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Saccharomyces cerevisiae and Kluyveromyces marxianus yeast co-cultures modulate the ruminal microbiome and metabolite availability to enhance rumen barrier function and growth performance in weaned lambs
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Zixuan Xu, Lan Yang, Hui Chen, Shixiong Liu, Xueqiang Li, Songjian Li, Chun Ying, Xiao Li, Rui Du, Dacheng Liu*
Animal Nutrition | 2024, 19(1) : 139 - 152
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Animal Nutrition | 2024, 19(1): 139-152
• Original Research Article •
Saccharomyces cerevisiae and Kluyveromyces marxianus yeast co-cultures modulate the ruminal microbiome and metabolite availability to enhance rumen barrier function and growth performance in weaned lambs
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Zixuan Xu, Lan Yang, Hui Chen, Shixiong Liu, Xueqiang Li, Songjian Li, Chun Ying, Xiao Li, Rui Du, Dacheng Liu*
Affiliations
  • College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China
Published: 2024-12-10 doi: 10.1016/j.aninu.2024.06.005
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In lambs, weaning imposes stress that can contribute to impaired rumen epithelial barrier functionality and immunological dysregulation. In this study, the effects of a yeast co-culture consisting of Saccharomyces cerevisiae and Kluyveromyces marxianus (NM) on rumen health in lambs was evaluated, with a focus on parameters including growth performance, ruminal fermentation, and epithelial barrier integrity, ruminal metabolic function, and the composition of the ruminal bacteria. In total, 24 lambs were grouped into four groups of six lambs including a control (C) group fed a basal diet, and N, M, and NM groups in which lambs were fed the basal diet respectively supplemented with S. cerevisiae yeast cultures (30 g/d per head), K. marxianus yeast cultures (30 g/d per head), and co-cultures of both yeasts (30 g/d per head), the experiment lasted for 42 d. Subsequent analyses revealed that relative to the C group, the average daily gain (ADG) of lambs in the NM group was significantly greater and exhibited significant increases in a range of mRNA relative expression including monocarboxylate transporter 1 (MCT1), (Na+)/hydrogen (H+) exchanger 1 (NHE1), (Na+)/hydrogen (H+) exchanger 3 (NHE3), proton-coupled amino acid transporter 1 (PAT1), vacuolar H+-ATPase (vH+ ATPase), claudin-1, occludin in the rumen epithelium (P < 0.05). Compared with the C group, the pH of the rumen contents in the NM group was significantly decreased , and the concentrations of acetate, propionate, and butyrate were significantly increased (P < 0.05). Analysis of the rumen bacteria showed that the NM group exhibited increases in the relative abundance of Prevotella, Treponema, Moryella, Fibrobacter, CF231 and Ruminococcus (P < 0.05). Metabolomics analyses revealed an increase in the relative content of phthalic acid and cinnamaldehyde in the NM group as compared to the C group (P < 0.05), together with the greater relative content of L-tyrosine, L-dopa, rosmarinic acid, and tyrosol generated by the tyrosine metabolic pathway (P < 0.05). Spearman's correlation analyses revealed relative abundance levels of Fibrobacter and Ruminococcus were positively correlated with the mRNA relative expression levels of PAT1, NHE3, and zonula occluden-1 (ZO-1), as well as with tyrosol, phthalic acid, and cinnamaldehyde levels (P < 0.05). Ultimately, these results suggest that dietary supplementation with NM has a wide range of beneficial effects on weaned lambs and is superior to single bacterial fermentation. These effects include improvements in daily gain and rumen epithelial barrier integrity, as well as improvements in the composition of the rumen microbiome, and alterations in tyrosine metabolic pathways.

Weaned lamb  /  Rumen bacteria  /  Co-culture  /  Metabolic profile  /  Rumen function
Zixuan Xu, Lan Yang, Hui Chen, Shixiong Liu, Xueqiang Li, Songjian Li, Chun Ying, Xiao Li, Rui Du, Dacheng Liu. Saccharomyces cerevisiae and Kluyveromyces marxianus yeast co-cultures modulate the ruminal microbiome and metabolite availability to enhance rumen barrier function and growth performance in weaned lambs[J]. Animal Nutrition, 2024 , 19 (1) : 139 -152 . DOI: 10.1016/j.aninu.2024.06.005
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1.　Introduction
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In the meat sheep husbandry field, early weaning has conclusively been shown to improve ewe fertility while reducing the costs associated with feeding (Cloete et al., 2021). However, after weaning, some lambs exhibit diarrhea, reduced feed intake, slow growth, and impaired immune function (Cheng et al., 2021). While the addition of antibiotics to lamb feed has been effective as an approach to abrogating these undesirable outcomes (Estrada-Angulo et al., 2023), antibiotic administration can incur negative effects on the gastrointestinal homeostasis in recipient animals, resulting in harm while also contributing to environmental contamination (Kraemer et al., 2019). The ruminal environment in mammals has been shown to harbor a wide array of microbes that serve as essential regulators of immune function, metabolic activity, and the maintenance of the ruminal epithelial barrier (Liu et al., 2021). The use of dietary modulation to improve ruminal microbes and metabolites has demonstrated promising efficacy as a means of improving the health of animals (Redoy et al., 2020). In weaned lambs, yeast cultures have been demonstrated to influence their gastrointestinal microflora, stimulate growth, and improve immune activity. As they can be readily stored and offer a high degree of economic value, these cultures are commonly used in feed preparations (Amin and Mao, 2021). The impacts of different yeast strains on ruminal function, however, differs markedly (Pang et al., 2022). Saccharomyces cerevisiae cultures can reportedly contribute to increased ruminal microbial abundance and enhanced ruminal homeostasis (Suntara et al., 2021). Takemura et al., for example, conducted a study wherein the supplemental administration of S. cerevisiae cultures to calves fed an alfalfa hay diet resulted in an increase in the abundance of the dominant genera detected within ruminal contents after a feeding interval of 7 to 16 weeks (Takemura et al., 2020). Monteiro et al. further studied weaned lambs and found that the inclusion of S. cerevisiae cultures in their diets led to the successful mitigation of lactic acid accumulation in the rumen and an increase in pH consistent with the potential therapeutic value of this supplementation strategy (Monteiro et al., 2022). Kluyveromyces marxianus culture fermentation has been shown to produce a wide array of small peptides and organic acids (Leonel et al., 2021), and many organic acids have been shown to positively benefit ruminal regulation, including butyrate, propionate, and acetate (Zhen et al., 2023). The ability of animals to directly absorb small peptides produced by K. marxianus cultures can also modulate a wide array of metabolic and physiological activities that ultimately promotes greater ruminal health and animal growth, thus contributing to improved livestock-derived product quality (Cui et al., 2023; Intanoo et al., 2020).
