Effects of dietary Bacillus coagulans on the productive performance, egg quality, serum parameters, and intestinal morphology of laying hens during the late laying period

Abstract The aim of this study was to investigate the effects of Bacillus coagulans (B. coagulans) on the productive performance, egg quality, blood biochemistry, antioxidant capacity, reproductive hormones, immune function, and intestinal morphology of laying hens (86 weeks old) during the late laying period. A total of 960 Hy-Line Brown layers were randomly assigned to five treatment groups, each with 6 replicates of 32 hens. Hens were grouped as follows: basal diet (control group) and a basal diet supplemented with 3.25 × 105, 6.5 × 105, 9.75 × 105, or 1.3 × 106 cfu/g B. coagulans. Dietary supplementation with B. coagulans significantly enhanced egg mass, egg weight, and laying rate and decreased the feed conversion ratio (FCR) in comparison with the control group. No significant difference was observed in the feed intake, breaking rate, albumen height, haugh unit, egg yolk colour, eggshell strength, and eggshell thickness among all groups. The serum total protein (TP) levels of laying hens fed 6.5 × 105 cfu/g B. coagulans were higher than those of laying hens in the control group. Dietary supplementation with 3.25 × 105 cfu/g and 6.5 × 105 cfu/g B. coagulans improved the activity of glutathione peroxide (GSH-Px) compared with that of the control group. The serum IgA concentration of laying hens in the 1.3 × 106 cfu/g B. coagulans treatment group was higher than that of the control. In the ileum, 6.5 × 105 cfu/g B. coagulans supplementation significantly increased the ratio of villus height to crypt depth (VH/CD) compared to that of the control group. It can be concluded that B. coagulans, at a supplementation level of 6.5 × 105 cfu/g feed, can be used as an effective feed additive to improve the production performance of laying hens during the late laying period by increasing the serum TP level, serum GSH-Px activity, and ileal VH/CD ratio. HIGHLIGHTS Bacillus coagulans improved the productive performance of laying hens during the late laying period. Inclusion of Bacillus coagulans in the diet might benefit laying hens’ health by improving blood biochemistry, enhancing antioxidant activity and intestinal morphology, and slightly enhancing immune ability. The optimum concentration of Bacillus coagulans as a supplement to the basal diet to promote production performance and improve host health is 6.5 × 105 cfu/g.


