The Effects of Dual-Yeast Compound Preparation on the Intestinal Health and Metabolism of Lambs

Simple Summary

With the aim of clarifying the effects and mechanisms of yeast microbiota preparations on the health of young ruminants, the influence of yeast culture on the growth, gut microbiota, and metabolism of weaned lambs was studied and analyzed. In this study, we divided twenty weaned lambs into four groups and fed each group separately with basic feed and different yeast cultures. The study continued for 40 days, and relevant indicators were detected. The results showed that the lambs added with composite yeast culture had significantly higher daily weight gain and feed intake, improved feed utilization efficiency, increased abundance of Bacteroidetes in the intestine, enrichment of specific signaling pathways, and increased levels of ketones and benzoic acid substances. Research has indicated that adding yeast culture to feed can have a positive effect on lamb productivity performance and intestinal health, providing a basis for the development of microecological preparations for ruminants, helping to improve lamb breeding efficiency and animal product quality, and having value for the development of animal husbandry.

Abstract

Microecological preparations exert beneficial effects on the health of young ruminant animals; however, the mechanism is unclear. As a result, the present study analyzed the effects of yeast cultures on the growth properties, microbiome, and metabolism of weaned lambs. In this study, a total of 20 weaned lambs were randomly, stochastically divided into four teams: the control group (Group A) were fed a basic diet; Group B were fed with Saccharomyces cerevisiae BC strain culture (30 g/head/d); Group C were fed with Kluyveromyces marquez XR4 strain culture (30 g/head/d); Group D were fed with a composite culture of the two yeast strains (30 g/head/d). The study lasted for 40 days, with daily records of lamb feed intake and weight. Lamb feces were collected regularly for metagenomic sequencing and metabolomics analysis. The average daily weight gain and average daily yield of Group D lambs were significantly higher than those of Group A lambs (p < 0.01). The feed utilization rate in the yeast-fed groups was considerably higher than in the control group (p < 0.05), indicating that the addition of yeast crops to lamb feed might improve lamb feed performance. Bacteroides and the mTOR signaling pathway were dramatically enriched in the intestines of weaned lambs in the yeast-culture-fed groups, and their expression levels of ketones and benzoic acid compounds were significantly upregulated. These results indicated that yeast culture had excellent effects on weaned lambs in regulating immunological functioning and the intestinal environment, protecting the enteric mucosal barrier, improving digestion and nutritional absorption, and enhancing antioxidant function. In summary, adding yeast culture to weaned lamb feed can generate a positive effect on its productivity performance and gut health. These findings provide novel insights into promoting the health of young ruminants.

1. Introduction

Beef, mutton, milk and other commodities produced by ruminants are important sources of animal protein for human beings. Improving the growth performance of ruminants is an important direction of research in the field of ruminants. Young ruminants face a series of changes, such as nutrition, body development and environment, during the growth process, generating long-term impacts for them [1]. Therefore, the health of young ruminants is particularly noteworthy. For young ruminants, the development level of the digestive tract during childhood determines the level of production performance in adulthood. Accordingly, ensuring the enteric health of young ruminant animals is extremely vital. At different stages of growth in young ruminants, the colonization of gut microbiota is crucial and is one of the factors affecting normal digestive function [2]. It could participate in digestion and nutritional absorption as well as regulating host metabolism and health [3]. After a decrease in the richness and diversity of the gut microbiota, the immune status of the gut also changes, and the body’s susceptibility to pathogenic microorganisms in the gut increases [4]. Establishing a stable symbiotic microbial community, which promotes intestinal development and improves intestinal barrier function, can lead to better utilization of intestinal microorganisms to enhance animal immunity, decrease the incidence of gastrointestinal diseases, and thereby ameliorate animal production performance [5]. The factors that affect the colonization of gastrointestinal microbiota in young ruminants include solid feed, host genotype, feeding management, and environment. In recent years, researchers have suggested that the early regulation of the colonization process of gastrointestinal microbiota through feed, additives, and probiotics may improve the growth performance of ruminants and generate long-term impacts [6].

Microecological preparations are live microbial preparations made from normal microorganisms or substances that promote microbial growth, mainly including live bacteria, dead bacteria, and bacterial metabolites. Microecological preparations have the irreplaceable advantages of drugs, namely “treating diseases, preventing diseases before they occur, and providing health care without diseases” [4]. Numerous studies have indicated that microecological preparations have multiple effects. For instance, microecological preparations could stimulate the activity of dominant intestinal microbial communities (e.g., anaerobic bacteria) such as anaerobic bacteria in the intestine and maintain the balance of gut microbiota [6]. Meanwhile, it could secrete short-chain fatty acids, such as acetic acid, propionic acid and butyric acid, in order to inhibit the growth of pathogens and intestinal inflammation [7]. Furthermore, it could secrete proteases, lipases, and cellulases to accelerate macromolecule digestion and absorption and thereby enhance growth performance, ameliorate the activity of various enzymes of digestion in the gut, facilitate the digestion and absorption of nutrients, and improve growth productivity [8]. Therefore, adding yeast microbiota preparations in the early stages of lambs might promote microbial colonization and fibrinolytic potential in the lambs’ gastrointestinal tract, improve intestinal development and digestibility, and promote the growth and development of lambs [9]. Specifically, adding Saccharomyces cerevisiae culture to the diet has been proven to increase the average daily weight gain of beef cattle and regulate the gut microbiota of calves [10]. In lambs, yeast cultures have the effect of improving weaning stress and promoting the digestion of nutrients [11].

