antibiotics, ATAs) that might facilitate the abolishment of continuous use of in-feed AGPs and coccidiostats has increased during the recent years. Numerous new feed additives have reached the global poultry feed market. Different ATAs, including products based on probiotics, prebiotics, phytogenics and/or organic acids, claim to exert beneficial effects related to productivity, intestinal functions and intestinal health in broiler chickens.

Probiotics are based on non-pathogenic and non-toxigenic live microorganisms (e.g., bacteria or yeasts) supposed to provide health benefits to the host. Possible modes of action of probiotics include colonization of the intestine, competitive exclusion of other microorganisms, production of specific metabolites and stimulation of the immune system [24]. Two categories of probiotics are non-spore forming bacteria (e.g., *Lactobacillus* spp., *Enterococcus* spp., *Bifidobacterium* spp.) and bacterial spore formers (e.g., *Bacillus* spp.) [25]. Regulatory agencies have been reluctant to approve undefined microbial products due to the uncertainty of a consistent composition of the products. This concern has paved the way for defined probiotic products based on one or a few known strains.

Prebiotics are non-digestible feed ingredients assumed to stimulate proliferation and/or activity of intestinal microorganisms, which leads to beneficial physiological responses in the host [26]. Intake of prebiotics may increase the number of specific microbes and change the composition of the intestinal microbiota [27]. Examples of prebiotic compounds are complex carbohydrates derived from plants or yeasts, such as fructooligosaccharides (FOS), mannanoligosaccharides (MOS) and β-glucans [28,29]. In addition to selective promotion of beneficial bacteria, suggested modes of action of prebiotics are blocking pathogen adhesion, altering gene expression, affecting gut morphological structure and immunomodulation [29].

Phytogenic feed additives are based on bioactive compounds derived from plants, and a multitude of such plant products can broadly be classified as herbs or spices [28]. Examples of biologically active components and substances from plants are essential oils, oleoresins, tannins, saponins, flavonoids, alkaloids and resin acids. Various functions among plant-based products have been suggested, including antimicrobial, antiviral, antioxidative, anti-inflammatory and flavoring effects [30]. The compositional variation is considerable due to biological factors such as plant species, growing conditions, climate, harvest and manufacturing processes, and it is thus challenging to identify and evaluate the functional basis of this broad group of active components [31].

Organic acids of various lengths and their corresponding salts or esters are widely used as feed additives in livestock production and can be used individually or as blends of multiple acids. They may vary considerably in functionality due to number of carbon atoms and may be aliphatic or aromatic. Many organic acids consist of carboxylic acids and are natural constituents of animal or plant tissue or products of microbial fermentation. Industrially produced organic acids often come as salts or esters and in a coated or encapsulated form [31]. Carboxylic acids with an aliphatic chain are designated fatty acids. The subgroup short-chain fatty acids (SCFAs, 1–5 carbon atoms; C1–C5) are aliphatic compounds produced in nature by microbial fermentation of carbohydrates in the hindgut of humans and animals. The subgroup medium-chain fatty acids (MCFAs, 6–12 carbon atoms; C6–C12) are aliphatic compounds formed in nature predominantly in plants and extra-intestinal animal tissues. Suggested effects of organic acids are antibacterial activity through pH-regulation and changes in microbiota composition, immunomodulatory action and stimulation of the gut mucosa [28,29,31]. The heterogeneity of this feed additive category makes it difficult to define common properties and function, and the effects of different organic acids may vary considerably. It has been proposed that SCFAs can act directly upon the cell wall of gram-negative bacteria, and that fatty acids with longer chains can incorporate themselves into the cell membrane of gram-positive bacteria and promote leakage [32].

A multitude of studies on the impact of alternative feed additives in broiler chickens have been published. However, most studies focus on only one or a few additives within one or two ATA classes. Furthermore, these studies often differ with regard to a number of factors that may influence the results (e.g., housing of chickens, number of replicates and challenge), which makes it difficult or impossible to compare results across studies. Another problematic issue is publication bias that occurs when only results that show significant findings are reported [33]. These considerations make it relevant to study the effect of ATAs under uniform testing conditions.

The present study was conducted in order to examine the effect of commercially available ATAs from four different product classes on production performance and cecal CP counts. Feed additives were selected on the basis of being marketed with claimed beneficial effects on production performance, intestinal function and/or intestinal health in poultry. Production performance was recorded during two separate age levels; days 0–14 and days 14–28. CP counts were recorded during the fourth week of rearing, four to six days after challenge with *Eimeria* spp.

The aims of the study were to (a) evaluate the performance of the collective ATA group, (b) compare effects of classes of ATAs (probiotics/prebiotics/phytogenics/organic acids) and (c) identify active components or component combinations with beneficial effects on production performance and CP counts, with emphasis on the time span following *Eimeria* challenge.

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

#### *2.1. Animals and Housing*

Six trials were carried out at Scandinavian Poultry Research in Våler, Hedmark, Norway, using one-day-old Ross 308 broiler chickens obtained from a commercial hatchery (Nortura Samvirkekylling, Våler, Norway). The chickens were housed in floor pens of 5.6 m<sup>2</sup> on new wood shavings in a climate-controlled poultry research facility, with a 50/50 female-to-male ratio per pen. Water and pelleted feed were given *ad libitum*. The chickens were exposed to light for 23 h a day on the first two days. For the rest of the experimental period, the chickens were exposed to light during 2 × 8 h a day, interrupted by 4 h periods of darkness. Apart from a 10-fold dose of Paracox-5 vet. on day 17 or 18, no vaccines were administered throughout the study. The study period lasted from day of hatch until day 28. Animal experiments were approved by the national animal research authority (Norwegian Food Safety Authority, approval ID 8179), and performed in accordance with national and international guidelines for the care and use of experimental animals.

#### *2.2. Experimental Design*

In each of the six trials, a total of 5280 one-day-old Ross 308 broiler chickens were randomly allocated into six experimental groups, each group comprising 11 replicate pens with 80 chickens per pen. All trials had similar design, and included four treatment groups receiving feed with a specific ATA product or a combination of two ATA products, a positive control group (NAR) receiving feed with the polyether ionophore and coccidiostat narasin (Monteban, Elanco Animal Health, Greenfield, IN, USA), and a negative control group (NEG) receiving feed with neither antimicrobial feed additives (AGPs or coccidiostats) nor ATA products. Feed additives were added to the feeds at an inclusion rate recommended by the manufacturers. No AGP products were included in this study, and narasin was used as a sole coccidiostat in the NAR group. The chickens were fed wheat-based starter and grower diets based on Ross Broiler Nutrition Specifications adapted to Norwegian broiler production from 0 to 14 and 14 to 28 days of age, respectively (Table 1).

In the five initial trials, 20 commercially available ATA products were evaluated individually for their effect on production performance and cecal CP counts. In the sixth trial, combinations of two ATA products per treatment group were evaluated using the same outcome variables. Products included in the sixth experiment were selected for testing due to promising impact on either production performance or CP counts in the five initial experiments. Products with positive effects on production performance were combined with products with CP reducing effect in order to study potential synergy effects. Descriptions of active ingredients and dose levels of the feed additives and feed additive combinations tested are listed in Table 2. Composition of the products and dosage levels are based on information given by the feed additive manufacturers on their web sites or as a response to our request.

On day 17 (one trial) or 18 (five trials) post hatch, all treatment groups in all six trials were challenged with a 10-fold dose of Paracox-5 vet. (MSD Animal Health, Boxmeer, the Netherlands) containing live, sporulated oocysts from five attenuated strains of *Eimeria* spp. (one precocious line each of *Eimeria acervulina* [approximately 5750 oocysts per broiler], *Eimeria mitis* [approximately 11,500 oocysts], and *Eimeria tenella* [approximately 5750 oocysts], and two precocious lines of *Eimeria maxima* [approximately 3450 oocysts]) in the drinking water.


**Table 1.** Diet composition 1.

<sup>1</sup> Mean values from diets in six trials. <sup>2</sup> Vitamins and minerals: Cu 15 mg/kg; Zn 82 mg/kg; Mn 126 mg/kg; Se 0.27 mg/kg; I 1.04 mg/kg; Fe 52 mg/kg; Vit.A 9575 IU; Vit.E 96 IU; Vit.D3 4994 IU; Vit.K 7.0 mg/kg; Vit.B1 4.2 mg/kg; Vit.B2 7.3 mg/kg; Vit.B3 59.7 mg/kg; Vit.B5 20.0 mg/kg; Vit.B6 12.0 mg/kg; Vit.B12 0.02 mg/kg; biotin 2.1 mg/kg; folic acid 2.9 mg/kg; choline chloride 1726 mg/kg. <sup>3</sup> Vitamins and minerals: Cu 15 mg/kg; Zn 82 mg/kg; Mn 128 mg/kg; Se 0.27 mg/kg; I 1.05 mg/kg; Fe 53 mg/kg; Vit.A 9488 IU; Vit.E 81 IU; Vit.D3 4983 IU; Vit.K 5.6 mg/kg; Vit.B1 3.6 mg/kg; Vit.B2 6.8 mg/kg; Vit.B3 54.0 mg/kg; Vit.B5 18.0 mg/kg; Vit.B6 11.0 mg/kg; Vit.B12 0.02 mg/kg; biotin 2.4 mg/kg; folic acid 2.7 mg/kg; choline chloride 1500 mg/kg. <sup>4</sup> Non-starch polysaccharide enzymes.

**Table 2.** Treatment ID, class of feed additives, active components and inclusion rate of feed additive products.



**Table 2.** *Cont.*

<sup>1</sup> Treatment ID number. <sup>2</sup> NEG = negative control, NAR = positive control, PRO = probiotics, PRE = prebiotics, PFA = phytogenics, and OA = organic acids. <sup>3</sup> Based on available information from the product manufacturers. <sup>4</sup> Amount added product given as grams/ton feed in starter and grower diets.

#### *2.3. Clostridium Perfringens Quantification*

On days 4, 5 and 6 after *Eimeria* challenge, 11 chickens per treatment group (1 chicken from each replicate pen) were randomly selected and humanely euthanized by cranial stunning immediately followed by cervical dislocation before necropsy. Samples of cecal contents were collected in sterile stomacher bags and directly subjected to cultivation in order to quantify CP. In brief, the samples were diluted 1:100 in peptone saline water (0.1% peptone, Difco Laboratories Inc., Detroit, US and 0.85% NaCl) and homogenized for 30 s in a stomacher (Bagmixer 400 CC, Interscience, Saint Nom, France). Serial dilutions were made with non-buffered peptone water until a dilution of 10–6 was reached. Aliquots of 100 μL from the dilutions 10–2, 10–4 and 10–6 were plated onto sheep blood agar plates (Oxoid Blood Agar Base No.2 and 5% sheep blood, manufactured by the Norwegian Veterinary Institute, Oslo, Norway). The plates were incubated anaerobically at 37 ◦C for 24 h (Genbox anaer, Biomérieux, Marcy-l'Étoile, France). Single colonies with double hemolysis were counted, and colony-forming units per gram (cfu/g) cecal contents were calculated based on the given dilution. Typical colonies were selected for pure cultivation and later confirmed as CP by a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltonics, Bruker Corp., Billerica, MA, USA).

#### *2.4. Post Mortem Examination*

The small intestine of all chickens that were sampled for CP quantification was opened longitudinally and examined for pathological changes indicating NE, and scored as follows (modified from [34]): necrotic enteritis negative with no macroscopic mucosal ulcers or pseudomembranes, or necrotic enteritis positive with minimum one mucosal ulcer or pseudomembrane.

#### *2.5. Production Performance Measurements*

The amount of feed per pen was weighed when allocated and remaining feed was weighed before being discarded at feed change and at the end of the experiment. Accumulated feed intake per pen from days 0 to 14, 14 to 28 and 0 to 28 was calculated. Total live chicken weights per pen were recorded on days 0, 14 and 28, and mean body weight gain (BWG, g/chicken) and mean feed conversion ratio (FCR, g feed intake/g weight gain) per pen were calculated.

#### *2.6. Statistical Analysis*

Data on production performance and CP counts were examined on three different levels; (a) the impact of ATAs as one collective group (group level), (b) the impact of classes of ATAs (class level), and (c) the impact of individual ATA treatments (treatment level). On all levels, ATAs and the positive control with narasin-supplemented feed (NAR) were compared against the negative control with no feed additive (NEG). Frequencies of broilers with NE lesions were analyzed only on group level using Pearson's chi-squared test in Stata version 14.2 (StataCorp LLC, College Station, TX, USA). Production performance and CP count data were analyzed using regression analyses in R version 3.5.3 (R Foundation for Statistical Computing, Vienna, Austria).

Production performance data were analyzed with pen as the unit of concern. Body weight gain and feed conversion ratio was obtained in the periods 0–14 days, 14–28 days and 0–28 days for groups, classes and treatments tested in six trials. The outcome from the six different trials could not be compared directly due to intertrial variability. In order to validly compare results from six different trials, it was necessary to control for the effect of trial in the statistical analysis. The principle approach to achieve such control was to use the results from NEG in each of the six trials as indicators of trial effect. A mixed-effects model (1) with only intercept (*a*) was used to obtain a trial-specific random effect (ε*Trial*) for each outcome variable (*yNeg*) per trial based on results from NEG using the package *lme4* in R [35].

$$y\_{N\text{eq}} = a + \varepsilon\_{T\text{nil}} \tag{1}$$

For each of the outcome variables (*y*), results achieved in the different trials were adjusted with a value equal to the random effect obtained for the respective trial. Results across trials were compared using regression analysis (2) with ATA group/class/treatment (*x*) as fixed-effect variable and trial-specific random-effects from NEG as offset variable (ε*Trial*). *b* represents the estimated parameters in the model.

$$y = \ \varepsilon\_{Trial} + b \cdot \mathbf{x} \tag{2}$$

The necessity of adjustment for trial effect was calculated by the intraclass correlation coefficient (ICC), which is variance explained by the random effect divided by total variance of the residuals for the model based on all observations from NEG. Extreme outlier pens that were highly influential on the estimated regression results were identified using the function *outlierTest* from the package *car* in R [36]. Residuals from the regression models were visually inspected using the functions *qqnorm* and *qqline* in R and found to follow a normal distribution. The production performance results were reported in tables as means with standard deviation. Differences from NEG with *p* < 0.05 were accepted as statistically significant differences.

CP counts in cecal samples were analyzed with individual chicken samples as unit of concern. Since the residuals from the regression model did not follow a normal distribution, the CP count

numbers were log transformed in order to fulfil this requirement. The effect of trial was controlled by adjusting for obtained random effect as described above, and subsequently regression analysis with ATA group/class/treatment as fixed-effect variable and trial-specific random-effects from NEG as offset variable was conducted. The results were reported in tables as mean log10 colony forming units per gram cecal content. Estimated mean log10 CP counts with 95% confidence interval for each treatment were presented in a graph where feed additive classes are indicated with different colors.

#### **3. Results**

#### *3.1. Impact of the Collective ATA Group on Necrotic Enteritis, Intestinal CP Counts and Production Performance*

Broilers with necrotic enteritis lesions during days 4–6 after *Eimeria* challenge constituted 8.1% among chickens from the NEG group (no feed additive, *n* = 198 chickens), 4.4% in the collective ATA group (24 ATA treatments, *n* = 792 chickens) and 0.5% in the NAR group (in-feed narasin, *n* = 198 chickens). Statistical analyses indicated significant difference in NE occurrence between the NEG group and the ATA group (*p* < 0.05), and between the ATA group and the NAR group (*p* < 0.01).

The ATA group reduced CP counts in intestinal contents from log10 6.09 to log10 5.63 cfu/g (*p* = 0.005), corresponding to a 65% reduction in non-transformed counts (Table 3). This substantial reduction was, however, moderate as compared to the very strong effect of narasin (from log10 6.09 to log10 2.92 cfu/g (*p* < 0.001), corresponding to a 99.9% reduction in non-transformed counts).

**Table 3.** Body weight gain, feed conversion ratio and *Clostridium perfringens* counts for negative control, narasin and alternatives to antibiotics 1.


<sup>1</sup> Results are reported as means ± standard deviation. Body weight gain (BWG) in grams/chicken, feed conversion ratio (FCR) in grams feed intake/grams weight gain and *Clostridium perfringens* (CP) counts as log10 colony forming units/gram cecal content. <sup>2</sup> Negative control (no feed additive); production performance data based on *n* = 66 pens, and CP data based on *n* = 198 individual chicken samples. <sup>3</sup> Narasin; production performance data based on *n* = 66 pens, and CP data based on *n* = 198 individual chicken samples. <sup>4</sup> Alternatives to antibiotics treatments; production performance data based on *n* = 264 pens, and CP data based on *n* = 792 individual chicken samples. <sup>5</sup> Intraclass correlation coefficient.

Both the ATA group and the NAR group had strongest beneficial impact on production performance during days 14–28, i.e., the age interval characterized by intestinal stress induced by *Eimeria* challenge on day 17 or 18. The collective ATA group demonstrated a 1.6% improvement (*p* < 0.001) in FCR during days 14 to 28 (FCR14–28) and a 2.8% increase (*p* < 0.001) in BWG during days 14 to 28 (BWG14–28) compared to the NEG group (Table 3). The beneficial effect of the ATA group on production performance was not as pronounced as the positive effect of narasin (4.9% improved FCR14–28 and 7.8% increased BWG14–28).

#### *3.2. Impact of ATA Classes on Intestinal CP Counts and Production Performance*

Four ATA classes (probiotics, PRO; prebiotics, PRE; phytogenics, PFA; organic acids, OA), a set of treatments each based on more than one ATA class (mixed products, MIX) and NAR (i.e., narasin) were compared with NEG (i.e., no feed additive) (Table 4). Although all ATA classes demonstrated

a reducing effect on numbers of CP per gram intestinal contents, only two classes (PFA and PRO) showed statistically significant reduction (*p* < 0.05). The estimated reducing impacts of PFA and PRO were 87% and 75% in non-transformed CP counts, respectively, when compared to NEG.


**Table 4.** Body weight gain, feed conversion ratio and *Clostridium perfringens* counts for negative control, narasin and classes of alternatives to antibiotics 1.

<sup>1</sup> Results are reported as means ± standard deviation. Body weight gain (BWG) in grams/chicken, feed conversion ratio (FCR) in grams feed intake/grams weight gain and *Clostridium perfringens* (CP) counts as log10 colony forming units/gram cecal content. <sup>2</sup> Negative control (no feed additive); production performance data based on *n* = 66 pens, and CP data based on *n* = 198 individual chicken samples. <sup>3</sup> Narasin; production performance data based on *n* = 66 pens, and CP data based on *n* = 198 individual chicken samples. <sup>4</sup> Probiotics (PRO); production performance data based on *n* = 33 pens, and CP data based on *n* = 99 individual chicken samples. <sup>5</sup> Prebiotics (PRE), phytogenics (PFA), organic acids (OA); production performance data based on *n* = 44 pens, and CP data based on *n* = 132 individual chicken samples. <sup>6</sup> Mixed products (MIX), i.e., treatments based on more than one ATA class; production performance data based on *n* = 99 pens, and CP data based on *n* = 297 individual chicken samples.<sup>7</sup> Intraclass correlation coefficient.

Three ATA classes (PRO, PRE and MIX) improved FCR14–28 (1.3%–2.7% improvement, *p*<0.01), and four classes (PRO, PRE, OA and MIX) increased BWG14–28 (2.8%–3.9% increase, *p* < 0.01). Accumulated feed conversion during days 0 to 28 (FCR0–28) was improved by all ATA classes (1.1%–1.5%, *p* < 0.01). However, only the OA class improved feed conversion during days 0 to 14 (FCR0–14) significantly (3.3%, *p* < 0.001). Narasin outperformed the ATA classes at all age intervals, except for body weight gain during days 0 to 14 (BWG0–14) and FCR0–14, where the OA class performed similarly.

#### *3.3. Impact of Treatments on Intestinal CP Counts and Production Performance*

Intestinal CP counts were significantly reduced (*p* < 0.05) by 8 out of 24 ATA treatments (ID 3, 5, 15, 16, 18, 20, 21 and 24) as shown in Table 5. Estimated reduction in non-transformed CP counts among these eight treatments ranged from 84% to 97% when compared to NEG. Phytogenic components were present in 5/8 treatments (ID 15, 18, 20, 21 and 24), prebiotic components in 3/8 treatments (ID 5, 16 and 24), probiotic components in 2/8 treatments (ID 3 and 5) and OA components were present in 2/8 treatments (ID 15 and 24). Mean log10 CP counts with 95% confidence interval for each ATA treatment are shown in Figure 1.


**Table 5.** Body weight gain, feed conversion ratio and *Clostridium perfringens* counts for negative control, narasin and alternatives to antibiotics treatments 1.