Introduction
In recent years, probiotics have been widely used as substitutes for antibiotics in livestock and poultry farming.Probiotics are defined as 'live microorganisms which when administered in adequate amounts confer a health benefit on the host' by the World Health Organization (Adami and Cavazzoni 1999).Furthermore, microorganisms that can be classified as probiotics should meet the following requirements: (1) to survive in the gastrointestinal environment, that is, to be acid and bile resistant; (2) to adhere to intestinal epithelial cells; (3) to grow rapidly, colonise the intestinal tract, persist there and then leave the hosts; (4) to stabilise the intestinal microbiota; (5) to have no signs of pathogenicity; and (6) to maintain vitality both in food and pharmaceutical processes (Gibson and Roberfroid 1995;Fuller and Gibson 1998).Antibiotic abuse has had severe effects on human health and has been forbidden in laying hens' production, and probiotics have been widely used in the animal husbandry industry as an alternative to antibiotics.
Probiotics, including Lactobacillus, Bacillus, Bifidobacterium, and Enterococcus, have become increasingly popular worldwide due to their promotion of growth effects in animals (Jin et al. 1997;Mountzouris et al. 2007), especially after the European Union ban on antibiotic growth promoters (AGP) in 2006.In China, some Bacillus spp strains have been approved as animal feed supplements.Bacillus spp.are promising feed supplements because their metabolically dormant spores are stable, resistant to heat and have low pH extremes and low levels of toxic chemicals (Setlow 1994).Bacillus coagulans (B.coagulans), known as 'the king of probiotics', is a gram-positive, spore-forming, lactic acid-producing bacillus that does not encode enterotoxin (Jurenka 2012).As a new feed additive, Bacillus coagulans is a popular probiotic because this species has the same functions as lactic acid bacteria and possesses a protective, spore-like protein coating, which allows it to resist high temperature, survive stomach acid and bile salts, reach the small intestine, germinate, and multiply.Moreover, the safe application of B. coagulans has been supported by a toxicological safety assessment by Endres et al. (2009).They proved that B. coagulans is safe after conducting a one-year chronic oral toxicity study combined with a one-generation reproduction study (Endres et al. 2011).It can replace Lactobacillus application in livestock and poultry production (Chen et al. 2018;J€ ager et al. 2018;Kapse et al. 2018).Therefore, B. coagulans, as a potential probiotic, has been used in cattle, broilers, and weaned piglet production (Ripamonti et al. 2009;Zhou et al. 2010;Hung et al. 2012;Pu et al. 2020).
Studies by Zhen et al. (2018) showed that the addition of B. coagulans to broilers' diet improved the growth performance, intestinal microbiota, and villus structure of broilers and repaired the intestinal inflammation and structural damage caused by Salmonella enteritis infection.Liu et al. (2022) found that B. coagulans could maintain the intestinal mucosal barrier by improving intestinal flora, enhancing innate immunity, and promoting intestinal epithelial proliferation.Although many studies of the genus Bacillus in poultry have been published (Zhang et al. 2012(Zhang et al. , 2013;;Ahmed et al. 2014;Park and Kim 2014;Latorre et al. 2015), reports about B. coagulans in laying hens are still limited.Recently, Xu et al. (2022) found that dietary supplementation with B. coagulans X26 significantly improved the production performance and egg quality of laying hens by increasing the villus height of the ileum and the ratio of villus height to crypt depth as well as changing the intestinal microbiota composition and the short-chain fatty acid (SCFA) content of the intestinal contents.These effects are equivalent to the effects of the antibiotic chlortetracycline hydrochloride and indicate that B. coagulans can be used as an alternative to antibiotics in the animal husbandry industry (Xu et al. 2022).Because eggs are a 'high-quality and cheap' nutriment, their proportion in the food consumption structure of the human population is gradually increasing.However, in the process of breeding aged laying hens, there are often problems such as stress stimulation, decreased resistance and intestinal dysfunction, which lead to the decline of egg production performance, poor egg products, high mortality and egg contamination.However, antibiotics have been forbidden in the production of laying hens, while probiotics have been widely used in the animal husbandry industry as an alternative to antibiotics.The benefits of B. coagulans in the animal husbandry industry have been widely studied, especially in pigs and broilers, and B. coagulans has been approved in China and used as a feed additive in the animal husbandry industry.Whether and how B. coagulans affects the production performance, egg quality, antioxidant capacity, reproductive hormones, immunity, and intestinal morphology of aged laying hens remain unkown.Considering the potential probiotic effects of B. coagulans, we hypothesised that B. coagulans would enhance the production performance and host health of laying hens during the late laying period.The purpose of this study is thus to add different doses of high-yield and stable Bacillus coagulans to the diet, to clarify their effects on the productive performance, egg quality, serum biochemistry, antioxidant capacity, reproductive hormones, immunity and intestinal morphology of laying hens and to further analyse its mechanism to provide a theoretical basis for the research and application of Bacillus coagulans, which is of great significance for promoting healthy breeding of laying hens and producing safe, high-quality eggs.

Materials and methods
All experimental procedures in this trial were conducted following the Chinese Guidelines for Animal Welfare and approved by the Zhejiang University Institutional Animal Care and Use Committee (No. ZJU2013105002) (Hangzhou, China).

