Effects of Different Fermented Feeds on Production Performance, Cecal Microorganisms, and Intestinal Immunity of Laying Hens

Simple Summary Fermented feed exerts beneficial effects on intestinal microorganisms, host health, and production performance. However, the effect of fermented feed on laying hens is uncertain due to the different types of inoculated probiotics, fermentation substrates, and fermentation technology. Hence, this experiment was conducted to investigate the effects of fermented feed with different compound strains on the performance and intestinal health of laying hens. Supplement fermented feed reduced the feed conversion ratio and promoted egg quality. Both dietary treatment (fermented feed A produced Bacillus subtilis, Lactobacillus, and Yeast and fermented feed B produced by C. butyricum and L. salivarius) influenced intestinal immunity and regulated cecal microbial structure. This may be because the metabolites of microorganisms in fermented feed and the reduced pH value inhibited the colonization of harmful bacteria, improved the intestinal morphology, and then had a positive impact on the production performance and albumen quality of laying hens. Abstract This experiment was conducted to investigate the effects of different compound probiotics on the performance, cecal microflora, and intestinal immunity of laying hens. A total of 270 Jing Fen No.6 (22-week-old) were randomly divided into 3 groups: basal diet (CON); basal diet supplemented with 6% fermented feed A by Bacillus subtilis, Lactobacillus, and Yeast (FA); and with 6% fermented feed B by C. butyricum and L. salivarius (FB). Phytic acid, trypsin inhibitor, β-glucan concentrations, and pH value in fermented feed were lower than the CON group (p < 0.05). The feed conversion ratio (FCR) in the experimental groups was decreased, while albumen height and Haugh unit were increased, compared with the CON group (p < 0.05). Fermented feed could upregulate the expression of the signal pathway (TLR4/MyD88/NF-κB) to inhibit mRNA expression of pro-inflammatory cytokines (p < 0.05). Fermented feed promoted the level of Romboutsia (in the FA group) Butyricicoccus (in the FB group), and other beneficial bacteria, and reduced opportunistic pathogens, such as Enterocooccus (p < 0.05). Spearman’s correlations showed that the above bacteria were closely related to albumen height and intestinal immunity. In summary, fermented feed can decrease the feed conversion ratio, and improve the performance and intestinal immunity of laying hens, which may be related to the improvement of the cecal microflora structure.


Introduction
Corn and soybean meal (SBM) have been widely applied to animal feed because of their high nutritional value and organic matter digestibility. However, various antinutritional factors (ANFs) in feed, such as phytic acid and trypsin inhibitor (TI), decrease the digestibility and absorption of animals [1,2]. These ANFs may compromise the nutritional value, utilization, and digestibility of feed materials [3]; cause animal digestive supplemented into mixed diet, which poured dilute bacteria for a 30% moisture content. Thirdly, the mixed diet was put into a sealed bag, and the air was exhausted. Anaerobic fermentation was allowed to occur at 37 • C for 5 days. Finally, 6% FF was added to the basal diet to obtain the experimental diet. The FF was prepared once a week and adjusted according to the feed intake of layers. Feed samples were analyzed to establish the contents of routine nutrients and pH value according to AOAC International guidelines. Phytic acid, TI, and β-glucan in feed were determined using a commercial kit (Jianglai Bio Company, Shanghai, China).

Experimental Design
A total of 270 Jing Fen No.6 laying hens (22-weeks-old) with a similar body weight and good health were randomly divided into 3 groups with 6 replicates in each group and 15 hens in each replicate. Three dietary treatments were as follows: basal diet (CON), basal diet supplemented with 6% compound bacteria I FF (FA), and basal diet supplemented with 6% compound bacteria II FF (FB). The experimental period was 8 weeks. According to the feeding standard of NRC (1994) and the nutritional level recommended by Hailan Company, the corn-SBM-based diet was designed (shown in Table 1).