<sup>1</sup> Results are reported as means ± standard deviation. Body weight gain (BWG) in grams/chicken, feed conversion ratio (FCR) in grams feed intake/grams weight gain and *Clostridium perfringens* (CP) counts as log10 colony forming units/gram cecal content. <sup>2</sup> Negative control (no feed additive); production performance data based on *n* = 66 pens, and CP data based on *n* = 198 individual chicken samples. <sup>3</sup> Narasin; production performance data based on *n* = 66 pens, and CP data based on *n* = 198 individual chicken samples. <sup>4</sup> Probiotics (PRO), prebiotics (PRE), phytogenics (PFA), organic acids (OA), mixed products (MIX); production performance data based on *n* = 11 pens, and CP data based on *n* = 33 individual chicken samples. <sup>5</sup> Intraclass correlation coefficient.

**Figure 1.** Cecal *Clostridium perfringens* (CP) counts with 95% confidence intervals. Negative control (NEG) is treatment 0, narasin (NAR) is treatment 1, probiotics (PRO) are treatments 2–4, prebiotics (PRE) are treatments 6, 7, 16 and 19, phytogenics (PFA) are treatments 9, 18, 20 and 21, organic acids (OA) are treatments 10, 12, 13 and 14, and mixed products (MIX) are treatments 5, 8, 11, 15, 17 and 22–25.

FCR14–28 was improved (*p* < 0.05) by 10/24 tested ATA treatments (Table 5). Five of these treatments (ID 4, 6, 7, 8 and 9) achieved FCR14–28 improvements (3.2% to 5.2%, *p* < 0.001) that returned the same significance level as narasin (4.9% improvement, *p* < 0.001). These five treatments had active components classified as probiotics (ID 4), prebiotics (ID 6 and 7), phytogenics (ID 8 and 9) or organic acids (ID 8). In total, 13/24 ATA treatments improved FCR0–28 (1.4% to 3.2% improvement, *p* < 0.05). Seven of these treatments (ID 6, 7, 8, 9, 11, 13 and 24) achieved improvements in FCR 0–28 that returned the same significance level (2.1% to 3.2% improvement, *p* < 0.001) as narasin (4.5% improvement, *p* < 0.001).

BWG14–28 and body weight gain during days 0 to 28 (BWG0–28) were increased by 10/24 and 8/24 ATA treatments, respectively. Two treatments (ID 11 and 13) excelled in increasing both these parameters, with a significance level similar to narasin (*p* < 0.001).

In the sixth trial, two-product combinations of treatments with predominantly CP-reducing impact (ID 5, 16 and 21) and treatments with predominantly production performance-promoting impact (ID 7, 11 and 13) were evaluated (comprising treatment ID 22–25 in Table 2). Treatment 16 did not appear to reduce the FCR-improving effect of treatments 11 and 13 (Table 5) but tended to diminish the growth promoting impact of these treatments. Treatment 5 seemed to diminish the FCR-improving effect and remove the growth-promoting effect of treatment 7. Treatment 21 appeared to reduce or remove the improvement in FCR and to remove the growth-promoting effect of treatment 7. On the other hand, treatment 7 seemed to remove the CP-reducing impact of treatments 5 and 21, and treatment 13 appeared to remove the CP-reducing impact of treatment 16. In contrast to these

results, treatment 11 did not appear to impair the CP-reducing impact of treatment 16. As a result of these interactions between predominantly CP-reducing and production performance-promoting single treatments, treatment 24 was the only one of four tested product combinations (ID 22–25, Table 5) with beneficial effects on production performance variables as well as CP counts.

#### *3.4. Active Components with Combined Beneficial E*ff*ects on FCR14–28 and CP Counts*

In total, 10/24 and 8/24 tested treatments improved (*p* < 0.05) FCR14–28 and CP counts, respectively. Collectively these treatments comprised a group of 16 treatments; 14 treatments either improved FCR14–28 or reduced CP counts, and only two treatments (ID 3 and 24) influenced both FCR14–28 and CP counts in a beneficial way. One of the two superior treatments according to these criteria was a probiotic (ID 3) with the *Bacillus subtilis* PB6 strain as the only active component. The other treatment (ID 24) was a combination of three ATA classes (OA/PFA/PRE) and two products; one product (ID 16) containing whole cells of *Saccharomyces cerevisiae* and its metabolites, and another (ID 11) being a mixture of SCFAs (including C4), MCFAs (including C12) and a phenolic compound.

#### **4. Discussion**

The collective group of 24 ATA treatments tested in this study reduced the occurrence of NE and reduced intestinal CP counts after *Eimeria* challenge. Production performance measured as BWG and FCR was improved by the collective ATA group, but not significantly during the phase prior to *Eimeria* challenge. These results indicate a beneficial effect of several ATAs when chickens are exposed to mild to moderate intestinal stress. The favorable effects of the polyether ionophore narasin on CP counts and production performance were numerically and in part significantly stronger than the effect of the collective group of ATA treatments.

In this study, the chickens were challenged orally with five precocious lines of *Eimeria* spp. as a predisposing factor for NE. NE is expected to appear during oocyst excretion, which begins between three and four days following inoculation with precocious *Eimeria acervulina* [37] and *Eimeria mitis* lines, and presumably later with precocious *Eimeria maxima* and *Eimeria tenella* lines [38]. In a previous study with a similar type of *Eimeria* challenge, most gut damage was detected four to six days after inoculation [39]. Postmortem examinations in this study confirmed the presence of NE during this time span after *Eimeria* challenge. Intestinal CP counts are strongly associated with NE [19,34,40]. Furthermore, increased occurrence of NE in commercial broiler flocks has been associated with impaired accumulated FCR at slaughter [20]. Weakened production performance is likely to be most pronounced during the part of the rearing phase that is affected by NE. Based on the considerations mentioned above we chose to emphasize CP counts on days four to six after *Eimeria* challenge and FCR14–28 in our analyses of effect of ATAs on intestinal health.

The evaluation of ATAs can rest on different criteria, depending on point of view, practical circumstances and current health problems. Disease conditions associated with increased metabolism and rapid growth in broilers (in particular cardiovascular and musculoskeletal disorders) are generally important, and it has been claimed that such conditions cause greater economic loss than infectious agents [41]. In a recent study, it was found that chickens with higher body weight and BWG were predisposed to develop more severe NE lesions when challenged with CP [42]. Although attractive in the short run, increased weight gain may therefore come at a cost not only to chicken health and welfare but also to the farmers' economy and a sustainable use of feed resources. In light of this consideration, we have emphasized the effect on FCR as production performance parameter, because it is an indicator of intestinal health as well as resource efficiency.

The only two ATA treatments (ID 3 and 24) with a combined beneficial effect on CP counts (84 to 89% reduction, *p* < 0.05) and FCR14–28 (2.3% improvement, *p* < 0.05) were based on different types of active components. One of the treatments (ID 3) was a mono-strain (*Bacillus subtilis* strain PB6) spore-forming bacterial probiotic. This probiotic strain has been reported to inhibit CP in vitro [43] and improve FCR [44], which is in agreement with our findings.

The other treatment (ID 24) was based on a heterogeneous collection of active components including short- and medium-chain fatty acids, a phenolic compound and dehydrated whole cells and metabolites of the yeast *Saccharomyces cerevisiae* (SC). This treatment comprised two commercial products that were also tested individually (ID 11 and 16). Whereas the yeast product (ID 16) alone demonstrated a 95% reduction (*p* < 0.001) in non-transformed CP counts, the product containing a blend of organic acids and a phenolic compound (ID 11) had no reducing impact on CP. Viewed against this background it seems probable that the CP reducing effect of treatment 24 was mainly associated with one or several yeast components found in treatment 16. In addition to treatment 24, three other ATA treatments based on the yeast SC were tested. These treatments, which were based on SC cell wall extracts (ID 6, 7 and 19), did not reduce CP counts to an extent that was significant with the sample size and/or feed additive dosage used in our study, whilst the treatments based on SC whole cells and metabolites (ID 16 and 24) did. No previous reports on the effect of SC metabolites and SC whole cells on CP counts in broilers have been found. Regarding yeast cell wall extracts, previously published literature has indicated both significant [45] and non-significant [46] CP-reducing impact. Our results indicate that whole cells and/or metabolites of SC inhibited intestinal CP growth more efficiently than SC cell wall extracts with the product inclusion levels used in this study.

One of the active components in treatment 11 was lauric acid (C12), a MCFA that has been demonstrated to inhibit CP in vitro [47]. Treatment 11 did, as mentioned above, not reduce CP counts when used as sole feed additive in this study. Possible explanations include too low concentration of lauric acid and/or interfering effects by other treatment components.

Regarding production performance, the combination of treatments 11 and 16 (i.e., treatment 24) had a significantly beneficial effect on FCR14–28. However, neither of these two treatments improved FCR14–28 when tested individually. This finding suggests a synergy effect with regard to FCR14–28 between active components present in the two products. Beneficial effects of dietary supplementation of whole cells and metabolites of SC on production performance in broilers have been reported by others [48].

The combination of SCFAs (including butyric acid-C4), MCFAs (including lauric acid-C12) and a phenolic compound in treatment 11 generated the numerically highest weight gain (BWG0–14 and BWG14–28) of all ATA treatments in this study but had no apparent impact on CP counts. This result suggests that rapid growth is possible in the presence of relatively high cecal CP counts. A possible explanation could be that this treatment reduced the counts of virulent CP strains (e.g., strains harbouring the *netB* gene) or the expression of virulence factors (e.g., the NetB toxin), but not the total CP counts. Treatment 11 might also have influenced the intestinal microbiota in a way that neutralized the negative impact of high CP counts.

Six of 24 ATA treatments (ID 5, 15, 16, 18, 20 and 21) were associated with reduced CP counts (at least 83% reduction in non-transformed counts, *p* < 0.05) without improving FCR14–28 significantly. Treatment 21 had a very strong reducing impact on CP counts (a 97% reduction, *p* < 0.001) and improved FCR0–28 (1.8%, *p* < 0.01), but had only a numerically (1.3%, non-significant) beneficial impact on FCR14–28. Active components of treatment 21 included oleoresins from turmeric (*Curcuma longa*) and chili peppers (genus *Capsicum*). These results are in agreement with reports on inhibitory activity against CP of turmeric extracts [49], reduced gut lesion scores in CP-challenged broilers treated with *Capsicum* and *Curcuma* oleoresins [50,51] and improved cumulative FCR of turmeric powder [52]. Treatment 20 was based on tall oil fatty acids from coniferous trees including resin acids. Resin acids have been reported to inhibit CP in vitro [53], and our results suggest similar effects in vivo.

Treatments 22–25 were tested in a final trial intended to evaluate two-product combinations of treatments improving production performance and treatments with CP-reducing impact. Treatment 24 was the only combination with beneficial impact on both CP counts and production results. These results suggest that the interaction between predominantly CP-reducing and production-promoting components vary substantially. Among three tested CP-reducing treatments in the final trial (ID 5, 16 and 21), a treatment based on dehydrated SC culture with whole cells and metabolites (ID 16) was the least impairing with regard to the production-promoting effects of its combination treatment. Among the three tested production performance-improving treatments (ID 7, 11 and 13), treatment 11 based on short- and medium-chain fatty acids and other components was the only one that did not impair the CP-reducing impact of its combination treatment. More work is needed to identify the role of the different components in treatments 11 and 16, and whether the beneficial interaction of these components also can be extended to include other CP-reducing and production-promoting components.

Our findings indicate that a reduction of CP counts induced by ATAs was not always associated with improved production performance. Lack of a positive impact on feed efficiency and growth rate has also been documented with regard to ionophores under certain conditions [54], in spite of these compounds' suppressing effect on CP counts. However, when used at recommended concentrations in broiler flocks exposed to coccidia, the net effect of ionophores is usually improved performance. In our study, considerably improved performance combined with a strong CP-reducing effect of the ionophore narasin was present. These results confirm that our challenge model worked as expected, and that the in-feed concentration of narasin was within the optimal range. The reason why some of the ATAs with CP-reducing effect in our study did not induce a significantly positive net impact on production performance under the same test conditions remains unclear. Possible explanations may be that the inhibiting effect on CP was accompanied by reduced ability to utilize feed efficiently and/or establishment of another performance-impairing intestinal microbiota.

Eight of 24 ATA treatments (ID 2, 4, 6, 7, 8, 9, 13 and 25) improved FCR14–28 (at least 2.0% improvement, *p* < 0.05) without reducing CP counts significantly. One of these treatments (ID 4) was a mono-strain *Bacillus subtilis* probiotic. Data from other studies demonstrate the capacity of *Bacillus subtilis* strains to suppress the growth of CP and improve production performance and intestinal morphology [55–57]. However, the favorable impact on FCR14–28 in this study might have been caused by other mechanisms than inhibition of CP growth. Suggested modes of action associated with probiotics are maintenance of balanced microbial populations, modulation of the host immune system, promotion of epithelial barrier integrity and alteration of villus length and crypt depth [44,58–61].

Two (ID 6 and 7) of the treatments improving FCR14–28 but not CP counts contained cell wall extracts from the yeast SC. Both treatments 6 and 7 had a considerably beneficial impact on FCR14–28 (estimated 4.3 and 5.2% reduction, respectively). Of the SC cell wall-based treatments, the products with the apparently highest content of β-glucans (ID 6 and 7) had the best effect on FCR14–28 as compared with the other yeast cell wall-based treatment in our study (ID 19). Treatment 7, containing minimum 60% purified β-1.3/1.6 glucans, even outperformed narasin numerically with regard to FCR14–28. These findings suggest that SC-derived β-glucans are potent when it comes to improvement of FCR in broilers exposed to *Eimeria* spp. Beneficial effects of yeast β-glucans on performance in broilers are supported by some [62,63] and in contradiction with results from other previous reports [64,65]. Possible explanations for the FCR14–28-promoting effect of feed additives containing β-glucans are modulations of the immune response [62,64].

Two other treatments (ID 8 and 9) with favorable effect on FCR14–28 without significant reduction of CP counts contained essential plant oils. Essential oil components in treatments 8 and 9 included thymol (in both treatments), eugenol and piperine (in treatment 8) and carvacrol, anethol and limonene (in treatment 9). In treatment 8, essential oils were combined with benzoic acid. Published results on effects and mode of action of essential oils suggest that several of these compounds inhibit the growth of CP [66–68], although the findings are not always clear cut [69], or they show no effect on CP counts [70]. Reports on mitigation of gut lesions in chickens challenged with CP [67,71] underpin the view that at least thymol and carvacrol suppress the pathogenic action of CP. Studies on the effect of essential oil components on production performance reveal variable results. One study reports a negative effect on broiler performance using a blend of thymol, eugenol, curcumin and piperine [70], another describe a non-significant tendency of improved FCR14–28 using a blend of carvacrol and thymol [71], and a third study presents significant improvement of FCR0–28 by carvacrol but not by thymol [72]. The lack of standardization of studies, including variable feed additive dosage and different combinations of

active compounds, makes comparison of results from different studies difficult. The interpretation of results is further complicated by the multiple suggested effects of different essential oils, including antibacterial and antioxidant properties, enhancement of the immune system, and stimulation of digestive secretions and blood circulation [73,74]. Regardless of mechanism, these two predominantly phytogenic feed additives (ID 8 and 9) had a pronounced beneficial effect on FCR14–28 on par with narasin in the current study.

The apparent lack of a CP-reducing effect of yeast cell wall extracts, essential oils and other active components associated with improved FCR14–28 in this study may in part be related to experimental design. As observed from our results (Table 5), estimated CP count reductions of 76% or less (e.g., treatment ID 2 and 14) returned non-significant (>0.05) *p*-values. The main reason for this low statistical power was high variance of CP counts in individual observations within each treatment, leading to imprecise estimates. Our experiments were designed with 33 replicates of individual CP counts per ATA treatment, and this sample size returned relatively wide confidence intervals (as shown in Figure 1). The statistical analysis involving the whole ATA group (Table 3) indicated that when 792 individual samples with a log10 5.63 CP estimate were compared with the NEG group with 198 individual samples and a log10 6.09 estimate (corresponding to a 65% difference), this difference was significant (*p* = 0.005).

The ATAs did not suppress CP counts to the same extent as the ionophorous coccidiostat narasin. The superior results of narasin in this respect were most likely due to the strong antibacterial effect of this compound. Narasin has been reported with inhibitory effect on CP growth similar to or better than antibiotics used as drinking water medication for poultry [75].

Different ATAs can add value to the broiler chicken industry in several ways. Some improve BWG and/or FCR, others inhibit growth of CP or have a beneficial effect on both production performance parameters and intestinal CP counts. The use of specific ATAs could possibly be targeted to specific age intervals or current health status in the flock. Future studies of the impact of ATAs on intestinal CP counts would most likely benefit from modified sampling protocols and quantification methods. Study designs that were useful for investigating the effect of AGPs and ionophorous coccidiostats should not be copied without reservation when studying non-antibiotic alternative feed additives. Finally, a less pronounced effect than narasin of selected ATAs on production performance and/or CP counts in this study does not necessarily mean that the impact is of no importance to broiler health and production economy.

In this study, ATA classes displayed distinct performance profiles. The probiotic class reduced CP counts and improved production performance during the time period with intestinal stress (days 14–28), but impaired weight gain during days 0–14. The prebiotic class improved production performance during days 14–28 and had a non-significantly reducing impact on CP counts. The phytogenic class had a markedly reducing impact on CP counts and improved FCR0–28. The organic acid class increased weight gain throughout the study period and improved FCR0–14 but did not reduce CP counts significantly. These findings suggest that employing ATA classes for specific purposes may be useful. As an example, combining probiotic and organic acid treatments might boost production performance throughout the grow-out period and at the same time reduce CP counts during intestinal stress. In this study, we tested other ATA class combinations with variable results, indicating the need for testing of specific combinations of active components within the ATA classes.

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

**Funding:** This research was funded by the Norwegian Research Council, grant number 244635.

**Acknowledgments:** We are grateful to Tone M. Fagereng and Gro S. Johannessen (Norwegian Veterinary Institute) for *Clostridium perfringens* analysis, Charles Albin-Amiot and Elliot Lambert (Norwegian Veterinary Institute) for *Eimeria* spp. analysis, Torfinn Moldal, Reidun Haugerud Bolstad and Inger Helene Kravik (Norwegian Veterinary Institute) for contribution to sample collection, Franciska Steinhoff (Felleskjøpet Fôrutvikling AS) for involvement in feed analysis, Felleskjøpet Agri SA and Centre of Feed Technology for production of feed and Benny Berg with colleagues at Scandinavian Poultry Research for assistance during trials.

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

#### **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 Housing System and Rosemary and Cinnamon Essential Oils on Layers Performance, Egg Quality, Haematological Traits, Blood Chemistry, Immunity, and Antioxidant**

**Mahmoud M. Abo Ghanima 1, Mohamed F. Elsadek 2,3,\*, Ayman E. Taha 4, Mohamed E. Abd El-Hack 5, Mahmoud Alagawany 5, Badreldin M. Ahmed 2, Mona M. Elshafie <sup>2</sup> and Karim El-Sabrout 6,\***


Received: 29 December 2019; Accepted: 22 January 2020; Published: 4 February 2020

**Simple Summary:** The current study aimed to investigate the effects of a housing system, and dietary supplementation of rosemary and cinnamon essential oils on layers performance and egg quality. A factorial arrangement (2 × 3) was performed including two housing systems (floor and cage) and three different types of essential oils (0, 300 mg/kg diet of rosemary and 300 mg/kg diet of cinnamon essential oils) to study their effects on the productive performance, egg quality, immunity, oxidative stress and haematology of laying hens during the production stages. The data suggested that the supplementation of rosemary and cinnamon essential oils in laying hen diet showed significant positive effects on hen performance and egg production. Additionally, the different housing systems did not result in any positive or negative impact on these traits.