Birds, diets, and management
A total of 960 Hy-Line Brown laying hens (86 weeks old) were randomly assigned to five groups including the control group (Con, fed a basal diet), BC50 group (basal diet supplemented with 50 g/t B. coagulans containing 3.25 Â 10 5 cfu/g B. coagulans), BC100 group (basal diet supplemented with 100 g/t B. coagulans containing 6.5 Â 10 5 cfu/g B. coagulans), BC150 group (basal diet supplemented with 150 g/t B. coagulans containing 9.75 Â 10 5 cfu/g B. coagulans), and BC200 group (basal diet supplemented with 200 g/t B. coagulans containing 1.3 Â 10 6 cfu/g B. coagulans), each with 6 replicates of 32 hens (4 birds/cage).There were no significant differences in laying rate and/or egg weight among all groups before the start of the formal trial (Day 0).B. coagulans was provided by Xiamen HuiYing Animal Technology Co., Ltd., and the concentration of live bacteria of B. coagulans was larger than 6.5 Â 10 9 cfu/g.The strain was deposited in the China Centre for Type Culture Collection (CCTCC ATCC 7050).The actual presence of B. coagulans in the feed was calculated by using a coagulation Bacillus count medium after the experimental feed was prepared.The actual amounts of B. coagulans in the five groups were 0 cfu/g, 3.0 Â 10 5 cfu/g, 6.25 Â 10 5 cfu/g, 9.35 Â 10 5 cfu/g, and 1.26 Â 10 6 cfu/ g.The composition and nutritional level of the basal diet based on corn and soybean meal are shown in Table 1.This study lasted 9 weeks, including a oneweek acclimation period and an 8-week experimental period.The environmental conditions were the same for all groups.All hens were raised in a naturally ventilated, windowed poultry house with a temperature of approximately 23 C, and the light was provided from incandescent bulbs for 16 h each day.The laying hens were allowed ad libitum access to feed and water.Mortality and health status were checked daily, and dead birds were recorded to adjust estimates of laying rate, feed intake, and feed conversion ratio.

Sample collection
Two hens were randomly selected from each replicate (12 hens in each group; a total of 60 hens) at the end of the nine weeks trial.From an animal welfare point of view, laying hens were deprived of feed but no water for 12 h, and the blood samples (5 mL/ bird) were collected from the vein under the wing using a procoagulant tube so that the quantity of blood was sufficient for experimental analysis.Serum samples were acquired by centrifugation at 3,000 rpm for 15 min and stored at À80 C until analysis.Then, birds were euthanised by cervical dislocation.The intestine segments of the jejunum and ileum were carefully collected along with the liver, snap-frozen in liquid nitrogen, and stored at À80 C for further analysis.

Laying performance and determination of egg quality
During the feeding trial, eggs were collected daily, and data on egg production, egg weight, dirty eggs, broken eggs, soft eggs, and malformed eggs were recorded on a replication basis.Meanwhile, the laying rate and breaking rate were calculated.Feed intake was recorded once a week on a replication basis.The feed conversion ratio was calculated as grams of feed intake per gram of egg mass produced.Thirty eggs (5 eggs per replicate, 6 replicates per treatment) per group were picked out at the end of the feeding trial for subsequent laboratory determination of egg quality parameters.Egg weight, eggshell strength, albumen height, haugh unit, and egg yolk colour were measured with a digital egg tester (DET-6000, NABEL, Kyoto, Japan).Eggshell thickness (without the shell membrane) was measured in the middle of the egg with an eggshell thickness gauge (ESTG-1, Orka Food Technology Ltd., Ramat Hasharon, Israel).vitamin D3, 1200 IU;vitamin E, 6 mg;vitamin B1, 1.4 mg; vitamin B2, 3 mg; vitamin B6, 1.0 mg; vitamin B12, 0.01 mg; pantothenic acid, 7.5 mg; choline chloride, 500 mg; biotin, 0.15 mg; Ca,7500 mg; P, 3000 mg; Mn, 72 mg; Zn, 56 mg; Fe, 60 mg; Cu, 25 mg; I, 0.50 mg; Se, 0.10 mg; phytase, 2000 FTU (phytase unit).

Serum biochemical analysis
The content of total protein (TP), albumin (ALB), calcium (Ca), phosphorus (P), uric acid (UA), total cholesterol (T-CHO), and triglyceride (TG) and the activities of alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum was determined and calculated using the commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).All determinations were carried out according to the instructions of the manufacturer.

Serum and liver antioxidant capacity assays
The liver samples were cut into small pieces, added to ice-cold physiological saline at a ratio of 1:9 (weight: volume) to prepare 10% tissue homogenate mechanically, and then centrifuged at 12,000 rpm x g for 5 min at 4 C to separate supernatant.The supernatant was collected and stored at À80 C for further enzyme activity assays.Briefly, after thawing the serum and homogenates and allowing them to equilibrate to room temperature, the activities of glutathione peroxide (GSH-Px), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and catalase (CAT) and the content of malondialdehyde (MDA) in the serum were assayed using commercially available assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions of the manufacturer.The T-AOC content and the SOD, CAT, and GSH-Px activities of hepatic supernatants were also measured using kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Determination of serum hormone levels
Concentrations of serum follicle-stimulating hormone (FSH), oestradiol (E 2 ), and luteinising hormone (LH) were measured by ELISA using commercial kits provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's protocol.

