With an increase in large-scale layers, especially under conditions of high-density rearing, aged layers are susceptible to various stress factors that can lead to several problems such as imbalance in intestinal microbiota, reduction in antioxidant capacity (Liu et al., 2018), increase in immune inflammatory responses (Attia et al., 2020), and a decline in performance (Bar et al., 1999), egg quality (Bar et al., 1999), and reproductive efficiency (Liu et al., 2018). These changes lead to economic losses to the poultry industry. Although antibiotics are often used to control and treat pathogenic bacterial infections, such as those caused by pathogenic Escherichia coli, Clostridium perfringens, and Salmonella in aged hens, their use has been gradually banned during the laying period in many countries due to egg safety, antibiotic resistance, and environmental pollution issues (Wang et al., 2015). Thus, it is important and interesting to maintain gut health, enhance antioxidant activity, and delay senescence of aged hens through nutritional strategies, thus improving their laying performance and egg quality, and extending their lifespan.

Yeast culture (YC) is a type of microecological product produced under specific conditions of yeast fermentation. It contains yeast and various metabolites. YC is rich in proteins, small peptides, oligosaccharides, vitamins, minerals, enzymes and a variety of unknown growth factors which can provide abundant nutrition for gut microbes, stimulate the proliferation of beneficial bacteria and inhibit the growth of harmful bacteria (Liu et al., 2019). Several experiments have suggested that YC has a positive effect on layers and broilers. For example, Zhang J.C. et al. (2020) reported that YC addition at 3.0 g/kg of feed improves the performance of aged layers by upregulating intestinal digestive enzyme activity and intestinal health-related gene expression. Another study in broilers showed that dietary YC supplementation at 0.1 g/kg increases daily weight gain and reduces Campylobacter in the cecum (Froebel et al., 2019). In addition, yeast bioactive substances can alleviate the negative effects of Eimeria infection on the growth of poultry, improving the structure of the jejunum mucosa and increasing the content of IgA in the egg yolk (Lu et al., 2019). The addition of YC at 2.5 g/kg activates macrophages, thereby increasing lysozyme content in the serum of laying hens, thus contributing to their resistance of pathogenic bacteria and enhancing their immunity (Gao et al., 2008). Further, studies have also reported that this immunity induced by YC is passed on to offspring and reduces symptoms of coccidiosis infection (Lu et al., 2019; Sun and Kim, 2019). In addition, some previous studies have shown that YC administration positively affects poultry laying performance and intestinal health (Zhang J.C. et al., 2020). We suspect that this is because of improvements within the intestinal microbiota community, which then increase the proportion of beneficial microorganisms and inhibit the reproduction of harmful microorganisms. However, at present, there is insufficient research on the influence of YC supplementation on the productive and reproductive performance of aged layers, and it is unclear whether YC affects these parameters by regulating the layers’ intestinal microbiome. Therefore, this study aimed to explore whether YC supplementation can improve laying performance, egg quality, and reproductive performance of breeder-aged layers by improving the intestinal microbial flora structure.

Yeast culture did not significantly affect the laying performance of layers, a result that was not in agreement with some previous studies. Zhang J.C. et al. (2020) reported that adding 3.0 g/kg YC to the feed of 67-week-old hens can improve their egg-laying rate and the total egg weight, and reduce the feed/egg ratio. Lu et al. (2019) found that the addition of YC alleviated laying performance loss and intestinal damage caused by Eimeria in broiler breeders. The variable results of the present study could be attributed to the good health of the hens used in this experiment. Additionally, some reports have indicated that differences may be due to the changes in the treatment of YC, especially factors such as administration time and period, diet, and environment (Gadde et al., 2017).