**Abstract:** Housing system and nutrition are non-genetic factors that can improve the well-being of animals to obtain higher quality products. A better understanding of how different housing systems and essential oils can influence the performance of layers is very important at the research and commercial levels. The current study aimed to investigate the effects of a housing system and dietary supplementation of rosemary and cinnamon essential oils on layers' performance and egg quality. A factorial arrangement (2 × 3) was performed include two housing systems (floor and cage) and three different types of essential oils (0, 300 mg/kg diet of rosemary and 300 mg/kg diet of cinnamon essential oils) to study their effects on the productive performance, egg quality, immunity, oxidative stress and haematology of ISA brown laying hens during the production stages (from 28 to 76 weeks of age). Birds were randomly divided into two groups each comprising of 1500 birds; the first group was moved from the litter to reared laying cages while the second group was floor reared. Each group was randomly divided into three groups, the first was considered as a control group, the second treated with rosemary essential oil, and the third with cinnamon essential oil. The differences in egg production and weight, egg quality, feed intake and conversion, blood picture and chemistry, immunity, and antioxidant parameters between the different housing systems (floor and cage) were not significant at (*p* < 0.05 or 0.01). On the other hand, the egg production and weight, Haugh

unit, feed intake and conversion, blood cholesterol, Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), urea, Ca, P, immunity, and antioxidant parameters were significantly (*p* < 0.05 or 0.01) better in rosemary and cinnamon groups than in the control group. Furthermore, the results of dietary supplementation with rosemary and cinnamon were very close. Regarding egg production and weight, there were no significant differences due to the interactions. The differences in egg mass among the interactions were also not significant except at 68–76 weeks, where the cage × cinnamon group was the highest. Under the floor rearing system, birds that were fed a diet supplemented with or without essential oils (EOs) consumed more feed than those raised under the cage system. Regarding feed conversion rate (FCR), the differences among the interactions were not significant except at 44–52, 52–60 and 68–76 weeks, where the cage × cinnamon group was the lowest. Excluding glutathione peroxidase (GPx) activity (*p* < 0.001), all immunity and antioxidant indices were not statistically different as a consequence of the interaction among EOs and housing systems. Additionally, the highest levels of phosphorus were observed for layers fed diets enriched with cinnamon oil with the cage or floor system. In conclusion, the data suggested that supplementation of rosemary and cinnamon essential oils in laying hen diet showed significantly positive effects on hen performance and egg production. Cholesterol, liver and kidney functions, immunity, and antioxidant parameters improved with rosemary and cinnamon supplementation when compared to the control. Additionally, the different housing systems did not result in any positive or negative impact on these traits.

**Keywords:** antioxidant; essential oils; housing system; immunity; ISA brown; production

#### **1. Introduction**

Housing systems have always had an impact on animal welfare and performance [1–3]. Housing systems, as a non-genetic factor, can improve the well-being of animals to obtain higher quality products. A better understanding of how different housing systems can influence the performance of layers is required. The housing system could influence both the laying hen's performance and egg quality traits [4]. Englmaierová et al. [4] found that the highest egg production, lowest daily feed consumption, and feed conversion ratio were measured in cages compared to litter. Moreover, higher eggshell, yolk index, and albumen qualities were observed in cages. El-Deek and El-Sabrout [5] concluded that the maintenance of hens in enriched cages and with outdoor access would make it easier for the layers to express their natural behaviour, which has a favourable effect on their welfare and production. Additionally, consumers are recently interested in poultry products originating from alternative housing systems [6], which are natural, organic and have less content of substances that can endanger human health.

In recent decades, plants' oils have been used routinely in chicken farms for keeping chickens healthy and enhancing their productive performance because they contain active components which exert positive effects on physiological processes and have medicinal effects such as antibacterial, anti-inflammatory, and antioxidant [7–10]. There is a global trend in restricting the use of antibiotic growth promoters in the animal diet [11] and finding alternative solutions to maintain current animal production efficiency. Essential oils (EOs) have great potential among the alternatives and are generally regarded as safer and residue-free [12]. Due to their preventive and curative properties, species of the Labiatae family have enjoyed a rich tradition of flavoring and pharmaceutical use. Rosemary (*Rosmarinus o*ffi*cinalis*) is an herb that belongs to this family and has been recognized as the plant with the highest antioxidative activity [13]. Rosmarinic acid, camphor, and the antioxidants carnosic acid and carnosol are the most important organic chemicals, which have been already extracted from rosemary [14,15]. The supplementation of rosemary oil (200 mg/kg) in the laying hen's diet led to a significant improvement in feed conversion and an increase in the Haugh unit (key indicators of

internal egg quality) of the egg as well as a larger egg weight [16]. It was also determined that rosemary oil exhibited higher antimicrobial activity than the control (commercial diet) by reducing the *E. coli* concentration in feces. Additionally, using rosemary as a natural antioxidant can decrease plasma total lipids when compared to the control, while LDL-cholesterol and total cholesterol can be insignificantly decreased [7]. Supplementation of 1% rosemary can also improve feed conversion and decreased malonaldehyede (MDA) formation in egg yolk, and has been shown to have a positive impact on oxidative stability of eggshell storage [7]. Moreover, Alagawany and Abd El-Hack [17] concluded that rosemary supplemented up to 6 g/kg diet can be used as an effective feed additive to improve performance, immunity and antioxidant status in laying hens.

On the other hand, cinnamon (*Cinnamomum zeylanicum*) is a common herb and is produced from the bark of the cinnamon tree. Cinnamon herb or its derivatives can serve as a hepatic stimulant by increasing bile secretion, removing toxins, and regulating hydration and can be used as a growth enhancer. Additionally, nutritional aspects of cinnamon powder or its derivatives include positive impacts regarding growth curve, digestion, absorption, activity of gut microbiota, immunity, as well as improved feed utilization and public health of poultry [18]. As a conclusion of ¸Sim¸sek et al.'s [9] study, working on laying quails, cinnamon essential oil supplementation into a diet with a 200 ppm level increased egg production, eggshell quality, and improved the feed conversion ratio. On the other hand, rosemary supplementation with the same amount did not result in any positive or negative effects on egg production traits while the mixture of both of them had a negative effect on egg weight.

The data concerning the effect of different essential oils (rosemary and cinnamon) under different housing systems (floor and cage) on production, egg quality, immunity, haematology, blood biochemical and antioxidant parameters of layers are rare. Therefore, the objective of this research was to investigate the effects of a housing system, and the supplementation of rosemary and cinnamon essential oils on layers' performance, haematological traits, blood chemistry, immunity, egg quality, and antioxidants.

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

All procedures were implemented according to the Local Experimental Animal Care Committee and approved by the ethics of the institutional committee of Damanhour University, Egypt.

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

Three thousand 27-week old ISA brown laying hens were obtained from Al Waha poultry industry (Damo—El Basyounia—El Fayoum—Egypt). A factorial arrangement (2 × 3) was performed, which included two housing systems and three different types of essential oils (0, 300 mg/kg diet of rosemary and 300 mg/kg diet of cinnamon essential oils) to study their effects on the productive performance, egg quality, immunity, oxidative stress and haematology of laying hens during the production stages (from 28 to 76 weeks of age). Birds were randomly divided into two groups each comprising 1500 birds; the first group was moved to laying cages, while the second group was floor reared. Each group was randomly divided into three groups, the first were considered as a control group, the second treated with rosemary essential oil, and the third with cinnamon essential oil. Each group was divided into five equal replicates each of 100 birds. Rosemary essential oil was obtained from Quanao, Shaanxi, China; and cinnamon essential oil was obtained from YiSenYuan, Jiangxi, China (the purity of both oils was 100%). The birds were housed in an open sided farm and each replicate of floor reared layers were housed in a separate pen of about 10 m2 space. The pens were separated by nets of 2 m height to avoid group mixing and to avoid interference with air movement, while caged birds in each replicate were housed in separate cages divided into pens, where each pen had dimensions of 40 cm × 50 cm × 40 cm suitable for five birds. The cage consisted of two levels, with each level containing 10 pens (5 on each side).

#### *2.2. Dietary Treatments*

Composition of basal diet and their calculated analysis is presented in Table 1. The hens were fed diets in mash form during the experiment (28–76 weeks). The diets were formulated to meet or exceed NRC [19] recommendations.


**Table 1.** Ingredients and calculated analysis of layer basal diet.

\* Each diet was supplied with 3 kg/ton Vitamins and Minerals Mix (commercial source B. p. Max) Each 3 kg contains, Vit. A 10,000,000 MIU, Vit. D 2,000,000 MIU, Vit. E 10,000 mg, Vit. K3 1000 mg, Vit. B1 1000 mg, Vit. B2 5000 mg, Vit. B6 1500 mg, Biotin 50 mg, BHT 10,000 mg, Pantothenic 10,000 mg, folic acid 1000 mg, Nicotinic acid 30,000 mg Mn 60 g, Zinc 50 g, Fe 30 g, Cu 4 g, I 3 g, Selenium 0.1 g and Co 0.1 g. \*\* The diets were formulated to meet or exceed NRC [19] recommendations.

#### *2.3. Estimation of Laying Performance Parameters and Egg Quality*

Hen-day egg production (HDEP), feed consumption and egg weight was recorded daily on a replicate basis. Feed intake was calculated by subtracting the remaining feed daily from the offered feed. Feed conversion ratio was calculated as grams of feed intake per gram of egg mass produced. Average egg mass (per hen per day in grams) = % HDEP × Average egg weight in grams. The parameters relative to egg quality were evaluated at 72 weeks of age. Fifteen eggs were randomly collected per treatment to determine these parameters. The collected eggs were weighed using a digital balance. On breaking, the egg contents were poured. The Haugh unit (HU) was measured for the internal quality of the eggs [20]. The height, correlated with the weight, determined the HU. The higher the number, the better the quality of the egg (fresher, higher quality eggs have thicker whites). Eggshell, albumin, and yolk percentages were also measured. Eggshell thickness (without the shell membrane) was measured from the middle part of the egg using a micrometer. Yolk index was calculated by formula: Yolk height/Yolk width × 100; while the egg index was calculating using the following formula: (Egg width/egg length) × 100.

#### *2.4. Estimation of Blood Haematological and Biochemical Parameters*

Blood samples (*n* = 25) were collected from each group as five samples from each replicate from the wing vein at 56 weeks of age. After collecting the blood samples, the tubes were left in the slope position until serum samples were separated through centrifugation at 3000 rpm for 15 min. Red blood cells (RBCs) and white blood cells (WBCs) counts were determined according to Stoskopf [21] using haemocytometer. Blood hemoglobin (HB) was assessed by cyanomta-hemoglobin method [22]. Packed cell volume (PCV) was carried out by using microhaematocrit capillary tubes centrifuged at 12,000 rpm for 5 min. The reading was made with the aid of a microhaematocrit reader and expressed as the volume of erythrocytes per 100 cm3 according to Blaxhall and Daisley [23]. Differential leucocytic counts were defined using a blood film that was prepared according to the method described by Lucky [24]. Ten drops from May-Grunwald stain stock solution on a dry, unfixed smear were added to an equal amount of distilled water, then mixed and left for 1 min for staining. The dye was decanted without rinsing. Diluted Giemsa's solution (10 drops of the dye were added to 10 mL of distilled water) was poured over the film as a counter stain and left for 20 min, then rinsed in water current and examined by oil immersion lens. The percentage and absolute value for each of the type of cells were calculated according to Schalm et al. [25]. The sera were collected and preserved in a deep freezer at (−20 ◦C) until the time of analysis. All the following studied parameters were calorimetrically evaluated. Estimation of blood cholesterol content was determined by cholesterol kit of Bio-diagnostic according to Richmond [26] and Allain et al. [27]. Total protein was determined by kits of Bio-diagnostic according to the method of Gornal et al. [28]. Alanine Aminotransferase (ALT) was determined by the ALT kit of Bio-diagnostic according to the method of Reitman and Frankel [29], while Aspartate Aminotransferase (AST) was determined by the AST kit of Bio-diagnostic according to the method of Retiman and Frankel [29]. Creatinine was defined according to the method of Bartles et al. [30], while urea was defined according to the method of Fawcett and Scott [31]. Calcium (Ca) and Phosphorus (P) were measured spectrophotometrically by using commercial kits (Spectrum chemical company, PO. Box 30, Obour City—Cairo, Egypt).

#### 2.4.1. Estimation of Malondialdehyde, Glutathione Peroxidase and Super Oxide Dismutase

Estimation of blood Malondialdehyde (MDA) concentration was measured by the method of Jo and Ahn [32]. Determination of Glutathione peroxidase (GPx) activity measured using the Paglia and Valentine [33] spectrophotometric method based on the Northwest Life Science Specialties (NWLSS™) Glutathione peroxidase assay kits protocol NWK-GPX01. Determination of Super Oxide Dismutase (SOD) activity was assessed using the NWLSS™ Superoxide dismutase activity assay, which provided a simple, rate method for determining SOD activity. This method is based on monitoring the auto-oxidation rate of haematoxylin as originally described by Martin Jr. et al. [34].

#### 2.4.2. Estimation of Phagocytic Index, Phagocytic Activity and Cellular Immunity

Blood and serum samples were collected at 56 days of age (five samples per replicate and total 25 samples per each group) as mentioned above according to Stott and Fellah [35] and used for the determination of Phagocytic activity and phagocytic index was determined according to Kawahara et al. [36]. Fifty micrograms of Candida albicans culture was added to 1 mL of citrated blood from each sample and incubated in a water bath at 25 ◦C for five hours, and then blood smears from each tube were stained with Giemsa stain. Phagocytosis was estimated by determining the proportion of macrophages, which contained intracellular yeast cells in a random count of 300 macrophages and expressed as percentage of phagocytic activity (PA). The number of phagocytized organisms was counted in the phagocytic cells and called the phagocytic index (PI).

Phagocytic activity (PA) = Percentage of phagocytic cells containing yeast cells.

Phagocytic index (PI) = Number of yeast cells phagocytized/Number of phagocytic cells.

#### *2.5. Statistical Analysis*

Data were analyzed by the statistical analysis system SAS [37]. A 2 × 3 factorial design was used to analyze data of performance as a response to two housing systems and three different types of essential oils. Differences among means were detected using two-way analysis of variance (ANOVA). The differences among means were determined using the Duncan test (*p* < 0.05). The model used was:

$$\mathbf{Y}\_{\mathbf{i}\mathbf{j}} = \boldsymbol{\mu} + \mathbf{D}\_{\mathbf{i}} + \mathbf{A}\_{\mathbf{j}} + \mathbf{D} \mathbf{A}\_{\mathbf{i}\mathbf{j}} + \mathbf{e}\_{\mathbf{i}\mathbf{j}}$$

where: Yij = an observation, μ = the overall mean, Di = fixed effect of housing system, Aj = fixed effect of essential oils, DAij = fixed effect of interaction between housing system and essential oils and eij = random error associated to each observation.

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

The effects of different housing systems, essential oils supplementations and their interaction on the egg production of laying hens is shown in Table 2. The differences in egg production percentages between the different housing systems (floor and cage) were not significant at *p* < 0.05 or 0.01. This finding was in agreement with the results of Zita et al. [38]. They indicated no effect of housing system on the egg production of hens. Egg production percentages were significantly (*p* < 0.05 or 0.01) higher in the rosemary and cinnamon groups than in the control group, whereas there were no significant interactions. ¸Sim¸sek et al. [9] found that cinnamon supplementation helped to increase the egg production. Moreover, Ding et al. [39] revealed that hen-day egg production was significantly improved (*p* < 0.05) at 58 to 61 weeks with the diet supplemented with essential oils Enviva commercial product (50, 100, and 150 mg/kg) including thymol 13.5% and cinnamaldehyde 4.5%.



Means in the same column within each classification bearing different letters are significantly different. (*p* < 0.05 or 0.01). <sup>1</sup> EOs: Essential oils, EO: Essential oil. <sup>2</sup> SEM: standard error mean.

The effects of different housing systems, essential oils supplementations and their interaction on egg weight of laying hens are shown in Table 3. The differences in egg weight between the different housing systems (floor and cage) were not significant at (*p* < 0.05 or 0.01). This finding was also in agreement with the results of Zita et al. [38]. The egg weights were significantly (*p* < 0.05 or 0.01) higher in rosemary and cinnamon groups than in the control group at 28–36 and 52–60 weeks while there were no significant interactions. The highest egg weight was found in the rosemary group at 44–52 weeks and 60–68 weeks. In agreement, ¸Sim¸sek et al. [9] reported that the highest egg weight in quail was found in the rosemary group. On the other hand, Alagawany and Abd El-Hack [17] reported that there were no differences in egg weight due to adding rosemary to laying hens. Furthermore, Ding et al. [39] and Cufadar [40] reported that egg weight was not affected by the diet supplemented with essential oils.


**Table 3.** Egg weight of laying hens as affected by different housing systems, essential oils and their interaction during the experiment.

Means in the same column within each classification bearing different letters are significantly different. (*p* < 0.05 or 0.01). <sup>1</sup> EOs: Essential oils, EO: Essential oil. <sup>2</sup> SEM: standard error mean.

The effects of different housing systems, essential oils supplementations and their interaction on egg mass of laying hens are shown in Table 4. The differences in egg mass between the different housing systems (floor and cage) were not significant at (*p* < 0.05 or 0.01). The egg masses were significantly (*p* < 0.05 or 0.01) higher in rosemary and cinnamon groups than in the control group at 28–36, 44–52, 52–60, 60–68 and 68–76 weeks, while the differences among the interactions were not significant except at 68–76 weeks, and the cage × cinnamon group was the highest. In agreement, Alagawany and Abd El-Hack [17] reported that egg mass linearly increased with rosemary supplementation, while Cufadar [40] indicated that there were no differences in egg mass due to the addition of rosemary in laying hen diet.



Means in the same column within each classification bearing different letters are significantly different. (*p* < 0.05 or 0.01). <sup>1</sup> EOs: Essential oils, EO: Essential oil. <sup>2</sup> SEM: standard error mean.

The effects of different housing systems, essential oils supplementations and their interaction on feed intake of laying hens are shown in Table 5. The differences in feed intake between the different housing systems (floor and cage) were not significant at *p* < 0.05 or 0.01. The feed intakes were significantly (*p* < 0.05 or 0.01) lower in the rosemary and cinnamon groups than in the control group at 60–68 and 68–76 weeks. Apart from feed intake at 44–52 weeks, there were no significant differences due to the interaction effect between EOs and housing system. Under the floor system, birds fed diet supplemented with or without EOs consumed more feed (127.40 to 128.62 g) than those raised under the cage system (119.50 to 124.60 g). Feed intake was found to be similar between the rosemary and cinnamon groups as mentioned before by ¸Sim¸sek et al. [9]. Although, essential oils are perceived as growth promoters in poultry diets [41], recent studies on poultry feeding intakes [42,43] have indicated that dietary incorporation of essential oils did not significantly affect the bird feed intake or they could decrease it insignificantly. However, there is a possible explanation for the reduced intake of feed is the irritating scent of essential oils, which makes the diet unpleasant to birds.


**Table 5.** Feed intake (g) of laying hens as affected by different housing systems, essential oils and their interaction during the experiment.

Means in the same column within each classification bearing different letters are significantly different. (*p* < 0.05 or 0.01).1 EOs: Essential oils, EO: Essential oil. <sup>2</sup> SEM: standard error mean.

The effects of different housing systems, essential oils supplementations and their interaction on feed conversion of laying hens are shown in Table 6. The differences in feed conversion between the different housing systems (floor and cage) were not significant at *p* < 0.05 or 0.01. The feed conversions were significantly (*p* < 0.05 or 0.01) lower in the rosemary and cinnamon groups than in the control group at 28–36, 36–44, 52–60, 60–68 and 68–76 weeks, while the differences among the interactions were not significant at (*p* < 0.05 or 0.01) except at 44–52, 52–60, and 68–76 weeks, with the lowest rates being observed for layers fed diets enriched with EOs in the cage. The cage × rosemary group achieved the best values of FCR at 44–52 and 52–60 weeks; while at 68–76 weeks, the best value was recorded by the cage × cinnamon group. It can be concluded that the interactions among cage and EOs system achieved the good results for the FCR in comparison with the floor system. ¸Sim¸sek et al. [9] found that the best feed conversion was obtained in the cinnamon group. Ding et al. [39] reported that the hen feed conversion ratio was significantly improved (*p* < 0.05) at 58 to 61 weeks with the diet supplemented with essential oils. On the other hand, Alagawany and Abd El-Hack [17] reported that there were no differences in the feed consumption and feed conversion ratio due to adding rosemary to laying hens. However, there are two acceptable mechanisms to understand the effect of these essential oils. The first one considers the promotion of digestive enzyme secretion, and the second deals with the gut microflora ecosystem stabilization, leading to enhanced utilization of food and decreased exposure to growth-depressing disorders that could be related to the metabolism and the digestion processes [44–46]. Several chicken studies have documented positive effects of essential oils on the digestive enzyme (pancreatic α-amylase and intestinal maltase) secretion and intestinal mucosa [47,48]. In broilers, the ileal activity of trypsin and chymotrypsin was significantly increased in the thymol group at day 21 compared with the control group [49]. In the in vitro study, Mathlouthi et al. [50] stated that rosemary essential oils had different antimicrobial impacts against pathogenic microbes however, had the same effect on avilamycin as a growth promoter when added to broiler rations. Furthermore, the latter authors found that in vivo growth promotion effects were due to the alterations in the gut microbiota rather than antimicrobial activities against a single bacterial species and genus. Decreased microbes in the gastrointestinal tract may enhance the proliferation ability of epithelial cells and thus improve intestinal absorptive capacity [51]. These effects have been confirmed by increased nutrient digestibility, however, this has not resulted in improved growth performance [52,53].


**Table 6.** Feed conversion of laying hens as affected by different housing systems, essential oils and their interaction during the experiment.

Means in the same column within each classification bearing different letters are significantly different. (*p* < 0.05 or 0.01). <sup>1</sup> EOs: Essential oils, EO: Essential oil. <sup>2</sup> SEM: standard error mean.