The determination of immunoglobulins
Immunoglobulin (Ig) levels, including IgA, IgG, and IgM were determined using enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions.The concentrations of IgA, IgG and IgM were determined using standard curves constructed from the standards run on the plate.

Histopathological analysis
Approximately 1 cm segments of the jejunum and ileum fixed with 4% paraformaldehyde were trimmed and embedded in paraffin wax.The paraffin sections were cut into 5-6 lm thick sections using a microtome (Leica Microsystems, RM2016) and then stained with haematoxylin and eosin (H&E) for histomorphological observation.The villus height, villus width, villus area, and crypt depth of 8 villi from each intestinal sample were calculated by optical microscopy (Nikon Eclipse 80i, Nikon, Tokyo, Japan).

Statistical analysis
The data were statistically analysed by one-way ANOVA using SPSS 20.0 (SPSS Inc., Chicago, IL) and are expressed as the means and standard error of the means (SEM).When significant differences were found (p < 0.05), Tukey post hoc tests were performed.Orthogonal polynomials were used for linear and quadratic response analysis.

Productive performance
Effects of B. coagulans on the production performance of laying hens are presented in Table 2. Egg mass, laying rate, and egg weight showed a linear or quadratic increase with the increase in dietary B. coagulans levels (p < 0.05).However, broken egg rate and feed conversion ratio (FCR) were significantly decreased (p < 0.05), whereas it had no effect on feed intake (p > 0.05).

Egg quality parameters
As shown in Table 3, no significant differences in albumen height, haugh unit, egg yolk colour, eggshell strength, and eggshell thickness were observed among all groups throughout the experiment (p > 0.05).
The effects of B. coagulans treatment on serum biochemical indices are summarised in Table 4. Serum ALT, AST, ALB, TC, TG, P, and UA were not influenced by dietary B. coagulans treatments (p > 0.05), whereas serum Ca level showed a significant decrease in a quadratic manner with increasing dietary B. coagulans levels.Total protein was significantly increased with increasing dietary B. coagulans levels (p < 0.05).However, the concentration of ALP was decreased in the group supplemented with 100 g/t of B. coagulans compared with the other groups (p < 0.05).

Serum hormone and antioxidant enzyme activities
The effects of B. coagulans treatment on serum hormone levels and antioxidant enzyme activities are listed in Table 5.The serum concentration of LH, FSH, E 2 and MDA, and the activities of T-AOC, SOD, and CAT were not influenced by dietary B. coagulans treatment (p > 0.05).However, dietary supplementation with 50 and 100 g/t B. coagulans significantly improved the serum activity of GSH-Px compared with that of hens fed the control diet (p < 0.05).Therefore,    The effects of B. coagulans supplementation on hepatic antioxidant enzyme activities are illustrated in Table 6.The hepatic CAT level was higher in laying hens fed 200 g/t B. coagulans than in those fed 100 g/t and 150 g/t groups (p < 0.05).No significant difference was observed in hepatic T-AOC, GSH-Px, and MDA levels among all treatments (p > 0.05).

Systemic immunity
The effects of B. coagulans on immune ability are shown in Table 7. Dietary supplementation with 200 g/t B. coagulans significantly improved the serum IgA concentration compared with that of laying hens fed the control diet (p < 0.05).However, serum IgG and IgM concentrations were not influenced by dietary B. coagulans treatment (p > 0.05).

Intestinal morphology
Histological changes in the small intestine were evaluated using a light microscope.As shown in Table 8, in the jejunum, a significant difference in villus height was obtained between the 50 g/t B. coagulans and 200 g/t B. coagulans groups (p < 0.05), but B. coagulans treatment did not affect crypt depth and VH/CD ratio (p > 0.05).In the ileum, a significant difference in crypt depth was observed between the 100 g/t B. coagulans and 200 g/t B. coagulans groups (p < 0.05).Supplementation with 100 g/t B. coagulans significantly increased VH/CD ratio compared to that of the control group (p < 0.05).Significant differences were not detected in villus height (p > 0.05).