In this study, YC was found to significantly increase eggshell strength, egg albumen height and Haugh unit. However, it significantly reduced the b^∗ value of egg shell color. In line with our findings, several previous studies have demonstrated that adding yeast or yeast extract can significantly improve egg weight, albumen height, Haugh unit, eggshell strength, and nutrient content (Zhong et al., 2016; Özsoy et al., 2018; Gaboardi et al., 2019); however, other studies have shown that dietary YC supplementation has no significant effect on eggshell strength, egg weight, albumen height, Haugh unit, and yolk color (Yalçin et al., 2008; Zhang J.C. et al., 2020). The positive effect of YC on egg quality may be associated with the presence of bioactive substances such as enzymes, vitamins, amino acids, polypeptides, and oligosaccharides in it (Jensen et al., 2008). Moreover, the presence of many carotenoids in YC, which promote pigment deposition in the egg yolk, may also be a factor in its effectiveness (Kot et al., 2019). As our results indicate improvements in egg quality, the increased hatching rate, the improved fertilization rate, and the healthy chick rate can also be associated with the egg quality and the increased eggshell strength and density, which prevent pathogenic microorganisms from entering the eggs. Moreover, YC also contains a large amount of trace elements, such as selenium and zinc that improve embryonic and early postnatal development of the aged layer (Hostetler et al., 2003; Sun et al., 2012). A previous study has shown that adding yeast bioactives significantly improves the level of IgA in the egg yolk (Lu et al., 2019), resulting in enhanced chick immunity and improved reproductive performance of the aged layers.

Hens in the late laying stage are considered to be affected by oxidative stress and ovarian aging; furthermore, an increase in lipid and protein oxidation substantially influences the normal physiology of layers (Liu et al., 2018). To counter such oxidation, T-AOC is an important integrative index that reflects antioxidant capacity (Zhang S. et al., 2020). Our results indicated that YC supplementation significantly increased lysozyme and T-AOC in serum and reduced serum AST. Similarly, a previous study showed that supplemented yeast hydrolysates tended to increase serum T-AOC (Fu et al., 2019). Thus, improvements in serum T-AOC and lysozyme activity indicated that YC administration can improve antioxidant capacity and enhance innate immunity (Min et al., 2015) of layers. AST normally exists in liver cells, and plasma AST and alanine aminotransferase contents are the most sensitive indicators of liver injury in poultry (Lumeij, 1997; Yousefi et al., 2005). Under normal circumstances, the upper limit of AST content in poultry plasma is 230 IU/L (Jones, 1999). In our study, AST content in both groups was within the normal range; however, it showed a trend of significant decrease after YC addition, which is a positive signal for liver function.

Feed digestibility and absorption gradually decreased as the age of layer hens increased (Duan et al., 2015). Thus, improving feed digestibility and availability through nutritional strategies is important to maintain constant egg production, egg quality, and health in aged hens. The results of this study showed that YC supplementation significantly improved the digestibility of crude fat, but its effect on the digestibility of energy or crude protein was of little significance. The digestion, absorption, and utilization of fat largely depend on trypsin digestive enzymes and emulsifiers (Aloulou et al., 2015). Additionally, studies have shown that the addition of YC can significantly increase the activity of duodenal enzymes in aged layers. Moreover, YC is known to contain enzymes and organic acids. Thus, our results suggest that increased fat digestion and absorption is possibly related to stimulating endogenous enzyme secretion or enzymes contained in YC (Dawood et al., 2020; Zhang J.C. et al., 2020). Intestinal microorganisms can produce bioactive substances and substantially influence health, nutrition, and immunity (Hooper et al., 2002; Ley et al., 2008; Xu M. et al., 2020). Our study showed that adding YC could alter the ileum microbial community structure in aged layers. The results of this study showed that Firmicutes, Bacteroides, and Proteobacteria were the dominant microbial species in the ileum, which was confirmed by other studies (Wang et al., 2018; Spring et al., 2020). After YC supplementation, the relative abundance of Firmicutes decreased (from 90.76 to 77.64%) while that of Proteobacteria (from 5.92 to 17.08%) and Bacteroidetes (from 2.31 to 3.94%) increased. The proportion of Firmicutes/Bacteroides (F/B) also increased (from 100.12 to 332.29). Studies have shown that the F/B ratio is related to inflammatory bowel disease (Frank et al., 2007), obesity (Turnbaugh et al., 2008), and type 2 diabetes mellitus (Remely et al., 2014). In this study, the decrease in Firmicutes and the increased F/B values indicated that YC might have a regulating effect on lipid metabolism and gut health and also reduce fat deposition in aged layers.