Biological science has achieved tremendous technological advances in the past few decades, ushering in a new era of research that encompasses systems biology. Systems biology seeks to investigate organisms as integral wholes or systems and to comprehensively analyze the responses of biological systems to diseases, genes, and environmental factors through omics-based analyses [12]. Metagenomics can be used to characterize the taxonomic structure and functional potential of microbial assemblages, as well as to reconstruct complete genome sequences, thereby enabling the inference of microbial community biological functions and the exploration of correlations between the gut microbiota and their hosts [13]. Non-targeted metabolomics primarily compares the metabolomes (all metabolites produced by a specific organism) of control and experimental groups to identify differences in their metabolite profiles. Through reverse engineering of known metabolic pathways, regulatory enzymes and genes can then be identified, which facilitates research into disease pathogenesis, the mechanisms of effective drug therapies, and other related fields [14]. The integration of metagenomics and metabolomics allows for a more systematic characterization of differences in the host gut microbiota, their functional capacities, and metabolic features [15].

Saccharomyces cerevisiae BC and Kluyveromyces marxianus XR4 are two yeast strains isolated by the College of Veterinary Medicine, Inner Mongolia Agricultural University, from naturally fermented mare’s milk and silage collected in the pristine grasslands of Inner Mongolia [16]. Yeast cultures fermented from these strains exhibit unique biological functions, including enhancing the digestive and absorptive capacity of young ruminants, regulating the gut microbiota, maintaining intestinal health, and modulating glucose and lipid metabolism. However, the intestinal health of lambs is highly susceptible to various stressors during early life, such as weaning, long-distance transportation, and gut microbiota dysbiosis [17]. Additionally, the digestive tract of ruminants has a complex structural organization, and research into the effects of yeast microecological preparations on the intestinal microbiota and metabolism of young ruminants remains limited and, thus, warrants urgent investigation. The impacts of yeast cultures on the gut microbiota and metabolic profiles of young ruminants are still not fully elucidated, particularly when novel yeast strains and multi-omics analytical approaches are employed. We hypothesize that supplementing the diet of weaned lambs with yeast cultures may uniquely alter their gut microbiota and metabolic profiles, thereby improving growth performance and intestinal health. The present study aims to evaluate these effects using an integrated metagenomic and metabolomic approach, which offers a novel perspective in this research field.

We postulate that the dietary supplementation of yeast microecological preparations for weaned lambs may improve growth performance and intestinal health by modulating the gut microbiota and their metabolite profiles and that these changes may exert a positive regulatory effect on intestinal homeostasis in post-weaned lambs. Therefore, the primary objective of this study is to assess the impacts of dietary yeast microecological preparation supplementation on the growth performance, gut microbiota, and metabolic profiles of weaned lambs.

2. Materials and Methods

2.1. Yeast Culture Preparation

S. cerevisiae BC strain and K. marxianus XR4 strain, which exhibit good fermentation characteristics, were isolated in the laboratory from naturally fermented mare’s milk and silage, and pilot production was implemented at Kehong Feed Co., Ltd. (Inner Mongolia, China). The base substrate formulation includes 12% bran, 12% spray corn bran, 10% corn, 10% rice bran, 10% cottonseed meal, 8% red dog wheat, 28% corn germ meal, and 10% soybean meal. S. cerevisiae BC (3 × 10⁸ CFU/g), K. marxianus XR4 (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 °C. The nutritional components of the mixed feed and yeast cultures were tested according to national standards and are presented in

Table 1. Nutrient composition of mixed feed and yeast culture (feed-based).

Item	MF	B	C	D
Crude protein %	19.23	20.35	20.55	20.39
Dry matter %	92.04	93.23	92.58	93.21
Neutral detergent fiber %	34.11	32.45	33.72	34.23
Acid detergent fiber %	20.02	19.42	19.23	19.97
Live yeast cells, CFU/g	-	6.8 × 104	1.5 × 104	7.2 × 104
Lactic acid mmol/kg	-	371.13	391.57	380.31
Cellulase U/g	-	0.03	0.88	0.91
Protease U/g	2.67	9.56	8.23	9.88
Amylase U/g	3.69	6.11	7.28	7.65

MF = mixed feed; B = mixed feed inoculated with the S. cerevisiae BC strain; C = mixed feed inoculated with the K. marxianus XR4 strain; D = mixed feed inoculated with the mixed bacteria of BC strain and XR4 strain.