Intestinal Morphology
At the end of the experiment, about 2-3 cm as taken from the duodenum, jejunum, and middle ileum; the chyme was rinsed with a syringe containing 0.9% normal saline; and then was fixed in a 10 mL centrifuge tube with 4% paraformaldehyde solution. Then, 5-mm-thick intestinal tissue was selected to make sections and stored at room temperature. The fixed segments were sectioned and observed under the light microscope, and the villus height (VH) and crypt depth (CD) of each segment were measured.

RNA Extraction and Polymerase Chain Reaction (PCR) Amplification
Total RNA was extracted from each jejunal mucosa sample using a CWBIO ® Ultrapure RNA Kit according to the manufacturer's instructions. The total RNA concentration was detected by a microplate reader (Bio-Tek, Winooski, VT, USA), and 1% of agarose gel electrophoresis was used to determine RNA purity. RNA samples were diluted to 1 ng/µL and subsequently reverse transcribed into cDNA. Diluted cDNA was used to measure the expression levels of ZO-1, Occludin, and Mucin-2, TNF-α, IL-8, TLR 4, and related signal proteins MyD88 and NF-κB. Primers used for quantitative real-time PCR are shown in Table 2.

16S rDNA Gene Sequencing and Bioinformatics Analysis
Genomic DNA of the cecal content was extracted by the CTAB method. Agarose gel electrophoresis was used to detect the purity and concentration of DNA. An appropriate sample of DNA was applied to the centrifuge tube and diluted with sterile water to 1 ng/µL. Diluted genomic DNA as template and phusion ® High fidelity PCR master mix with GC buffer (New England Biolabs) was used for PCR amplification. PCR products were detected by electrophoresis with 2% agarose gel. A Truseq ® DNA PCR-Free Sample Preparation Kit provided by Illumina was used to construct the gene library. After Qubit and Q-PCR quantitation, the qualified library was sequenced by novaseq6000. The samples were clustered into operational taxonomic units (OTUs) using Uparse software (Uparse v7.0.1001, http://www.drive5.com/uparse/, accessed on 29 August 2020), and the representative principle of OTUs was selected according to the algorithm principle. The Chao1, Shannon, and ACE index were calculated with Qiime software (version 1.9.1) to analyze the microbial Animals 2021, 11, 2799 5 of 16 alpha diversity. Using the UniFrac phylogenetic distance, the bacterial diversity was assessed to detect the structural changes of microbial communities among the treatment groups, and principal coordinate analysis (PCoA) was used for visualization.

Statistical Analysis
In order to further search for the species of different groups under different classification levels (phylum, family, genus), and to perform the T-test among the groups, species with significant differences were found. The Spearman correlation coefficient was used to evaluate the changes in the production performance, intestinal immunity, and microbiota. Data of laying hens were analyzed by one-way ANOVA and Duncan's multiple range test using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). Results are presented as means with standard error of the mean (SEM). p-value of less than 0.05 was considered statistically significant.

Fermented Feed Characteristics
As shown in Table 3, the content of crude protein (CP) in FA and FB was 15.08% and 13.09% higher than CON, respectively (p < 0.05). Crude fiber (CF) and pH value also decreased significantly in FF (p < 0.05). Moreover, the concentration of TI in FA and FB decreased by 2.48% and 11.66%, respectively, during fermentation (p < 0.05). Furthermore, the content of β-glucan in FA and FB was 202.38 and 183.33 pg/mL respectively, which was significantly different from 207.52 pg/mL in the CON group (p < 0.05). After fermentation, the number of viable bacteria in the FA group was 1.74 × 10 7 CFU/g for B. subtilis, 2.33 × 10 5 CFU/g for Yeast, and 1.62 × 10 7 CFU/g for Lactobacillus, respectively, while that in the FB group was 1.32 × 10 7 CFU/g for C. butyricum and 1.89 × 10 7 CFU/g for L. salivarius, respectively.

Laying Performance and Egg Quality
The laying performance and egg quality are summarized in Table 4. Although no distinct difference was found for the ADFI, laying rate, yolk color, eggshell thickness, and strength, FF dramatically reduced the FCR (p < 0.05), compared with that in the CON group. Besides, dietary FF treatment increased the laying rate by 3%. In terms of egg quality, adding 6% of FF presented an upward trend in albumen height and Haugh unit (p < 0.05).

