Research Article
Life Sciences
Livestock science

Laying performance, immunity and digestive health of layer chickens fed diets containing a combination of three probiotics

Takio Inatomi1

Abstract

This study examined the use of probiotic supplementation of layer chicken feed. Two hundred layer chickens were divided randomly into two groups: group 1, fed on a conventional diet, and group 2, fed on a diet supplemented with probiotic bacteria (Bacillus mesentericus TO-A, Clostridium butyricum TO-A and Streptococcus faecalis T-110). Several important indicators of chicken health and production efficiency were measured as chickens were reared from age 18 to 56 weeks of age. Feed conversion ratios, laying performance and measures of egg quality were improved by inclusion of probiotics in the diet. Feed was converted into egg mass more efficiently in layer chickens given feed supplemented with probiotics. Egg shell thicknesses and strength was also improved for chickens receiving probiotic supplemented feed. Measures of immune function were improved by inclusion of probiotics in the diet. Serum retinol and α-tocopherol concentrations were higher as were plasma reactive oxygen metabolites and biological antioxidant potentials. Phagocytic activity and IgA titer in jejunum and ileum were higher in hens receiving probiotics. Probiotic inclusion in the diet improved the digestive health of chickens based on several parameters. Plasma glucagon-like peptide-2 titers were higher. The pH of the contents from the duodenum (45- and 56-week-old), the jejunum (30- and 56-week-old), the ileum (45-week-old) and the caecum (45- and 56-week-old) were significantly lower in probiotic fed hens. The n-butyric acid concentration was higher in the contents of the ilea and caeca, while acetic acid was found in higher concentrations in the caecal contents of 30-, 45-, and 56-week-old hens fed probiotics. The results of this study clearly suggest that supplementation of layer chicken diet with probiotics improved laying performance, egg shell quality, immunity and their intestinal environment.

Keywords: laying performance; immunity; digestive health; layer chicken; probiotics

Author and Article Information

Affiliation 1) Toa Pharmaceutical Co., Ltd.

RecievedDec 19 2015 Accepted: Feb 29 2016 Published: Mar 16 2016

CitationInatomi T (2016) Laying performance, immunity and digestive health of layer chickens fed diets containing a combination of three probiotics. Science Postprint 1(2): e00058. doi: 10.14340/spp.2016.03A0001.

Copyright©2016 The Authors. Science Postprint published by GH Inc. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 2.1 Japan (CC BY-NC-ND 2.1 JP) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Competing interest None

Financial Disclosure None

Ethics Ethical approval was obtained from the ethics committee of Toa Pharmaceutical Co., Ltd.

Corresponding author Takio Inatomi

Address 2-1-11, Sasazuka, Shibuya, Tokyo, 151-0073, Japan.

E-mail takato.inatomi@gmail.com

Reviewers Mohammed Hashim1 and Reviewer B
1 Poultry Science Department/Texas A&M University

Introduction

The sub-therapeutic use of antibiotics in animal nutrition has been beneficial for the improvement of growth performance such as weight gain, feed efficiency and mortality rate [1]. Antibiotics are used to improve the health and productive performance of animals used for human consumption but also result in the emergence of drug-resistant microorganisms. The European Union has banned the use of antibiotics as growth-promoting agents in the poultry industry, and many countries are increasingly restricting the prophylactic use of antibiotics in animals raised for food. Therefore, the poultry industry is actively searching for alternatives to antibiotics. Many different functional preparations such as herbs, essential oils, organic acids and probiotics have been tried.

Probiotics are living microorganisms that improve animal health when provided in the diet. Probiotics act by balancing the intestinal flora, influencing intestinal villi and improving nutrient digestion and absorption [2]. Supplementation of probiotics to the feed of layer hens has been found to improve: i) hen performance, including feeding efficiency, egg production and egg quality [3–11], ii) nutrient digestibility [12–14], iii) modulation of intestinal microflora [15–17], iv) pathogen growth inhibition [14], v) immunomodulation and gut mucosal immunity [9,14,16,17], and vi) antioxidant status [18]. A variety of microorganisms have been used as probiotics, including species of Bacillus, Bifidobacterium, Enterococcus, Lactobacillus, Lactococcus, Streptococcus, and numerous yeast strains. Although there have been many studies on the beneficial effects of probiotics in layer chickens, few studies have examined the symbiotic effects of the combined use of three or more probiotics. The present study was undertaken to determine the effect of feeding three probiotics in combination, on productive performance, immunity, and antioxidant status in laying hens.