All the results of this study indicated that Lactobacilli were the dominant genus in the ileum, accompanied by a significant decrease in Romboutsia in the YC group. A study by Li et al. (2018) found that in day-old sika deer, Halomonas (48.9%), Lactobacilli (21.4%), and Escherichia Shigella (19.2%) were the dominant genera in the jejunum and ileum; with increasing age, the abundance of Lactobacilli began to reduce gradually from 21.4 to 6.0%. Romboutsia appeared and gradually became the dominant species with a relative abundance of 22.9%. Since our results suggest higher abundance of Lactobacilli in the intestinal tract after YC supplementation, it may be a good indicator of aged layers as Lactobacilli are involved in metabolic activity, such as decomposing proteins and sugars in food, synthesizing vitamins and promoting the fermentation and degradation of fat (Wu et al., 2015). Moreover, carbohydrates can be converted by Lactobacilli into lactic acid for further use by other bacteria (Wu et al., 2018). Lactobacilli have been shown to be negatively correlated with lipid metabolism. They accelerate the synthesis of lipid peroxidation metabolites and are positively related to intestinal health (Chen et al., 2020). Some experiments related to the addition of Lactobacilli also revealed that they have the potential to improve the performance, digestion, and feed utilization efficiency of broilers and laying hens (Saleh et al., 2017, 2020). Romboutsia can metabolize carbohydrates, synthesize amino acids and vitamins, and are sensitive to bile acids (Gerritsen et al., 2017). Some studies suggest that Romboutsia may be linked to obesity (Hu et al., 2019) and liver injury (Yu et al., 2020). Furthermore, a large number of studies have directly or indirectly proved that Romboutsia and Lactobacilli show opposing trends in relative abundance (Chen et al., 2019; Lee et al., 2019; Qiao et al., 2019; Xu S. et al., 2020). Based on these results, we can speculate that these two bacteria have especially strong competitive exclusion effects. The increased abundance of Lactobacilli following a decrease in Romboutsia may be due to polysaccharides in YC being used by Lactobacilli to produce a large number of short-chain fatty acids and lactic acid, which reduce intestinal pH. Another reason may be the increased shedding of Romboutsia due to the occupied effect of Lactobacilli (Baldwin et al., 2018; Qiao et al., 2018; Bunte et al., 2020).