In recent years, studies have demonstrated the benefits of co-cultured yeast cultures for animals, which includes the interactions between the fermentation strains, the production of organic acids and antimicrobial compounds, the regulation of immune function, and the ability to improve the integrity of the rumen epithelial barrier. Jia et al. determined that Bacillus subtilis and S. cerevisiae co-cultures were sufficient to increase the utilization of nitrogen by sheep ruminal microbes while altering fermentation patterns therein and increasing the relative abundance of members of the Fibrobacter genus (Jia et al., 2018). Xie et al. determined that Lactobacillus acidophilus and B. subtilis co-cultures were similarly capable of enhancing mucosal barrier integrity in weaned piglets via the modulation of intestinal microflora-derived short-chain fatty acids (Xie et al., 2022). Such co-cultures thus appear to offer a range of positive benefits to animal gastrointestinal health (Arowolo and He, 2018).
This study hypothesized that supplementation of S. cerevisiae and K. marxianus yeast co-cultures (NM) in the diets of weaned lambs may improve growth performance and improve rumen health by altering the rumen bacteria and metabolites. This information would have beneficial effects on the rumen homeostasis of weaned lambs. Therefore, the objective of this study was to evaluate the effects of dietary supplementation with NM on growth performance, rumen fermentation indices, epithelial barrier function, rumen bacteria, and metabolomics.
2.　Materials and methods
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2.1.　 Animal ethics statement
The procedures used in the study were approved by the Institutional Animal Protection and Utilization Committee of Inner Mongolia Agricultural University (202212006). These experiments were performed as per the guidelines established by the National Research Council (2022-6-10/SYXK 2022-0031).
2.2.　 Yeast culture preparation
Strains of S. cerevisiae and K. marxianus exhibiting good fermentation characteristics were isolated in the laboratory from naturally fermented horse milk, and pilot production was performed in Kehong Feed Co., Ltd. (Inner Mongolia, China). The base matrix contained 12% bran, 12% spraying corn bran, 10% corn, 10% rice bran, 10% cottonseed meal, 8% wheat shorts, 28% corn germ meal, and 10% soybean meal. S. cerevisiae (3 × 108 CFU/g), K. marxianus (3 × 108 CFU/g), and a 1:1 mixture of the two yeasts (3 × 108 CFU/g) were inoculated at a concentration of 8% per 1000 kg wet mixed matrix, with the addition of sterile water while stirring, resulting in a total moisture content in the system of 40%. Aerobic fermentation was then conducted for 72 h at 30℃. The nutrient compositions of the yeast cultures are presented in Table 1.
2.3.　 Experimental design and diets
This study was conducted at Fuchuan Farm (Bayannur, Inner Mongolia, China). Using a complete randomized design, 24 weaned 2-month-old lambs (Dorper × Thin-tailed Han) in good health (23.5 ± 2.85 kg) were assigned at random into four equally sized groups (n = 6) such that the average weights of animals in all groups were comparable. These lambs were individually housed in areas separated by fences (approximately 2 m2). Control animals (C group) were fed a basal diet, while animals in N group received the basal diet that had been supplemented with a 30 g/d per head culture of S. cerevisiae, animals in M group received the basal diet that had been supplemented with a 30 g/d per head culture of K. maximus, and NM group received the basal diet that had been supplemented with 30 g/d per head of S. cerevisiae and K. maximus co-cultured culture. These diets were used to feed animals in equal portions at 08:00 and 19:00 each day. The experiment lasted for 42 d, during which the first 7 d represented a period of adaptation. During this period, animals were allowed to freely access feed and water. Composition and nutrient levels of diets are presented in Table 2. Lambs were weighed at the beginning and end of the experiment before morning feeding and the average daily gain (ADG) was calculated by dividing the weight gain (final weight − initial weight) by the number of days on feed. The daily feed intake of each lamb is continuously recorded to calculate the average daily feed intake (ADFI). The ratio of feed intake to body weight gain calculates the ratio of feed intake to body weight gain (F/G). Total mixed ration (TMR) was collected every two weeks and frozen for further analysis.
2.4.　 Sample collection and chemical analysis
The dry matter (DM) of feed samples was determined by drying at 105℃ for 24 h in a forced air oven (AOAC, 2000; method 930.15). The total nitrogen (N) content in the feed was determined by the micro-Kjeldahl method (K1100, Hanon instruments, Shandong, China) (AOAC, 2000; method 976.05), and crude protein (CP) was calculated as N × 6.25. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined by an automatic fiber analyzer (Ankom Technology, Fairport, NY, USA) according to Van Soest et al. (1991), respectively. The lactic acid contents were evaluated using a commercial assay kit (Nanjing Jiancheng Bio Co., Nanjing, China), according to the manufacturer's instructions.
After slaughter, the epithelial tissue from the rumen was collected, frozen in liquid nitrogen, and subsequently stored at −80℃ for further analysis. The rumen contents were filtered using gauze, and ruminal pH were then measured with a portable pH meter (Ecoscan pH 5, Eutech Instruments, USA). Volatile fatty acids (VFA) were detected via gas chromatography (Agilent 8860 GC, USA). For VFA concentrations analyses, a capillary DB-FFAP column was used with a column temperature of 130℃, a gasification temperature of 180℃, and a detector temperature of 180℃ with a hydrogen ion flame detector. The carrier gas consisted of nitrogen (60 kPa), oxygen (50 kPa), and hydrogen (50 kPa). The sensitivity and attenuation values were 101 and 3.0, respectively. As an internal standard for these analyses, crotonic acid was selected. For metaphosphoric acid-crotonic acid solution preparation, deionized water was used to dissolve 25 g of metaphosphoric acid, followed by the addition of 0.6464 g of crotonic acid and adjustment to a final 100 mL volume.