The effects of different housing systems, essential oils supplementations and their interaction on egg quality of laying hens are shown in Table 7. The differences in egg quality traits between the different housing systems (floor and cage) were not significant at *p* < 0.05 or 0.01. In contrast, Tumova and Ebeid [54] found that the egg quality characteristics were better in eggs produced in cages when compared to alternative systems, and eggs produced from cage systems had higher values of Haugh units, albumen and yolk indices. Furthermore, higher eggshell and albumen qualities were observed in conventional cages by Englmaierová et al. [4]. The differences between the study's results could be due to differences in the strain used or the environmental conditions surrounding it (such as cages indoors or outdoors), as well as differences in the cages design. However, the differences in shell thickness among the different essential oil groups (including the control group) were also not significant (*p* < 0.05 or 0.01). On the other hand, Cufadar [40] reported that eggshell thickness was significantly increased with rosemary essential oil supplementation (250 mg/kg) in the diet of NOVOgen White laying hens. In general, Ding et al. [39] reported that eggshell thickness was significantly increased at 65 weeks with the diet supplemented with essential oils. Haugh unit scores were higher in rosemary and cinnamon groups than in the control group, while the control group was the highest in yolk index among the interactions at *p* < 0.05 or 0.01. It means that rosemary and cinnamon supplementation (0.3 g/kg) decreased the egg yolk index for Isa Brown laying hens. On the other hand, Alagawany and Abd El-Hack [17] found that adding rosemary (up 6 g/kg) to Hi-sex Brown laying hen' diets resulted in a linear increase in yolk percent and yolk-to-albumen ratio. While, Botsoglou et al. [55] indicated that diets supplemented with rosemary oil (5 g/kg) for Lohmann laying hens had no effects on the yolk index neither Haugh units. These conflicting results may be due to the different supplementation ratio or/and the strain used. Furthermore, Ding et al. [39] reported that Haugh units, generally, were not affected by the diet supplemented with essential oils.


**Table 7.** Egg quality of laying hens as affected by different housing systems, essential oils and their interaction during the experiment.

Means in the same column within each classification bearing different letters are significantly different (*p* < 0.05 or 0.01). <sup>1</sup> EOs: Essential oils, EO: Essential oil. <sup>2</sup> SEM: standard error mean.

The effects of different housing systems, essential oils supplementations and their interaction on the blood picture (haematological traits) of laying hens are shown in Table 8. The differences in blood picture between the different housing systems (floor and cage) and among the different essential oils groups were not significant at *p* < 0.05 or 0.01 except in some differential WBCs (basophils; lymphocytes and monocytes %).


**Table 8.** Blood picture of laying hens as affected by different housing systems, essential oils and their interaction during the experiment.

Means in the same column within each classification bearing different letters are significantly different (*p* < 0.05 or 0.01). <sup>1</sup> WBC: White blood cells, RBC: Red blood cells, PCV: Packed cell volume, HB: Hemoglobin, Eosino: Eosinophils, Lympho: Lymphocytes, Heter: Heterophils, Baso: Basophils, Mono: Monocytes. <sup>2</sup> EOs: Essential oils, Ros EO: Rosemary essential oil, Cinn EO: Cinnamon essential oil. <sup>3</sup> SEM: Standard error mean.

The effects of different housing systems, essential oils supplementations and their interaction on immunity and antioxidant parameters of laying hens are shown in Table 9. The differences in immunity and antioxidant parameters between the different housing systems (floor and cage) were not significant at *p* < 0.05 or 0.01 while among the different essential oils groups, they were significant in ND, AI H5, AI H9, MDA, and SOD. Excluding GPx activity (*p* < 0.001), all immunity and antioxidant indices were not statistically different as a consequence of the interaction among housing systems and EOs. The interaction between floor and basal diet gave the highest (26.25 U/gHb) activity of GPx in comparison with the other interactions. However, the lowest value (15.00 U/gHb) was found in birds fed with a control diet with the cage system.

In general, some designs of housing systems can cause some stress on hens. This stress can play an effective role in the bird's immune system resulting in failure of vaccination or increased disease during production [56,57]. However, essential oils are a total of volatile constituents, and therefore the effects of essential oils could be a complete product of all components and their interactions. Two or three components can account for up to 85% of the total mixture, and thereby contribute to the primary property of the essential oil mixture [58]. For example, phenols (thymol and carvacrol) account for more than 70% of plant essential oils and are primarily responsible for their antibacterial and antioxidant functions. Rosemary has been recognized as the plant with the highest anti-oxidative activity [13]. On the other hand, cinnamon or its oil play an important role in improving the growth, production, digestion, absorption, activity of gut microbiota, immunity, as well as feed utilization and public health of poultry [18].


**Table 9.** Immunity and antioxidant parameters of laying hens as affected by different housing systems, essential oils and their interaction during the experiment.

Means in the same column within each classification bearing different letters are significantly different (*p* < 0.05 or 0.01). <sup>1</sup> ND: Newcastle disease, AI H5: Avian influenza H5, AI H9: Avian influenza H9, MDA: Malondialdehyde, GPX: Glutathione peroxidase, SOD: Superoxide dismutase. <sup>2</sup> EOs: Essential oils, Ros EO: Rosemary essential oil, Cinn EO: Cinnamon essential oil. <sup>3</sup> SEM: Standard error mean.

Through supplementing essential oils, a bird's anti-oxidative stability can be enhanced [12]. Placha et al. [59] found that malondialdehyde concentration in the liver and kidney was significantly reduced by the supplementation of essential oils to bird diet. Moreover, Lee et al. [60] and Khan et al.[61] reported that thyme essential oil had a significant effect on avian-derived products (meat and eggs) retarding oxidant degradation. Antioxidant activity could be derived from the phenolic OH group, which acts as a hydrogen donor interacting with peroxy radicals during the initial process of lipid oxidation and thereby inhibiting the formation of hydroxyl peroxide [60]. The antioxidant effect differs from plant essential oil to others depending on the amount of total phenols in it. With its successful antioxidant activities in broiler meat, rosemary has been identified [62,63].

The effects of different housing systems, essential oils supplementations and their interaction on the blood chemistry of laying hens are shown in Table 10.

The differences in blood chemistry including the cholesterol between the different housing systems (floor and cage) were not significant at *p* < 0.05 or 0.01. Contrary to this, Zita et al. [38] found that the blood cholesterol level was higher in birds raised in cages than on litter. However, the cholesterol, ALT, AST, and urea levels were significantly (*p* < 0.05 or 0.01) lower in rosemary and cinnamon groups than in the control group. The effect of interaction between dietary the interaction among EOs and housing systems was not significant (*p* < 0.05) on blood chemistry tests except for the blood phosphorus content, the highest levels were observed for birds fed diets enriched with cinnamon oil with a cage or floor system. Alagawany and Abd El-Hack [17] found that serum constituents were not significantly influenced by rosemary supplementation, except for urea and cholesterol. Moreover, Torki et al. [64] reported that birds given rosemary exhibited lower serum cholesterol and triglycerides concentration. The Ca and P were higher in rosemary and cinnamon groups than in the control group.


**Table 10.** Blood chemistry of laying hens as affected by different housing systems, essential oils and their interaction during the experiment.

Means in the same column within each classification bearing different letters are significantly different (*p* < 0.05 or 0.01). <sup>1</sup> ALT: Alanine transferase. <sup>2</sup> AST; Aspartate transferase. <sup>3</sup> EOs: Essential oils, Ros EO: Rosemary essential oil, Cinn EO: Cinnamon essential oil. <sup>4</sup> SEM: Standard error mean.

#### **4. Conclusions**

The supplementation of rosemary and cinnamon essential oils had great impacts on egg production and weight, some egg quality traits, feed intake and conversion, some haematological traits and blood chemistry, immunity, and antioxidant parameters. On the other hand, the different housing systems did not result in any positive or negative impact on these studied traits. However, the cage × cinnamon group was the highest in egg mass, and the lowest in FCR. Therefore, we have recommended the usage of rosemary or cinnamon essential oils at 300 mg/kg in layer's diet to improve its productive performance and egg production.

**Author Contributions:** Conceptualization, M.M.A.G. and M.F.E.; data curation, M.M.A.G., M.F.E., A.E.T., M.E.A.E.-H. and M.A.; formal analysis, A.E.T., M.E.A. and M.A.; funding acquisition, M.F.E., B.M.A. and M.M.E.; investigation, M.M.AG., M.F.E. and M.A.; methodology, M.M.A.G., M.F.E., A.E.T., M.E.A.E.-H., M.A., B.M.A. and M.M.E.; resources, M.M.A.G., A.E.T., M.E.A.E.-H. and B.M.A.; supervision, M.M.A.G., M.F.E. and M.A.; visualization, K.E.-S.; writing–original draft, B.M.A., M.M.E. and K.E.-S.; writing–review and editing, K.E.-S. and M.A. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** Authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no. (RG-1439-81).

**Conflicts of Interest:** The authors declare no conflicts 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/).

### **Green Tea Powder Decreased Egg Weight Through Increased Liver Lipoprotein Lipase and Decreased Plasma Total Cholesterol in an Indigenous Chicken Breed**

### **Xingyong Chen 1,2,\*, Kaiqin He 1, Congcong Wei 1, Wanli Yang <sup>1</sup> and Zhaoyu Geng 1,2,\***


Received: 10 February 2020; Accepted: 14 February 2020; Published: 25 February 2020

**Simple Summary:** Tons of green tea powder (GTP) are produced and cast off during green tea processing. It is suggested that GTP could increase immunity and health, and so improve animal production performance. We demonstrated that one percent of GTP supplemented in the diet did not affect egg production. However, long time GTP inclusion resulted in decreased egg weight and increased feed-to-egg ratio. Combined with plasma lipid concentration, the decreased egg weight might be because of lower plasma lipid concentration, increased plasma orexin A, and liver lipoprotein lipase expression in chickens fed a diet containing GTP.

**Abstract:** Whether or not green tea promotes egg production is unclear. Huainan partridge chickens at 20 weeks of age were divided into two groups, with one group fed a basal diet (control) and one fed a basal diet plus 10 g/kg green tea powder (GTP) for 12 weeks. Egg production (EP) and feed intake (FI) were recorded daily. Plasma lipid parameters, and apolipoprotein-B (Apo-B), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), and lipoprotein lipase (LPL) expression were determined every four weeks. Egg production and FI showed no significant difference between the two groups (*p* > 0.05). Egg weight was 47.58 g in the control group, which was higher than that of the GTP group, and the feed-to-egg ratio (FCR) was 4.62 in the control group, which was lower than that of the GTP group after 12 weeks feeding. Compared with the control group, plasma orexin A (*p* < 0.05), high-density lipoprotein (HDL), apolipoprotein A (Apo A), and very high-density lipoprotein (VHDL) (*p* < 0.01, respectively) were increased. Plasma glucose (Glu), free fatty acid (FFA), apolipoprotein B (Apo B), triglyceride (TG), total cholesterol (TC) (*p* < 0.01, respectively), and low density lipoprotein (LDL) (*p* < 0.05) were decreased in the GTP group after 8 weeks feeding. The LPL expression in the liver was increased in the GTP group after 8 to 12 weeks feeding when compared to the control group (*p* < 0.05). Chickens fed GTP did not affect EP, but decreased egg weight, which might be because of lower plasma lipid concentration, increased plasma Orexin A, and liver LPL expression.

**Keywords:** egg production; green tea powder; huainan partridge chicken; lipoprotein lipase; plasma lipid

#### **1. Introduction**

Green tea is one of the most popular beverages worldwide and produces nearly 14,380,000 tons each year in China. Nearly 5% to 10% green tea powder is produced during green tea processing and cast off. Major components of green tea are polyphenols, including catechins (constitute about 30% of its dry weight), alkaloids, polysaccharide, etc. [1,2]. Tea polyphenols are natural antioxidants that can scavenge free radicals and protect magnum from damage [3]. Green tea could prevent dental caries and reduce cholesterol and lipid absorption in the gastrointestinal tract [4]. Koo and Noh [5] further demonstrate that green tea could lower body fat through interfering with intestinal absorption of dietary fat, cholesterol, and other lipids. Catechins, the major component in tea polyphenols, could decrease plasma and liver malondialdehyde (MDA) concentrations, plasma glucose, and total cholesterol level [6]. A meta-analysis of randomized clinical trials also suggested that green tea decreased plasma total cholesterol and low-density lipoprotein (LDL) cholesterol [1].

The egg is one kind of animal product that is predominantly consumed; it contains many easily absorbed nutrients, and is easy to digest [7]. However, egg production performance could be affected by many factors, including genetics, feed composition, age, etc. [8]. The consumption of eggs from indigenous breeds is widely looked for in the market [8]. The most important problem for an indigenous breed is low egg production performance. Egg production is mostly affected by follicle maturation and ovulation, which is regulated by synthesis and transportation of yolk precursors [9]. The main component for yolk precursors include yolk very low-density lipoprotein (VLDLy, more than 90%) and vitellogenin (VTG); meaning VLDLy and VTG synthesis determine the speed of follicular development [10]. As a rate-limited endogenous cholesterol synthesis enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) determines egg yolk cholesterol content [11]. Apolipoprotein B (ApoB) regulates the synthesis and secretion of VLDLy. Apolipoprotein B could also increase lipoprotein lipase (LPL), binding to cells, and promoting the degradation of Very low density lipoprotein (VLDL) [12].

Wang et al. [3] suggested that adding 200 mg kg−<sup>1</sup> tea polyphenols improved egg production performance in laying hens. Xia et al. mentioned that 10 g/kg green tea powder did not affect egg laying and feed conversion ratio, but a high amount of green tea powder (>20 g/kg) decreased egg production performance [13]. Our previous study also demonstrated that 10 g/kg green tea inclusion could improve meat color and Lactobacillus proliferation for broiler production [14].

It is well known that green tea could lower body fat in animals. However, lipids are one of the most important components in egg yolks, and determine yolk formation. Biswas et al. [15] reported that 6 g/kg of Japanese green tea powder (GTP) supplemented for laying hens tended to decrease egg weight and increase egg production rate. Thus, whether green tea powder could be used as feed additive in indigenous chicken breed during egg laying is still in doubt. The present study was conducted to evaluate the effect of 10 g/kg of green tea powder on chicken laying performance, plasma lipid content, and lipid synthesis related gene expression levels in Chinese indigenous Huainan partridge chickens.

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

All the experimental protocols involving care, handling, and treatment of broilers were approved by the Institutional Animal Care and Use Committee of Anhui Agricultural University, Hefei, Anhui, China. The permission number is No. SYDW-P2018110702.

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

A total of 1080 Huainan partridge hens at 18 weeks of age, with similar body weights (1.46 ± 0.13 kg), were raised in one row of battery cages. There were 30 battery cages (30 replicates, 6 tiers, and 6 cages per tier) in the row, and one hen per cage. One side of the battery cage was used as the control group and the other side was used as the experimental group. The two sides of the battery cages had equal distances to windows. The hens received 13 h light at 20 weeks old, which was extended to 16 h light at 32 weeks of age. Hens from the control group received a basal diet (without GTP), and hens from the experimental group received a basal diet plus 10 g/kg GTP, instead of bran. The feed ingredient and chemical composition are listed in Table 1. The experiment consisted

of a two-week acclimation period and a 12-week collection period. During the two-week acclimation period, the diet with 10 g/kg GTP was gradually applied to hens, instead of the basal diet in one week, and another week for adaptation. Mean body weight, egg production (EP), egg weight (EW) and feed intake (FI) were calculated every 2 weeks from 20 to 32 weeks of age. The feed conversion ratio (FCR) was calculated by the formula FCR = FI/EP × EW.


**Table 1.** Feed composition and nutrition level.

<sup>1</sup> Premix provided per kg of diet: Fe, 65 mg; Cu, 8 mg; Zn, 80 mg; Mn, 105 mg; I, 1 mg; Se, 0.3 mg; vitamin A, 9800IU; vitamin D3, 3100IU; vitamin E, 26 IU; vitamin B1, 2.5 mg; vitamin B2, 7 mg; vitamin B12, 0.018 mg; vitamin K, 2.2 mg; biotin, 0.09 mg; folic acid, 1 mg; pantothenic acid, 11 mg; niacin, 38 mg. <sup>2</sup> CON means chickens fed a basal diet as control group. GTP means chickens fed a basal diet plus 10 g/kg green tea powder. The same as Table 5. <sup>3</sup> The diet calcium was 2.0% at 20 weeks of age, and then gradually increased to 3.2% with the increased egg production.

At 20, 24, 28, and 32 weeks of age, thirty hens from each replicate of each group were randomly selected for blood sampling from the wing vein, and ten hens (one from three replicates) were slaughtered for liver and follicular membrane collection.

#### *2.2. Blood Plasma Orexin A and Lipid Content Determination*

Blood samples (2 mL) were separated by centrifugation (2500× *g* for 10 min at 4 °C). Separated plasma were frozen for lipids and orexin A analysis within one week. Commercial enzyme-linked immunosorbent assay (ELISA) kits were used for the measurement of very high density lipoprotein (JL21659), total cholesterol (JL21710), triglyceride (JL21645), low density lipoprotein (JL15965), high density lipoprotein (JL21648), apolipoprotein B (JL45582), apolipoprotein A (JL21703), free fatty acid (JL15893), plasma glucose (JL21700), and orexin A (JL25500) by using an automatic ELISA analyzer (Rayto RT-6100). All of the kits were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

#### *2.3. Determination of mRNA Expression Level by Real-time Reverse Transcription*

Liver and follicular membrane (stored in −80 °C) were used for total RNA extraction by using a commercial kit (Omega bio-Tek Inc., GA, American) according to the manufacturer's instructions. The quality and quantity of total RNA was determined by using Nanodrop2000 (Thermo Fisher, MA, American). After DNase treatment, 5 μg of total RNA was reverse transcribed by using RNase reverse transcriptase (Easyscript RT/RI Enzyme mix, TransGen Biotech, Beijing, China), random primers (Anchored Oligo[dT]18 Primer [0.5 μg/μL]), and random 6 mers. The mRNA expression level for each gene was determined by real-time reverse transcription according to Chen et al. [16]. β-actin was chosen as reference. The primers used for quantification were listed in Table 2.


**Table 2.** Primers for quantitative RT-PCR.

#### *2.4. Statistical Analysis*

The results from the egg production, egg weight, feed intake, feed conversion ratio, and gene expression were analyzed by two-way ANOVA, with treatment and feeding time as two variables. The results of the lipid parameters and orexin A analyses were subjected to Student *T*-tests by SAS 9.3. Data for gene expression were presented in mean ± SE, and other data were expressed as mean ± SD.

#### **3. Results**

#### *3.1. Laying Hens Performance*

The chicken performances during the feeding trial period were summarized in Table 3. Egg production, egg weight, and feed intake significantly increased with age (*p* < 0.01, respectively), and feed conversion ratio (FCR) significantly decreased with age (*p* < 0.01). No significant difference was observed in egg production performance between the control and GTP groups during the experimental period. Egg weight showed no significant difference between the two groups before 30 weeks of age. That is, after 10 weeks of the feeding diet, containing 10 g/kg GTP, egg weight was significantly lowered than that of the control group (*p* < 0.05). Feed intake tended to be lower in the GTP group than that of the control group at the first four weeks of feeding, and no significant difference was observed between the two groups (*p* > 0.05). Feed conversion ratio (FCR,) was relatively high before 24 weeks of age because of low egg production. A relatively higher feed intake caused a higher FCR in the control group when compared to the GTP group at 22 and 24 weeks of age (*p* < 0.05). Feed conversion ratio was higher in the GTP group than that of the control group after 10 weeks feeding (*p* < 0.05).



a, b Different lowercase letter in the same row within the same item indicates significant difference (*p* < 0.05). EP, egg production; EW, egg weight; FI, feed intake; FCR, feed conversion ratio.

#### *3.2. E*ff*ect of Green Tea Powder on Orexin A and Plasma Lipid Content of Huainan Partridge Chicken*

Compared with the control group, orexin A, apolipoprotein A (Apo A), and high-density lipoprotein (HDL) were significantly increased (*p* < 0.01, respectively), and glucose (Glu), free fatty acid (FFA), total cholesterol (TC), triglyceride (TG) (*p* < 0.01, respectively), and apolipoprotein-B (Apo B) (*p* < 0.05) were decreased in chickens fed diets with 10 g/kg GTP for 4 weeks. No significant difference was observed in the low-density lipoprotein (LDL) and very high-density lipoprotein (VHDL) of chickens fed a diet with GTP or not for 4 weeks. Compared with the control group, orexin A, HDL, Apo A, and VHDL significantly increased; Glu, FFA, Apo B, LDL, TG, and TC significantly decreased in chickens fed a diet with GTP for 8 or 12 weeks (Table 4).