. Using the Kjeldahl nitrogen analyzer (Kjeltec 8400, FOSS, Hilleroed, Denmark), the digestion, distillation, and titration of the sample were completed to calculate the crude protein content. Using oven drying method, we placed the sample in a 105 °C oven to dry to constant weight and calculated the proportion of dry matter based on the weight loss of the sample. Using the van der Waals washing fiber method, the separation and quantification of neutral washing fiber (NDF) and acidic washing fiber (ADF) components were achieved through a fiber analyzer (ANKOM A2000i, ANKOM Technology, Macedon, NY, USA). Using the plate coating method, the sample was gradient diluted and coated onto Bengal red medium. After 48 h of cultivation at 30 °C, the effective colony count (CFU/g) of live yeast cells was calculated. High-performance liquid chromatography (HPLC) was used, with Agilent ZORBAX SB Aq (Agilent Technologies, Santa Clara, CA, USA) (4.6 mm × 250 mm, 5 μm) as the chromatographic column, 0.01 mol/L potassium dihydrogen phosphate solution (pH = 2.5) as the mobile phase, flow rate of 0.8 mL/min, column temperature of 30 °C, UV detection wavelength of 210 nm, and external standard method for quantitative analysis of lactic acid. The activities of cellulase, protease, and amylase in the yeast cultures were assayed using corresponding kits from Boxbio (Beijing, China) in strict accordance with the manufacturer’s instructions.

2.2. Experimental Design and Diets

This experiment was carried out at Fuchuan Farm (Bayannur, Inner Mongolia, China). Using a fully randomized design, 20 two-month-old weaned male lambs (Dorper × Thin-tailed Han) in good health (23.5 ± 2.85 kg) were assigned at random to four equally sized groups (n = 5/group), such that the average weights of animals in all groups were comparable. Each lamb was enclosed separately in a fence (about 2 m²). The control group (Group A) was provided with a basic diet, while Group B received a basic diet supplemented with 30 g/head/day of BC culture, Group C received a basic diet supplemented with 30 g/head/day of XR4 culture, and Group D received a basic diet supplemented with 30 g/head/day of a mixed culture of BC and XR4 at a 1:1 ratio (50% each) [16]. The daily concentrate ration was served to lambs in equal portions at 08:00 and 19:00 every day, and forage was provided ad libitum for the lambs. At the same time, animals had free access to drinking water. The entire period of the experiment was 47 days, including 7 days of adaptation. The content of the main forages and nutrients is shown in

Table 2. Dietary composition and nutrient levels of the basal diet (%, DM basis).

Ingredients	A	B	C	D
Peanutvine	9.30	9.30	9.30	9.30
Corn stalk	10.00	10.00	10.00	10.00
Sunflower seed skin	4.00	4.00	4.00	4.00
Alfalfa meal	10.00	10.00	10.00	10.00
Corngrain	29.00	29.00	29.00	29.00
Soybean meal	6.00	6.00	6.00	6.00
Germ meal	8.00	8.00	8.00	8.00
Cottonmeal	6.00	6.00	6.00	6.00
DDGS Distillers Dried Grainswith Solubles	6.00	6.00	6.00	6.00
Sunflower Cakes	7.50	7.50	7.50	7.50
NaCl	0.50	0.50	0.50	0.50
Limestone	0.5	0.5	0.5	0.5
CaHPO4	0.50	0.50	0.50	0.50
4%Premix 1	2.7	2.7	2.7	2.7
Total	100.00	100.00	100.00	100.00
Nutrient levels
ME 2, MJ/kg	10.19	10.21	10.18	10.22
Dry matter %	89.77	88.25	88.13	87.75
Crude protein %	15.62	16.23	15.98	16.85
Neutral detergent fiber %	33.35	34.03	33.45	33.38
Acid detergent fiber %	21.40	20.99	21.38	21.59

¹ The premix provided the following per kg of the diet: Fe 60 mg, Cu 12 mg, Zn 60 mg, Mn 45 mg, nicotinic acid 60 mg, I 0.6 mg, Se 0.2 mg, vitamin A 3500 IU, vitamin D 1200 IU, vitamin E 20 IU, Ca 2 g, P 1 g, Co 20 mg, NaCl 5 g. A, B, C, and D represented the composition and nutritional components of the basal diet for lambs in the A, B, C, and D groups respectively. ² Metabolizable energy (ME) was calculated as described [12].

. Feed consumption and lamb weight were recorded daily. Fecal samples were collected from each group at three time points: day 0, 30, and 40.

2.3. Sample Collection

Fecal samples were collected from each lamb in all groups at 08:00 a.m. on days 0, 30 and 40 of the formal experiment (Section 2.2), following the actual operational procedures implemented in this study. Prior to sampling, the sampling area was sanitized and subjected to ultraviolet (UV) sterilization for 30 min. All sampling tools were sterilized under UV light on an ultra-clean workbench and individually labeled with the corresponding lamb identification numbers for subsequent use. Sampling personnel wore sterile lab coats, disposable face masks and hair covers; their hands were thoroughly washed and disinfected with 75% alcohol before donning disposable polyethylene (PE) gloves. Each lamb was securely fixed in a dedicated restraint frame: sterile cotton swabs were used to remove fecal debris and dirt around the anus, followed by spiral disinfection of the anus and its 2 cm surrounding area with 75% alcohol for three consecutive passes. After complete evaporation of the alcohol, the personnel changed into new sterile PE gloves, then inserted a gloved finger 3–5 cm into the lamb’s rectum to induce defecation, and collected 5–8 g of fresh fecal material directly into sterile 50 mL centrifuge tubes. Gloves were replaced between sampling each individual lamb to avoid cross-contamination. Immediately after collection, each centrifuge tube was tightly sealed and snap-frozen in liquid nitrogen for at least 10 min. All frozen fecal samples were then placed in a dry ice-insulated container and transported to the laboratory within 2 h of collection, followed by long-term storage in a −80 °C ultra-low-temperature freezer for subsequent metagenomic and metabolomic analyses. Sterile controls and procedural blank controls were included for each batch of sampled feces. Metagenomic sequencing was performed on all control samples to verify the absence of exogenous contamination; only samples with contamination-free controls were deemed qualified and included in subsequent bioinformatic analyses.