2.5.　 Real-time quantitative PCR (RT-qPCR)
Trizol (TIANGEN., Ltd., Shanghai, China) was used as directed to extract RNA from rumen epithelial samples, after which a Nanodrop 2000 spectrophotometer (Agilent, Cary 60 UV-Vis, USA) was used to assess RNA quality, with an OD260/OD280 ratio of 1.8 to 2.0 being indicative of RNA that was sufficiently pure for further use. An M5 Sprint Perfect RT Kit with gDNA Clean for qPCR II (RR037A, Takara Bio, Beijing, China) was used to prepare cDNA, and a LightCycler 480 Instrument (Roche, Basel, Beijing, China) was then used for RT-qPCR amplification with SYBR Premix Pro Taq HS qPCR Kit (CN830s, TaKaRa Bio Beijing, China). Each 15 μL reaction was composed of 7.5 μL of 2 × SYBR Green Pro Taq HS Premix, 1.5 μL of appropriate primers (F + R), 2 μL of cDNA, and 4.0 μL of RNase-free water. Thermocycler settings were as follows: 95℃ for 30 s, 40 cycles of 95℃ for 15 s, 60℃ for 30 s. The 2−△△Ct method was used to evaluate relative expression (Livak and Schmittgen, 2001), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a normalization control. Primer 3.0 (Applied Biosystems, CA, USA) was used to design primers, which were synthesized by Shenggong Biotech (Shanghai, China). All primers used for these analyses are presented in Table 3.
2.6.　 16S rRNA sequencing
The OMEGA Soil DNA Kit (M5635-02) (OMEGA Bio-Tek, GA, USA) was used to extract DNA from 24 rumen content samples, after which a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, MA, USA) and agarose gel electrophoresis were respectively used to assess DNA quantity and quality. The 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primer pair was used for the amplification of the V3–V4 region of the bacterial 16S rRNA gene, with primers having incorporated of sample-specific 7-bp barcodes. Each PCR reaction included 5 μL of 5 × buffer, 0.25 μL of Fast pfu DNA polymerase (5 U/μL), 2 μL of dNTPs (2.5 mmol/L), 1 μL each of forward and reverse primers (10 μmol/L), 1 μL of template DNA, and 14.75 μL of ddH2O. Thermocycler settings were as follows: 98℃ for 5 min; 35 cycles of 98℃ for 30 s, 53℃ for 30 s, 72℃ for 45 s; 72℃ for 5 min. Vazyme VAHTSTM DNA Clean Beads (Vazyme, Shanghai, China) were used to purify PCR products, after which a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, CA, USA) was used for quantification. Equal amounts of each amplicon were pooled, and end-to-end 2 × 250 bp sequencing was then performed with the Illumina NovaSeq platform by Personalbio Technology Co, Ltd. (Shanghai, China) using the NovaSeq 6000 SP Reagent Kit (500 cycles). Raw reads have been uploaded to the NCBI Sequence Read Archive (SRA) database (entry number: PRJNA1029322).
2.7.　 Bioinformatics analyses
QIIME2 2019.4 (Bolyen et al., 2019) was used to analyze the ruminal bacteria based on a slightly modified version of the official tutorial (https://docs.qiime2.org/2019.4/tutorials/). Briefly, raw sequencing data underwent demultiplexing with the demux plug-in, after which cutadapt was used to remove primer sequences (Martin, 2011). Data were then filtered based on quality, de-noised, merged, and chimeric sequences were removed with the DADA2 plug-in (Callahan et al., 2016). Non-monadic amplification sequence variants (ASV) were aligned using matt, and fasttre2 was used to construct a phylogenetic tree based on these results (Price et al., 2009). The RDP Classified algorithm was used to classify and analyze 16S rRNA gene sequences, and the Silva 132 database was used for comparisons which were conducted at a 70% comparison threshold.
2.8.　 Rumen metabolomics analyses
Samples of ruminal contents (about 45 mg) were combined with 500 mL of cold 70% (v/v) methanol containing 2-chlorophenylalanine (1 mg/mL) as an internal standard. The solution was then swirled for 3 min, followed by ultrasonication for 10 min in ice water. Samples were then centrifuged for 10 min at 12,000 × g, and supernatants were transferred into an automatic sampler for UPLC-MS/MS analyses (metware) performed with an appropriate UPLC-MS/MS system (UPLC, Shim-pack UFLC SHIMADZU CBM A system; MS, QTRAP® system). Samples were analyzed in positive and negative ESI modes at a 0.4 mL/min flow rate using solvent A (0.04% acetate in water) and B (0.04% acetic acetonitrile) with the following gradient settings: 95:5 v/v at 0 min, 5:95 v/v at 11 min, and 95:5 v/v at 12 min (A:B) v/v. The ESI source settings included a source temperature of 500℃ and an ion spray voltage of 5500 V or −4500 V. Source gas I, gas II, and curtain gas pressures were respectively set at 55, 60, and 25 psi. Collison gas (CAD) was high.
2.9.　 Statistical analyses
One-way ANOVA were used to analyze ruminal fermentation parameters and the expression of cytokines and other genes. Data analyses were conducted with SPSS 22.0, and the results were presented using GraphPad Prism (v 8.0). P < 0.05 was considered indicative of a significant difference, whereas 0.05 ≤ P < 0.10 was considered indicative of a significant trend. Analyses of microbial communities were performed at the ASV level. Wilcoxon rank sum tests were used to compute richness estimates (ACE and Chao1 indexes) and alpha diversity indexes. Taxa abundance at the genus and phylum levels was compared with python 2.8. Differences in the most abundant genera among groups were identified with the Wilcoxon rank sum test. P-values were false discovery rate (FDR)-corrected. Analyst 1.6.3 was used to analyze and process mass spectrum data. Peaks extracted from rumen samples during metabolomics analyses were analyzed with principal component analysis (PCA) and orthogonal projection latent structure discriminant analysis (OPLS-DA) approaches, selecting those metabolites that differed significantly in abundance among these groups based on a variable importance in projections (VIP) ≥ 1.00 and a fold-change (FC)≥1.2 or ≤0.5 between groups. Differences in the enrichment of different metabolic pathway modules were evaluated via online Kyoto Encyclopedia of Genes and Genomes (KEGG) database annotation. Spearman's correlation analyses were performed in R (v 3.2.1) to detect correlations among ruminal fermentation, inflammatory cytokine gene expression, barrier-related gene expression, and rumen microbiome-related metabolites in these experimental lambs.
3.　Results
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3.1.　 Growth performance
The growth performance of the lambs is shown in Table 4. The ADG of lambs in the NM group was significantly higher than that in the C group (P = 0.044). However, there were no significant differences in the other growth performance indicators (P > 0.05).