Materials and Methods

Ethical approval

This study was conducted at the commercial poultry farm in Kagoshima Prefecture, Japan, and carried out under the fundamental guidelines for the proper conduct of animal experiments and related activities at academic research institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology.

Ethical approval was obtained from the Ethics Committee of Toa Pharmaceutical Co., Ltd (Tokyo, Japan).

Birds, diets, and management

Layer hens were obtained from a local commercial flock and were previously vaccinated for infectious bronchitis and Newcastle disease virus. A total of two hundred 18 week-old HyLine Brown chickens with similar body weights (BW) were randomly divided into 2 treatment groups. Each treatment had 100 laying hens that were individually caged in double-sided, 3-tier battery cages (40 × 35 × 60 cm, with a floor slope of 12°, 740 cm² per hen). The different treatment groups were equally and randomly distributed between upper, middle, and lower tiers to minimize cage-level effect. All hens were housed in a windowless and environmentally controlled room, with the room temperature maintained at 20°C to 22°C. The illumination period was increased from 12 h d−1 at 18 weeks age to 16 h d−1 at the rate of 1 h per week (incandescent lighting, 12 lux). Each cage was equipped with an individual nipple drinker. A continuous, metal feed trough was divided by enclosure to ensure that the hens were not able to consume feed assigned to the adjoining replicate. A wire egg collector was installed in the front of each cage to prevent mixing of eggs between replicates. The trial started with 18-week-old hens and lasted for 38 weeks (until hens were 56 weeks old). The treatments were basal diet without supplement (group 1) or basal diet containing probiotics (group 2). The probiotic mixture (AVITEAM, TOA Pharmaceutical Co. Ltd., Tokyo, Japan) containing Bacillus mesentericus at 1 × 108 CFU g−1, Clostridium butyricum at 1 × 108 CFU g−1, and Streptococcus faecalis at 1 × 109 CFU g−1 in corn starch was supplemented to the feed at 0.02% (w/w). The basal diet consisted of commercially available antibiotic-free poultry feed (Kagoshima Agricultural Economic Federation) and was used for all experiments. The basal diet was in mash form and was formulated to meet the nutritional requirements of 18–56-week-old brown layer chickens under the guidelines given by Kagoshima Agricultural Economic Federation. The composition of the basal diet and nutrient content are shown in Table 1. Experimental diets and water were provided ad libitum throughout the study.

Table 1: Ingredient and nutrient analysis of basal diet

1 Nutrient: obtained by calculation
2 Vitamin premix supplied per kilogram of diet. vitamin A: 15,000 IU; vitamin D3: 3,000 IU; vitamin E: 15 IU; vitamin K3: 5 mg; vitamin B1: 4 mg; vitamin B2: 10 mg; vitamin B6: 6 mg; vitamin B12: 25 mg; pantothenic acid: 15 mg; folic acid: 2 mg; niacin: 20 mg; biotin: 10 mg; choline: 400 mg.
3 Mineral premix supplied per kilogram of diet. iron: 25 mg; manganese: 100 mg; copper: 10 mg; zinc: 60 mg; iodine: 0.5 mg; cobalt: 0.1 mg; selenium: 0.2 mg.

Laying performance

Eggs were collected daily and egg production was expressed on a hen-day basis (% hen-day: total number of eggs produced during the period / total number of hens in the same period). Individual eggs were weighed and recorded daily to calculate mean egg weight within experimental periods. Total egg mass was calculated by multiplying egg weight by egg production. Feed intake was measured per cage each week. Daily feed consumption per bird was calculated on a cage total feed consumption basis for the entire experimental period and for the number of days in the period. The feed conversion ratio (kg of feed/kg of eggs) for each period was calculated on a cage basis from egg production, egg weight, and feed consumption. Mortality rates were monitored every day.

