Effects of Different Probiotics on Laying Performance, Egg Quality, Oxidative Status, and Gut Health in Laying Hens

Simple Summary Resistance has developed against almost all the main classes of antibiotics, and finding efficient alternatives to these antibiotics is urgently required. Based on previous research, three types of probiotic bacteria were chosen to be administrated in a laying hen diet. We found that the performance, egg quality, and gut health of laying hens were improved after probiotic treatment. Abstract With recent bans on the growth-promoting use of antibiotics, alternative strategies are needed to improve the performance of agricultural animals. Here, the effects of dietary supplementation with Clostridium butyricum and a combination of Saccharomyces boulardii and Pediococcus acidilactici were assessed on laying performance, egg quality, oxidative status, and gut health in laying hens. A total of 8208 Lohmann pink laying hens were divided into 3 treatment groups, with each group replicated 12 times (n = 228). Hens in the control group (CON) were provided a basic diet devoid of added antibiotics and probiotics. Treatment group 1 (T1) received the same base diet supplemented with 0.5 g/kg C. butyricum, and the diets of treatment group 2 (T2) supplemented with S. boulardii (0.05 g/kg) and P. acidilactici (0.1 g/kg) for the entirety of the 5-week trial. The data indicated that C. butyricum supplementation resulted in a significant reduction in ADFI, a significant increase in feed conversion, eggshell strength, and the CP% of albumen (dry matter, DM) relative to CON. The probiotic-treated hens exhibited decreased reactive oxygen species (ROS) levels in ileum and cecum, and reduced malondialdehyde (MDA) in serum. In conclusion, dietary supplementation with C. butyricum may be beneficial with respect to hen performance, egg quality, and gut health.


Introduction
Antibiotics have been recognized as one of the most effective therapies in medicine [1,2]. However, due to the growing number of antibiotic-resistant bacteria, the agricultural use of antibiotics as growth promoters in livestock has being banned in many countries worldwide. Antibiotic growth promoters (AGPs) have been banned in the European Union since 2006 [3]. In spite of the prohibition, a large number of AGPs are being administered to laying hens, through individual treatments or water and diet supplementation, in an effort to reduce the occurrence of diseases and improve the performance of laying hens [4,5]. Not only can overuse of antibiotics at subtherapeutic doses lead to

Probiotic Strains
Three pure probiotic strains were used in this study. C. butyricum was provided by Hubei Lvxue Biotechnology Co., Ltd. (Xianning, Hubei, China) Powdered C. butyricum is provided. S. boulardii I-1079 and P. acidilactici MA18/5M were provided by Beijing Hilink International Biotechnology Co., Ltd. (Beijing, China) The dose of dietary probiotic supplement was added with reference to the companies' commercial recommendations.

Experimental Design
The protocol for the animal experimental procedures was approved by Institutional Animal Care and Use Committee of Huazhong Agricultural University (Wuhan, China). The ethical number of this study is HZAUCH-2017-009.
A total of 8208 Lohmann pink laying hens (180 days) were randomly assigned to 3 groups with 12 replicates of 228 birds each (2736 laying hens per group). The 3 groups were divided into one control group (CON) and 2 treatment groups (T1 and T2). The diets for T1 consisted of a standard feed supplemented with C. butyricum (0.5 g/kg), and that of T2 was supplemented with both S. boulardii (0.05 g/kg) and P. acidilactici (0.1 g/kg).

Birds, Diet and Management
This trial was carried out in Xian Ning, Hubei Province, during the month of January. Three birds were housed in individual cages, under a 16 h light/8 h dark cycle (lights on at 5:00, lights off at 21:00) and a constant temperature of 9 ± 2 • C. All birds were acclimated to a basal diet for 1 week. laying hens were caged in 3-tier battery cages, 3 hens per cage. The basal diets provided to the hens are presented in Table 1. The temperature, humidity, and light conditions in different chicken coops were similar. Water and feed were provided ad libitum during the 5-week study period.

Samples
Health condition, mortality, feed intake, laying performance, and egg quality were assessed daily. At the end of the trial, a single healthy laying hen was selected from each group replicate, 12 in each group, for a total of 48 hens, which were then sacrificed. Blood and intestinal samples were collected for examination. Blood samples were collected from the axillary vein into vacuum tubes (10 mL) containing coagulant. The blood was then centrifuged at 3000× g for 10 min at 4 • C. Serum was collected and stored at −80 • C until the time of further analysis. The gut of each hen was removed immediately after sacrifice, and segments of ileum and cecum were identified and ligated prior to excision. Digesta of the ileum and cecum were collected and stored at −80 • C until analysis. Intestinal tissues were fixed in 10% phosphate-buffered formalin and stored at room temperature (Huang et al. 2014) for histological examination.