3.2.　 Analysis of ruminal fermentation and epithelial gene expression
Analyses of the ruminal fermentation parameters in these experimental animals revealed a significant decrease in pH in the NM group (P = 0.024). While acetate concentration was significantly lower in C group as compared to the M and NM groups (P < 0.001), propionate concentration was significantly higher in the N, M, and NM groups (P < 0.001) as compared to C group. Moreover, C group exhibited butyrate concentration significantly lower than that in the M and NM groups (P < 0.001) (Table 5).
The results on the permeability of the rumen epithelium are shown in Fig. 1. The mRNA relative expression levels of monocarborxylat transporter 1 (MCT1) in M group (P < 0.05) and NM group (P < 0.01) differed significantly from those in C group. The mRNA relative expression levels of (Na+)/hydrogen (H+) exchanger 1 (NHE1) was significantly higher in the NM group as compared to the C (P < 0.001), N (P < 0.05), and M groups (P < 0.01). The NM group also exhibited significant increases in the mRNA relative expression levels of (Na+)/hydrogen (H+) exchanger 3 (NHE3) (P < 0.01), proton-coupled amino acid transporter 1 (PAT1) (P < 0.01), and vacuolar H+-ATPase (vH+ATPase) (P < 0.05) compared to C group, whereas no significant differences in mRNA relative expression levels of anion exchanger 2 (AE2), down-regulated in adenoma (DRA), monocarboxylate transporter 4 (MCT4), or (Na+)/hydrogen (H+) exchanger 2 (NHE2) were observed among these four groups (P > 0.05).
3.3.　 Analysis of tight junction protein and cytokine mRNA relative expression levels in the rumen epithelium
To determine the effects of co-cultured yeast on the rumen epithelium, the mRNA relative expression of tight junction proteins and cytokines were analyzed in the samples. The mRNA relative expression levels of genes encoding claudin-1 (P < 0.01), zonula occluden-1 (ZO-1) (P < 0.01), and interferon-γ (IFN-γ) (P < 0.05) were significantly elevated in the N group compared to C group, while the opposite was true for the gene encoding interleukin-6 (IL-6) (P < 0.01). Samples from M group exhibited significantly higher claudin-1 mRNA relative expression level (P < 0.01) and significantly lower IL-6 mRNA relative expression levels (P < 0.05), while the NM group exhibited significantly elevated mRNA relative expression levels encoding claudin-1 (P < 0.001) and occludin (P < 0.05) together with significantly lower levels of mRNA relative expression encoding transforming growth factor-β1 (TGF-β1) (P < 0.05) and IL-6 (P < 0.01). No significant changes in the levels of mRNA relative expression encoding claudin-4, IL-1β, interleukin-10 (IL-10), or tumor necrosis factor-α (TNF-α) were observed when comparing these four experimental groups (Fig. 2A and B). Increased mRNA relative expression of claudin-1, ZO-1, IFN-γ, and occludin is indicative of increases in rumen epithelial barrier function and cytokine production. N and NM groups showed better effects in regulating rumen epithelial barrier function and rumen immunity of weaned lambs.
3.4.　 16S rRNA sequencing analyses of the ruminal bacteria
To explore the potential effects of co-cultured yeast cultures on the ruminal microbiome in experimental lambs, 16s rRNA gene sequencing was performed using primers specific for the V3–V4 region. In total 2,077,312 effective sequences were obtained across 24 samples (average: 86,555 sequences/sample), leading to the identification of 253 ASV at the 97% nucleotide sequence identity level (Fig. 3A). In total, 82 ASV were represented across samples in different groups, while the C, N, M, and NM groups respectively harbored 42 (16.6%), 24 (9.49%), 22 (8.70%), and 20 (7.91%) unique ASV. Alpha diversity indexes revealed that the Observed_species index and Chao1 index in C group were significantly higher than those in the N, M, and NM groups (P < 0.05), while the Shannon and Simpson indexes in M group significantly exceeded those for C, N, and NM groups (P < 0.05) (Table 6).
Analyses of β-diversity were also conducted to assess structural differences among these microbial communities in different groups of experimental lambs. Discrete distributions of samples were observed in these four groups (Fig. 3B), with C group being spatially separated from the three other groups. Non-metric multidimensional scaling (NMDS) plots generated via the Jaccard method highlighted differences among groups in terms of bacteria composition. Firmicutes, Bacteroidetes, and Fibrobacteres were the predominant phyla in these samples (Fig. 3C), while the predominant genera were Prevotella, Succiniclasticum, and Fibrobacter (Fig. 3D). Compare to C group, N group exhibited significant increases in relative abundance of Succiniclasticum, Clostridium, and Selenomonas at the genus level (P < 0.05), while M group exhibited significantly increased relative abundance of Succiniclasticum, Butyrivibrio, and Clostridium (P < 0.05), and NM group exhibited significantly increased relative abundance of Prevotella and CF231 (P < 0.05). Significant increases in relative abundance of Fibrobacter, Treponema, Ruminococcus, and Moryella were evident in the N, M, and NM groups (P < 0.05) (Table 7). In general, the changes in the dominant genera were more significant in NM group relative to the other experimental groups.
3.5.　 Analyses of correlations between ruminal bacterial, barrier function, and inflammatory gene expression
Correlations between rumen barrier function, inflammatory cytokine gene expression, and the dominant genera detected in ruminal samples were next assessed. The relative abundance of Fibrobacter (P < 0.01), Succiniclasticum (P < 0.001), Treponema (P < 0.001), Ruminococcus (P < 0.01), and Clostridium (P < 0.01) were all found to be positively correlated with the mRNA relative expression of ZO-1, whereas the relative abundance of Succiniclasticum (P < 0.05) and Treponema (P < 0.05) were positively correlated with the mRNA relative expression of IL-10. In addition, NHE3 mRNA relative expression was positively correlated with the relative abundance of Fibrobacter (P < 0.01) and Ruminococcus (P < 0.05) abundance, whereas the mRNA relative expression of MCT4 was positively correlated with Ruminococcus relative abundance (P < 0.05) (Fig. 4). These data indicate that co-culture supplementation and associated modulation of the ruminal microbiome may contribute to improvements in rumen function.