2.4. Extraction and Detection of Metagenomic Samples

The OMEGA Soil DNA Kit (M5635-02) manufactured by OMEGA Bio-Tek (Norcross, GA, USA) was utilized for the isolation of DNA from 60 excrement specimen, followed by assessment of DNA concentration, integrity, and purity via the Agilent 5400 system.

2.5. Construction and Sequencing of Metagenomic Libraries

We used NEBNext according to the procedure recommended by the manufacturer ^® Ultra ™ DNA library preparation kits ((NEB, Ipswich, MA, USA) Catalog number: E7370L) and unique index code for building library sequencing. In short, the DNA sample of the genome was fragmented to 350 bp by ultrasound processing, and the fragments were recovered at the end and by drag, Illumina compound and further PCR expansion. AMPure XP (Beckman Coulter, Beverly, MA, USA) was used to clean PCR products. Library quality was inspected using an Agilent 5400 instrument, and quantitative evaluation was performed via qPCR at a concentration of 1.5 nM. Depending on the concentration of the effective library and the required amount of data on the Illumina platform, the PE150 sequencing strategy was used to collect and sequence the corresponding library.

2.6. Metagenomic Bioinformatics Analysis

This study used the high-performance Illumina Novas-eq sequencing platform for macro genomic sequencing to obtain primary data on the sequencing of bacteria, fungi and viruses in sheep excrement samples (baseline data). To ensure the reliability and accuracy of the information used for subsequent analysis, it is necessary to pre-process the resulting original sequenced data using Knead Data software (v0.12.0). Specific operational steps are as follows:

(1)

Removed connector sequences (based on Trimmatic, parameter:ILLUMINACLIP:adapters_path: 2:30:10) and low-quality sequences (default quality threshold ≤ 20) from the original data (based on Trimmatic, parameter:SLIDINGWINDOW: 4:20) and removed sequences with a final length less than 50 bp (based on Trimmatic, parameter: MINLEN: 50).

(2)

Given the likelihood of contamination by the host, it is necessary to compare the clean data with the host genome, and the Bowtie2 software (v2.5.4, http://bowtie-bio.sourceforge.net/bowtie2/index.shtml, accessed on 15 May 2025; Parameters:—Very sensitive) was used by default to filter sequences from the host and obtain valid sequences for subsequent analysis.

(3)

Finally, the rationality and effectiveness of quality control were tested through FastQC [18,19,20].

Kraken2 and the self-build microbial nucleic acid database (screening sequences belonging to bacteria, fungi, archaea, and viruses in the NCBI NT nucleic acid database and RefSeq whole genome database) were used to compare and compute the number of sequences containing species in the sample, and then Bracken was used to divine the actual relative abundance of species in the sample. Kraken2 is the newest comparison software based on K-mer. The local Kraken2 database was used to contain 16,799 known bacterial genomes [21,22,23,24].

The clean reads and de-host sequences were blasted against the database (UniRef90, based on DIAMOND) using HUMAnN2 software (v2.8.1), it is possible to obtain annotated information and a table of relative abundance for each functional database based on the match between the UniRef90 ID and each database [25,26,27,28]. Based on the species and functional abundance table, cluster sediment density analysis, primary coordinate analysis (PCOA) and hypometric analysis (species only) on non-metric multidimensional scales (NMDS) can be performed, as well as sample cluster analysis. Using cluster information, linear discriminatory analysis, effect magnitude biomarkers (LEfSe), and the Dangler test can be conducted to study discrepancies in species and functional composition between samples [29].

The DIAMOND software (v2.0.15) was employed to perform blast analysis and functional annotation of the quality-controlled, host-depleted clean reads from each sample against the Comprehensive Antibiotic Resistance Database (CARDS) (parameter: −e 0.001 (e-value < 1 × 10⁻³) −i 80 (percent identity > 80%), so as to obtain the relative abundance of potential antibiotic resistance genes in each sample.

2.7. Extraction and Detection of Metabolomic Samples

Fecal samples (100 mg) previously ground in liquid nitrogen were placed into a 1.5 mL centrifuge tube. Then, 500 µL of an 80% methanol aqueous solution was added [30]. The mixture was vortexed in an ice bath for 5 min, followed by centrifugation at 15,000× g and 4 °C for 20 min. The supernatant was diluted with mass spectrometry-grade water to a final methanol concentration of 53%. After a second centrifugation under the same conditions (15,000× g, 4 °C, 20 min), the supernatant was collected and subjected to LC-MS analysis [31]. The chromatographic conditions are as follows: column, Hypersil Gold column (C18); column temperature, 40 °C; flow rate, 0.2 mL/min. For positive-ion mode, mobile phase A is 0.1% formic acid and mobile phase B is methanol. For negative-ion mode, mobile phase A is 5 mM ammonium acetate (pH 9.0) and mobile phase B is methanol. The mass spectrometry conditions are as follows: scanning range, m/z 100–1500; ESI source settings: spray voltage, 3.5 kV; sheath gas flow rate, 35 psi; auxiliary gas flow rate, 10 L/min; capillary temperature, 320 °C; S-lens RF level, 60; auxiliary gas heater temperature, 350 °C; polarity, positive and negative. Data-dependent MS/MS scanning is employed [32].