Effects of FF on the Intestinal Immune-Related Genes of Layers
The results shown in Table 5 reveal that no noteworthy effect was observed on the concentration of ZO-1 (p > 0.05). However, the level of Mucin-2 in the FB group was extremely upregulated, compared with the CON group and FA (p < 0.05). Furthermore, compound bacteria FF made the concentration of Occludin higher than the CON group (p < 0.05), and no notable differences were found between FA and FB (p > 0.05). The data indicated that FF can decrease the content of pro-inflammatory genes IL-8 and TNF-α (p < 0.05). Supplementation of 6% FF significantly upregulated the content of TLR4 and MyD88 (p < 0.05), while striking promoted the NF-κB gene expression level (p < 0.05). Furthermore, the effects of FB were superior to FA.

Intestinal Morphology
In order to quantitatively evaluate intestinal morphology, the commonly used indexes for measuring the intestinal histology of hens were determined, as shown in Table 6, including villus height (VH), crypt depth (CD), and their ratio (VH/CD). From the table, FA and FB significantly improved the integrity of intestinal mucosa. Compared with the control group, the VH of the duodenum and the VH/CD ratio significantly increased (p < 0.05). Besides, probiotics treatment promoted the VH of the jejunum, while FF greatly decreased the crypt depth so that made the VH/CD increase significantly (p < 0.05).

Effects of FF on Cecal Microbial Composition
The community of gut diversity showed an upward trend (p = 0.068) in contrast with that in the control group. The increase of the Chao1 index and ACE index indicated the gut community richness significantly improved in the FB group (p < 0.05), compared with that of the FA and CON group. Additionally, as shown in Table 7, there were no distinct differences between the FA and CON group. In order to further analyze the differences of the cecal microbial community structure between the control group and the treatment group, the PCoA method was used to analyze the cecal microbial community structure according to weight UniFrac. As presented in Figure 1, FB is mainly concentrated in the first quadrant, which was significantly different from FA and CON. From what has been discussed above, compound probiotics II FF could significantly improve the species and evenness of cecal microorganisms in layers. lyze the cecal microbial community structure according to weight UniFrac. As pre in Figure 1, FB is mainly concentrated in the first quadrant, which was significant ferent from FA and CON. From what has been discussed above, compound probi FF could significantly improve the species and evenness of cecal microorganisms ers. The results of sequencing revealed that the dominant bacteria in the layers' c of the three groups were Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, an ryarchaeota (Table 8). Compared with the CON group, the RA of Bacteroidetes was s cantly increased in both experimental groups (p < 0.05), and the RA of Proteobacteria t to decrease in the experimental group (P = 0.07).  The results of sequencing revealed that the dominant bacteria in the layers' caecum of the three groups were Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, and Euryarchaeota (Table 8). Compared with the CON group, the RA of Bacteroidetes was significantly increased in both experimental groups (p < 0.05), and the RA of Proteobacteria tended to decrease in the experimental group (p = 0.07). At the genus level (Figure 2a,b), fermented feed significantly influenced the intestinal microflora of laying hens. The RA of Romboutsia (in the FA group), Butyricicoccus (in the FB group), and other beneficial bacteria for intestinal health was significantly higher than that in the CON group (p < 0.05). The relative abundance of opportunistic pathogens, such as, Alistipes, Enterococcus, and Alloprevotella, decreased significantly in contrast with the CON group (p < 0.05).

Correlation of Laying Performance, Gut-Associated Gene Expression, and Gut Bacteria
Spearman correlation analysis was applied to investigate the relationship among laying performance, expression of gut-associated genes, and gut bacteria (Figure 3). The abundance of Bacteroides was positively corrected with albumen height and strongly negatively corrected with TNF-α. As to the gut-associated gene expression, Alistipes was negatively correlated with Mucin-2, TLR 4, and MyD88, and positively correlated with TNF-α and IL-8 (p < 0.05). a,b Means within each row with different superscripts are statistically significantly different (p < 0.05). 1 CON, control group; FA, basal diet with 6% FF produced by B. subtilis, Lactobacillus, Yeast; FB, basal diet with 6% FF produced by C. butyricum, L. salivarius.
At the genus level (Figure 2a,b), fermented feed significantly influenced the intestinal microflora of laying hens. The RA of Romboutsia (in the FA group), Butyricicoccus (in the FB group), and other beneficial bacteria for intestinal health was significantly higher than that in the CON group (p < 0.05). The relative abundance of opportunistic pathogens, such as, Alistipes, Enterococcus, and Alloprevotella, decreased significantly in contrast with the CON group (p < 0.05).