3.6.　 Ruminal metabolic profiling
Changes in ruminal metabolic profiles in weaned lambs were analyzed using a UPLC-MS/MS approach. In total, 710 metabolites were detected as presented in the PCA score plots shown in Fig. 5A. The OPLS-DA score plots revealed good separation among the four groups, and a random permutation test confirmed that this model exhibited satisfactory accuracy (R2X = 0.272, R2Y = 0.995, Q2 = 0.914, Fig. 5B), enabling screening for differentially abundant metabolites (Fig. 6). Comparisons of the C and N groups, showed significantly higher relative contents of phthalic acid, tyrosol, rosmarinic acid, and cinnamaldehyde in the N group with a corresponding reduction in L-tyrosine acid (P = 0.005); when comparing the C and M groups, significantly higher relative contents of L-tyrosine, phthalic acid, and rosmarinic acid were present in the M group, whereas tyrosol and L-dopa relative contents were significantly decreased (P < 0.05); a comparison of the C and NM groups showed significantly increased relative contents of L-tyrosine, rosmarinic acid, phthalic acid, cinnamaldehyde, L-dopa, and tyrosol (P < 0.05) in the NM group; L-glutamine and phenylethylamine relative contents were found to be significantly lower in all experimental groups relative content to C group (P < 0.05) (Table 8). Pathway analyses suggested that relative to C group, the weaned lambs that underwent N and M dietary supplementation primarily exhibited changes in the biosynthesis of plant secondary metabolites and tyrosine metabolism, while animals in the NM group exhibited significant changes in tyrosine metabolism (Table 9). Dietary supplementation with co-cultured yeast cultures may thus significantly influence tyrosine metabolism.
3.7.　 Correlations between the bacteria and ruminal metabolites
Spearman correlation coefficients were used to explore relationships among ruminal microbes and differentially abundant metabolites. When comparing the C and N groups, significant positive correlations were observed between the relative abundance of Ruminococcus and Fibrobacter and the levels of phthalic acid, rosmarinic acid, cinnamaldehyde, and tyrosol (P < 0.05). Significant positive correlations were observed between the relative abundance of Coprococcus and the levels of phenylethylamine, naringin, tyrosol, and L-glutamine, whereas it was negatively correlated with L-tyrosine abundance (P < 0.05) (Fig. 7A). A comparison of the C and M groups also demonstrated positive correlations between the relative abundance of Ruminococcus and Fibrobacter and the levels of phthalic acid, rosmarinic acid, and tyrosol. Significant positive correlations were additionally detected between the relative abundance of Coprococcus and the levels of phenylethylamine, thymidine, pantothenic acid, tyrosol, and L-glutamine (P < 0.05) (Fig. 7B). Similarly, comparisons of the C and NM groups revealed significant positive correlations between the relative abundance of Ruminococcus and Fibrobacter and the ruminal levels of tyrosol, phthalic acid, and cinnamaldehyde. There was a significant positive correlation between the relative abundance of Coprococcus and the levels of phenylethylamine, isocitric acid, tyrosol, and L-glutamine, with a negative correlation with levels of L-tyrosine and creatine (P < 0.05) also shown (Fig. 7C).
4.　Discussion
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Levels of interest in the application of yeast cultures as an approach to improving growth performance, enhancing ruminal fermentation, modulating the ruminal microbiota, and alleviating adverse stress-related outcomes in post-weaned lambs have been rising in recent years (Ban and Guan, 2021). The strains most commonly used in this context include S. cerevisiae (Suntara et al., 2021), Candida utilis (Yang et al., 2021), and Lactobacillus (Karamad et al., 2020). However, there have been no prior reports examining the effects of S. cerevisiae and K. marxianus yeast co-cultures on ruminal dynamics and related processes in weaned lambs. In this study, NM yeast cultures increased the ADG in the lambs. Analyzing ruminal pH can provide an invaluable index for the assessment of the internal rumen microenvironment (Zhao et al., 2023). A pH of 6.0 to 6.5 is generally favorable for ruminal microbe proliferation, in turn contributing to the production of ample VFA that can meet the energy requirements of the host (Matthews et al., 2019). In this study, NM group supplementation was associated with significantly reduced ruminal pH in the post-weaned lambs, which may be a consequence of the rapid ruminal nutrient fermentation observed following co-culture supplementation, leading to the production of organic acids and a marked drop in the pH in the rumen. Shifts in rumen pH can affect the microbial communities present therein and the fermentation products that they generate (Therion et al., 1982). In prior research focused on weaned calves and lambs, yeast cultures were found to enhance both ruminal fermentation activity and VFA uptake (Hassan et al., 2020). Rackwitz et al. analyzed the permeability of the rumen epithelium and found that adding yeast cultures led to an increase in butyrate uptake per unit of epithelial area (Bertens et al., 2023). Here, significantly increased propionate concentrations were detected in the three experimental groups, with the M and NM groups showing markely higher increases in the levels of acetate and butyrate. Acetate, propionate, and butyrate are substrates for the synthesis of primary nutrients in sheep and are also related to the energy balance (Hartinger and Zebeli, 2021). These results suggest that the addition of co-cultured yeast cultures improved the growth performance and decreased rumen pH, the addition of K. marxianus cultures and co-cultured yeast cultures to post-weaning diets in lambs can improve the ruminal concentrations of acetate and propionate, thereby enhancing the function of the rumen.
In addition, an investigation of SCFA-related transporters has shown that the addition of yeast culture could promote the expression of MCT1 in sheep rumen epithelium (Kuzinski and Röntgen, 2011). Here, dietary supplementation with the M and NM cultures was associated with an increase in MCT1 expression, thus enhancing rumen epithelium-mediated butyrate uptake. The vH+ATPase transporter is expressed within the rumen epithelium where it is responsible for 30% of total H+ transport, thus shaping the pH within the rumen (Etschmann et al., 2006; Hu et al., 2021). Higher vH+ATPase expression in the NM group may be related to the synergistic enhancement of microbe-mediated secretion of active nutrients and substances that are fermented in the rumen, reducing H+ concentrations therein and maintaining the local pH balance via regulating vH+ATPase expression. The cell membrane NHE1 and NHE3 proteins are responsible for the extracellular excretion of most H+ from rumen epithelial cells, with these respective transporters accounting for 50% and 20%, of all transported H+ (Etschmann et al., 2006; Hu et al., 2021). All experimental groups in this study exhibited significant differences in NHE1 expression in the rumen epithelium as compared to control lambs, while significant increases in NHE3 and PAT1 expression were also observed in the NM group. Lamb dietary supplementation with yeast co-cultures may thus lead to at least some enhancement of the permeability of the rumen epithelium while promoting epithelial development in this following weaning.