2.8. Metabolomic Profiling Analysis

The acquired data files (.raw) are imported into CD 3.1 search software. Processing and preliminary screening of parameters such as retention time and mass-to-charge ratio for each metabolite are performed. Peak alignment is conducted with retention time and mass deviations set to 0.2 min and 5 ppm, respectively, to ensure accurate calibration. For peak extraction, parameters are set as follows: mass deviation, 5 ppm; signal intensity deviation, 30%; signal-to-noise ratio, 3, along with defined minimum signal intensity and ion addition criteria. Peak areas are quantified, and target ions are integrated. Molecular formulas are predicted based on molecular ion peaks and fragment ions, and the results are compared against the mzCloud https://www.mzcloud.org/ (accessed on 30 May 2025), mzVault, and Masslist databases. Background ions identified in blank samples are removed. The original quantitative results are normalized to finally obtain metabolite identification (using R 3.4.3) and relative quantitative results (using Python 3.5.0). Data processing is performed on a Linux operating system (CentOS version 6.6). Identified metabolites are annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database https://www.genome.jp/kegg/pathway.html (accessed on 3 June 2025), HMDB database https://hmdb.ca/metabolites (accessed on 20 June 2025), and LIPIDMaps database http://www.lipidmaps.org/ (accessed on 29 June 2025) (using Python 2.7.6 and R 3.4.3) [33].

2.9. Statistical Analyses

The data analysis was conducted using SPSS 22.0 software, and the statistical results were obtained using GraphPad Prism (v 8.0) software. p < 0.05 indicated significant difference, while 0.05 ≤ p < 0.10 indicated significant trend. We performed microbial community analysis at the ASV level and used Wilcoxon rank sum test to calculate richness estimates (ACE and Chao1) and alpha diversity index. The abundance of taxa at the genus and phylum levels was compared with Python 2.8. Wilcoxon rank sum test was used to identify the differences in the most abundant genera between groups. The p-value was corrected for false discovery rate (FDR). We used Analyst 1.6.3 to analyze and process mass spectrometry data. We used principal component analysis (PCA) (R 3.4.3) and discriminatory analysis of orthogonal projection (OPLS-DA) methods to analyze the extracted fecal sample peaks in metabolomics analysis. Based on the predictive variable importance (VIP) ≥ 1.00 and inter group fold-change (FC) ≥1.2 or ≤0.5, we selected metabolites with significant differences in abundance between groups. The enrichment differences of various metabolic pathway modules were assessed by annotating the KEGG database online and performed Spearman correlation analysis in R (v 3.2.1) to detect the correlation between gut microbiota and metabolites in experimental lambs [34].

2.10. Nucleotide Sequence Accession Numbers

The raw sequence data reported in this study were archived in the Genome Sequence Archive [35] in National Genomics Data Center [36], China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA018278), publicly accessible at https://ngdc.cncb.ac.cn/gsa (accessed on 6 July 2025).

3. Results

3.1. Growth Performance

The dynamic changes in the body weight of the lambs during the experimental period are summarized in

Table 3. The effect of yeast probiotics on the weight of weaned lambs.

Items	Groups				SEM	p-Value
	A	B	C	D
Initial mean weight, kg	22.26	21.79	21.57	22.45	0.204	1.2 × 10−1
Average body weight on day 40, kg	33.76	34.11	33.97	35.37	0.363	4.0 × 10−1
ADG, kg/d	0.29 a	0.31 ab	0.31 ab	0.32 b	0.007	1.2 × 10−4
ADFI, kg/d	1.52 a	1.54 ab	1.55 ab	1.64 b	0.027	2.0 × 10−3
F:G	5.24 a	4.97 b	5.00 ab	5.13 ab	0.062	1.4 × 10−2

ADG = average daily gain; ADFI = average daily feed intake; F:G = the ratio of feed intake to body weight gain. ^a,b Within a row, values with different superscripts differ significantly at p < 0.05. Data are expressed as means and SEM, n = 5/group.

. The ADG and ADFI values of lambs in Group D were significantly higher than those in Group A (p < 0.01); meanwhile, the feed-to-gain ratio (F:G) of experimental Groups B, C, and D showed significant statistical differences compared to Group A (p < 0.05).

3.2. Effects of Yeast Culture on Lamb Intestinal Microbiota Composition and Diversity

In this study, the microbial community maps of these samples were described using MetaPhlAn3. The relative intensity of bacterial genomes was calculated based on the average coverage of each metagenome and normalized by the overall readings of each sample. We selected the top 20 features in terms of abundance percentage among different groups. At the phylum level, Bacteroidota (60%) and Bacillota (17%) were the dominant bacterial phyla in lamb intestines (Figure 1A). As the experiment progressed, the proportion of Bacteroidota and Bacillota showed a decreasing trend but still dominated the relative content of intestinal bacteria in weaned lambs. The relative content of Bacillota remained relatively stable in the yeast culture groups (Group B, Group C, and Group D), and yet it showed significant fluctuations in the control group (Group A) (Figure 1C).