Egg and egg shell quality

Egg and egg shell quality examinations were conducted at four-week intervals. For this purpose, 15 eggs laid between 0900 and 1200 h were randomly picked from each group on the first day of the hens reaching 22, 26, 30, 34, 38, 46, and 54 weeks of age. Eggs were weighed individually and Haugh units, egg shell thickness, and egg shell strength were measured with a digital egg tester (NABEL, Kyoto, Japan). Internal and external quality analyses of eggs were completed within 24 h of the collection of eggs. After eggs were weighed and cracked open, egg quality evaluation was performed individually on each egg.

Plasma glucagon-like peptide-2 concentration

For the analysis of plasma glucagon-like peptide-2 (GLP-2) concentration, 20 birds per group were randomly selected and blood samples were collected from the wing vein at 30, 45, and 56 weeks of age. All blood samples were drawn into ice-cold tubes containing EDTA (3.9 mmol/L) for later determination of plasma GLP-2 concentrations. Blood samples were gently shaken and immediately centrifuged at 2000 × g at 4°C for 5 min to obtain plasma, which was stored at −20°C until analysis. Plasma GLP-2 concentrations were measured using Chicken GLP-2 ELISA Kits (My BioSource Inc, California, USA). The ELISA procedure was performed according to the manufacturer’s protocol.

Serum retinol and α-tocopherol concentrations

For the analysis of serum retinol and α-tocopherol concentrations, twenty birds per group were randomly selected and blood samples were collected from the wing vein at 30, 45, and 56 weeks of age and allowed to clot for 30 min. Blood clots were centrifuged at 3,000 × g for 15 min at 4°C; the top yellow serum layer was pipetted into two 1-mL conical tubes and held at −80°C. Retinol and α-tocopherol in serum were determined using HPLC [19].

Reactive oxygen metabolites and biological antioxidant potential

The d-ROMs (reactive oxygen metabolites- derived compounds) test provides a measure of the whole oxidant capacity of plasma against N, N-diethylparaphenylendiamine in acidic buffer. Such oxidant capacity is mainly due to hydroperoxides, with the contribution of other minor oxidant factors. The BAP (biological antioxidant potential) test evaluates the plasma antioxidant biological potential as the capacity of the plasma sample to reduce ferric ions to ferrous ions. BAP varies primarily as a function of the titers of the major oxidative barriers in plasma (vitamin C, vitamin E, uric acid, bilirubin, etc.). For analysis d-ROMs and BAP, twenty birds per group were randomly selected and blood samples were collected from the wing vein at 30, 45, and 56 weeks of age using EDTA-containing blood collection tubes and centrifuged (1,000 × g for 15 min). The plasma supernatants were stored at −80°C until assayed. d-ROMs and BAP were determined by commercial kits (Diacron, Grosseto, Italy) and FRAS4 (H & D, Parma, Italy), respectively [20].

Phagocytic activity

Blood phagocytic activity was assessed by measuring the carbon clearance rate as described previously [21]. On week 30, 45, and 56, 10 birds from each group were given colloidal carbon (black Indian ink, Shanghai Xinde Oriental Co., Shanghai, China) intravenously (0.1 mL 300 g−1 of BW). At 2- and 20-min intervals, 30 μL blood samples were collected from the wing vein following carbon injection. Each blood sample was haemolysed immediately by the addition of 2 mL of 0.1% Na2CO3 solution. The concentration of carbon particles was analysed in plasma samples by measuring optical density (OD) at a wavelength of 600 nm. Phagocytic activity was calculated in terms of the phagocytic index by using the following equations [22].

Rate of carbon clearance (K) = (log OD2 − log OD10)/(T2 − T1);

Phagocytic index = {K × {(BW of animal)/(liver weight + spleen weight)},

where OD2 is the log OD600 of blood at 2 min, OD10 is log OD600 of blood at 10 min, T2 is the last time point of blood collection, and T1 is the first time point of blood collection.