Rumen epithelial barrier integrity in the lambs in the NM group in the present study was significantly enhanced. ZO-1, occludin, claudin-1, and claudin-4 are currently regarded as key tight junction proteins responsible for shaping the composition and function of the intercellular barrier within the rumen (Kuo et al., 2022; Wang et al., 2022). Lambs in the NM group exhibited the upregulation of claudin-1 and occludin at the mRNA level. The integrity of the rumen epithelium is closely associated with inflammatory factors, and yeast cultures can inhibit rumen IL-1β, TNF-α, and IFN-γ expression by modulating signaling through TLR4-mediated pathways (Fang et al., 2017). TLR signaling activation can also stimulate NF-κB signaling, in turn leading to a reduction in the expression of pro-inflammatory mediators including IL-6, IL-1β, and TNF-α (Bu et al., 2019; Conrad et al., 2014). Yeast cultures were previously found to suppress ruminal TNF-α and IL-6 expression in weaned lambs (Izuddin et al., 2019). Here, NM supplementation was associated with a drop in TGF-β1 and IL-6 gene expression. Corresponding decreases in IL-6 expression were also evident in the N and M groups. Overall, the addition of the NM yeast culture could enhance the function of the rumen epithelial barrier.
To better understand how adding NM yeast cultures to the diet of weaned lambs can influence the rumen bacteria, 16S rRNA sequencing was performed. This approach revealed that the rumen microbial communities in these experimental lambs were dominated by Bacteroidetes and Firmicutes, in line with prior reports (Mizrahi et al., 2021). Significantly higher Fibrobacterium relative abundance was observed in the N, M, and NM groups as compared to the C group. In the gut of ruminants, Fibrobacter is known to serve as an efficient lignocellulose degrader (Ransom-Jones et al., 2012). Dietary yeast culture supplementation can contribute to improvements in the abundance of ruminal bacterial with the ability to degrade cellulose and starch as well as the ability to utilize lactate (Amin and Mao, 2021). Most notably, the NM group exhibited increases in the relative abundance of certain beneficial bacteria including Prevotella, Fibrobacter, Ruminococcus, and Butyrivibrio. Certain Ruminococcus strains have been identified as natural probiotics capable of promoting rumen health in the context of animal production (Hsieh et al., 2023; Mamuad et al., 2019; Yu et al., 2020). Consistently, in the present study, a positive correlation was noted between Ruminococcus relative abundance and the mRNA relative expression levels of PAT1, NHE3, MCT4, and ZO-1. Moreover, the relative abundance of Fibrobacter was positively associated with the expression levels of mRNAs encoding claudin-1, PAT1, NHE3, and ZO-1. These data thus indicate that the changes in rumen bacteria composition in these lambs fed an NM-supplemented diet may be associated with the enhancement of rumen epithelial barrier function and nutrient absorption.
Metabolomics analysis showed significantly higher cinnamaldehyde levels in the N and NM groups relative to those in C group. Cinnamaldehyde exhibits promising antibacterial efficacy and is also capable of exerting antioxidant and anti-inflammatory effects to alleviate external stress-related effects (Doyle et al., 2019). Significant reductions of the amino acid metabolism-related metabolite phenylethylamine were also detected. Phenylethylamine can be generated by the Ruminococcus gnavus-mediated dietary catabolism of phenylalanine, facilitating 5-hydroxytryptamine (5-HT) biosynthesis within the intestines via trace amine-associated receptor 1 (TAAR1) activation and thereby provoking irritable bowel syndrome-like symptoms including diarrhea (Zhai et al., 2023). Here, the observed reduction in the relative content of phenylethylamine may be indicative of altered ruminal digestive function and reduced incidence of diarrhea in weaned lambs. Tyrosine metabolism is another relevant process that was enriched in the metabolites that were differentially abundant in the N, M, and NM groups. As an essential amino acid, tyrosine is used by microbes and their hosts as a precursor to synthesize various metabolites (Schenck and Maeda, 2018). The NM group also presented with significant tyrosine-related metabolite enrichment, including elevated levels of rosmarinic acid, tyrosol, L-tyrosine, and L-dopa. Rosmarinic acid exhibits robust bioactivity, including reported antimicrobial, anti-inflammatory, and antioxidant properties (Zhao et al., 2022). Tyrosol can reportedly drive significant increases in the expression of Nrf2 anti-inflammatory pathway-related genes at the mRNA level while also contributing to increases in the relative abundance of beneficial thick-walled bacterial phyla within the small intestines of sheep, which is consistent with the results of the present study. Tyrosine hydroxylase catalyzes the processing of tyrosine to generate L-dopa, which is further processed by L-dopa decarboxylase to generate dopamine (Güvenç et al., 2019). Dopamine serves as a regulator of immunoinflammatory activity within the rumen through its ability to inhibit NLRP3 inflammasome activity and downstream inflammation via DRD1 signaling. Alternative tyrosine metabolism detected in this study may thus positively impact the ruminal health of weaned lambs (Bueno-Carrasco et al., 2022; Yan et al., 2015). Spearman correlation analyses revealed that Coprococcus relative abundance was significantly positively correlated with the levels of phenethylamine, tyrosol, and L-glutamine, whereas it was negatively correlated with L-tyrosine levels. Moreover, Fibrobacter and Ruminococcus relative abundance was found to be positively correlated with the levels of phthalic acid, cinnamaldehyde, and tyrosol. The tyrosine metabolism detected in the NM group may thus be associated with altered Coprococcus, Fibrobacter, and Ruminococcus relative abundance.
5.　Conclusion
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In summary, these results suggest that dietary supplementation with co-cultured yeast cultures can increase the average daily gain in lambs, alter the composition and metabolite profiles of the ruminal microbiome, promoting Prevotella, Coprococcus Fibrobacter, CF231, Butyrivibrio, and Ruminococcus enrichment while increasing the levels of metabolites including phthalic acid, L-dopa, L-tyrosine, tyrosol, cinnamaldehyde, and rosmarinic acid. These shifts in the makeup of the ruminal microflora may enhance rumen epithelial barrier function and nutrient uptake changes. These findings offer insights that can support future efforts to establish antibiotic alternative strategies aimed at fostering greater rumen health in weaned lambs (Fig. 8).