Gut microbes can convert indigestible glycans into short-chain fatty acids such as butyrate, propionate, and acetate as nutrients for hindgut intestinal epithelial cells (Koropatkin et al., 2012; Sun et al., 2016). YC contains a large amount of glucan and mannooligosaccharides, which can induce the upregulation of glycan biosynthesis and metabolism pathways in microorganisms. Glutathione metabolism is generally considered to be related to the promotion of cell redox balance. It has antioxidant and detoxifying effects and provides a protective response (Xu et al., 2015). Glutathione-S-transferase and glutathione peroxidase are two important enzymes involved in glutathione metabolism (Schneider et al., 2016). In this study, we found that serum T-AOC content in the YC group increased significantly, which may be associated with improvement in intestinal microbial glutathione metabolic function. Ubiquinone and other terpenoid-quinone usually refer to hydrocarbon or terpenoid derivatives, and their oligomers, such as coenzyme Q10, squalene, farnesol, vitamin A, E, and K, are necessary for life activities. The upregulation of ubiquinone and other terpenoid-quinone biosynthesis pathways in YC-treated hens may be related to improvement in laying performance. Lipopolysaccharides, also known as endotoxins, are not only the main component of the outer membrane of Gram-negative bacteria but are also the main cause of inflammation and part of the natural immune response of animals. The upregulation of the lipopolysaccharide biosynthesis protein pathway after YC supplementation may be associated with an increase in lysozyme activity in the serum. Furthermore, we found that the sporulation pathway was significantly downregulated by YC. Spores are formed by bacteria in a near-dormant state. They can store the bacteria’s hereditary material in an unsuitable and harmful environment (Huang and Hull, 2017; Bressuire-Isoard et al., 2018). Metabolism in the spore state is 10 million times slower than that in normally growing bacteria. YC supplementation may contribute toward improving the original harsh ecological environment within which microorganisms live, along with increasing the vitality of bacterial communities and strengthening network relationships. ABC transporters use the energy generated through ATP binding and hydrolysis to transport various substrates on the cell membrane. They have transport and excretion roles in prokaryotes and eukaryotes, and can remove toxins and drugs from cells. Research has shown that the ABC transporter system plays a very important role in the adaptation of Escherichia coli to unsuitable environments (Moussatova et al., 2008; Cario, 2017). Hence, the upregulation of the ABC transporter pathway in the DC group in our study could be due to the poor ecological environment, which need to excrete a large amount of toxic substrates.

Interestingly, we found that YC supplementation substantially improved the relationships among bacteria in the ileum. An increase in Lactobacilli abundance in the YC group led to more complex interactions among the bacteria. Such a complex network has been suggested to increase resistance against pathogen invasions because the pathogens would have to adapt to the external environment and compete for the original ecological niche with the existing bacteria (Wei et al., 2015; Mendes et al., 2018). As the DC group had very low bacterial connectivity, and based on its function prediction, we speculated that many bacteria may be in the resting state, and the loss of one niche will substantially influence the whole microbial community. On the contrary, the YC group had very high bacterial connectivity; even when it suffered from a pathogen invasion or was missing one niche, the neighboring niches supplemented any gaps. Moreover, connectivity among bacteria limits the nutrient supply for any invasive microorganisms, causing their extinction. Therefore, a complex network of microbes shows stronger resistance to external influences.

In addition, we found that within networks of intestinal microbial relationships, bacteria that were in high abundance showed a lower degree of correlation. Furthermore, the number of positive correlation was significantly more than negative correlation in the whole network relationship. Interestingly, common probiotics such as Lactobacilli, Enterococcus, and Acetobacter were mostly found to be negatively correlated with other bacteria, which may be because they produce bacteriocin to inhibit the growth and reproduction of other bacteria (Corr et al., 2009). Lactobacilli enhance the degree of correlation between bacteria and improve the consistency of bacterial flora. An increase in Lactobacilli abundance after YC administration was accompanied by a more complex correlation between bacterial communities. Lactobacilli could thus improve the intestinal microenvironment of layers and inhibit the growth of some pathogenic bacteria. Therefore, YC supplementation can result in good health of layers.

In conclusion, this study suggested that adding 2.0 g/kg YC to the diet of breeder-aged layers can improve their egg quality and reproductive efficiency. It may also be beneficial for their antioxidant capacity and systemic immunity. Moreover, it can improve the structure of aged layers’ intestinal microbial flora by increasing the abundance of Lactobacilli and the consistency of bacterial flora, resulting in improved feed digestion, absorption, and gut health.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, PRJNA675783.

The animal study was reviewed and approved by principle of the Animal care and Use Committee of China Agricultural University.

YL and XC performed the study, contributed to most of the experiments, and wrote the manuscript. WZ and DZ provided calculation and operation support. ZW, ZN, and LQ designed the study, evaluated the test details, and revised the manuscript. All authors contributed to this study and approved to submit the final manuscript.

DZ was employed by company Huayu Agricultural Science and Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.