Measurement of gastrointestinal tract parameters

Ten birds were selected randomly from each group at 30, 45, and 56 weeks of age. Gastrointestinal tracts were extirpated after the birds were killed by cervical dislocation. The jejunum, ileum, duodenum, and caecum were dissected from each bird and used for further analysis. Tissues and gut contents were processed as described below.

Jejunum and ileum IgA concentration

Jejunum and ileum IgA concentrations were measured as described previously [23]. Jejuna and ilea were excised and stored at −20°C until analysis. For analysis, samples were thawed to room temperature. Deionised water was added to 2 g of each sample; tissues were homogenized for 30 s using a mechanical homogenizer (VirTis, Gardiner, NY). An aliquot (5 mL) of the sample homogenate was centrifuged at 20,000 × g for 30 min. The supernatant was collected and stored at −20°C until analysis. Jejuna and ilea IgA concentrations were measured using a chicken-specific IgA ELISA quantitation kit (Jiancheng Biological Engineering Research Institute, Nanjing,China). The ELISA procedure was performed according to the manufacturer’s protocol.

pH of digestive tract contents

The pH in different parts of gastrointestinal tract were measured as described previously [24]. Gut content samples were collected from dissected gastrointestinal tracts. Gut contents (10 g) from the duodenum, jejunum and ileum of each bird were collected aseptically in 90 mL sterilized physiological saline (Terumo Corporation, Tokyo, Japan) (1:10 dilution) and the pH was determined using a pH meter (HORIBA, Ltd, Kyoto, Japan).

Short-chain fatty acid concentration of digestive tract contents

The short chain fatty acid (SCFA) concentration from the contents of the ileum, jejunum, duodenum, and caecum of each bird was measured by gas chromatography as described previously [25]. Approximately 0.5 g intestinal contents from the dissections described above were gently squeezed into a micro-centrifuge tube containing 1 mL of 10% meta-phosphoric acid with 0.4 mL of 4-methyl valeric acid per mL added as an internal standard. The solution was thoroughly mixed using a vortex mixer and centrifuged at 5,700 × g for 20 min at 4°C. The SCFA content of the supernatant was measured using a HP Agilent 6890 series gas chromatograph (Agilent Technologies Inc., CA) fitted with a HP 5973 series mass spectrometer (Agilent Technologies Inc., CA). The columns (Agilent Technologies, CA) used were HP-free fatty acid polyester stationary phase capillary columns of polyethylene glycol on Shimalite TPA 60/80, measuring 30 m long with a 0.25-mm internal diameter. The parameters were as follows: 1 µl injection volume, 240°C injector temperature, 12.15 psi pressure, with 1.1 mL min−1 constant flow using helium as a carrier. Fatty acids were eluted with the following oven program: 80°C initial temperature hold for 5 min, ramp 10°C min−1 to 240°C, held for 12 min. Individual SCFA concentrations were expressed in mg g-1 wet intestinal content.

Statistical analysis

Mann–Whitney U tests were performed using EZR software (Saitama Medical Center, Jichi Medical University). EZR is a graphical user interface for R (The R Foundation for Statistical Computing, version 2.13.0). A significance level of p <0.05 was used.

Results

Laying performance

The laying performance of each group is summarized in Table 2. There were no differences in the hen-day egg production and egg weights between group 1 (basal diet) and group 2 (basal diet plus probiotics) during this trial. The feed conversion ratio (kg of feed/kg of eggs) for 22–25-, 26–29-, 30–33-, 34–37-, 38–41-, 42–45-, 46–49-, 50–53-, and 54–56-week intervals was significantly lower in group 2 than in group 1 (p <0.05). Mortality rates were lower in group 2 than in group 1 during this trial.

Table 2: Laying performance in layer hens (mean ± SD)

wk: weeks of age
a, b: Different letters within rows indicate differences between treatment groups (p <0.05).

Egg and egg shell quality

Egg quality (Haugh units) of eggs from each group is shown in Table 3. There were no differences in the Haugh units between group 1 and group 2 during this trial. The quality of hen egg shells (egg shell thickness and egg shell strength) from each group is shown in Table 4 and Table 5. Egg shell thicknesses for 22-, 26-, 30-, 34-, 46-, and 54-week-old hens were significantly higher in group 2 than that in group 1 (p <0.05). Egg shell strength was significantly higher in group 2 than that in group 1 at all time points (p <0.05).