Discussion
Several recent studies have verified that probiotics, such as Bacillus spp., Lactobacillus spp., and Saccharomyces spp., can enhance livestock and poultry growth performance.(Cao et al. 2019;Lokapirnasari et al. 2019;Massacci et al. 2019).As shown by Fu et al. (2021), dietary B. coagulans and yeast hydrolysate supplementation improved the body weight and average daily gain of pigs.Alaqil et al. (2020) presented that dietary supplementation of the probiotic Lactobacillus acidophilus improved egg production, egg weight, egg mass, and feed efficiency.According to Liu et al. (2019), dietary supplementation with 9.0 Â 10 5 cfu/g B. subtilis C-3102 in laying breeder diets may be a feasible means of effectively increasing egg weight.Ribeiro et al. (2014) reported that dietary B. subtilis at a level of 8 Â 10 5 cfu/g could significantly improve the egg weight and egg mass of Hyland brown laying hens.Similar to our study, laying hens fed B. coagulans-supplemented diets significantly increased the egg weight, egg mass, and laying rate and decreased the feed conversion ratio with increasing dietary B. coagulans supplementation.Consistently, dietary supplementation with Bacillus coagulans X26 significantly increased the average laying rate and the survival rate, and decreased the feed intake and feed conversion ratio (Xu et al. 2022).In addition, Khajeh Bami et al. (2020) also found that dietary supplementation with B. coagulans could increase the body weight gain, body weight, and feed conversion ratio of broilers.In combination with the results above, these results clearly demonstrate that dietary supplementation with B. coagulans both effectively improves productive performance and ameliorates the feed conversion ratio.
Eggs are an important source of nutrients for humans and it is well known that their composition can be modified through the manipulation of laying hens' diets (Caston et al. 1994).In the present study, we found that dietary B. coagulans supplementation did not affect albumen height, haugh unit, egg yolk colour, eggshell strength, and eggshell thickness of laying hens.Zeweil et al. (2006) reported that dietary supplementation with probiotics did not significantly influence egg quality, which is consistent with our results.However, Ma et al. (2012) demonstrated that egg quality greatly improved when laying hens were fed Bacillus subtilis-supplemented diets.Lei et al. (2013), reported that B. licheniformis supplementation increased eggshell strength, whereas its effects on egg yolk colour were unclear.Panda et al. (2008) demonstrated that the haugh unit was not influenced by adding 100 mg probiotic (Lactobacillus sporogenes)/kg to the diet, but that eggshell strength significantly increased.Zhang et al. (2012) reported that no significant variance was found in yolk colour compared with the control group, while differences were observed in the haugh unit.On the contrary, Xu et al. (2022) found that dietary Bacillus coagulans X26 significantly improved the average egg weight and the content of egg white protein while decreasing the rate of softbroken eggshells, which is inconsistent with our results.A possible explanation is that these contradictory results are mediated by the difference in bacterial strains (Bacillus coagulans vs Bacillus coagulans X26) and/or the different ages of laying hens used in the experiment.These contradictory results indicate that the effect of B. coagulans on egg quality is uncertain and depends on various factors, such as the bacterial strains, dose, and growth period of experimental animals.Future research should consider the effects of the different bacterial strains on the egg quality of hens of various ages and in both breeds.
Biochemical blood parameters usually reflect the health of an animal.These parameters are vital indicators of the nutritional and physiological status of birds and animals (Alagawany and El-Hack 2015).Measurement of such indicators is routinely conducted to evaluate the response of animals to various feed additives (Yan et al. 2010).In this study, treatment with 6.5 Â 10 5 cfu/g B. coagulans was found to increase the TP content, suggesting that dietary B. coagulans may participate in host protein metabolism and future research will be required to define how protein metabolism is altered in B. coagulans-fed laying hens and to elucidate the role of B. coagulans in protein synthesis.The total protein content of poultry blood increases with age as a result of metabolic changes in the animal and reflects various disorders of nutritional character resulting from either insufficient or excessive intake of proteins in feed mixtures (Pavl ık et al. 2007).Our results regarding the protein content of the serum were within the range for laying hens, that is, between 32.0 and 62.0 g/L, which suggests that dietary supplementation with B. coagulans is non-toxic and may contribute to the increase of laying hens.ALT and AST are important amino acid transferases in animals and are indicators of protein metabolism in the body.They are released from the liver or cardiac cells into the plasma and the increase beyond the normal range indicates liver injury or damage (Hafeman et al. 1974).Fathi (2013) reported that probiotic (Lactobacillus) supplementation in broilers can decrease ALT and AST plasma concentrations.Therefore, this result indicated that probiotics are associated with the liver function of poultry and may promote the utilisation of amino acids by the body.However, in our study, no significant differences in the serum concentrations of ALT, AST, ALB, TC, TG, Ca, P and UA were observed during the whole experimental period in any experimental treatment, compared with  the control group.Consistently, other authors (Baidya et al. 1994;Capcarova et al. 2010) also found that ALT and AST were not affected by the addition of probiotic strains in broiler diets.These contradictory results may be attributed to differences in experimental design or probiotic strains.
