**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<EMAIL_ADDRESS>(A.V<EMAIL_ADDRESS>(A.C.B.)

**\*** Correspondence<EMAIL_ADDRESS>
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).

1 Soybean oil and soybean lecithin high in free fatty acids and monounsaturated acid oil in different blending proportions. 2 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. 1 S: Soybean oil; L: Soybean lecithin high in free fatty acids; AO: Monounsaturated acid oil. 2 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 + αi + 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. 1 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%. 2 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. 1 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%. 2 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. 1 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).

1 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%. 2 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. **1** 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).

1 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%. 2 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. 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 1 in diet (Experiments 1 and 2).

1 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%. 2 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 3 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 × 60 cm × 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).

1 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; 2 the nutrient levels were as fed basis; 3 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 (10−3, 10−4 and 10−5 for *Salmonella*; 10−4, 10−5 and 10−6 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 1 and Habib Rehman 1**

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).

1 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. 2 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<EMAIL_ADDRESS>(T.H<EMAIL_ADDRESS>(S.L<EMAIL_ADDRESS>(S.M<EMAIL_ADDRESS>(D.W<EMAIL_ADDRESS>(X.W<EMAIL_ADDRESS>(X.W.)

**\*** Correspondence<EMAIL_ADDRESS>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 × 109 CFU/g, *Bacillus licheniformis* 2.5 × 1010 CFU/g and *Saccharomyces cerevisiae* 1 × 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 × 109 CFU/g, *Bacillus licheniformis* 2.5 × 1010 CFU/g, and *Saccharomyces cerevisiae* 1 × 109 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.

1 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. 2 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.

1 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. 2 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.

1 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. 2 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.

1 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. 2 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.

1 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. 2 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.

1 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. 2 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<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>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.

1 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.

1 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.