Subsequently, discrepancies in the gut microbial community components at the genus level between the control group and the yeast culture-supplemented groups of weaned lambs were investigated (Figure 2A). At day 0, Prevotella (with an average relative content of 33%) was predominant in the intestinal tract of weaned lambs, followed by Bacteroides (with an average relative content of 10%). However, as the experiment progressed, Prevotella in the yeast culture groups decreased, while Bacteroides began to increase (Figure 2B,C). Bacteroides became the dominant bacterial genus in the yeast culture groups for weaned lambs (with an average relative content of 21%), wherein the Bacteroides in the gut of weaned lambs in the compound yeast culture group (group D) reached as high as 36% on day 40.

To further investigate the impact of yeast culture on intestinal bacteria in weaned lambs, we investigated the changes in bacteria at the species level. On day 0, the dominant bacterial species in the intestinal tract of weaned lambs were Prevotella compri (33%) and Phocaeicola vulgatus (5.8%). As the lamb gut developed, the dominant bacterial composition of the yeast culture group changed to Prevotella compri and Bacteroides uniformis, especially at 30–40 days, where the comparative content of Prevotella compri rapidly decreased, while the correlative content of Bacteroides uniformis increased, becoming the main dominant bacterial species in the lamb gut. Among them, the lamb gut microbiota in the compound yeast culture group (Group D) changed most dramatically (Figure 3).

We conducted a horizontal comparison of gut microbiota diversity at different time points and found that the XR4 yeast culture group (group C) and the composite yeast culture group (group D) had higher gut microbiota diversity than the control group (Figure 4A); this phenomenon disappeared at day 40, although the entire process was not significant (p > 0.05) (Figure 4B). Principal coordinate analysis (PCoA) was used to investigate the impact of yeast culture on gut microbiota diversity in weaned lambs, and the PCoA findings demonstrated that yeast culture did not exert an obvious effect on the variety of the gut microbial community in weaned lambs (Figure 4C,D).

3.3. Effect of Yeast Culture on the Function and Metabolism of Intestinal Microbiota in Lambs

The functional metagenome was annotated using KEGG to compare the functional differences in gut microbiota in different experimental groups. The results demonstrated that the relative abundance of alpha linolenic acid metabolism in Group A was higher than other groups on day 30. Ubiquinone and other terpenoid quinone biosynthesis were enriched in Group B. Type I diabetes mellitus was enriched in Group C, and the mTOR signaling pathway was enriched in Group D, as shown in Figure 5A.

On day 40, arachidonic acid metabolism, platinum drug resistance, primary bile acid biosynthesis, alpha linolenic acid metabolism, secondary bile acid biosynthesis, and beta lactam resistance were enriched in Group A. Shigellosis, bacterial invasion of epithelial cells, oxidative phosphorylation, and ubiquinone and other terpenoid quinone biosynthesis were enriched in Group B. Atrazine degradation was enriched in Group C. Napoleon degradation and synthesis and degradation of ketone bodies were enriched in Group D, as shown in Figure 5B.

The metabolic analysis results (Figure 6) indicated that the intestinal metabolism of weaned lambs could be divided into three main branches. Firstly, the first branch was the intestinal metabolites on day 0, mainly consisting of jasmone, 22 (S)-hydroxycholesterol, 1-(6-methyl-3-pyridyl) ethan-1-one O1-ethoxyxime hydrochloride, TKK, and Ip7G, indicating significant changes in metabolites as the lambs’ intestine developed.

Secondly, the second branch appeared on day 30, and there were significant differences in intestinal metabolites between the yeast culture groups and the control group of weaned lambs. The expression levels of metabolites in the yeast culture groups, including pyrithioxin, 3-benzyl-1-butyl-4-hydroxy-1,2-dihydroquinolin-2-one, 1-(2,4-dihydroxyphenyl)-2-(3,5-diethoxyphenyl) propan-1-one, 5,7-dihydroxy-2-phenyl-4H-chromen-4-one, phyloquinone, (2E)-3-(3,4-dihydroxyphenyl) prop-2-oic acid, 4-(hydroxymethyl) benzoic acid, 4-hydroxybenzaldehyde, epoxicin, and 5,6-dihydroindole-2-carbonylic acid, were considerably higher than those in the control group, wherein the expression levels of (2E)-3-(3,4-dimethylphenyl) prop-2-enoic acid and 4-(hydroxymethyl) benzoic acid in Group D were significantly higher than in Groups B and C. This suggested that the yeast culture might have a positive impact on the intestinal metabolism of lambs, promoting the production of some beneficial metabolites.

Finally, the third branch mainly appeared on the 40th day of the experiment, with a significant decrease in metabolic function in the yeast culture groups and a reduced difference compared to the control group. However, it is worth noting that the control group showed elevated levels of spermine, 3-hydroxyanthranilic acid, lithocholic acid, N1-[4-(cyanomethyl) phenyl]-4-chlorobenzamide, and cadaverine in intestinal metabolism on day 40. This might reflect some changes in intestinal metabolism in weaned lambs without yeast culture supplementation.