Correlation of Laying Performance, Gut-Associated Gene Expression, and Gut Bacteria
Spearman correlation analysis was applied to investigate the relationship among laying performance, expression of gut-associated genes, and gut bacteria (Figure 3). The abundance of Bacteroides was positively corrected with albumen height and strongly negatively corrected with TNF-α. As to the gut-associated gene expression, Alistipes was negatively correlated with Mucin-2, TLR 4, and MyD88, and positively correlated with TNFα and IL-8 (p < 0.05).

Discussion
The improvement of the chemical composition and nutritional value of poultry feed by fermentation has been widely reported. This feed processing technology aids the digestion and absorption of nutrients by the host and thereby improves animal growth performance [30]. The crude protein content, pH value, and living bacteria number are im-

Discussion
The improvement of the chemical composition and nutritional value of poultry feed by fermentation has been widely reported. This feed processing technology aids the digestion and absorption of nutrients by the host and thereby improves animal growth performance [30]. The crude protein content, pH value, and living bacteria number are important indexes to evaluate the nutritional value of FF [31]. Our results showed that fermentation with probiotics could significantly increase the CP content in the substrate. This may be because the loss of dry matter, especially carbohydrates, in fermented feed increases the relative concentration of other nutrients. The increase of CP may also be due to the hydrolysis of macromolecular proteins, especially antigen proteins [32]. At the same time, a large amount of probiotics in fermented feed could produce positive effects on the host, including inhibiting the proliferation of pathogenic microorganisms [33] and improving the intestinal barrier function [34]. A previous study demonstrated that microbial fermentation of SBM efficiently eliminates ANFs and enhances the nutrient value [35,36], which was consistent with our results. It was observed that the contents of crude fiber, phytic acid, and β-glucan were reduced following fermentation with compound probiotics. This may be due to the production of related enzymes, such as cellulase and phytase, which degraded these anti-nutrient substrates. Therefore, the elevated CP content, lower pH value, and reduced anti-nutrient factors in response to fermentation would be beneficial to the gut health and production performance of laying hens.
In pig production, results showed that FF can improve ADFI, FCR, and production performance [11,[37][38][39]. However, to date, the results in layers and broilers are limited and inconsistent. The data obtained in the present study demonstrated that adding compound probiotics to fermented feed significantly increased albumen height and Haugh unit and decreased FCR of layers, compared with the control group, but did not influence ADFI and other relevant indicators during the test stage, similar to Li [40]. Adding fermented SBM produced by B. subtilis could significantly improve the daily gain of broilers [41]. However, it was reported that probiotics had little effect on the growth performance of broilers [42]. This inconsistency may be related to the species of probiotics, preparation technology, dosage, feed composition, and bird age and health status. In addition, it has been reported that the combination of probiotics can exhibit synergistic effects [43]. The improvement of the growth performance and feed efficiency of layers by adding compound probiotics was considered to be caused by the cumulative effect of probiotics, such as C. butyricum, which can promote the growth of beneficial lactic acid-producing bacteria [44].
Albumen height (AH) and Haugh unit (HU) are important indexes of egg quality. Haugh unit is mainly determined by the content of thick protein [45], and the value represents the viscosity of egg protein: the higher the protein viscosity, the longer the egg stays fresh. AH and HU, as a measure of egg quality, may be related to protein synthesis and water transfer in yolk [46]. Feed with 1 × 10 8 CFU/kg B. subtilis significantly increased AH, HU, and eggshell thickness [47], which was consistent with our results. The changes of AH and HU were influenced by the electrolyte balance of birds, suggesting that the increase of AH was related to the increase of the divalent cation concentration [48]. These may be attributed to the addition of C. butyricum, L. salivarius, and Lactobacillus to create a favorable microbial intestinal environment in birds, which decreased the lumen pH and enhanced the solubility and absorption of cations [49]. Probiotics reduced the pH value in the gut, which helps dissolve more Ca and P, and has a positive impact on Ca deposition into the eggshell. In addition, the increased CP content in fermented feed may be another factor responsible for the elevation of the albumen height and Haugh unit.