Laying Performance
Daily feed intake, egg production, and egg weights were measured throughout the experimental period. The actual daily feed intake per group replicate was measured and used to calculate the average daily feed intake (ADFI). Laying rate is expressed as average hen-day production, calculated from the total number of eggs divided by the total number of days. Feed conversion ratio was expressed as grams of feed consumed per grams of eggs produced.

Egg Quality
Egg quality was evaluated at the end of trial. A total of 72 eggs were randomly collected from each treatment (n = 72, 6 per replicate) in order to evaluate egg quality. Egg weight, Haugh unit (HU), yolk color, shell strength (measured with an eggshell strength tester), shell thickness (measured with a spiral micrometer), and shape index (determined using Vernier calipers) were evaluated. Crude protein, water, crude fat, ash, calcium, phosphorus, and the cholesterol levels of the eggs were also evaluated. Reactive oxygen species (ROS), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX) activity, and malondialdehyde (MDA) concentrations in either ileum or cecum were measured to estimate the oxidative status. To assess ROS, chemiluminescence (CL) derived from luminol was measured as an indicator of radical formation. Determination of T-SOD, GSH-Px, and MDA levels was accomplished using an assay kit from the Nanjing Jiancheng Bioengineering Institute. The specific procedures were followed according to the manufacturer's instructions.

Gut Histological Analysis
Ileum and cecum were fixed in 10% phosphate-buffered formalin, and embedded in paraffin blocks. The samples were then sectioned to a thickness of 5 mm, and stained with hematoxylin and eosin. Villus height, villus width, and crypt depth were measured on the stained sections using a light microscope fitted with an image analyzer (Image Pro Plus 6.0, Media Cybernetics, Bethesda, MD, USA).

Statistical Analysis
All results were statistically compared with an ANOVA test using SAS 9.4. A one-way ANOVA, followed by Duncan's multiple comparison test, was used to evaluate different means among treatments. Differences were considered to be significant at p < 0.05.

Ethical Statement
This study is approved by the Scientific Ethic Committee of Huazhong Agricultural University (ethical approval number is HZAUCH-2017-009).

Laying Performance
Laying performance data are summarized in Table 2. Dietary supplementation with C. butyricum (T1) not only reduced ADFI, but also significantly increased feed conversion (p = 0.0341). However, there was no apparent effect on the mortality of hens during the experimental period (p = 0.3445). In addition, laying rate (p = 0.4777), average egg weights (p = 0.4923), and average cracked egg percentage (p = 0.0957) were not influenced by dietary probiotic supplementation compared with the CON group. Means without a common superscript with a row differ significantly (p < 0.05). CON = basal diet (BD), T1 = BD plus 0.5 g/kg diet C. butyricum preparation, T2 = BD plus 0.1 g/kg diet P. acidilactici and 0.05 g/kg S. boulardii preparation. ADFI = average daily feed intake.

Egg Quality
The external and internal egg qualities for layers are presented in Table 3. Compared with CON, there were no significant differences in egg quality in T2. In T1, dietary C. butyricum supplementation decreased eggshell strength (p = 0.0044) and yolk color (p = 0.055), and increased the CP% of albumen (DM) (p = 0.0809), but no statistically significant changes were noted in any of the other egg quality traits examined. Means without a common superscript with a row differ significantly (p < 0.05). CON = basal diet (BD), T1 = BD plus 0.5 g/kg diet C. butyricum preparation, T2 = BD plus 0.1 g/kg diet P. acidilactici and 0.05 g/kg S. boulardii preparation. CP = crude protein, DM = dry matter. Table 4 shows the antioxidative stress status of the serum, ileum, and cecum of laying hens. The data indicate that dietary S. boulardii and P. acidilactici (T2) supplementation did not have an effect on the biomarkers of antioxidative stress. However, C. butyricum supplementation (T1) decreased ROS levels in both ileum (p < 0.01) and cecum (p < 0.01), as well as reduced serum MDA (p < 0.05). No statistically significant differences were observed for T-SOD and GSH-PX in laying hens (p > 0.05). Means without a common superscript with a row differ significantly (p < 0.05). CON = basal diet (BD), T1 = BD plus 0.5 g/kg diet C. butyricum preparation, T2 = BD plus 0.1 g/kg diet P. acidilactici and 0.05 g/kg S. boulardii preparation. ROS = reactive oxygen species, T-SOD = total superoxide dismutase, GSH-PX = glutathione peroxidase, MDA = malondialdehyde.