References
Less
Amin AB, Mao SY. Influence of yeast on rumen fermentation, growth performance and quality of products in ruminants: a review. Anim Nutr 2021;7:31-41.
AOAC. Association of official analytical chemists. 17 th ed. Arlington, VA, USA: AOAC international; 2000.
Arowolo MA, He JH. Use of probiotics and botanical extracts to improve ruminant production in the tropics: a review. Anim Nutr 2018;4:241-9.
Ban YJ, Guan LL. Implication and challenges of direct-fed microbial supplementation to improve ruminant production and health. J Anim Sci Biotechnol 2021;12:109.
Bertens CA, Mutsvangwa T, Van Kessel AG, Penner GB. Effect of sodium concentration and mucosal ph on apical uptake of acetate and butyrate, and barrier function of the isolated bovine ruminal epithelium. J Dairy Sci 2023;106: 7310-9.
Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, et al. Reproducible, interactive, scalable and extensible microbiome data science using qiime 2. Nat Biotechnol 2019;37:852-7.
Bu XY, Lian XQ, Wang Y, Luo CZ, Tao SQ, Liao YL, et al. Dietary yeast culture modulates immune response related to TLR2-MyD88-NF-kβ signaling pathway, antioxidant capability and disease resistance against Aeromonas hydrophila for Ussuri catfish (Pseudobagrus ussuriensis). Fish Shellfish Immunol 2019;84:711-8.
Bueno-Carrasco MT, Cuéllar J, Flydal MI, Santiago C, Kråkenes TA, Kleppe R, et al. Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by ser40 phosphorylation. Nat Commun 2022;13:74.
Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. Dada2: highresolution sample inference from illumina amplicon data. Nat Methods 2016;13:581-3.
Cheng L, Cantalapiedra-Hijar G, Meale SJ, Rugoho I, Jonker A, Khan MA, et al. Review: markers and proxies to monitor ruminal function and feed efficiency in young ruminants. Animal 2021;15:100337.
Cloete SWP, Muller A, Steyn S, Van Der Merwe DA, Nel CL, Cloete S, et al. The effect of tree shade on ambient conditions and heat stress indicator traits of new-born south African Mutton Merino and Dormer lambs: preliminary results. J Therm Biol 2021;99:103024.
Conrad M, Schothorst J, Kankipati HN, Van Zeebroeck G, Rubio-Texeira M, Thevelein JM. Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 2014;38:254-99.
Cui YY, Guo P, Ning MG, Yue Y, Yuan YH, Yue TL. Kluyveromyces marxianus supplementation ameliorates alcohol-induced liver injury associated with the modulation of gut microbiota in mice. Food Funct 2023;14:9920-35.
Doyle AA, Krämer T, Kavanagh K, Stephens JC. Cinnamaldehydes: synthesis, antibacterial evaluation, and the effect of molecular structure on antibacterial activity. Results Chem 2019;1:100013.
Estrada-Angulo A, Escobedo-Gallegos LDG, Arteaga-Wences YJ, Ramos-Méndez JL, Quezada-Rubio JA, Vizcarra-Chávez CA, et al. Effect of combining the ionophore monensin with natural antimicrobials supplemented in the last phase of finishing of lambs: growth performance, dietary energetics, and carcass characteristics. Animals 2023;13:2547.
Etschmann B, Heipertz KS, Von Der Schulenburg A, Schweigel M. A vH+-atpase is present in cultured sheep ruminal epithelial cells. Am J Physiol Gastrointest Liver Physiol 2006;291:G1171-9.
Fang WS, Bi DC, Zheng RJ, Cai N, Xu H, Zhou R, et al. Identification and activation of tlr4-mediated signalling pathways by alginate-derived guluronate oligosaccharide in RAW264.7 macrophages. Sci Rep 2017;7:1663.
Güvenç M, Cellat M, Özkan H, Tekeli İO, Uyar A, Gökçek İ, et al. Protective effects of tyrosol against dss-induced ulcerative colitis in rats. Inflammation 2019;42: 1680-91.
Hartinger T, Zebeli Q. The present role and new potentials of anaerobic fungi in ruminant nutrition. J Fungi 2021;7:200.
Hassan FU, Arshad MA, Ebeid HM, Rehman MSU, Khan MS, Shahid S, et al. Phytogenic additives can modulate rumen microbiome to mediate fermentation kinetics and methanogenesis through exploiting diet-microbe interaction. Front Vet Sci 2020;7:575801.
Hsieh JC, Chuang ST, Hsu YT, Ho ST, Li KY, Chou SH, et al. In vitro ruminal fermentation and cow-to-mouse fecal transplantations verify the interrelationship of microbiome and metabolome biomarkers: potential to promote health in dairy cows. Front Vet Sci 2023;10:1228086.
Hu ZX, Lin M, Ma XY, Zhao GQ, Zhan K. Effect of tea tree oil on the expression of genes involved in the innate immune system in goat rumen epithelial cells. Animals 2021;11:2460.
Intanoo M, Kongkeitkajorn MB, Suriyasathaporn W, Phasuk Y, Bernard JK, Pattarajinda V. Effect of supplemental Kluyveromyces marxianus and Pichia kudriavzevii on aflatoxin M1 excretion in milk of lactating dairy cows. Animals 2020;10:709.
Izuddin WI, Loh TC, Foo HL, Samsudin AA, Humam AM, Postbiotic L. Plantarum RG14 improves ruminal epithelium growth, immune status and upregulates the intestinal barrier function in post-weaning lambs. Sci Rep 2019;9:9938.
Jia P, Cui K, Ma T, Wan F, Wang WY, Yang D, et al. Influence of dietary supplementation with Bacillus licheniformis and Saccharomyces cerevisiae as alternatives to monensin on growth performance, antioxidant, immunity, ruminal fermentation and microbial diversity of fattening lambs. Sci Rep 2018;8:16712.
Karamad D, Kianoushkhosravi-Darani K, Hosseini H, Tavasoli S, Miller AW. Assessment of the process variables for degradation of oxalate by lactobacillus acidophilus atcc 4356 using simulated rumen fluid media and tea. Appl Food Biotechnol 2020;7:195-204.
Kraemer SA, Ramachandran A, Perron GG. Antibiotic pollution in the environment: from microbial ecology to public policy. Microorganisms 2019;7:180.