**Table 4.** Effect of Green tea powder on plasma parameters and orexin A of Huainan partridge chickens.

a, b A different lowercase letter in the same row within the same item indicates significant difference (*p* < 0.05). A, B A different uppercase letter in the same row within the same item indicates significant difference (*p* < 0.01). Glu, glucose; FFA, free fatty acid; Apo-B, apolipoprotein B; Apo-A, apolipoprotein A; HDL, high density lipoprotein; LDL, low density lipoprotein; TG, triglyceride; TC, total cholesterol; VHDL, very high-density lipoprotein.

#### *3.3. E*ff*ect of Green Tea Powder on Lipid Metabolize Related Gene Expression*

The expression level of genes related to lipid metabolize was listed in Table 5. The expression of Apo-B in the liver of the control group showed no significant difference at different ages (*p* > 0.05). While the Apo-B expression in the liver of the GTP group significantly increased after 4 weeks of GTP feeding, significantly decreased after 8 weeks of feeding, and kept decreasing after 12 weeks of feeding (*p* < 0.05). The expression of Apo-B in follicular membrane of the control group was significantly higher in chickens at 20 and 24 weeks of age as compared to the chickens at 28 and 32 weeks of age (*p* < 0.05). The expression of Apo-B in the follicular membrane of the GTP group significantly decreased after GTP feeding for 4, 8, or 12 weeks as compared to chickens before GTP feeding (*p* < 0.05). The expression of HMGR in the liver of the control group was higher at 20 and 24 weeks of age as compared to 28 and 32 weeks of age (*p* < 0.05). The expression of HMGR in the liver of the GTP group was decreased after GTP feeding for 4, 8, or 12 weeks as compared to chickens before GTP feeding (*p* < 0.05). The expression of HMGR in follicular membrane showed no significant difference in both control and GTP groups (*p* > 0.05). The LPL expression in the liver showed no significant difference within the control and GTP groups during the experiment (*p* > 0.05). However, the LPL expression in the liver significantly increased in chickens fed GTP for 8 to 12 weeks, as compared to control groups during the same time

(*p* < 0.05). The LPL expression in the follicular membrane showed no significant difference in both control and GTP groups (*p* > 0.05).


**Table 5.** Expression of Apo-B, HMGR, and LPL in the liver and follicular membrane in Huainan partridge chickens fed GTP, or not, for different times.

a, b Means with no common superscripts are different within the same row (*p* < 0.05).

#### **4. Discussion**

In our previous research, it has been demonstrated that 10 g/kg of green tea powder as a feed additive could promote intestinal health and meat quality, and did not affect the body weight of broilers [14]. Xia et al. [13] also suggested that 1% green tea powder was beneficial on egg quality from Chinese local chicken breeds, but a high amount of green tea powder (>20 g/kg) inclusion in the diet could decrease the egg weight and increase the feed-to-egg ratio. Thus, 10 g/kg of green tea powder inclusion was selected to analyze its effect on egg production performance, chicken blood parameters, and lipid synthesis related genes (Apo-B, HMGR, and LPL) expression level.

The results of laying performance suggested that 10 g/kg of green tea powder did not affect egg laying in chickens. A digitally higher feed intake in chickens from the GTP group was observed, which might be because of increased orexin A caused by intake of green tea powder. However, Soori et al. [17] stated that green tea could reduce orexin A in overweight and obese women. Chickens before egg laying are usually under strict feed restriction to control body weight. In addition, green tea could trigger feed consumption especially under starvation. Combined with a lower palatability of diet with GTP, a digitally lower feed intake was observed in chickens fed GTP at the first four weeks. However, we also detected increased orexin A in chickens fed diets with GTP for 4 weeks, which further suggests that green tea might trigger feed consumption in laying hens after 8 weeks of feeding.

In this research, a lower level of glucose, FFA, Apo-B, LDL, TG, and TC were detected in chickens fed diets with GTP when compared to chickens from the control group. The main components in green tea powder are tea polyphenol, caffeine, catechins, crude fiber, etc. [13]. Although some intervention research suggested no significant effects on plasma lipids after six cups of green tea per day for 4 weeks [18], there were still many studies that insisted that catechins decreased blood glucose, triglycerides, and total cholesterol content in humans and poultry [6,19]. Tea could suppress the glucose transport activity in the intestinal epithelium to lower the absorption of sugar and then reduce blood sugar levels [20]. Green tea has been demonstrated to reduce cholesterols and lipids absorption in the gastrointestinal tract [5]. It is also widely accepted that green tea consumption could reduce blood LDL and glucose levels [1,21]. A higher Apo-A, HDL, and VHDL were also observed in chickens

fed diets with GTP as compared to chickens fed basal diets. Catechins from green tea could decrease the apolipoprotein B-100 (ApoB) level of human plasma in a radical reaction initiated by Cu2<sup>+</sup> [22], which explained the decreased level of ApoB in chickens fed diets with GTP. In vitro experiments also demonstrated that green tea could inhibit ApoB secretion via the proteasome-independent pathway [23]. Increased HDL and apolipoprotein A (Apo A) was observed [21] in Portuguese adults who were given 1 liter of green tea per day for 4 weeks, which is in accordance with the data obtained from this experiment. During egg laying, lipogenic, and genes related to egg yolk formation in the liver were highly expressed, which caused high plasma lipid concentration in layers [24]. High plasma VLDL will then pass through the granulose basal lamina and reach the receptors located on the oocyte surface for providing yolk precursors [25]. Reduced plasma VLDL concentration might decrease egg yolk weight. The yolk weight is positively correlated with egg weight [25]. Thus, a significantly lower egg weight detected in chickens from the GTP group might be because of the decreased plasma lipid concentration. The increased feed intake and decreased egg weight caused a higher FCR (feed-to-egg ratio).

HMGR is a key rate-limiting enzyme in the synthesis of endogenous cholesterol. When dietary cholesterol absorption increased, the expression of HMGR decreased and resulted in a reduced endogenous cholesterol synthesis [11]. In this experiment, the expression of HMGR was in large quantity in the liver for the rapid development of oocytes before egg laying, and then decreased after egg laying. Under the stimulation of estrogen, large number of lipoproteins Apo-B and Apo-2 were synthesized in the liver, the secreted apolipoproteins bind with cholesterol to form VLDLy for the rapid growth of oocytes [26]. Before egg laying, plasma VLDL increased rapidly under the regulation of estrogen and the Apo-B accounts for more than 70 percent of lipoproteins [27]. Thus, it is clear that the high expression of Apo-B in liver and follicular membranes before egg laying is to promote the rapid development of follicles. As a key enzyme for regulating lipid metabolism, LPL can catalyze the hydrolysis of triglyceride to glycerol and FFA, and can remove TG-rich lipoproteins [28]. In this experiment, a significant difference was observed in the expression of LPL between the two treatment groups. It is stated that the expression of the high level of VLDL could inhibit the activity of lipoprotein lipase in laying birds; thus, allowing greater lipid transport to the ovary for egg yolk formation [24]. It is further demonstrated that higher LPL expression in chickens from the GTP group inhibited VLDL formation and yolk synthesis.

#### **5. Conclusions**

In conclusion, green tea powder inclusion decreased plasma lipid concentration and increased orexin A content in Huainan partridge chickens. Green tea powder might decrease egg production after long-time inclusion through decreasing plasma TG content by increasing liver LPL expression.

**Author Contributions:** Conceptualization, X.C. and Z.G.; Methodology, X.C., C.W., W.Y., and K.H.; Formal analysis, X.C. and K.H.; Investigation, C.W., W.Y., and K.H.; Writing and editing, X.C. and Z.G.; Funding acquisition, X.C. All authors have read and agreed to the published version of the manuscript

**Funding:** This work was financially supported by Major Scientific and Technological Special Project in Anhui Province (18030701174) and Key Natural Science Research Projects in Universities of Anhui Province (KJ2018A0951).

**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*

### **Detoxification Impacts of Ascorbic Acid and Clay on Laying Japanese Quail Fed Diets Polluted by Various Levels of Cadmium**

**Diaa E. Abou-Kassem 1, Mohamed E. Abd El-Hack 2,\*, Ayman E. Taha 3,\*, Jamaan S. Ajarem 4, Saleh N. Maodaa <sup>4</sup> and Ahmed A. Allam <sup>5</sup>**


Received: 6 February 2020; Accepted: 20 February 2020; Published: 25 February 2020

**Simple Summary:** The present study aimed to evaluate the impacts of ascorbic acid and clay supplementation on laying Japanese quail fed diets polluted by various levels of cadmium (Cd). Results revealed that consuming polluted diets with Cd causes harmful impacts on the productive performance of laying Japanese quail. The supplementation of ascorbic acid or natural clay to layer diets had beneficial effects on productive performance, improved egg quality and diminished the toxic effect of Cd.

**Abstract:** A total number of 360 laying Japanese quail (8 weeks of age) were randomly divided into 12 groups. Birds in all groups had nearly the same average initial body weight. A factorial arrangement (4 × 3) was performed including four levels of dietary cadmium (Cd) as cadmium chloride (0, 50, 100, and 150 mg/kg diet) and three levels of feed additives (without, 300 mg/kg ascorbic acid and 1.50% natural clay). Results revealed that Cd contaminated feed caused significant (*p* < 0.01) retardation in body weight, lower egg number and egg mass and worse feed conversion. On the other hand, the addition of ascorbic acid or natural clay to quail diets caused a significant (*p* < 0.01) improvement in all studied traits. With respect to the interaction among Cd and the experimental additives, results showed that within each Cd level, ascorbic acid or clay supplementation recorded the highest body weight, egg number, egg weight and mass in addition to improved feed conversion. Cadmium levels decreased (*p* < 0.05) blood total protein, albumen and A/G ratio. Both 300 mg ascorbic acid and 1.50% clay increased (*p* < 0.05) blood total protein and albumen compared to non-supplemented groups. It could be concluded that the consumption of polluted diets Cd causes deleterious effects on the productive performance of laying Japanese quail. The addition of ascorbic acid or natural clay to the diets causes beneficial effects on productive performance traits, improves egg quality criteria and diminishes the toxic effects of Cd.

**Keywords:** cadmium; ascorbic acid; clay; performance; blood biochemical parameters

#### **1. Introduction**

For commercial use, domestic quails are available in both laying and meat strains [1–3]. The reproductive performance of Japanese quail is important in the overall management of the flock [4]. The contamination of the diets and the environment with heavy metals remains a big problem. It affects food safety and subsequently affects the consumers. The contamination of poultry rations with heavy metals causes a high reduction in feed efficiency and egg production, which finally result in a great economic loss for poultry farmers. All potential feed ingredients contain some kinds of heavy metals. Cadmium is considered one of the major environmental pollutants [5]. It is a highly toxic and reactive element which is distributed sparsely in the most of agricultural ecosystems. Once absorbed by humans or animals, Cd is poorly excreted. Great efforts are being made to protect the human food-chain from the entry of cadmium. The excess of Cd intake more than 2 mg/kg diet resulted in elevation in metallothionein synthesis, disturbance of the metabolism of Zn, Ca, and Fe [6]. Cd toxicity induced altered energy, altered behavioral responses, metabolism, kidney damage, anemia, adrenal hypertrophy and cardiac dysfunction [7]. Some reports found that kidney changes occurred independent of bone disease, while others concluded that Cd induced renal dysfunction is the secondary cause of skeletal deterioration [8]. Recently, Saleemi et al. [9] concluded that Cd leads to hepatotoxic and gonadotoxic effects in the quails with dose of 150 and 300 mg/kg feed. As well, researchers found that Cd affects the biochemical parameters of birds. It may decline blood concentrations of total protein and total albumen, A/G ratio and increase the activity of liver enzymes and levels of blood uric acid, urea, and alkaline phosphatase [8,9].

Ascorbic acid is a potent water soluble antioxidant capable of neutralizing and scavenging an array of reactive oxygen species viz., alkoxyl, hydroxyl, superoxide anion, peroxyl, hydroperoxyl radicals, and radicals of reactive nitrogen such as nitroxide, nitrogen di-oxide, and peroxynitrite at very low concentrations [10–12]. Ascorbic acid is required for the conversion of vitamin D into its metabolite form (calciterole) which is essential for calcium regulation and the calcification process [13]. It is also is an antioxidant and water-soluble vitamin that is found intra- and extra cellular as ascorbate [14]. In addition, it is a natural antioxidant that prevents the increased production of free radicals induced by oxidative damage to lipids and lipoproteins in various cellular compartments and tissues [15]. Ascorbic acid supplementation at 200 ppm is beneficial for enhancing the immunity, performance, and exploiting the broilers' full genetic potential [16]. Sharaf et al. [17] concluded that ascorbic acid has potent antioxidant activity against lead and Cd toxicity. They added that the consumption of foods rich in ascorbic acid is a highly recommended to reduce the damage caused by the toxicity with cadmium.

Supplementing poultry diets with natural clay improves growth performance and egg production rate which is a consequence of the improvement in the nutrient digestibility, feed conversion, ability to make rations more available to the bird and nitrogen retention in bird body, and retarded the absorption of toxic products of digestion that reduce toxicity [18]. Ability of clay to diminish the harmful effects of radiation may have a role in this respect [18]. Abou-Kassem et al. [19] reported that clay supplementation diminished the toxic effects of Cd with levels up to 120 mg/kg of quail diet. The present study aimed to investigate the role of ascorbic acid or natural clay on laying performance, some blood biochemical components and Cd residues in egg components of Japanese quail layers fed diets polluted by Cd at various levels.

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

A total number of 360 laying Japanese quail at 8 weeks of age were randomly divided into 12 groups (30 birds/group). Each group was sub-divided into five replicates (6 females each). Each replicate was housed in one cage with area of (2400 cm2). A factorial arrangement (4 <sup>×</sup> 3) was applied including four levels of cadmium chloride (CdCl2); the first group (G0) received zero cadmium level and served as a control group, the other three groups G1, G2, and G3 received 50, 100 and 150 mg/kg diet, respectively). Each group was subdivided into three kinds of feed additives (control, 300 mg ascorbic acid/kg diet

and 1.50% natural clay) to study the effect of cadmium, feed additives and their interactions on the productive performance, egg quality and cadmium residues in eggs of laying Japanese quails.

Basal experimental diet was formulated to meet the laying Japanese quail nutrient requirements as recommended by National Research Council (NRC) [20] as shown in Table 1. Vitamin C (Rovimix® Stay-C 35 (Obour city, Egypt); specifically produced for the use as a stabilized source of vitamin C in feed; L ascorbic acid), according to the manufacturers' guidelines. Cadmium chloride (CdCl2, 2.5 H2O) imported from Chem-Lab NV, Industriezone, De Arend 2, B-8210 Zedelgem, Batch Nr.: 23.5852506, Belgium. Natural clay (from Adwia Company LTD, Obour city, Egypt, Batch No. E09860200) analysis as soluble cations and anions (milligram equivalent (meq)/ 100 gm dry matter soil) were Mg++ 0.25, Ca++ 0.75, K+ 0.10, Na+ 0.05, So4 0.30, Cl 0.55, and HCO3 0.75. Exchangeable cations (meq/100 g dry matter soil) was 2.65 and available nutrients (mg /100 g dry matter soil) were K 1.2, Mn 2.4, P 5.0, Cu 0.30, Zn 0.74, and Fe 0.55 mg [21]. The birds were fed the contaminated diets from 8 to 20 weeks of age. While, at the period from 21 to 24 weeks of age, birds were fed diets without cadmium addition. Birds were reared during the experimental periods under the same management, hygienic and environmental conditions. The bird's health status was monitored throughout the trial. Quails were exposed to 16 hours of light per day; fed ad-libitum and fresh water was available during the experimental periods. Drinkers and feeding troughs were daily cleaned.


**Table 1.** Composition and chemical analysis of experimental diet (8–24 w).

\* Layer Vitamin and Mineral premix Each 3 kg consists of: Vit A. 12 Mi.U, Vit E. 15 IU., Vit. D3 4 Mi.U, Vit, B1 1g, Vit, B2 8g, Pantothenic acid 10.87g, Nicotinic acid 30g, Vit. B6 2g, Vit. B12 10 mg. Folic acid 1 g, Biotin 150 mg, Copper 5g, Iron 5g, Manganese 70 g Iodine 0.5g, Selenium 0.15 g, Zinc 60 g. Antioxidant 10 g; \*\* Calculated according to NRC [20].

#### *2.1. Laying Performance*

Egg number and egg weight were daily recorded per replicate in each pen from 8 weeks of age up to the end of the experiment (24 weeks of age). Egg mass (g) was obtained by multiplying egg number by the average egg weight in each pen per day (8–24 weeks of age). Feed conversion during the experimental period (8–24 weeks of age) was calculated for egg production as follows:

Feed conversion for egg produced = g feed/ g egg produced.

Mortality rate of laying experimental periods was calculated as follows:

$$\mathbf{M}.\mathbf{R} = \mathbf{N}\_1 - \mathbf{N}\_2/\mathbf{N}\_1\tag{1}$$

where, M.R—Mortality rate; N1—The number of birds at the beginning of laying period; N2—The number of birds at the end of laying period.

#### *2.2. Blood Biochemical Components*

At 20 and 24 weeks of age, blood samples were collected from sacrificed quails into heparinized sterile tubes. Samples then centrifuged at 3500 rpm for 15 min and serum was separated, collected and stored at −20 ◦C till examination. Total albumin, protein, urea-N, creatinine, uric acid, aspartate transaminase (AST), alanine transaminase (ALT), and alkaline-phosphatase activities (ALP) were assessed using biodiagnostic commercial kits provided from Biodiagnostic Company (29 El-Tahrir St. Dokki, Giza, Egypt) Batch No: ALT (cat#AL1031), AST (cat#AS1061) according to the manufacturers' guidelines (REF: 264 001, 264 002) and a spectrophotometer (Shimadzu). The globulin concentrations and A/G ratio were calculated from the difference between the concentrations of total protein and albumin.

#### *2.3. Cadmium Residue in Egg Components*

At 20 and 24 weeks of age, Cd residues in egg components were determined. Twenty eggs from each treatment group were randomly taken, broken, and egg components were separated (white, yolk and shell) using a separation funnel and directly put in plates then transferred to an oven at 70 ◦C for 24 h or till a constant dray mass was achieved [22]. The dried samples were perfectly ground and homogenized to be prepared for digestion and residues determination. Cadmium residues were estimated in eggs collected from each treatment group (μg/g wet weight, ppm) in the Central Laboratory of the Faculty of Veterinary Medicine, Zagazig University, Egypt. One gram of each egg component sample (white, yolk, and shell) was placed in a clean screw capped glass bottle and digested with a 4 mL of digesting solution (nitric/per chloric acids, 1:1). Initial digestion was carried out for 24 h at room temperature followed by heating at 110 ◦C for 2 h. After cooling, deionized water was added, then the solutions were warmed in water bath for 1 h to expel nitrous gases. Digests were then filtered (Whatman No. 1, Ashless, grade 42, cat# 1442-110), and diluted in water to 25 ml deionized water [23]. The obtained solutions were analyzed by flame atomic absorption spectrophotometer (FAAS), (PerkinElmer, 520 South Main St., Suite 2423, Akron, Ohio 44311, USA, Model 2380, Serial No. 131865) to measuring the level of Cd residues in each egg components for all treatment groups. The laboratory had established a calibration programme, which reviewed and adjusted as necessary in order to maintain confidence in the status of calibration, whereas, the laboratory follows international standard Iso/IEC, 17025.

#### *2.4. Statistics*

Data were statistically analyzed on a 4 × 3 factorial arrangement basis according to Snedecor and Cochran [24] using the following model:

$$\mathbf{Y}\_{\rm ijk} = \mu + \mathbf{A}\_{\rm i} + \mathbf{S}\_{\rm j} + \mathbf{A}\mathbf{S}\_{\rm ij} + \mathbf{e}\_{\rm ijk} \tag{2}$$

where Yijk = an observation, μ = the overall Mean, Ai = effect of cadmium level (i = 1 to 4), Sj = effect of feed additives (j = 1 to 3), ASij = the interaction between cadmium level and feed additives (ij = 1 to 12) and eijk = random error. Differences among means within the same factor were tested using Duncan's New Multiple Range test [25].

#### **3. Results**

#### *3.1. Productive Performance*

Significant (*p* < 0.05 and *p* < 0.01) reduction was shown in live body weight with increasing the dietary Cd level at 12, 20, and 24 weeks of age compared to the control (Table 2). Final body weight of laying Japanese quail fed diet supplemented with ascorbic acid or clay was significantly (*p* < 0.05 or 0.01) increased comparing to those fed the control diet (Table 2).


weightchanges,eggnumber,eggweightandmortalityrateofJapanesequaillayersasaffectedbydietarycadmiumlevels,somefeed

567

#### *Animals* **2020** , *10*, 372

#### *3.2. Mortality Rate:*

Mortality rate was significantly (*p* < 0.01) affected by Cd polluted diet during the experimental period of 8–20 weeks of age. The highest value was 12.50% with the high Cd contaminated level (150 mg/kg diet). Results in Table 2 showed that mortality rate was not significantly affected by the addition of ascorbic acid or natural clay to the diet during all experimental periods. Mortality rate was not affected by the interaction among Cd level and ascorbic acid or natural clay in the diet at 20 w.