Table 3: Egg quality (Haugh units) (mean ± SD)

Table 4: Egg shell thickness (mm) (mean ± SD)

a, b: Different letters within columns indicate differences between treatment groups (p <0.05).

Table 5: Egg shell strength (kg/cm²) (mean ± SD)

a, b: Different letters within columns indicate differences between treatment groups (p <0.05).

Plasma GLP-2 concentration

The plasma GLP-2 concentration in serum from each group is shown in Table 6. Plasma GLP-2 concentrations were significantly higher in group 2 than in group 1 at 30, 45, and 56 weeks (p <0.05).

Table 6: Plasma GLP-2 concentration (pmol/l) (mean ± SD)

a, b: Different letters within columns indicate differences between treatment groups (p <0.05).

Serum retinol and α-tocopherol concentrations

The serum retinol and α-tocopherol concentrations from each group are shown in Tables 7 and 8, respectively. The serum retinol and tocopherol concentrations were significantly higher in group 2 than in group 1 (p <0.05).

Table 7: Serum retinol concentration (μg mL-1) (mean ± SD)

a, b: Different letters within columns indicate differences between treatment groups (p <0.05).

Table 8: Serumα-tocopherol concentration (μg mL-1) (mean ± SD)

a, b: Different letters within columns indicate differences between treatment groups (p <0.05).

Reactive oxygen metabolites and biological antioxidant potential

Plasma d-ROM levels in plasma from each group are shown in Table 9. There were no differences in reactive oxygen metabolites between groups 1 and 2 during this trial. The BAP in each group is shown in Table 10. Differences in BAP between group 1 and 2 were statistically significant at 30, 45, and 56 weeks, with group 2 being higher than group 1 (p <0.05).

Table 9: Reactive oxygen metabolites in plasma (U.Carr) (mean ± SD)

Table 10: Biological antioxidant potential in plasma (μmol L-1) (mean ± SD)

a, b: Different letters within columns indicate differences between treatment groups (p <0.05).

Phagocytic activity

The phagocytic index from each group is shown in Table 11. Phagocytic indices at 30, 45, and 56 weeks were significantly higher in group 2 than that in group 1 (p <0.05).

Table 11: Phagocytic index (mean ± SD)

a, b: Different letters within columns indicate differences between treatment groups (p <0.05).

Jejunum and ileum IgA concentration

The IgA concentrations from jejuna and ilea of each group are shown in Tables 12 and 13, respectively. At 30, 45, and 56 weeks, IgA concentrations were higher in group 2 than group 1 in both jejunum and ileum (p <0.05).

Table 12: Jejunum IgA concentration (mg mL-1) (mean ± SD)

a, b: Different letters within columns indicate differences between treatment groups (p <0.05).

Table 13: Ileum IgA concentration (mg mL-1) (mean ± SD)

pH of digestive tract contents

The pH of the digestive tract contents from the duodena, jejuna, ilea and caeca from each group is shown in Table 14. The pH of the contents from the duodenum of 45 and 56 week-old hens was significantly lower in group 2 than that in group 1 (p <0.05). The pH of the contents from the jejunum of 30- and 56-week-old hens was significantly lower in group 2 than that in group 1 (p <0.05). The pH of the contents from the ileum of 45-week-old hens was significantly lower in group 2 than that in group 1 (p <0.05). The pH of the contents from the caecum of 45- and 56-week-old hens was significantly lower in group 2 than that in group 1 (p <0.05).

Table 14: pH of digestive tract contents (mean ± SD)

a, b: Different letters within rows indicate differences between treatment groups (p <0.05).

Short-chain fatty acid concentration of digestive tract contents

The short-chain fatty acid concentration of the contents from the duodena, jejuna, ilea, and caeca of each group is shown in Tables 15-18. The n-butyric acid concentration of the contents from the ilea of 30-, 45-, and 56-week-old hens was significantly higher in group 2 than that in group 1 (p <0.05). Both n-butyric and acetic acid concentrations of the caecal contents from 30-, 45-, and 56-week-old hens were significantly higher in group 2 than that in group 1 (p <0.05).