Kuo WT, Odenwald MA, Turner JR, Zuo L. Tight junction proteins occludin and ZO-1 as regulators of epithelial proliferation and survival. Ann N Y Acad Sci 2022;1514:21-33.
Kuzinski J, Röntgen M. The mrna and protein expression of ruminal MCT1 is increased by feeding a mixed hay/concentrate diet compared with hay ad libitum diet (Short Communication). Arch Anim Breed 2011;54:280-6.
Leonel LV, Arruda PV, Chandel AK, Felipe MGA, Sene L. Kluyveromyces marxianus: a potential biocatalyst of renewable chemicals and lignocellulosic ethanol production. Crit Rev Biotechnol 2021;41:1131-52.
Liu KZ, Zhang YD, Yu ZT, Xu QB, Zheng N, Zhao SG, et al. Ruminal microbiota-host interaction and its effect on nutrient metabolism. Anim Nutr 2021;7:49-55.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-∆∆Ct method. Methods 2001;25:402-8.
Mamuad LL, Kim SH, Biswas AA, Yu ZT, Cho KK, Kim SB, et al. Rumen fermentation and microbial community composition influenced by live enterococcus faecium supplementation. Amb Express 2019;9:123.
Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 2011;17:10-2.
Matthews C, Crispie F, Lewis E, Reid M, O’Toole PW, Cotter PD. The rumen microbiome: a crucial consideration when optimising milk and meat production and nitrogen utilisation efficiency. Gut Microb 2019;10:115-32.
Mizrahi I, Wallace RJ, Moraïs S. The rumen microbiome: balancing food security and environmental impacts. Nat Rev Microbiol 2021;19:553-66.
Monteiro HF, Agustinho BC, Vinyard JR, Harden T, Bennett SL, Arce-Cordero JA, et al. Megasphaera elsdenii and saccharomyces cerevisiae as direct fed microbials during an in vitro acute ruminal acidosis challenge. Sci Rep 2022;12:7978.
NRC. Nutrient requirements of dairy cattle. 7th ed. Washington, DC: National Academies Press; 2001. 2001.
Pang YX, Zhang HL, Wen HY, Wan HB, Wu H, Chen Y, et al. Yeast probiotic and yeast products in enhancing livestock feeds utilization and performance: an overview. J Fungi 2022;8:1191.
Price MN, Dehal PS, Arkin AP. Fasttree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol Biol Evol 2009;26:1641-50.
Ransom-Jones E, Jones DL, McCarthy AJ, McDonald JE. The Fibrobacteres: an important phylum of cellulose-degrading bacteria. Microb Ecol 2012;63: 267-81.
Redoy MRA, Shuvo AAS, Cheng L, Al-Mamun M. Effect of herbal supplementation on growth, immunity, rumen histology, serum antioxidants and meat quality of sheep. Animal 2020;14:2433-41.
Schenck CA, Maeda HA. Tyrosine biosynthesis, metabolism, and catabolism in plants. Phytochemistry 2018;149:82-102.
Suntara C, Cherdthong A, Wanapat M, Uriyapongson S, Leelavatcharamas V, Sawaengkaew J, et al. Isolation and characterization of yeasts from rumen fluids for potential use as additives in ruminant feeding. Vet Sci 2021;8:52.
Takemura K, Shingu H, Ikuta K, Sato S, Kushibiki S. Effects of saccharomyces cerevisiae supplementation on growth performance, plasma metabolites and hormones, and rumen fermentation in holstein calves during pre- and postweaning periods. Anim Sci J 2020;91:e13402.
Therion JJ, Kistner A, Kornelius JH. Effect of pH on growth rates of rumen amylolytic and lactilytic bacteria. Appl Environ Microbiol 1982;44:428-34.
Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 1991;74:3583-97.
Wang KX, Lei Q, Ma HM, Jiang MC, Yang TY, Ma QB, et al. Phloretin protects bovine rumen epithelial cells from LPS-induced injury. Toxins 2022;14:337.
Xie ZJ, Li M, Qian MQ, Yang ZR, Han XY. Co-cultures of Lactobacillus acidophilus and Bacillus subtilis enhance mucosal barrier by modulating gut microbiota-derived short-chain fatty acids. Nutrients 2022;14:4475.
Yan YQ, Jiang W, Liu L, Wang XQ, Ding C, Tian ZG, et al. Dopamine controls systemic inflammation through inhibition of NLRP3 inflammasome. Cell 2015;160: 62-73.
Yang ZG, Wang Y, He TL, Ziema Bumbie G, Wu LT, Sun ZH, et al. Effects of dietary yucca schidigera extract and oral Candida utilis on growth performance and intestinal health of weaned piglets. Front Nutr 2021;8:685540.
Yu SB, Shi WB, Yang B, Gao G, Chen HW, Cao L, et al. Effects of repeated oral inoculation of artificially fed lambs with lyophilized rumen fluid on growth performance, rumen fermentation, microbial population and organ development. Anim Feed Sci Technol 2020;264:114465.
Zhai LX, Huang CH, Ning ZW, Zhang YJ, Zhuang M, Yang W, et al. Ruminococcus gnavus plays a pathogenic role in diarrhea-predominant irritable bowel syndrome by increasing serotonin biosynthesis. Cell Host Microbe 2023;31: 33-44.e5.
Zhao JC, Xu LW, Jin D, Xin Y, Tian L, Wang T, et al. Rosmarinic acid and related dietary supplements: potential applications in the prevention and treatment of cancer. Biomolecules 2022;12:1410.
Zhao MY, Zhang XA, Chen Y, Ren CH, Sun YM, Wang PH, et al. Stall-feeding of sheep on restricted grazing: effects on performance and serum metabolites, ruminal fermentation, and fecal microbiota. Animals 2023;13:2644.
Zhen YK, Xi ZN, Nasr SM, He FY, Han ML, Yin JL, et al. Multi-omics reveals the impact of exogenous short-chain fatty acid infusion on rumen homeostasis: insights into crosstalk between the microbiome and the epithelium in a goat model. Microbiol Spectr 2023;11:e0534322.
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Year 2024 volume 19 Issue 1
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doi: 10.1016/j.aninu.2024.06.005
  • Receive Date：2024-01-15
  • Online Date：2026-01-28
  • Published：2024-12-10
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  • Received：2024-01-15
  • Revised：2024-04-27
  • Accepted：2024-06-03
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    College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China

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Corresponding author. E-mail address: (D. Liu).
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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