#### *3.3. Egg Number*

Egg number was significantly (*p* < 0.05) decreased in quails as the concentration of the Cd increased at all the experimental periods. Ascorbic acid or clay supplementation significantly (*p* < 0.05) increased egg number comparing to hens fed on a diet without supplementation during all the experimental periods (Table 2). The results revealed that egg number was not significantly affected by the interaction between feed additives and Cd pollution during the whole experimental period (Table 3). Within any Cd level, ascorbic acid or clay supplementation increased egg number compared to the other groups.

#### *3.4. Egg Weight*

Egg weight was significantly (*p* < 0.05) decreased with increasing Cd level during the experimental period (Table 2). Ascorbic acid or clay supplementation (*p* < 0.01) increased egg weight comparing to the control during the whole period (8–20) and 20–24 weeks of age. Whilst, egg weight values were insignificantly improved by ascorbic acid or clay addition during the other periods (Table 2). Results showed that egg weight was not significantly affected by the interaction between feed additives and Cd contamination during the whole experimental period (Table 3).

#### *3.5. Egg Mass*

Quails exposed to Cd had decreased (*p* < 0.01) egg mass as compared to the control throughout the experimental periods and the whole experimental period (Table 4). Results in Table 4 showed that ascorbic acid or clay supplementation significantly (*p* < 0.01) improved egg mass.

While, the interaction between main the two factors insignificantly affected egg mass values. Results indicated that quails fed diet without Cd with 300 mg ascorbic acid/kg diet produced higher egg mass value (8.45 g/day/bird). Hens fed diet contained 150 mg cadmium/kg supplemented with 1.50% clay produced the lowest egg mass value (4.39 gm/day/bird).

#### *3.6. Feed Conversion Ratio*

With increasing cadmium level in quail diets, feed conversion became significantly worst per egg unit (*p* < 0.05) throughout the experimental periods and the whole period except the insignificant effect during 13–16 weeks of age (Table 4). A significant improvement (*p* < 0.01) was found in feed conversion as a result to ascorbic acid or clay supplementations during the whole experimental period (8–20 weeks of age) and the period of 21–24 weeks of age. However, the diet had 300 mg ascorbic acid recorded the best (*p* < 0.05) feed conversion (3.27 and 3.15 gm feed/gm/egg) values during 8–20 and 21–24 weeks of age, respectively compared to the other experimental groups.


*Animals* **2020** , *10*, 372



*Animals* **2020** , *10*, 372

#### *3.7. Blood Biochemical Components*

Cadmium levels at 100 and 150 mg/kg diet decreased (*p* < 0.05) blood total protein, total albumen and A/G ratio compared to the control and 50 mg cadmium/kg diet at 20 weeks of age. Meanwhile, cadmium did not affect total globulin. On the other hand, creatinine level decreased (*p* < 0.05) at various cadmium levels compared to the control (Tables 5–8). Concerning the effect of feed additives at 20 weeks of age, both 300 mg ascorbic acid and 1.50% Clay increased (*p* < 0.05) total protein and albumen compared to the non-supplemented group but did not affect total globulin and A/G ratio level at both 20 and 24 weeks of age. Increasing cadmium levels increased (*p* < 0.05) ALT, AST, ALP, urea-N, and creatinine as compared to the control at 20 and 24 weeks of age. At 20 weeks of age, both 300 mg ascorbic acid and 1.50% Clay decreased (*p* < 0.05) serum ALT, AST and urea-N compared to the non-supplemented group but did not affect ALP and creatinine level at both 20 and 24 weeks of age. The interaction between cadmium level and feed additives had no effect on various serum parameters.

**Table 5.** Some blood parameters of Japanese quail layers as affected by dietary cadmium levels, feed additives and their interactions at 20 weeks of age.


a–c Values followed by different letters within each effect in each column are significantly different (*p* < 0.05).


**Table 6.** Some blood parameters of Japanese quail layers as affected by dietary cadmium levels, feed additives and their interactions at the end of (experiment 24 weeks of age).

a–c Values followed by different letters within each effect in each column are significantly different (*p* < 0.05).

**Table 7.** Some blood parameters as affected by dietary cadmium levels, feed additives and their interactions at 20 weeks of age.



**Table 7.** *Cont*.

a–c Values followed by different letters in each column are significantly different (*p* < 0.01).



a–d Values followed by different letters within each effect in each column are significantly different (*p* < 0.05).

#### *3.8. Cadmium Residues in Egg Components*

Pollution of quail diets with Cd had a highly significant (*p* < 0.01) effect on cadmium residues in egg components (white, yolk and shell) at 20 and 24 weeks of age as shown in Table 9. Data showed that ascorbic acid or natural clay had a significant (*p* < 0.01) effect on cadmium residues in egg components. Results showed that Cd residues in egg components at 20 and 24 weeks of age were significantly (*p* < 0.01) affected by the interaction between Cd level and the dietary supplementation of ascorbic acid or natural clay. The highest value recorded (37.59 ± 2.03 mg) was found in yolk with high dietary Cd level (150 ppm) and without additives at 20 weeks of age.

**Table 9.** Cadmium residual in tissues of Japanese quail layers as affected by dietary cadmium levels, feed additives and their interaction at 20 and 24 weeks of age.


a–g Values followed by different letters within each effect in each column are significantly different (*p* < 0.05).

#### **4. Discussion**

The body weight loss produced by Cd polluted diets (Table 2) might be due to the wide toxic effect of Cd on the whole-body processes in the bird. It has been reported that long term Cd exposure causes depletion of liver and muscular glycogen due to its action on the enzymes involved with glycogenesis and energy metabolism and inducing the oxidative stress in liver and kidney [26]. Also, decreasing body weight might be mediated by the Cd effect on the small intestine, where the nutrients are absorbed or through a dysfunction in the renal tubules resulted from the destructive effect of Cd on kidney. It would lead to an enhanced urinary excretion of some food nutrients which causes a decrease in the utilization of these nutrients in the body [26,27]. Decreasing body weight was also recorded in Japanese quail layers exposed to graded dietary Cd concentration of 40, 80, and 120 ppm [28]. Author attributed this result to the different doses of administration. Some studies on poultry have shown that supplemental ascorbic acid given in feed or by injection improved performance of chickens. Dietary supplementation with ascorbic acid increased the growth rate by about 4.5% [29]. These results agree with Shit et al. [30] who found that dietary L-ascorbic acid supplementation significantly (*p* < 0.05) increased body weight as of laying Japanese quail compared to the control group. On the other hand, these results disagree with those of Attia et al. [31] who reported that the addition of ascorbic acid had no effect on the body weight of broiler chicks until 42 d of age. The improvement in live body weight by clay may be due to delaying the transit time of digesta through the digestive tract by 2 to 2.5 h and promoting absorption of nutrients.

Digestibility of organic matter, fat, and nitrogen free extract and the nitrogen utilization were increased by supplementary Zeolite (aluminosilicate mineral) [32]. The present results agree with those obtained by Ayyat et al. [33] who found that supplementing clay (1%) in silver Montazah layers diet improved body weight. At any Cd level, ascorbic acid or clay supplementation in quail layer diets recorded higher body weight than the un-supplemented one. The results showed that Cd poisoning can be partially reduced by providing supplementary ascorbic acid or natural clay.

According to our results, Cd polluted diets caused 12.50% mortality rate. This result is in disagreement with Bokori et al. [7] who stated that dietary Cd pollution of 25 or 75 ppm did not cause indicative signs of mortality rate in chickens as compared to the control during the experimental period for nine months ago. Due to Klassen and Liu [34], the lethality associated with exposure to cadmium is related to cardiotoxicity and hepatotoxicity. Contrarily, Erdogan et al. [15] clarified that mortalities were not related to toxicity of Cd because no abnormal signs were observed during clinical observation or at autopsy examination in broiler chicks. Ascorbic acid has been reported to protect cells involved in the immune response such as lymphocytes, macrophages and plasma cells against oxidative damage of quail layer cells. Some studies on poultry have shown that supplemental ascorbic acid given in feed or drinking water or by injection of chickens reduced the mortality percentage by about 5% [29].

The significant decrease in egg number in the Cd polluted diets Japanese quail found in the present study (Table 2) could have resulted from alterations in the egg formation pathway or may be through the suppression of calcium metabolism [35]. Similarly, Rahman et al. [35] showed reduced egg production in quails. Decreased egg production was also observed in laying hens with single dietary exposures of 60 ppm radioactive Cd [36]. The dose and period of Cd exposure may be an important factor altering egg production potentialities which could be confirmed by the findings that showed no effect of Cd contamination on the egg production of pheasants given drinking water containing 1.5 mg cadmium/L for only 12 weeks [37]. Ascorbic acid or clay supplementation significantly increased egg number (Table 2). These results agree with Njoku and Nwazota [38] who clarified that ascorbic acid supplementation to laying hens' diets caused a proportional increase in the number of eggs laid. The presence of antioxidant (ascorbic acid) could adversely inhibit the oxidative protein denaturation and improve nutrients digestibility and feed efficiency. It could be also a reason for rising layers performance [39]. Present results agree with Moghaddam et al. [40] who found that dietary Zeolite addition at levels of 1.5%, 3%, and 4.5% caused a significant (*p* < 0.01) increase in egg production of Hy-Line hens. Therefore, supplementation of Zeolite can be used to extract ammonia by ion exchange

which helps to remove stress-forming ammonia from the hen intestine and thereby increasing egg production [41]. On the other hand, Lemser et al. [42] indicated that supplementation of rye-based broiler diets with natural clay minerals showed a negative effect on egg production.

Generally, the results indicated that the use of the cation exchange is capable to reduce the uptake and influence the distribution of heavy metals in poultry tissues. Evans et al. [43] concluded that the use of synthetic and natural Zeolite improved the performance of poultry. In line with our results, Rahman et al. [35] showed that egg weight of Japanese quail layers was markedly affected by Cd administration with injection levels of 0.1, 0.3, 1, 3, and 10 mg/kg body weight/day up to the end of the experimental period (14 days). Authors attributed this action to alterations in the egg formation pathway. This is may also returned to that quails exposed to Cd pollution produced fewer eggs and had lower egg weights than controls. Ascorbic acid or clay supplementation significantly (*p* < 0.01) increased egg weights (Table 2). This is because quails fed diets supplemented with ascorbic acid or clay produced more eggs and had higher egg weights than those fed diet without feed additives. These results are confirmed by Njoku and Nwazota [38] who clarified that ascorbic acid supplementation to laying hens' diets (250 ppm) showed highest mean egg weight but statistically no variation was drawn among the groups. According to Elliot and Edwards [44], natural Zeolite as a Clinoptolite bearing rock material was found to increase egg weight by incorporation in the hens' diet at an inclusion rate of less than 10%. Moghaddam et al. [40] found that dietary added Zeolite in levels of 1.5, 3 and 4.5% caused a significant (*p* < 0.05) increase in egg weight of Hy-Line hens. In contrast, Ozturk et al. [45] found that dietary Zeolite (aluminosilicate mineral) supplementation of 0%, 2%, 4%, 6%, and 8% as Clinoptilolite had no significant effect on egg weight of Babcock laying hens. The obtained results reported decreased egg mass due to Cd pollution (Table 4). It is worth noting that egg mass significantly decreased (*p* < 0.01) with increasing dietary Cd level which agree with results reported by Baykov et al. [46]. In a converse trend, results showed increased egg mass due to ascorbic acid or clay supplementation. These results agree with Moghaddam et al. [41] who demonstrated that Zeolite at levels of 1.5, 3 and 4.5% caused significant (*p* < 0.01) increases in egg mass of Hy-Line hens.

The poor feed conversion due to Cd pollution could be due to the highly significant decrease in egg mass as exposed to Cd pollution. Long-term cadmium exposure causes depletion of liver and muscular glycogen. This effect might reduce nutrient metabolism and feed utilization [35]. Feed conversion value depends mainly on the amount of feed consumed and the egg mass. In this context, it is worth noting that egg mass in ascorbic acid or clay groups were significantly (*p* < 0.05) higher than un-supplemented group (Table 4). These results are in harmony with Denli et al. [47] who found that dietary ascorbic acid improved feed conversion ratio in laying hens under stress. On the contrary, Soltani et al. [48] postulated that values of feed conversion rate of laying hens were not affected by dietary ascorbic acid (*p* > 0.05) with level of 250 mg/kg as compared to the control group.

For blood parameters, our results in Tables 7 and 8 fully agree with those obtained by Abou-Kassem et al. [19] who found that the increased levels of cadmium in quail diets significantly decreased (*p* < 0.05) total protein, total albumen and A/G ratio compared to the control group with no effects of various levels of clay or vitamin E supplementations. Also, Hashem et al. [49] found that cadmium levels at 100 mg/kg diet statistically decreased serum total proteins, albumin and globulins values. It was clear that cadmium levels at 100 and 150 mg/kg diet significantly increased (*p* < 0.05) ALT, AST, uric acid, urea-N, creatinine, and alkaline phosphatase levels compared to control and 50 mg cadmium/kg diet at 20 and 24 weeks of age of Japanese quails. Urea-N appears to be the most useful variable for the detection of pre-renal causes of renal failure with Cd toxicity. But the effects of feed additive and the interaction between cadmium level and feed additive at 20 and 24 weeks of age were not affecting (*p* > 0.05) on these traits (Tables 5 and 6). In a partially disagreement, Rambeck and Kollmer [50] showed that the addition of dietary ascorbic acid had a great protective effect on kidney damage from cadmium intake. Hashem et al. [49] reported that group received cadmium at level of 100 mg /kg diet significantly increased (*p* < 0.05) blood ALT, AST, uric acid and creatinine level compared to control group. The increase in ALT and AST enzyme levels and the outflow of

these enzymes to the blood from the liver due to Cd hepatotoxic effect is considered as an indicator of hepatocellular damage. ALT activity was significantly increased, while ALP activity was significantly decreased in plasma of rats fed 5 mg Cd /kg [51]. Cadmium revealed a significant increase in ALP activities when compared to the control group (Table 6). Our results assure the prophylactic potential of ascorbic acid or clay to prevent or decrease cadmium-induced toxic manifestations. ALP in blood is considered as an indicator of mineral status and bone mineralization especially Ca and P which plays an important role in the homeostasis of the body and ensures appropriate conditions for biological activities such as energy utilization, nucleic acid synthesis and bone mineralization [52].

The hepatocellular injury due to cadmium could be attributed to the cadmium-induced generation of free radicals [53]. The activity of AST and ALT enzymes used as stress indicators of evaluation for impairment and damage of tissue liver which produced from Cd toxicity and attributed the elevated activities of ALT and AST enzymes to the outflow from the liver cytosol to the blood. Also, increasing AST and ALT attributed to the hepatotoxic effect, hepatocellular damage or cellular degradation by Cd in quail liver. Toxicity with cadmium may be due to changes in liver carbohydrate metabolism especially activation of liver glycogenolysis and glycolysis as well as increased levels of plasma glucose [54]. From these explanations, the linkage between liver damage, energy metabolism, productive traits, and the residues of cadmium in quail egg components is clarified. Conversely, Erdogan et al. [15] reported that different Cd levels had no effects on AST and ALT activities.

Long-term exposure to cadmium leads to pathological changes in the quail kidneys which contains morphological changes are initially limited to proximal tubular epithelial cell degeneration followed by cellular atrophy, interstitial fibrosis, and glomerular sclerosis and finally associated with biochemical evidence of renal tubular dysfunction. These explanations ensured by Abdo and Abdalla [55] and Abou-Kassem et al. [19] who reported that kidney function tests elevated in Cd treated groups indicating the toxic effect of cadmium which was presumably due to nephrotoxic effect of cadmium on renal tubules and glomeruli.

Sato et al. [56] postulated that Cd transfer from laying hen to eggs was restricted after the maternal bird was exposed to Cd. furthermore; Cd accumulates in the follicle walls of ovary. These results suggest that the follicle walls might play a role in protecting the follicle yolks against Cd toxicity. These findings agree with Herzig et al. [57] who found that Cd level of all investigated tissues (liver and leg muscle) were significantly (*p* < 0.05) increased when chicken received 1.47 mg Cd within 10 days. Results of this study are in disagreement with Toman et al. [37] who found that the hepatic Cd concentration of pheasants exposed to Cd (1.5 mg/ L water) were under the limit of detection (0.01 mg/kg) in all sample collections (after 4, 8, and 12 w of the experiment).

Our results showed that ascorbic acid or natural clay had a significant (*p* < 0.01) effect on cadmium residues in egg components. On the contrary, Erdogan et al. [15] showed that ascorbic acid supplementation to the diet did not prevent Cd accumulation in several organs of chicks. These results agree with Attia et al. [31] who reported that natural clay addition to lead contaminated diets clearly reduced the level of lead residues in the viscera and muscles. Natural clay prevents the lead toxicity by reducing lead absorption in the intestinal tract and increasing fecal excretion [31].

#### **5. Conclusions**

It can be concluded that the consumption of polluted diets with heavy metals such as Cd causes deleterious effects on the productive performance of laying Japanese quails. The addition of ascorbic acid or natural clay to the diet of laying Japanese quails caused beneficial effects on productive performance and diminished the toxic effect of Cd on productive results during the treatment period.

Compliance with Ethical Standards: All procedures by this study were in accordance with international ethical standards. The research involved no human participants.

**Author Contributions:** D.E.A.-K. designed the study plan. M.E.A.E.-H. helped in conducting the experiment and collected literature. D.E.A.-K, A.E.T. and M.E.A.E.-H. analyzed the data and drafted the manuscript. J.S.A., S.N.M. and A.A.A. provided a technical help in writing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** Authors extend their appreciation to the Dean of Scientific Research, King Saud University, for funding the work through Researchers supporting project no RSP-2019/149.

**Conflicts of Interest:** No conflict of interest, financial, or otherwise, is declared by the authors.

#### **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*

### **Impact of Multienzymes Dose Supplemented Continuously or Intermittently in Drinking Water on Growth Performance, Nutrient Digestibility, and Blood Constituents of Broiler Chickens**

### **Youssef Attia 1,\*, Mahmoud El-kelawy 2, Mohammed Al-Harthi <sup>1</sup> and Ali El-Shafey <sup>3</sup>**


Received: 10 February 2020; Accepted: 18 February 2020; Published: 26 February 2020

**Simple Summary:** Supplementing enzymes in diet can improve animal performance, carcass traits, physiological status, and reduce cost of feeding due to improving feed utilization. Enzyme supplementation in water is simple to apply, disseminates, and the contact with the substrates is faster. In addition, water supplementation may lessen the negative effects of aggressive heat exposure on enzyme activities when the pelleting temperature exceeds 85 ◦C and can replace expensive post-pelleting spraying systems. We investigated the effect of different doses of enzymes supplemented in water either continuously or intermittently on growth performance, digestibility of nutrients, and blood profiles. Results indicated that intermittent supplementation of enzymes at 1 mL/L drinking water and continuous supplementation at 0.5 mL/L drinking water can be investigated in further experiments as a tool to improve broiler performance and European production index.

**Abstract:** The aim of this work was to study the continuous or intermittent impact of a multienzyme supplement on growth performance, nutrient digestibility, and blood metabolites of broilers, and to evaluate production index of dietary supplementation. A total of 315 unsexed day-old Arbor Acres broiler chicks were randomly distributed to seven treatments groups, keeping initial body weights similar, in 35-floor pens (replicates) of nine chicks per replicate (pen) and five experimental units per treatment. All experimental groups were fed the same basal diet and administered seven multienzyme treatments: the 1st group (control) did not receive any enzyme supplementation; the 2nd, 3rd, and 4th groups were administered multienzymes at 0.5, 1.0, and 1.5 mL/L drinking water, respectively. Each enzyme supplemented-group was divided into two subgroups, with additives being applied either continuously (24 h/day) or intermittently (12 h/day) from 1 to 35 days old. Regardless of administration method, multienzyme supplements at 1.0 mL/L water along with a corn-soybean meal diet increased the body weight gain (BWG) by 7.8% compared to 0.5 mL/L water during days 1–21 of age. In addition, 1.5 mL/L water significantly improved BWG by 5.1% of broilers compared to 0.5 mL/L water during days 1–35 of age. Enzyme supplementation at 1.5 mL/L water significantly enhanced feed conversion ratio (FCR) by 4.3% during days 1–21 of age, and FCR by 5.2% and European production index (EPI) by 10.4% during days 1–35 of age compared to the group on 0.5 mL/L water. For the whole period, there were improvements of beneficial consideration in BWG (4.0%), FCR (4.0%), and European production index (8.2%) due to continuous multienzyme supplementation at 0.5 mL/L water compared to the same dose added intermittently. A similar trend was observed due to intermittent multienzymes at 1 mL/L drinking water that resulted in increased BWG by (6.4%) and improved FCR by (6.7%) and EPI by (12.7%). Intermittent administration significantly

increased feed intake of broilers during 22–35 days of age compared to continuous supplementation. Multienzymes at different doses did not significantly affect the digestibility of nutrients, blood serum biochemical constituent, inner body organs, and markers of functions of liver and renal organs. In conclusion, the highest BWG and the best FCR and EPI for the whole period were from broilers given continuous 1 and 1.5 mL/L drinking water or intermittent multienzyme supplementation at 1.5 mL/L drinking water. Furthermore, intermittent supplementation of enzymes at 1 mL/L drinking water and continuous supplementation at 0.5 mL/L drinking water can be investigated in further experiments as a tool to improve broiler growth performance and economic traits and to decrease the cost of enzyme application.