Gramicidin produced by B. subtilis and the stimulation of the maturation and differentiation of dendritic cells of L. salivarius stimulated immune regulation in a non-inflammatory way [50], maintained metabolic homeostasis, and ultimately regulated host performance. The jejunal-associated immune gene expression reflected the physiological and immune status of laying hens. Cytokines were divided into pro-inflammatory cytokines and antiinflammatory cytokines. In this study, the expression of MyD88, NF-κB, and TLR-4 was significantly upregulated and the content of IL-8 and TNF-α were dramatically downregulated in the FF groups, in contrast with those in the CON group, suggesting that FF can regulate the immune function of laying hens. It was reported that the expression of pro-inflammatory genes was downregulated in response to fermented diets [51,52]. Similar results were found in this study. Tight junctions (TJS) are one of the ways in which cells connect to the endothelium, which plays a key role in the integrity of the barrier and the permeability of the endothelium [53]. TJS are composed of Occludin, Mucin-2, and ZO-1, which maintain the stability of intercellular junctions [54]. Our results showed that the expression of Occludin and Mucin-2 was significantly increased by FF. Similar to Al-Sadi et al. [55], they reported that most proinflammatory factors, such as IL-1β and TNF-α, can lead to destruction of the epithelial tight junction barrier. Pan et al. [56] reported that downregulation of inflammatory gene expression (IL-1, IL-6, IL-8, and TNF-α) was negatively correlated with the expression genes (Occludin, ZO-1, and Mucin-2) involved in tight junction protein biosynthesis. The TLR signaling pathway can be divided into two types: MyD88-dependent pathway and MyD88-independent pathway [57]. In the MyD88-independent signaling pathway, TRIF can only be recruited by TLR-3 and TLR-4 to activate the NF-κB signaling pathway [58]. The concentration of NF-κB, MyD88, and TLR-4 has a striking upward trend, indicating high expression of the classical pathway (NF-κB/MyD88/TLR-4) inhibited the expression of proinflammatory factors, so as to promote expression of tight junction protein and maintain intestinal health.
Previous evidence suggested that there is a close relationship between the growth performance and intestinal flora of different species of animals Erratum in [37] and [59]. VH, CD, and VH/CD are crucial in the intestinal health of animals, which are directly related to the absorption capacity of intestinal mucosa. Studies have reported that morphological changes of the small intestine, such as increasing VH and decreasing CD, enhanced gut digestion and absorption [60,61], which was also found in the current study. Our results showed that FF increased VH of each intestinal segment, decreased jejunal CD, and increased the VH/CD ratio. The main reason might be that the combination of probiotics reduced the relative abundance of Enterococcus and other pathogens at the genus level, which may injure intestinal mucosal epithelial cells (IECs). Another reason may be that butyric acid, the main metabolite of C. butyricum, can nourish intestinal goblet cells and repair and protect IECs [17] in the FB group.
The composition of intestinal microorganisms could be modulated by dietary composition [62]. The mechanism of probiotics for improving the production performance and egg quality of hens may include the change of intestinal microbiome, partly by inhibiting the proliferation of pathogens and promoting the growth of non-pathogenic facultative anaerobes and Gram-positive bacteria producing lactic acid [63], and promoting the digestion and utilization of nutrients. Intestinal flora assumed enormous importance in nutrition, physiology, and mucosal morphology [64]. In the present study, ACE and Chao1 richness estimators were significantly increased in FB layers, indicating higher species richness in the cecal digestive tract. Intestinal microorganisms with a higher abundance and quantity are more favorable for animals to cope with different environmental disturbances [65]. Beta diversity was used to further estimate the changes in cecal microbiota with FF addition. The average distance between the test groups showed that different treatments had significant differences in the intestinal bacterial community of laying hens. This evidence showed that after adding C. butyricum and L. salivarius to the fermented feed, the intestinal microorganisms of laying hens gradually reached a stable state and dynamic equilibrium. However, there was no significant distinction between the FA and CON groups, which need to be investigated in a future study, and further analysis was conducted on alterations of the microbiota composition following FF addition.