To our knowledge, few studies have investigated the supplemental effects of B. coagulans on the reproductive hormones of laying hens.In the present study, no significance was observed in the serum concentrations of LH, FSH and E 2 among the five groups.This finding is inconsistent with previous reports that different levels of B. amyloliquefaciens supplementation in the basal diet significantly increased the FSH and E 2 concentrations, while no significant differences were observed with respect to LH levels in any of the groups (Zhou et al. 2020).Understanding the role of B. coagulans in the regulation of reproductive hormones will be important to address in the future.
Oxidative stress refers to the imbalance of oxidation and antioxidation in the body, and it could produce varieties of reactive oxygen species (ROS) such as hydroxyl free radicals and superoxide anions, which can damage proteins, nucleic acids and other biological macromolecules leading to tissue damage and tissue mitochondrial damage, thus contributing to the development of diseases.With the development of modern intensive farming, laying hens are prone to oxidative stress (Salami et al. 2015).In particular, aged laying hens during the late laying period commonly show enhanced oxidative stress and are sensitive to the changing environment.Numerous studies have found that dietary probiotics are beneficial in oxidation resistance, scavenging ROS and promoting antioxidant capability (Chen et al. 2013;Singh et al. 2014).Antioxidant enzymes (SOD, CAT, GSH, and GSH-Px) serve as the first line of defense in suppressing oxidative damage.In the present study, supplementation with B. coagulans significantly improved the activity of GSH-Px in serum, but therewere no differences in the activities of T-AOC, CAT, and SOD, and the content of MDA among all groups.The hepatic CAT activity was higher in laying hens fed with 1.3 Â 10 6 of B. coagulans compared with that of other groups.No significant difference was observed in hepatic activities of T-AOC and GSH-Px, and MDA levels among the five treatments.These results suggest that dietary B. coagulans could partly enhance the antioxidant capacity of laying hens.As revealed by Lin et al. (2014), dietadded B. coagulans significantly increased yellow broilers' serum SOD and CAT activity and markedly reduced serum MDA concentration, which is comparable to our results.Similarly, Zhang et al. (2021) reported that the activities of GSH-Px, SOD, and CAT significantly increased and the content of MDA significantly decreased when B. coagulans was added to the diet of laying hens.These results are partly consistent with our animal study findings and suggest that dietary intake of a moderate proportion of B. coagulans increases the antioxidative capacity of laying hens during the late laying period.In summary, B. coagulans can effectively protect the body from oxidative stress injury, which is also an important reason why it can improve the productive performance of poultry.
Most immunostimulants stimulate the production of immune response compounds, such as immunoglobulin and complement proteins that activate the immune system after binding with cell surface receptor proteins of phagocytes or lymphocytes (Zou et al. 2016).Serum immunoglobulin concentration, as a parameter that reflects the animal humoral immune status, plays an important role in the fight against various infections (Liu et al. 2019).In the present study, we found that supplementation with B. coagulans significantly improved IgA concentration, whereas there were no differences in IgG and IgM concentrations among all groups.Wang et al. (2017) reported that Bacillus spp.can improve the systemic immunity of laying hens by modulating specific and non-specific immunity.On the one hand, the increased serum immunoglobulin levels of chickens were due to the immunomodulatory activities of probiotics (Paturi et al. 2007).On the other hand, the small intestine usually acts as an immune protection barrier in individuals.The body's immune status is improved by regulating intestinal mucosal cytokines (Patterson and Burkholder 2003).However, some studies found that dietary probiotics showed little beneficial effects on immune status, and these differing outcomes might be due to the different experimental designs and the different times of collection (Mountzouris et al. 2010;Zhang et al. 2012).In summary, our results demonstrated that dietary supplementation with B. coagulans could partly promote the immune function of laying hens by stimulating the production of IgA.However, further studies are still required to draw any conclusions on the dietary effects of probiotics on the immune response of laying hens during the late laying period.
The small intestine is the main site for nutrient digestion and absorption (Abdelnour et al. 2019).The basic function of the small intestinal mucosa, which consists of a single layer of epithelial cells, is to digest and absorb nutrients and block pathogenic bacteria and toxic substances in the intestinal cavity (Mowat and Agace 2014).The crypt is a tubular gland formed by the small intestinal epithelium descending into the lamina propria at the root of the villi (Clevers 2013).A higher villus height to crypt depth ratio (VH/CD) indicates a higher rate of digestion and absorption function; it is more representative when measuring individuals (Chee et al. 2010).In the current trial, we found that dietary B. coagulans supplementation produced positive effects on some villus morphological characteristics in both the jejunum and ileum, especially in the 100 g/t (6.5 Â 10 5 cfu/g) group.This result indicates that dietary B. coagulans supplementation contributes to improving intestinal morphology by decreasing crypt depth and increasing the villus height to crypt depth ratio.The changes in the VH/CD ratio indicated that B. coagulans may improve intestinal structure and function by promoting the proliferation of intestinal epithelial cells.