Histological Analysis of the Gut
As presented in Figure 1 and Table 5, dietary probiotic supplementation improved the health and microscopic structure of the ileum and cecum. The villi were observed to be short and thin in the ileum of the CON, whereas they were taller and a greater proportion were intact as observed in the ileum of groups A and B alike. Similarly, the villi in the cecum of hens from groups A and B were higher than in CON. Furthermore, villus height and villus height/crypt depth ratio (villus/crypt) were significantly increased in groups A and B compared with CON in the ileum and cecum (p < 0.05) ( Table 5).
ileum of the CON, whereas they were taller and a greater proportion were intact as observed in the ileum of groups A and B alike. Similarly, the villi in the cecum of hens from groups A and B were higher than in CON. Furthermore, villus height and villus height/crypt depth ratio (villus/crypt) were significantly increased in groups A and B compared with CON in the ileum and cecum (P < 0.05) ( Table 5).  a-c Means without a common superscript with a row differ significantly (p < 0.05). CON = basal diet (BD), T1 = BD plus 0.5 g/kg diet C. butyricum preparation, T2 = BD plus 0.1 g/kg diet P. acidilactici and 0.05 g/kg S. boulardii preparation.

mRNA Levels of Pro-Inflammatory Cytokines in the Intestinal Tract
Gene expression levels of four major inflammatory cytokines involved in intestinal tissues were measured. Included in the assays were interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α and/β (TNF-α/β). There were no statistically significant differences in gene expression observed between T1, T2, or CON.

Discussion
In recent years, the demand for eggs is no longer an issue of quantity, but has become increasingly focused on egg quality. The extensive use of antibiotics in poultry farms has not only increased the safety risks associated with egg consumption, but has also decreased egg quality. Moreover, microbial antibiotic resistance, caused by the overuse of antibiotics, is one of the biggest threats to global health, food security, and development [26]. Therefore, alternatives to antibiotics are critically needed. An important alternative to antibiotics, probiotics have been receiving more and more attention in the field of animal husbandry. Previous studies have suggested that C. butyricum and S. boulardii could improve growth performance of broiler chickens [18,[27][28][29]. A few studies have  a-c Means without a common superscript with a row differ significantly (p < 0.05). CON = basal diet (BD), T1 = BD plus 0.5 g/kg diet C. butyricum preparation, T2 = BD plus 0.1 g/kg diet P. acidilactici and 0.05 g/kg S. boulardii preparation.

mRNA Levels of Pro-Inflammatory Cytokines in the Intestinal Tract
Gene expression levels of four major inflammatory cytokines involved in intestinal tissues were measured. Included in the assays were interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α and/β (TNF-α/β). There were no statistically significant differences in gene expression observed between T1, T2, or CON.