Table 15: Short-chain fatty acid concentration of the contents from the duodenum (mg g−1) (mean ± SD)

Table 16: Short-chain fatty acid concentration of the contents from the jejunum (mg g−1) (mean ± SD)

Table 17: Short-chain fatty acid concentration of the contents from the ileum (mg g−1) (mean ± SD)

a, b: Different letters within rows indicate differences between treatment groups (p <0.05).

Table 18: Short-chain fatty acid concentration of the contents from the caecum (mg g−1) (mean ± SD)

a, b: Different letters within rows indicate differences between treatment groups (p <0.05).

Discussion

In the current study, dietary supplementation with a probiotic mixture of B. mesentericus, C. butyricum, and S. faecalis did not show any adverse effects on layer chickens. The probiotics did not increase the overall number of eggs produced or egg weight, whereas they significantly improved the feed conversion ratio (feed mass into egg mass). Many studies have shown that probiotics improved feed conversion ratios in layer chickens [3–11]. In contrast, other studies have shown that probiotics do not improve feed conversion ratios in layer chickens [3,5,26]. The difference in feed conversion ratios among the various studies might be due to differences in the composition of bacteria in the probiotic mixture. In general, different complements of probiotic species have different effects on layer hens. The mechanisms by which probiotics improve feed conversion ratios are considered to be complex. One possibility is that probiotics improve the structure of the intestinal mucosa, which is conducive to improved feed conversion. Xu et al. suggested that increased villus length of the intestinal mucosa implies that here would be an increased surface area for nutrient absorption and improved feed efficiency [27]. For example, it was shown that probiotics containing B. mesentericus TO-A, C. butyricum TO-A, and S. faecalis T-110 improved the structure of intestinal mucosa in broiler chickens [28].

Although probiotics did not improve egg quality (Haugh units), egg shell quality (egg shell thickness and egg shell strength) was improved. With respect to egg quality (Haugh units), Lei et al. [29] showed that probiotics improved egg quality, while Aghaii et al. found that they did not [30]. These contrasting results may be due to differences in the ability of probiotics to increase protein synthesis and the transfer of water from the yolk. With respect to egg shell quality (egg shell thickness and egg shell strength), a similar increase in egg shell thickness and egg shell strength has been found in layers fed diets supplemented with probiotics [29]. Xu et al. found that lower pH is required for the dissolution of calcium and phosphorus and promotes intestinal absorption and utilization of both [31]. Addition of probiotics to the basal media caused significantly lower pH in duodenal, jejunal, ileal, and caecal contents than basal media alone. Consistent with past studies, these results suggest that the probiotics decrease intestinal pH and increase egg shell thickness and strength by promoting calcium absorption in the intestine of layer chickens.

Glucagon-like peptide 2 (GLP-2) is a member of a family of peptides derived from the expression of proglucagon and is secreted into circulation by enteroendocrine L cells in the small and large intestine [32]. GLP-2 has been widely reported as a potent gut trophic factor [33–36]. GLP-2 prevented mucosal damage in multiple experimental models of intestinal injury and enhanced mucosal barrier function [37, 38]. In mice, plasma GLP-2 bolsters gut barrier integrity. Probiotic treatment increases endogenous GLP-2 production, and consequently improves gut barrier functioning during obesity and diabetes [39]. Sodium butyrate addition in diets tended to increase plasma GLP-2 concentration in weanling piglets [40]. In broiler chickens, GLP-2 injection reversed the negative effects of stress on weight, morphology and the absorptive function of the small bowel, having a positive effect on growth performance [41]. There have been studies on GLP-2 in chickens, but studies on the relationship between GLP-2 and probiotics in chickens are limited. To examine the effect of the probiotics on intestinal structure of layer chickens, GLP-2 concentration was measured. Supplementation of the media with probiotics elevated both plasma GLP-2 titers and ileal and caecal n-butyric acid concentrations. C. butyricum is known to produce butyrate and may have been the probiotic agent responsible for producing butyrate in the intestine, possibly leading to increased plasma GLP-2 concentration.