**Keywords:** broilers; multienzymes; administration method; enzyme dose; performance; blood constituents

#### **1. Introduction**

Diet composition is a key factor affecting the response to enzyme supplementation in poultry [1–3]. Vegetable based-diets contain anti-nutritive substances such non-starch polysaccharides (NSP), tannins, trypsin inhibitor, and phytic acid that negatively influence animal performance, digestibility of nutrients, environment, and decrease gut health and feed utilization [4–7].

Enzymes are commonly employed to decrease anti-nutritional substances and to improve animal performance [8–11]. Multienzymes containing carbohydrases and phytase were found to enhance the utilization of energy, protein, and minerals by chickens [12–14], suggesting that higher amounts of alternative feedstuffs could be used in the presence of enzymes [15]. Rye, wheat, and barley grains, which contain high levels of soluble-NSP, particularly arabinoxylans (pentosans) pectin, and β-glucans may reduce the rate of gut emptying and affect small intestinal transit time, block fats from digestion and thus absorption, therefore, cannot be incorporated into chickens' diets at high concentrations unless exogenous enzymes are adequately applied. It has been well documented that the high level of soluble NSP in rye increases digesta viscosity and the stickiness of droppings, which results in poor poultry performance [16,17].

Evidently, enzyme additions to corn, wheat, barley, and rye diets have been shown to significantly improve body weight gain (BWG) and feed conversion ratio (FCR) in broilers [11,16,18]. Generally, these enzymes are hypothesized to work in two steps, described as an ileal phase and a cecal phase [19,20]. During the ileal phase, enzymes remove fermentable substrates. During the cecal phase, degradation products of sugars, such as xylose and xylo-oligomers, are fermented by cecal bacteria, stimulating the production of volatile fatty acids (VFA) and the growth of specific beneficial bacteria [6]. In the process of depolymerization various polysaccharides in the diet, enzymes may produce manno-, galacto-, gluco-, or xylo-oligomers, which are similar to prebiotics and which may facilitate the proliferation of health-promoting bacteria such as *Lactobacillus* and Bifidobacterium [21]. Cellulase is a viscosity-reducing enzyme and is a group of enzymes that hydrolyze cellulose or β-(1,4)-glucan [2,14]. Protease enhanced degradation of soybean meal protein in the gut notably, and the mode of action of protease are wholly allied with the digestibility [14]. This observation has been evidenced by a significant increase in growth and an improvement in gut health and FCR when broilers were fed corn-based diets supplemented with enzymes [20,22,23].

In the available literature, there is a large body of results on feed supplementation with enzymes on broiler performance in contrast to the use of enzymes in drinking water. However, enzyme supplementation in water is more simple to apply, disseminate, and contact with the substrates is faster [24]. In addition, water supplementation of enzymes may lessen the harmful effects of aggressive heat exposure on enzyme activities when the pelleting temperature exceeds 85 ◦C and can replace expensive post-pelleting sparing systems [25]. Enzyme supplementation in drinking water significantly increased the body weight of broilers during different periods of growth, from 14 to 35 days of age, from 20 to 41 days, and from 20 to 40 days of age [26–29]. In addition, broilers that received multienzymes through drinking water recorded the highest weekly weight gain when compared to those given enzymes through the feed, and both groups had higher growth than unsupplemented controls [24]. Thus multienzymes through drinking water at 0.5 g/L had a positive and growth-boosting effect in broiler chickens. Recently, feed additives may be used intermittently and resulted in similar growth performance and more economic benefits than continuous supplementation [30].

The enzymes' influence on blood constituents may shed light on the impact of enzymes on metabolic processes. However, contradictory results were found in the literature. Multienzyme supplementations at 0.5–1 mL/L water significantly decreased alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST), showing an improvement in liver leakage markers [26–29]. On the other hand, plasma total protein was higher in enzyme supplemented-groups than of controls, but albumin, globulin and albumin/globulin ratio, ALT, and AST were not affected [13]. Enzymes also increased total protein, albumin, globulin and albumin/globulin ratio compared to controls, but liver leakage markers (AST and ALT) were not affected by enzymes [3,31]. However, an enzyme cocktail did not influence the plasma biochemical constituents or the indices of the liver function of broilers [23,32].

Hence, this study aimed to evaluate the effects of multienzymes given in water, either continuously or intermittently, on productive performance, nutrient digestibility, carcass characteristics, and indices of the liver and renal functions of broiler chicks from 1 to 35 days of age.

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

All procedures were approved by the Deanship of Scientific Research (DSR), King Abdulaziz University under proposal number D-182-155-1440 H, that recommends animal rights, welfare and minimal stress, and did not cause any harm or suffering to animals according to the Royal Decree number M59 in 14/9/1431H.

#### *2.1. Chick, Supplement, Design, and Husbandry*

A total of 315 unsexed day-old Arbor Acres broiler chicks were obtained from the Cairo hatchery, wing banded, and randomly distributed to seven treatment groups, keeping initial body weight similar, in 35-floor pens (experimental unit) of nine chickens per pen and five replicates per treatment. All experimental groups were fed the same basal diet (Table 1) and were given seven multienzymes (Caplix® is a product of Vetoquinol India; Caplix® contains multienzymes 100,000,000 U/L cellulase, 1,500,000 U/L xylanase, 250,000 U/L amylase, and 400,000 U/L protease) treatments as follows: the 1st group (control) did not receive enzyme supplementations; the 2nd, 3rd, and 4th groups were given multienzymes (Caplix®) at 0.5, 1.0, and 1.5 mL/L drinking water, respectively. Each supplemented group was divided into two subgroups, in which the additives were administrated continuously (24 h/day) or intermittently (12 h/day), respectively, during the 1st through to the 35th day of age. The enzyme was added to water tank daily for those given continuous access, while it was added from 8 am to 8 pm for those intermittently supplemented. The water system (tank, tube, and cups) was flushed with clean water in-between enzyme applications to avoid the residual of enzymes in the water system. The water used was tap water recommended for human consumption in Egypt. Water intake was not determined herein due to limited research facility.


**Table 1.** The ingredients and determined and calculated composition of the experimental diets.

\* Vitamins and minerals mix. provides per kg diet: Vit. A, 12,000 IU, vit. E (dl-α-tocopheryl acetate) 20 mg, menadione 2.3 mg, Vit. D3, 2200 ICU, riboflavin 5.5 mg, calcium pantothenate 12 mg, nicotinic acid 50 mg, Choline 250 mg, vit. B12 10 μg, vit. B6 3 mg, thiamine 3 mg, folic acid 1 mg, d-biotin 0.05 mg. Trace mineral (mg/kg of diet): Mn 80 Zn 60, Fe 35, Cu 8, Selenium 0.1 mg. <sup>1</sup> Calculated,2 Determined.

#### *2.2. Housing and Husbandry*

Chicks were housed in floor brooders in a semi-opened house. Their light schedule was 23 h light up to 21 days of age, followed by 20 h of light until slaughter. The average outdoor minimum and maximum temperature and relative humidity during the experimental period were 22.3 and 25.6 ◦C and 55.4% and 58.3%, respectively. The housing temperature was 32 ◦C during the 1st week and declined gradually by 2 ◦C each week and was then stabilized at 28 ◦C until slaughter. Chicks were vaccinated against the most common diseases, such as Newcastle disease (ND), infectious bursal disease (IBD), and infectious bronchitis (IB), under veterinary care. The experimental diets were formulated to meet the nutrient requirements of broiler chickens [33]. The ingredients and the calculated [33] and determined [34] composition of the experimental basal diets fed during the two phases of broiler production (starter and growing periods) are shown in Table 1. Crumble feed and water were given ad libitum.

#### *2.3. Response Traits*

Broilers in each replicate were weighed (g) at 1, 21, and 35 d of age, and the BWG (g/chick) was calculated. Feed intake was recorded for each replicate (g/chick) and thereby FCR (g feed/g gain), and survival rate (SR, 100 - mortality rate) during 1–21, 22–35, and 1–35 d of age were calculated. The European production efficiency index was calculated [8].

The apparent digestibility of crude protein (CP), ether extract (EE), crude fibre (CF), and ash was performed using five replicates of three males housed in metabolic metal cages per treatment in a separate trial [8]. The excreta of the total tract were collected using the total tract apparent digestibility method during 36–40 days of age [8]. To test the apparent nutrient digestibility, the experimental

period was 5 days: 2 days of adaptation and 3 days of the collection period in which non-marked excreta during days 38 and 40 of age was discarded. During the collection period, feed intake and voided excreta were recorded daily. To identify the excreta derived from the tested diets, 1% of ferric oxide was supplemented to the tested diet at the 1st and last day of collection, 38 and 40 days of age, respectively [35]. Hence, non-marked excreta of the 1st and 3rd days of collection period were discarded. The excreta was collected from each replicate, cleaned from feed, feather, and scales and pooled for the three days, dried in a force ventilated oven, stored in glass jars, and kept for further analyses. Nitrogen as a faecal nitrogen was determined after separation of urinary nitrogen according to [36] while EE, CF, and the ash content of the excreta as well as that of the feed were determined according to [34]. The digestibility of any nutrient was calculated using (input-output/input) × 100.

Five animals were taken randomly from each treatment group to represent all treatment replications and slaughtered according to the Islamic method after being fasted overnight. The carcass and inner organs were separated, weighed, and expressed as relative to live BWG as cited by [9]. The empty gizzard and intestinal were used to estimate gizzard and intestinal percentage.

At 35 days of age, five blood samples (5 mL per sample) of each treatment were collected in clean non-heparinized tubes. The serum was separated by centrifugation at 1500× *g* for 10 min at 4 ◦C, and stored at −18 ◦C until analysis. The serum profiles were determined using commercial diagnostic kits (Diamond Diagnostics Company, Cairo, Egypt). The serum total protein and albumin concentrations (g/dL) was established, according to Henry et al. [37] and Doumas et al. [38], respectively. The globulin concentrations (g/dL) were calculated as the difference between total protein and albumin. The activities (μ/L) of the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes were determined according to the method described by Reitman and Frankel [39]. Serum creatinine and urea were determined by [40]. Total cholesterol was determined, as defined by Watson [41].

#### *2.4. Statistical Analysis*

Statistical analyses were performed using the GLM procedure of the statistical analysis software of the SAS Institute [42] using one-way ANOVA to test for the effects of seven treatments. In addition, a contrast analysis was used after exclusion of the control group to compare intermittent vs. continuous treatment. The linear and non-linear effects of enzymes dose were tested. In addition, a factorial analysis was run after excluding the control-unsupplemented treatment to check the result of enzyme level, administration method, and the interaction. Before analysis, all percentages data of digestibility measurements, carcass traits, and inner organs were transformed to arcsines to normalize data distribution. The pens were the experimental units. The mean differences at *p* ≤ 0.05 were tested using the Student–Newman–Keuls test. The *p* value between 0.05 and 0.10 was considered a trend.

#### **3. Results**

It should be noted that results are presented according to the effect of contrasting intermittent vs. continuous methods of application, and then to the effect of treatments effects.

#### *3.1. Growth, Feed Intake, and Survival Rate*

The impact of administering multienzymes in drinking water on the growth and survival of broiler chickens are summarized in Table 2. Differences between (intermittent vs. continuous) were not significant for growth, FCR, and EPI during the experimental periods.

Treatments had no significant effects on BWG during 1–21 days (*p* < 0.143) days of age but the effect approached meaningful (*p* < 0.099) during 22–35, and (*p* < 0.06) during the whole period showing the highest growth of groups on 1.5 mL/L drinking water administrated either intermittently or continuously.

Regardless of administration method, enzyme level showed a significant effect on the growth of broilers during days 1–21 of age showing that groups supplemented with 1 mL/L enzymes increased growth compared to the group on 0.5 mL/L water. During days 22–35 of age, chicken that received the level of enzymes at 1.5 mL/L exhibited numerically (*p* < 0.061) higher BWG than those only given levels of 0.5 and 1 mL/L. For the whole period, 1.5 mL level of enzymes resulted in the growth of broilers more than 0.5 mL/L water.


**Table 2.** Effects of multienzymes supplemented continuously or intermittently on body weight gain and feed intake of Arbor Acres broiler chicks during 1–35 days of age.

a,b: Differences among means within a column within each factor not sharing similar superscripts are significant at *p* < 0.05; Int: intermittent; Con: continuous; SEM: standard error of the mean.

There no significant effect of administration method and the interaction between enzyme level and administration method on the growth of broilers during different periods.

Feed intake of broiler chickens during most of the experimental periods was significantly affected by the administration method (intermittent vs. continuous), except for days 22–35 of age in which broilers in the intermittent group consumed significantly more feed than the continuous one. Treatments also had no significant impact on feed intake during the different experimental period, nor there were significant effects due to the enzyme level and the interaction between enzyme level and administration method. There were no deaths in this experiment; the survival rate was 100% during the experimental period, and thus death-rate data are not presented.

#### *3.2. Feed Conversion Ratio and European Production Index*

The effects of administering multienzymes in drinking water on the FCR and EPI of broiler chickens are summarized in Table 3. The FCR and EPI were not significantly affected by the administration method (intermittent vs. continuous) during the experimental period. In addition, treatments did not affect FCR during most of the trial period except that FCR during days 1–35. In addition, the difference within 0.5, 1, and 1.5 mL levels was not significant between the two application methods. The results revealed that the group supplemented continuously with 1.5 mL/L water either continuously or intermittently show the best FCR, while the worst FCR was from a group that was supplemented with 0.5 mL/L intermittently.


**Table 3.** Effects of multienzyme supplemented continuously or intermittently on feed conversion ratio and European production index of Arbor Acres broiler chicks during 1–35 days of age.

a,b: Differences among means within a column within each factor not sharing similar superscripts are significant at *p* < 0.05; EPI: European production index; Int: intermittent; Con: continuous; SEM: standard error of the mean.

Irrespective of administration method, enzyme level significantly affect FCR during days 1–21 and FCR and EPI during days 1–35 of age, showing 1.5 mL/L water enhanced FCR and EPI compared to 0.5 mL/L water. The EPI was not significantly affected by administration method (intermittent vs. continuous) as well as treatments.

#### *3.3. Apparent Digestibility of Nutrients*

Data concerning the effects of the administration method and different enzyme treatments on the apparent digestibility of the nutrients of broiler chicks are shown in Table 4. There were no significant effects for the administration method (intermittent vs. continuous) and for the treatments on the apparent digestibility of nutrients. The results showed that there was a trend (*p* ≤ 0.11) for higher crude fiber digestibility of the group supplemented with enzymes continuously compared to that on intermittent supplementation. A trend towards increasing digestibility of crude protein (*p* ≤ 0.10), ether extracts (*p* ≤ 0.11), and ash (*p* ≤ 0.07) was shown because of different enzyme treatments compared to the control.

#### *3.4. Carcass Traits and Inner Body Organs*

The carcass characteristics and body organs of broilers as affected by the method of administration and different enzyme treatments are shown in Table 5. The percentages of the liver, heart, pancreas, abdominal fat, spleen, bursa, and thymus were not significantly affected by method of administration (intermittent vs. continuous). However, there was a trend (*p* < 0.09) of increased dressing (%) because

of constant supplementation compared to the intermittent one, but reduced gizzard (*p* < 0.089) and pancreas (*p* < 0.059) percentage. There were no significant differences in the carcass traits and body organs due to different enzyme treatments.


**Table 4.** Effects of multienzymes supplemented continuously or intermittently on apparent nutrient digestibility (%) during 36–40 days of age of Arbor Acres broiler chicks.

Int: intermittent; Con: continuous; SEM: standard error of the mean.



Int: intermittent; Con: continuous; SEM: standard error of the mean.

#### *3.5. Lymphoid Organs*

The lymphoid organs of broilers as affected the method of administration (intermittent vs. continuous) and different enzyme treatments are shown in Table 6. The percentages of the spleen, bursa,

and thymus were not significantly affected by method of administration (intermittent vs. continuous). There were no significant differences in the lymphoid organs due to different enzyme treatments.


**Table 6.** Effects of multienzymes supplemented continuously or intermittently on lymphoid organs of Arbor Acres broiler chicks during 1–35 days of age.

Int: intermittent; Con: continuous; SEM: standard error of the mean.

#### *3.6. Blood Serum Biochemical Constituents*

There was no significant effect of the administration method (intermittent vs. continuous) on albumin, globulin, albumin/globulin ratio, total cholesterol, but a trend towards (*p* < 0.075) increasing serum protein was shown in the continuous group compared to intermittent group Table 7.

**Table 7.** Effects of multienzymes supplemented continuously or intermittently on biochemical constituents of blood serum of Arbor Acres broiler chicks during 1–35 days of age.


Int: intermittent; Con: continuous; SEM: standard error of the mean.

#### *3.7. Markers of the Liver and Renal Functions*

Results for liver and renal functions of the broiler chicks as they were influenced by the administration method (intermittent vs. continuous) and different enzyme treatments are shown in Table 8. There was no significant effect of the administration mehtod (intermittent vs. continuous) on most of the indices of the liver and renal functions except for serum AST (*p* < 0.079) and Urea (*p* < 0.044). Both were higher in the group given enzymes continuously than those of the group supplemented with enzymes intermittently. There was no significant effect of different enzyme treatments on the markers of the liver and renal functions of the broiler chicks.


**Table 8.** Effects of multienzymes supplemented continuously or intermittently on liver function and renal function of Arbor Acres broiler chicks during 1–35 days of age.

Int: intermittent; Con: continuous; ALT: alanine aminotransferase; AST: aspartate aminotransferase; SEM: standard error of the mean.

#### **4. Discussion**

The use of enzymes in feed to overcome the anti-nutritional factors, to increase feed utilization, and to improve performance as well as to decrease stress has received considerable attention [3,4,8,11,16,43]; so, also is the use of enzymes in water [26–29], with water application inducing superior performance [24]. This advantage may be due to faster distribution, application, and availability [24,25] and greater use of water as well [27]. However, the effect of the enzymes depends on diet composition (target substrate), the dose of the enzyme, and the age of chickens [5,9]. However, the activity of enzymes was not determined herein by direct enzymatic method, but by growth performance and digestibility of diets instead, showing some positive effects depend on the age of broilers, and dose of enzymes.

The use of enzymes is also restricted by its cost-benefit ratio [44]; hence, we investigated the effect of different doses of multienzymes continuously (24 h/d) or intermittently (12 h/d) administered with the aim of improving the performance of the animals and reducing the costs of supplementation on broiler performance and the EPI. The results indicate that at 1 and 1.5 mL of enzyme supplementation either continuously or intermittently yields superior effects on biological and economic value than the low dose of enzymes with a low dosage and intermittent administration could yield better economic benefits under similar production performance due to low cost of supplementation.

The results indicate that the effect of the method of administration (intermittent vs. continuous) and different enzyme treatments are dependent on the age of the chickens (*p* < 0.034). It was observed

that the level of enzyme supplemented at 1 mL/L water improved growth (7.8%) of chickens during only days 1–21 of age compared to 0.5 mL/L supplementation. During days 22–35 and 1–35 of age, enzyme level at 1.5 mL resulted in higher growth of broilers by 5.9% and 5.1%, respectively, compared to 0.5 mL. This indicates that enzymes supplemented continuously at 1.5 mL/L water of a corn-soybean diet at 22–35 days of age was adequate to improve growth and FCR. On the other hand, the dose of multienzymes given intermittently to induce similar improvements in FCR was 1 mL/L. Either continuous or intermittent supplementation with 1.5 mL/L water was more efficient for increasing growth (8.6%–9.9%) and improving FCR (7.7%) and EPI (19.3%) for the whole experimental period compared to unsupplemented control even not significantly different from the 1 mL multienzyme treatments, but obviously economically beneficial. Similar results were reported previously [17,45]. The linear and non-linear components of enzymes concentration showed a weak linear effect on BWG during 1–21 days of age (*p* < 0.089) and FCR during 1–21 days of (*p* = 0.076). In the literature, various cereals responded differently to enzyme supplementation with a greater effect on viscous grains such as rye [9,18,23]. These results indicate that the enhancing effect of increasing dose of enzymes on growth and FCR of broilers occurs during the 1–21 days of age period.