In order to explore the intrinsic link between dominant intestinal bacterial genera and differential metabolites in weaned lambs, and further clarify the prominent disparities in metabolic products between the yeast culture groups and the control group, and the reasons for the positive effects on intestinal metabolism of yeast culture in lambs, we conducted an association investigation between dominant bacterial genera and differential metabolites in the intestinal tract of lambs through Procrustes analysis. In the figure, the (genus level, top 30, p < 0.05) nodes of dominant bacteria in all samples were connected to all significantly correlated metabolites (VIP > 1.0, p < 0.05). These consequences indicated that the four metabolites, lithocholic acid, 22-S-hydroxycholesterol, 4-hydroxybenzaldehyde, and 4-hydroxymethyl benzoic acid, had significant differences. Among them, 22-S-hydroxycholesterol and 4-hydroxymethyl benzoic acid were considerably positively correlated with Escherichia and Prevotella in the intestine, respectively. The four differential metabolites had significant negative correlations with various bacteria in the gut, such as 22-S-hydroxycholesterol with Prevotella, as well as Faecalibacterium, 4-hydroxymethyl benzoic acid with Faecalibacterium and Escherichia, and so forth. These results indicated that there might be some correlations between microorganisms and metabolites, which needed to be explored and discussed further (Figure 7).

4. Discussion

As one of the densest microecologies on Earth, gut dysbiosis may predispose hosts to a wide range of chronic conditions, including metabolic disorders and nutritional imbalances [37]. Supplementation with microecological agents in ruminant diets exerts a positive regulatory effect on the gut microbial community of young ruminants. Yeast culture, a microecological preparation produced via specific processes, has been shown to promote the establishment of intestinal microbiota and reduce the incidence of intestinal diseases in young ruminants [12]. To fully explore the functionality of yeast cultures, their effects on the intestinal tract of young ruminants, and the underlying mechanisms, rigorous scientific evidence derived from extensive clinical trials is essential. Metagenomic and metabolomic analyses have become widely used tools for investigating animal gut microbiota. In the present study, we integrated metagenomic sequencing and metabolomics to evaluate how two proprietary strains S. cerevisiae BC and K. marxianus XR4 affect the composition, interactions, functions, and metabolic profiles of gut microbiota in weaned lambs. Our findings characterize the changes in gut microbiota following dietary yeast culture supplementation and identify a large number of previously uncultured microbial sequences.

Weaning stress often leads to reduced forage intake in young ruminants, which hinders their growth and development. Yeast, as a beneficial fungus, possesses diverse biological functions, including improving animal growth performance [38], enhancing the transcriptional levels of anti-inflammatory factors, and promoting the proliferation of beneficial intestinal bacteria [39]. In this study, we continuously recorded the body weight and feed intake of weaned lambs over a 40-day period. The results indicated that dietary yeast culture supplementation significantly increased growth performance indices, including average daily gain (ADG) and average daily feed intake (ADFI), compared with the control group; concurrently, feed utilization efficiency was also substantially improved. Notably, weaned lambs receiving the compound yeast culture (Group D) exhibited significantly higher ADG and ADFI than those in the control group (p < 0.01), although no significant differences were observed among the three yeast culture-supplemented groups. These findings demonstrate that dietary yeast culture can enhance the production traits and feed utilization efficiency of weaned lambs, with the compound yeast culture exerting a particularly pronounced effect. However, the lack of significant variation between the individual yeast strain groups suggests that strain type contributes minimally to the observed phenotypic improvements.

The colonization and establishment of gut microbiota play a pivotal role in the development and function of young ruminants and are closely associated with their daily weight gain and feed utilization efficiency. Multiple factors directly or indirectly influence microbial colonization and intestinal development in ruminants, including maternal pregnancy, drinking water quality, weaning stress, the type and timing of roughage intake, and age [40]. Following weaning, lambs transition from milk or milk replacers to a solid diet, requiring reshaping of their gut microbiota to adapt to new nutrient sources [9]. Weaning is, thus, a critical disturbance point for the intestinal microecology of lambs [41]. In the present study, lambs in the control group (fed only the basal diet) exhibited significant fluctuations in the abundance of Bacillota, a core intestinal microbiota phylum, due to inadequate alleviation of weaning stress. In contrast, the yeast culture-supplemented groups maintained stable Bacillota abundance by secreting short-chain fatty acids (SCFAs) and digestive enzymes, which stabilized the intestinal environment [40]. In the control group, reduced Bacteroidetes abundance and insufficient activation of the mTOR signaling pathway disrupted the synergistic metabolism between Bacillota and intestinal immune homeostasis, exacerbating fluctuations in Bacillota abundance and leading to instability in bacterial community metabolism and survival. Conversely, yeast culture supplementation enriched Bacteroidetes and activated the mTOR pathway, ensuring the stability of bacterial community metabolism and survival. The more pronounced fluctuations in Bacillota abundance in the control group compared with the yeast-supplemented groups were confirmed at the functional level, highlighting the regulatory value of yeast microecological preparations in enhancing intestinal bacterial community stability and nutritional metabolic efficiency in lambs [42]. Typically, Prevotella is a member of the normal gut microbiota in humans and animals, with a primary function in carbohydrate digestion, particularly the breakdown of complex polysaccharides. Consequently, the relative abundance of Prevotella, which is closely linked to plant polysaccharide degradation, increases significantly in response to high-fiber diets; similarly, carbohydrate-digesting genera such as Megasphaera and Blautia also exhibit increased abundance [43]. Consistent with these observations, Prevotella was the dominant genus in the intestines of weaned lambs across all groups at the start of the experiment. However, previous studies have shown that high Prevotella abundance is often associated with lower Bacteroidetes abundance in the gut, as these two genera directly compete for intestinal niches [44]. Importantly, Bacteroides benefits hosts by regulating immune, metabolic, and nutritional functions, and reduced Bacteroidetes abundance post-weaning may increase the risk of intestinal diseases in young animals [45]. In the present study, as the experiment progressed, Prevotella abundance decreased, while Bacteroides abundance increased in the yeast culture-supplemented groups. By the end of the experiment, Bacteroides abundance reached as high as 36% in the compound yeast culture group (Group D). This finding indicates that S. cerevisiae BC and K. marxianus XR4 strains can optimize the compositional distribution and structural organization of the intestinal microbiota in weaned lambs, increase Bacteroides relative abundance, and reduce disease risk, effects that are particularly prominent with the compound yeast culture. These results suggest that S. cerevisiae BC and K. marxianus XR4 strains have the potential to regulate immune function in weaned lambs.