Mitsuoka indicated that irregular intestinal microflora can cause malabsorption of vitamins [42]. In mouse, it has been shown that rotavirus infection caused acute diarrhoea and the vitamin A deficiency [43]. In the current study, serum vitamin A and E (retinol and α-tocopherol, respectively) concentrations were significantly higher in group 2 than those in group 1. Probiotics containing B. mesentericus, C. butyricum, and S. faecalis increased serum vitamin E concentration in cattle and dog by improvement of the intestinal environment [44, 45]. This study showed that probiotics containing these bacteria increased serum vitamin A and E concentrations by improvement in digestive health in layer chickens.

Reactive oxygen metabolites are produced as a by-product of oxidative metabolism or exposure to oxidants in food or the environment. Oxidant exposure leads to the production of toxic reactive oxygen species, such as free radicals, which in turn modify biological macromolecules. Vitamin E is known as an excellent biological chain-breaking antioxidant that protects cells and tissue from lipid peroxidation induced by free radicals [46, 47]. Vitamin E has also been shown to increase plasma BAP in sheep exposed to heat stress [48]. However, there have been few studies linking BAP and probiotic supplements in chickens. In the current study, d-ROM and BAP were measured to determine the antioxidative effects of the probiotic supplements. No differences in d-ROM were found between groups 1 and 2 suggesting that probiotic supplements did not alter the level of ROS in the digestive tract. On the other hand, the biological antioxidant potential was significantly higher in group 2 than that in group 1. These data indicate that layer chickens in group 1 and 2 were under the same oxidative stress conditions but probiotic supplements increased antioxidative activity in layer chickens. Considering the increased serum vitamin E in the current study, it is likely that probiotics improved antioxidative activity of layer chicken by increasing antioxidant absorption in intestine, in support of past studies.

The phagocytic index is an important indicator of immune function in the blood. The phagocytic index was significantly higher in group 2 than that in group 1. One possible reason for this is that the probiotics may have increased serum vitamin E levels. Puthpongsiriporn et al. demonstrated that the antioxidant properties of vitamin E may have a role in the development of immune responses in chickens [49]. Vitamin E has been reported to protect cells involved in immune response, such as lymphocytes macrophages, and plasma cells, against oxidative damage and to enhance the function and proliferation of these cells [50, 51]. Gebremichael et al. demonstrated that vitamin E improves proliferation and phagocytosis by macrophages [52]. Kramer et al. also showed that vitamin E supplementation enhances lymphocyte proliferation in livestock animals [53].

Havenaar and Spanhaak [54] demonstrated that probiotics stimulate the immunity of chickens in two ways: 1) flora from the probiotic migrate throughout the gut wall and multiply to a limited extent and 2) antigens released by dead microorganisms are absorbed thus stimulating the immune system. It has been reported that probiotics containing B. mesentericus TO-A, C. butyricum TO-A and S. faecalis T-110 cause an increase in immunoglobulin production from the mesenteric lymph nodes in rats [55], stimulates the T-helper 1 immune response in peripheral blood mononuclear and dendritic cells [56], and cause an influx of CD8+ T cells into the intestinal mucosa. These changes may enhance intestinal immunity by CD8+ T cells in young chicks [57] and an increase IgA concentrations of the jejunum and ileum in broiler chickens [23]. In the current study, jejunal and ileal IgA concentrations were significantly higher in group 2 than that in group 1 and mortality rates were lower in group 2 than in group 1. The mechanisms responsible remain unclear; however, the results of this study are consistent with the hypothesis that the probiotic supplement improved gut immunity and decreased mortality rates.

Conclusions

In conclusion, these results indicate that probiotics containing B. mesentericus TO-A, C. butyricum TO-A and S. faecalis T-110 promote laying performance (feed conversion ratio), egg shell quality (egg shell thickness and egg shell strength), gut mucosal immunity (jejunum and ileum IgA concentration) and antioxidative activity (biological antioxidant potential) in layer chickens. It is therefore suggested that use of the probiotics would be advantageous to the poultry industry.

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