Multienzymes increased the digestibility of CP, EE, and CF and ash numerically of the corn-soybean diet containing 5% rye, and the saturation response was attained in the group supplemented with 1 mL/L water due to lack of response to further increase in enzyme concentration. This could explain the positive effect of enzymes on the performance of broilers found herein. The positive response of the corn-soybean meal diet to multienzymes indicates the presence of insoluble components, such as NSP in rye, which are not digested by broiler chickens and may limit the utilization of some nutrients or negatively influence gut health, nutrient digestibility, and dietary metabolizable energy [4,7,43,46]. The results cited herein are similar to those reported by [8,20,43] and can be explained by the improvement in gut architecture and health [4,6,47].

Dressing percentage increased (1.6%) due to enzyme supplementation continuously in comparison to the intermittent method, showing the positive effect of continuous enzyme supply on the availability of nutrients. It seems that increasing nutrient availability due to continuous supplementation will be used for tissue growth.

The results of this study indicate that enzyme supplementation through different methods at different doses did not affect lymphoid organs (spleen, bursa, and thymus) of broilers. There was an increase in serum protein and albumin (no specific immune protein) due to enzyme supplementation when continuously administered; this may be a reflection of the increase in digestibility of CP observed herein. This positive relationship between enzymes and immunity was recently reported [48,49]. Further evidence indicates a lack of adverse effects of enzymes on the markers for liver leakage and renal function, as enzyme supplementation in water significantly decreased AST and ALT, showing a decrease in liver leakage enzymes, showing an improvement in liver function [26–29].

#### **5. Conclusions**

The highest BWG and the best FCR and PI for the whole experimental period were from broilers given continuous or intermittent multienzyme supplementation at 1.5 mL/L drinking water suggesting further experiments should be undertaken to test the possibility of constant vs. intermittent application of multienzymes due to 50% saving in the cost of enzyme supplementation. In addition, intermittent supplementation of enzymes at 1 mL/L drinking water and continuous supplementation at 0.5 mL/L drinking water can be investigated in further experiments as a tool to improve broiler performance and European production index.

**Author Contributions:** M.E.-k. and M.A.-H. designed the experiment and MIE, and A.E.-S. carried it out. All authors contributed to the preparation of the manuscript and Y.A. revised it. All authors approved the final copy and the galley proof. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant No.)D-182-155-1440 H). The authors, therefore, gratefully acknowledge the DSR technical and financial support.

**Conflicts of Interest:** The authors declare that they have 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/).

### **A Mixture of Exogenous Emulsifiers Increased the Acceptance of Broilers to Low Energy Diets: Growth Performance, Blood Chemistry, and Fatty Acids Traits**

**Ahmed A. Saleh 1, Khairy A. Amber 1, Mahmoud M. Mousa 1, Ahmed L. Nada 1, Wael Awad 2, Mahmoud A.O. Dawood 3,\*, Abd El-Moneim E. Abd El-Moneim 4, Tarek A. Ebeid <sup>1</sup> and Mohamed M. Abdel-Daim 5,6**


Received: 9 February 2020; Accepted: 3 March 2020; Published: 5 March 2020

**Simple Summary:** Since fat energy is cheaper than carbohydrate energy, it is profitable to increase fat content in broiler diets. One of the factors that limits using high levels of fat in broiler diets is the indigestion of fat, because bile secretion in broilers is not efficient in the first days of age. In this sense, using exogenous emulsifiers in the high-fat diet enhanced fat utilization and digestive metabolism. In the current study, birds fed the basal diet and another two low-energy diets (−50 kcal/kg than control) with or without emulsifiers (500 g/ton). The obtained results revealed that the emulsifier's supplementation to low-energy diets enhanced fat utilization and resulted in positive effects on growth performance, nutrients utilization, lipid peroxidation, and modified plasma lipid profiles in broilers.

**Abstract:** To investigate the influence of emulsifiers on broilers fed low-energy diets, the birds were distributed into three sets—the control was fed the basal diet, the second group was fed diets 50 kcal/kg less than control, and the third group was fed diets 50 kcal/kg less than control and supplemented with 500 g/ton of emulsifiers. The used mixture of exogenous emulsifiers contains phosphatidyl choline, lysophosphatidyl choline, and polyethylene glycol ricinoleate. Although the feed intake was not meaningfully affected by dietary low-energy level with emulsifier inclusion (*P* = 0.42), the weight gain and FCR were clearly enhanced (*P* = 0.005 and *P* = 0.044, respectively). Protein and lipids utilization were decreased by reducing energy level, but they were increased by emulsifier supplementation (*P* = 0.022 and *P* = 0.011, respectively). Liver thiobarbituric acid-reactive substances (TBARs) and muscle palmitic acid concentrations were decreased by reducing the energy level and emulsifier's supplementation (*P* = 0.014 and *P* = 0.042, respectively). However, muscle total lipids and α-tocopherol, oleic acid, linoleic acid, and α-linolenic acid were not affected by dietary treatments (*P* > 0.05). Interestingly, the plasma total cholesterol, HDL-cholesterol, total protein, and globulin were decreased in the low-energy group without emulsifier but they were increased by emulsifier supplementation (*P* = 0.008, *P* = 0.005, *P* = 0.037, and *P* = 0.005, respectively). It could be concluded that the mixture of emulsifier supplementation to low-energy diets enhanced fat utilization

and resulted in positive effects on the growth performance, nutrient utilization, lipid peroxidation, and modified plasma lipid profiles in broilers. Getting such benefits in broilers is a necessity to reduce the feed cost and consequently the price of the product, which will lead to improved welfare of mankind.

**Keywords:** broilers; blood chemistry; emulsifiers; growth; nutrient utilization; TBARs

#### **1. Introduction**

Fats and oils are the most essential energy sources in broilers' diets as a worthy way for gathering the high energy demands for the highest growth rates of broiler chickens [1,2]. In addition, the evident merits of high-caloric-density lipids result in an excellent caloric impact [3,4]. The failure of the broilers to gain lipids is assigned to bad emulsification rather than the shortage in lipase secretion, which led to great interest in the possibility of using exogenous emulsifiers to improve the utilization of lipids in broiler chickens [5]. Several previous studies indicated that supplementation with bile acids or bile salts improve the utilization of dietary fat by chicks because of limited endogenous secretion [6,7].

Big amounts of fat are used in broilers feed, especially strains that require high-energy diets [8]. In various situations, the actual utilization of lipids is specified by the cost linkage between energy content and yellow corn energy content [9]. Since fat energy is cheaper than carbohydrate energy, it is profitable to increase fat content in broiler diets. One of the factors that limits using high levels of fat in broiler diets is the indigestion of fat, because bile secretion in broilers is not efficient in the first days of age [10]. In this time, using exogenous emulsifiers in the high-fat diet enhances fat utilization and digestive metabolism [3,5]. Moreover, San Tan et al. [11] recommended that supplementation of emulsifiers in the early stages of age improved digestion and absorption of the fats and enhanced growth performance in broilers. Although, supplemented bile acids (including cholic acid and chenodeoxycholic acid) and bile salts (taurocholate) improved the absorption of fat in broilers [10]. However, supplemented bile acid in the diets was not economically applicable due to the high cost. Thus, emulsifiers might be included in broilers' diets to reduce the surface tension of water [5]. Bontempo et al. [12] illustrated that emulsifier supplementation in broiler diets consisting of a vegetal bidistilled oleic acid and glycerol polyethylene glycol ricinoleate had a positive effect on growth performance, feed efficiency, carcass dressing, and plasma lipid metabolism. Furthermore, Siyal et al. [5] showed that the supplementation of emulsifiers improved the growth performance of broiler chickens by increasing fatty acid digestibility. However, the effects of emulsifiers (in association with low-energy diets) on growth performance, blood chemistry, and fatty acids traits have been rarely thoroughly investigated, even though the interest in using exogenous emulsifiers has increased in the last several decades. Thus, in this study, it could be hypothesized that the utilization of fats can be increased by emulsifiers, which in turn could enhance the growth performance of broilers. In this sense, the current investigation evaluated the influence of emulsifier supplementation into low-energy diets by the attenuation of growth, feed efficiency, and muscle fatty acid profiles in broilers.

#### **2. Material and Methods**

#### *2.1. Experimental Design and Diet Preparation*

The study was approved by the Ethics Committee of Local Experimental Animals Care Committee and conducted in accordance with the guidelines of Kafrelsheikh University, Egypt (Number 4/2016 EC). Three hundred one-day-old male birds (Ross 308) were kept in bens and divided randomly into 3 treatments, and each treatment divided into 4 replicates (25 birds/rep). The first treatment was served as control and fed on control diets containing the optimized energy requirements (3000, 3100, and 3180 kcal/kg) for starter, grower, and finisher diets, respectively. The second treatment was fed

diets 50 kcal/kg less than control and the third treatment was fed diets 50 kcal/kg less than control and supplemented with 500 g/ton of emulsifiers (Table 1). The emulsifiers used in this study were called Liposorb® and provided from CEVA POLCHEM PVT. LTD., India. Liposorb® contains three types of emulsifiers (Phosphatidyl Choline (PC), Lysophosphatidyl Choline (LPC), PolyEthylene Glycol Ricinoleate (PEGR)), and the optimum dose is 500 g Liposorb®/ton feed. The dose used in the current study was selected based on the study of Bontempo et al. [12] and Zhao et al. [13].

The birds were placed inside a room equipped with 12 pens (3 treatments/4 replicates each) with a chain feeder system and automatic nipple cup drinker in a completely randomized design. Feed and water were provided for ad libitum consumption for 35 days. The light cycle and temperature were the same in the experimental groups. The photoperiod was 24 h of light from day 0 to day 7 and 23 h of light from day 7 to the end of the trial. Room temperature was 25–29 °C with proportional moisture between 50% and 70% during the trial. Birds' body weighing was on record every three days, and feed consumption was on file every day over the empirical period.

#### *2.2. Final Sampling*

At the end of the trial period, 36 chicks (12 birds/treatment) were randomly chosen, weighed, and gently slaughtered to collect the breast muscle, liver, abdominal fat, and heart organs for offal weight. The blood samples were collected into heparinized test tubes and centrifuged at 5900 × g for 10 min at 4 ◦C to collect the plasma and finally kept at −20 ◦C for further analysis. Three days before the end of the trial, 20 birds per group were housed in batteries for digestibility tests where the excreta and feed were collected. Subsequently, the samples were desiccated by the drying kiln at 60 ◦C for 24 hrs. Then, the dried samples were grinded and kept for protein, lipid, and fiber analysis by following the standard procedure [14].

#### *2.3. Blood Biochemical Analysis*

Plasma triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-cholesterol), low-density lipoprotein cholesterol (LDL-cholesterol), glucose, glutamic oxalacetic transaminase (GOT), total protein, albumin, and globulin were tested calorimetrically by using trade kits (Diamond Diagnostics, Egypt) according to the steps outlined by the manufacturer.

Muscle total lipid content and fatty acid profile analysis were measured using gas-liquid chromatography (GLC) according to the method of Saleh [15].

Liver thiobarbituric acid retroactive substances (TBARs) concentration was tested by the process of Goodla et al. [16]. The α-tocopherol concentration in muscle was tested by the HPLC according to the method of Faustman et al. [17].

#### *2.4. Statistical Analysis*

Shapiro–Wilk and Levene's tests confirmed normal distribution and variance homogeneity. All statistical differences were assessed by one-way analysis of variance tests (SPSS version 17, SPSS Inc., Ill., USA) with Tukey's multiple test where differences in experimental groups occurred. The level of significance was accepted at *P* < 0.05. All data are presented as means ± standard error (SE).



26; (μg/kg feed): menadione sodium bisulfite, 650; D-biotin, 70; choline chloride, 780;

pteroylglutamic

 acid, 520;

cyanocobalamin,

 26;

cholecalciferol,

 13.

#### **3. Results**

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

Feed intake was not influenced by a low-energy diet supplemented with an emulsifier (*P* = 0.42), however, body weight gain and FCR were enhanced (*P* = 0.005 and *P* = 0.044, respectively) (Table 2). Crude protein utilization and ether extract utilization were decreased by reducing the energy level, but they were increased by emulsifier supplementation (*P* = 0.022 and *P* = 0.011, respectively) (Table 2). However, crude fiber utilization and mortality rate were not influenced by dietary treatments (*P* = 0.32) (Table 2). Carcass % and abdominal fat % were decreased by reduced energy level, but they were increased by adding emulsifier (*P* = 0.045 and *P* = 0.018, respectively). On the other hand, breast and thigh muscles, heart, and liver percentages were not affected by the reduction of energy or addition of emulsifier (*P* > 0.05) (Table 2).


**Table 2.** Effect of test diets on growth performance, nutrient utilization, and organ weights in broilers.

a,b Values expressed as means ± SE. Means within the same row with different superscripts differ (*P* < 0.05).

#### *3.2. Blood Biochemical Parameters*

Plasma total cholesterol, HDL-cholesterol, total protein, and globulin were decreased in the low-energy diet group without emulsifier but they were increased by emulsifier supplementation (*P* = 0.008, *P* = 0.005, *P* = 0.037, and *P* = 0.005, respectively) (Table 3). However, plasma LDL-cholesterol, triglycerides, glucose, GOT, albumin, creatinine, and uric acid were not influenced by dietary treatments (*P* > 0.05) (Table 3).



a,b Values expressed as means ± SE. Means within the same row with different superscripts differ (*<sup>P</sup>* <sup>&</sup>lt; 0.05). <sup>1</sup> HDL, high-density lipoprotein; <sup>2</sup> LDL, low-density lipoprotein; <sup>3</sup> GOT, glutamic oxaloacetic transaminase.

#### *3.3. Fatty Acid Profiles*

Liver TBARs and muscle palmitic acid concentrations were decreased by reducing the energy level and supplementation with emulsifier (*P* = 0.014 and *P* = 0.042, respectively). However, muscle total lipids and α-tocopherol, oleic acid, linoleic acid, and α-linolenic acid were not affected by the tested diets (*P* > 0.05) (Table 4).

**Table 4.** Effect of test diets on liver thiobarbituric acid retroactive substances (TBARs), muscle total lipid, α-tocopherol, and fatty acid profile contents in muscle.


a,b Values expressed as means ± SE. Means within the same row with different superscripts differ (*P* < 0.05).

#### **4. Discussion**

The growth performance was influenced by the reduction of the energy rate in this study. Normally, birds utilize the energy for life maintenance and body building. Subsequently, when birds were fed low-energy diets, the priority of energy utilization can be used for life preservation and in turn, the growth performance might be negatively affected. Following this hypothesis, the results of the present study displayed reduced body weight gain and increased FCR following feeding with a low-energy diet. However, dietary emulsifier presented a practical strategy to increase the growth performance and feed utilization in broilers fed low-energy diets in the present study (high body weight with low FCR). These results are in line with previous studies [11,12,18]. The obtained results revealed that feed intake was not affected by the experimental diets in the present study. Similarly, Kaczmarek et al. [19] reported that dietary emulsifier did not influence the feed intake in broilers. The increased growth performance in the present study might be related to the improvement in crude protein and fat utilization. San Tan et al. [11] and Zhang et al. [18] also illustrated that the inclusion of emulsifiers improved fat digestion and absorption as well as the nutrient digestibility and consequently resulted in enhancing the growth performance in broilers. The emulsifiers are reported to increase the integration of micelles in the gut lumen, which in turn increases the fat digestibility [13]. Thus, it might be hypothesized that the improved digestibility of the fat is an effect of emulsifiers on proteolysis [5,7].

The abdominal fat % was significantly decreased by reduced energy level, but it was increased by emulsifier inclusion in the present study. Indeed, a higher rate of dietary metabolizable energy can increase abdominal fat [20,21]. Similarly, Zaman et al. [22] reported that abdominal fat was increased by using diets with high metabolizable energy content. High-energy diets could increase the bulk of fat in broilers' bodies, and in turn, raises the level of abdominal fat when compared with low-energy diets [23].

By including an emulsifier in low-energy diets, plasma HDL-cholesterol and globulin concentrations were increased, while, plasma LDL-cholesterol, triglycerides, and glucose concentrations were decreased in the current study. Plasma GOT, albumin, creatinine, and uric acid concentrations were not influenced by the tested diets. To the knowledge of the authors, the impact of emulsifier on HDL:LDL levels in the blood plasma of broilers has not been documented yet. Emulsification could reduce the level of free fatty acids and total cholesterol in plasma by lowering the secretion of lipoprotein molecules in the blood [24]. It has been reported that total cholesterol, triglycerides, and HDL-cholesterol of broilers fed feed including plant oil or animal fat were not influenced by dietary emulsifiers [25,26]. On the contrary, broilers fed dietary emulsifier (sodium stearoyl-2-lactylate) displayed low blood triglycerides in comparison with those fed high-energy diets without the addition of emulsifier [27].

The discrepancy of the production and removal of oxidants from the organism cells is called oxidative stress [28]. Poor nutritional value of the poultry feeds is among the main reasons for the oxidative stress [29]. However, including feed additives such as antioxidants and emulsifiers in poultry diets has been recognized as an effective strategy to alleviate the impaired effects induced by oxidants on broiler performance [5,30]. TBARs are an indirect marker of oxidative stress, but they are a direct marker of lipid damage caused by increased oxygen under stressful conditions, and α-tocopherol has a crucial role as an antioxidant protecting the lipids from peroxidation [31,32]. In the present study, dietary emulsifiers lowered the TBAR levels in the liver of birds, which confirms that the lipid peroxidation was decreased by feeding emulsifiers [33]. In addition, the level of α-tocopherol is relatively increased by feeding emulsifier, which might be involved in reducing the lipid peroxidation in the liver [34,35]. These results mention that the inclusion of emulsifier in the feed of broilers could enhance the meat quality variables, such as drip loss and tenderness. Outcomes of the current experiment are in accordance with Attia and Kamel [36], who documented that TBARs were reduced by increasing soy lecithin rate in rabbit diets. Such refinements in this study agreed with Al-Daraji et al. [37]. Similarly, emulsifiers have neuroprotective and antioxidative properties, and they diminishes liver damage and enhances oxidative strength [27]. King et al. [38] stated that oxidative stabilization might be related to the ability of phospholipids to pool a hydrogen atom from the amino group that moves the oxidized phenolic molecule of the real antioxidant. Moreover, Judde et al. [39] elucidated that the antioxidative properties of emulsifiers depend on fatty acid structure and tocopherol concentration.

The muscle palmitic acid content as saturated fatty acids was decreased by reduced energy and emulsifier, however, muscle oleic acid, linoleic acid, and α-linolenic acid as unsaturated fatty acids were not significantly affected in this study. The emulsifiers can aid to fix the free fatty acids that are seldom soluble by themselves in bile salt micelles, and in this way they raise the digestibility of saturated fatty acids [25].

In the present study, we found that the inclusion of exogenous emulsifiers in broilers' diets with low energy clearly enhanced the weight gain and reduced the feed conversion ratio. Further, the protein and lipid utilization was increased by emulsifier supplementation. Interestingly, the liver TBARs and muscle palmitic acid concentrations were decreased by reducing energy level and supplementation with emulsifier, which confirms the protective role of emulsifiers against oxidative stress. The plasma total cholesterol, HDL-cholesterol, total protein, and globulin were also increased by emulsifier supplementation. The obtained results are in agreement with previous studies investigating the importance of using emulsifiers in poultry diets [12,40–43]. However, further experiments are needed by using different alternative fat sources with different emulsifier additions and doses to explore their effects on the fatty acid profiles and the performances of broilers in different periods of age.

#### **5. Conclusions**

It could be concluded that dietary supplementation of a mixture of emulsifiers in low-energy diets exhibited similar or more effective effects on growth performance, nutrient utilization, lipid peroxidation, and modified plasma lipids than the high-metabolizable energy diet in broiler chickens. Due to the continuous rise of ingredient price and energy cost, the obtained results confirm the concept of using emulsifiers in low-energy diets to reduce the cost of poultry feeding and in turn increase the profitability as well as reduce the price of the product, leading improved welfare of mankind. However, future studies are required to clarify the mechanistic role of emulsifiers in improving the performances of broiler feed diets with low energy using advanced molecular tools.

**Author Contributions:** Conceptualization, A.A.S., K.A.A., and M.M.M.; formal analysis, M.M.M., and M.M.M.; funding acquisition, M.A.O.D. and M.M.A.-D.; investigation, M.A.O.D., M.M.A.-D., and A.L.N.; methodology, W.A. and A.E.-M.E.A.E.-M.; project administration, A.A.S.; supervision, K.A.A.; writing—original draft, A.A.S., M.A.O.D., and T.A.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by Researchers Supporting Project number (RSP 2019/ 121), King Saud University, Riyadh, Saudi Arabia.

**Acknowledgments:** The authors thank all members of the Poultry Production Department, Faculty of Agriculture, Kafrelsheikh University, Egypt for their assistance during the trial. This work was funded by Researchers Supporting Project number (RSP 2019/ 121), King Saud University, Riyadh, Saudi Arabia.

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

#### **References**


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