Discussion
In recent years, the demand for eggs is no longer an issue of quantity, but has become increasingly focused on egg quality. The extensive use of antibiotics in poultry farms has not only increased the safety risks associated with egg consumption, but has also decreased egg quality. Moreover, microbial antibiotic resistance, caused by the overuse of antibiotics, is one of the biggest threats to global health, food security, and development [26]. Therefore, alternatives to antibiotics are critically needed. An important alternative to antibiotics, probiotics have been receiving more and more attention in the field of animal husbandry. Previous studies have suggested that C. butyricum and S. boulardii could improve growth performance of broiler chickens [18,[27][28][29]. A few studies have also demonstrated beneficial effects of dietary C. butyricum supplementation on the laying performance of hens [11,30]. However, there are very limited studies examining the effects of S. boulardii on laying hens. Conversely, several studies have shown that the addition of butyric acid bacteria to the diet has no significant effect on improving the performance of agricultural animals [31].
In the present study, diets supplemented with C. butyricum or a combination of S. boulardii and P. acidilactici were examined. Diets supplemented with C. butyricum reduced ADFI and increased feed conversion, which is similar to the results in broilers in Mervat et al. [32]. However, the combination of probiotics had no effect on laying performance. Compared with other studies, the scale of the animal experiments performed here was lager, lending confidence in our conclusion that C. butyricum exerts positive effects on laying performance in hens. The significant increase in the crude protein content of albumen means the egg quality of C. butyricum-treated laying hen was improved.
Antioxidative stress is one of the important factors affecting the performance and egg quality of laying hens. Reactive oxygen species are widely accepted to be detrimental for health [33,34]. Studies have indicated that dietary probiotics are effective in counteracting the adverse influences of antioxidative stress [35], and promoting the activities of antioxidant enzymes [36]. Here, it was observed that C. butyricum significantly decreased the levels of ROS in both the ileum and cecum. Although the major antioxidant enzymes (T-SOD and GSH-PX) were apparently unaffected by probiotic supplementation, the levels of MDA, a cellular endproduct of lipid peroxidation and an important indicator of stress [37], were significantly decreased. These results may indicate that C. butyricum is capable of reducing oxidative stress in the intestines of the laying hens by reducing the degree of intestinal lipid oxidation as opposed to by accelerating the depletion of ROS.
The health status of the mucosa and the microscopic structure can be good indicators of the response of the intestinal tract to active substances in feed [38]. Previous studies have indicated that probiotics may be useful for improving the gut health of laying hens [35] and broiler chickens [16]. Here, it was observed that probiotic supplementation was able to significantly improve the intestinal histological morphology of laying hens ( Figure 1 and Table 5). In our study, we found that dietary probiotic supplementation increased the villus height and villus height/crypt depth ratio in both ileum and cecum, which may reflect better epithelial growth after probiotic supplement [39,40].
The levels of pro-inflammatory cytokines in the intestinal tract were within normal ranges ( Figure 2). These results indicate that all hens were in good health, which would explain the lack of difference in the observed mortality (p = 0.3445, Table 2). Deng et al. (2012) demonstrated that the level of serum TNF-α in hens was reduced if probiotics were included in the diet under heat stress [35]. As this study was conducted during winter, none of the hens were under heat stress. In addition, our results also show that not all layers were experiencing oxidative stress. also demonstrated beneficial effects of dietary C. butyricum supplementation on the laying performance of hens [11,30]. However, there are very limited studies examining the effects of S. boulardii on laying hens. Conversely, several studies have shown that the addition of butyric acid bacteria to the diet has no significant effect on improving the performance of agricultural animals [31].
In the present study, diets supplemented with C. butyricum or a combination of S. boulardii and P. acidilactici were examined. Diets supplemented with C. butyricum reduced ADFI and increased feed conversion, which is similar to the results in broilers in Mervat et al. [32]. However, the combination of probiotics had no effect on laying performance. Compared with other studies, the scale of the animal experiments performed here was lager, lending confidence in our conclusion that C. butyricum exerts positive effects on laying performance in hens. The significant increase in the crude protein content of albumen means the egg quality of C. butyricum-treated laying hen was improved.
Antioxidative stress is one of the important factors affecting the performance and egg quality of laying hens. Reactive oxygen species are widely accepted to be detrimental for health [33,34]. Studies have indicated that dietary probiotics are effective in counteracting the adverse influences of antioxidative stress [35], and promoting the activities of antioxidant enzymes [36]. Here, it was observed that C. butyricum significantly decreased the levels of ROS in both the ileum and cecum. Although the major antioxidant enzymes (T-SOD and GSH-PX) were apparently unaffected by probiotic supplementation, the levels of MDA, a cellular endproduct of lipid peroxidation and an important indicator of stress [37], were significantly decreased. These results may indicate that C. butyricum is capable of reducing oxidative stress in the intestines of the laying hens by reducing the degree of intestinal lipid oxidation as opposed to by accelerating the depletion of ROS.
The health status of the mucosa and the microscopic structure can be good indicators of the response of the intestinal tract to active substances in feed [38]. Previous studies have indicated that probiotics may be useful for improving the gut health of laying hens [35] and broiler chickens [16]. Here, it was observed that probiotic supplementation was able to significantly improve the intestinal histological morphology of laying hens (Figure 1 and Table 5). In our study, we found that dietary probiotic supplementation increased the villus height and villus height/crypt depth ratio in both ileum and cecum, which may reflect better epithelial growth after probiotic supplement [39,40].
The levels of pro-inflammatory cytokines in the intestinal tract were within normal ranges ( Figure 2). These results indicate that all hens were in good health, which would explain the lack of difference in the observed mortality (p = 0.3445, Table 2). Deng et al. (2012) demonstrated that the level of serum TNF-α in hens was reduced if probiotics were included in the diet under heat stress [35]. As this study was conducted during winter, none of the hens were under heat stress. In addition, our results also show that not all layers were experiencing oxidative stress.

Conclusions
According to our findings, we concluded that the use of probiotics containing both C. butyricum and a combination of S. boulardii and P. acidilactici in the laying hen diet was beneficial in enhancing the development of the intestine. In particular, dietary C. butyricum has significant beneficial effects