The gut microbiota co-develops with the host from birth and plays a crucial role in regulating host metabolic pathways and mediating complex interactions between host physiology, microbial metabolism, and signal transduction. A comprehensive understanding of these host–microbiota interactive mechanisms is a prerequisite for optimizing gut health, manipulating the microbiota to combat diseases, and improving overall host health [46]. These interactions are largely driven by coordinated metabolic exchanges. In this study, functional analysis revealed a significant enrichment of the mTOR signaling pathway in the gut microbiota of the compound yeast culture group. Since the mTOR pathway is known to be involved in diverse processes, including protein synthesis, autophagy, immune modulation, and bone growth [47], its enrichment here suggests that the compound yeast culture may exert beneficial effects on the host, potentially including the regulation of immune function, through this key signaling pathway.

The integrity of the intestinal mucosal epithelium is a critical prerequisite for efficient nutrient absorption and the formation of a robust immune barrier. In young animals, weaning stress can compromise this integrity, leading to pathogenic invasion, impaired nutrient utilization, and growth delays [48]. Therefore, maintaining mucosal structure is fundamental to health. Research shows that ketone compounds preserve the intestinal epithelium through multiple mechanisms, including scavenging free radicals [49], regulating enzyme activity [50], and promoting cell proliferation [51]. Their broad pharmacological potential—encompassing anti-allergic, anti-diarrheal, anti-ulcer, and anti-inflammatory effects—has drawn significant research interest [52]. In line with this, our differential metabolite analysis revealed that the levels of specific ketone metabolites were significantly upregulated in lambs supplemented with yeast culture, compared to the control group. This finding indicates that yeast culture contributes to protecting intestinal mucosal integrity and function in weaned lambs. In addition, the expression levels of benzoic acid compounds, such as 4-(hydroxymethyl)benzoic acid, were significantly upregulated in the yeast culture-supplemented groups. Notably, 4-hydroxybenzaldehyde, which was also significantly upregulated, can be converted to benzoic acid compounds via oxidation reactions. Its expression level was particularly higher in the compound yeast culture group than in the single-strain groups (B and C). Benzoic acid is known to possess multiple biological functions. As a weak acid with a high dissociation constant, it can lower intestinal pH, activate digestive enzymes, enhance their activity, and improve nutrient utilization [41]. Additionally, benzoic acid promotes the growth and reproduction of beneficial bacteria such as Lactobacillus while inhibiting the proliferation of pathogenic microorganisms. This reduces competitive nutrient absorption by pathogens and increases intestinal nutrient uptake [53]. Furthermore, benzoic acid can scavenge free radicals by increasing the activity of antioxidant enzymes and through direct binding of its carboxyl groups to free radicals, thereby exerting antioxidant effects [54]. These findings indicate that yeast culture supplementation significantly increases the expression levels of benzoic acid metabolites in the intestines of weaned lambs. This promotes nutritional utilization, improves the intestinal environment, and enhances antioxidant capacity, all of which positively impact lamb health. The more pronounced effects observed with the compound yeast culture may be attributed to a symbiotic relationship between the two yeast strains, a mechanism that requires further exploration.

5. Conclusions

In summary, supplementing yeast culture in the diet of weaned lambs improves their average daily gain and feed intake, thereby enhancing overall production performance. It modulates the gut microbiota composition and metabolic profile, promoting a more balanced microbial structure, enriching beneficial Bacteroides, and activating the mTOR signaling pathway. These changes collectively contribute to improved immune regulation. Furthermore, yeast culture supports intestinal mucosal barrier function, enhances nutrient utilization, and strengthens antioxidant capacity through the upregulation of ketone and benzoic acid metabolites. These findings provide a theoretical basis for developing effective ruminant microecological preparations aimed at improving intestinal health in weaned lambs.