At the phylum level, dominant bacteria in the caecum of all groups included Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, and Euryarchaeota. In addition, feeding FF significantly increased RA of Bacteroidetes, and decreased RA of Proteobacteria at the phylum level, which was similar to that of Li et al. [40]. Several gut bacteria can utilize complex polysaccharides as a source of carbon and energy, and in this process, end products released by these bacteria also provided nutrition and other characteristics that were beneficial to the host [66]. Therefore, we speculated that the presence of a large number of Bacteroides in layers fed with FF may contribute to the adaptation of these components, so as to improve the digestibility of nutrients and improve the production performance. In this study, the RA of Proteobacteria in the two experimental groups was much lower than that in the CON group, which was consistent with previous studies [67]. Studies have shown that undigested dietary protein in the gut promotes reproduction of some harmful bacteria [68].Therefore, we conjectured that the low abundance of Proteobacteria might be related to the increased CP digestibility of fermented feed. Meanwhile, Lactobacillus in FF reduced the intestinal pH value by producing organic acids, and prevented the colonization of intestinal pathogens through competitive rejection, antagonistic activity, and bacteriocin production, which also helps to reduce the abundance of Proteobacteria [64].
Results of the T-test showed that cecal microorganisms could be modified in a different manner by these two kinds of FF at the genus level. The RA of Romboutsia was increased in the FA group, which was reported to be involved in carbohydrate utilization, single amino acid fermentation, anaerobic respiration, and metabolic end products. The RA of Alistipes was decreased in the FA group, a bacterial genus closely related to intestinal diseases. It has been isolated from appendicular and brain abscesses, highlighting their potential opportunistic role [69]. Supplementation of C. butyricum and L. salivarius resulted in an increasing trend of some beneficial bacteria, such as Butyricicoccus, which can balance the structure of mucosal flora, produce butyric acid with anti-inflammatory properties, and strengthen the intestinal epithelial barrier, thus favoring intestinal health [70]. The reduced RA of Alloprevotella and Gallibactergiella and other conditional pathogens would alleviate local or systemic inflammation, contributing to the improved growth performance [71].
Correlation analysis revealed the relationship between production performance, gut immune function, and intestinal microorganisms, and clarified the potential roles of intestinal microorganism structure in the production performance and gut immune functions of laying hens. It was worth noting that an increase in Bacteroides abundance was accompanied by the improvements of albumen height, which was also discovered in previous studies [72]. Bacteroides is widely regarded as one of the beneficial genera, which is an effective degraders of nondigestible carbohydrates and SCFA producers, and plays an important role in the maturation of the immune system [73]. Besides, some bacteria, such as Roseburia, Butyricicoccus, Intestinimonas, and Faecalibactium, were reported to participate in regulation of the immune response and their close relationship was also confirmed in our study. In addition, a decreased abundance of Alistipes was accompanied by an enhancement of tight junction protein. Therefore, the intestinal microbiota composition could be modified by FF addition, while the richness of microbiota was only enriched in the FB group, which may contribute to the improved gut health and production performance of laying hens.

Conclusions
In conclusion, fermented feed produced by compound probiotics (FA, B. subtilis, Lactobacillus and Yeast; FB, C. butyricum and L. salivarius) improved the intestinal morphology, epithelial barrier functions, and immune status, thus favoring feed efficiency and albumen quality of laying hens. The greater performance and improved gut health could be in part explained by the enhancement of bacterial richness in the FB group and minor microbiota shifts characterized by the enrichment of Bacteroidetes in both the FA and FB groups. These findings may provide insights into the regulatory roles of fermented feed in laying hens.