Conclusion
In conclusion, the results of this study demonstrate that supplementation with BC100 (6.5 Â 10 5 cfu/g B. coagulans) in laying breeders can significantly improve egg mass, egg weight, and laying rate, and decrease FCR.Additionally, B. coagulans can enhance the level of TP, the activity of GSH-Px, the concentration of IgA in the serum and the activity of CAT in the liver.Moreover, 6.5 Â 10 5 cfu/g B. coagulans supplementation significantly increased the VH/CD ratio of the ileum compared to that of the control group.Our data suggest that the optimum concentration of B. coagulans in the basal diet is 6.5 Â 10 5 cfu/g.It is speculated that B. coagulans can improve the production performance of laying hens by enhancing immunity and antioxidant enzyme activity and improving intestinal tissue morphology.The role of probiotics in different fields has been widely reported, but research on the mechanisms underlying the effects of B. coagulans as a probiotic is not comprehensive, and systematic in-depth research is needed.

Table 1 .
Composition and nutrient levels of the experimental basal diet for laying hens.
a Premix provided the following per kilogram of diet: vitamin A, 4000 IU;

Table 2 .
Effects of B. coagulans on productive performance of laying hens (n 32).Means sharing different letters in the same row are significantly different (p < 0.05).BC: Bacillus coagulans; FCR: feed conversion ratio; SEM: standard error of the means; PL: p-Value of linear analysis; PQ: p-Value of quadratic analysis.

Table 3 .
Effects of B. coagulans on egg quality of laying hens (n ¼ 30).
BC: Bacillus coagulans; SEM: standard error of the means; PL: p-Value of linear analysis; PQ: p-Value of quadratic analysis.Serum biochemistry.

Table 5 .
Effects of B. coagulans on serum hormone and antioxidant enzyme activities of laying hens (n ¼ 12).

Table 6 .
Effects of B. coagulans on hepatic antioxidant enzyme activities of laying hens (n ¼ 12).Means sharing different letters in the same row are significantly different (p < 0.05).T-AOC: total antioxidant capacity; CAT: catalase; GSH-Px: glutathione peroxide MDA: malondialdehyde; BC: Bacillus coagulans; SEM: standard error of the means; PL: p-Value of linear analysis; PQ: p-Value of quadratic analysis.

Table 8 .
Effects of B. coagulans on villi morphology of the small intestine in laying hens (n ¼ 12).Means sharing different letters in the same row are significantly different (p < 0.05).VH/CD: villus height/crypt depth; BC: Bacillus coagulans; SEM: standard error of the means; PL: p-Value of linear analysis; PQ: p-Value of quadratic analysis.

Table 7 .
Effects of B. coagulans on immune response in laying hens (n ¼ 12).Means sharing different letters in the same row are significantly different (p < 0.05).Ig: Immunoglobulins; SEM: standard error of the means; PL: p-Value of linear analysis; PQ: p-Value of quadratic analysis.