This study delves into the impact of yeast culture (YC) on rumen epithelial development, microbiota, and metabolome, with the aim of investigating YC’s mechanism in regulating rumen fermentation. Thirty male lambs of Hu sheep with similar age and body weight were selected and randomly divided into three groups with 10 lambs in each group. Lambs were fed a total mixed ration [TMR; rough: concentrate (R:C) ratio ≈ 30:70] to meet their nutritional needs. The experiment adopted completely randomized design (CRD). The control group (CON) was fed the basal diet with high concentrate, to which 20 g/d of YC was added in the low dose YC group (LYC) and 40 g/d of YC in the high dose YC group (HYC). The pretrial period was 14 days, and the experimental trial period was 60 days. At the end of a 60-day trial, ruminal epithelial tissues were collected for histomorphological analysis, and rumen microorganisms were analyzed by 16S rDNA sequencing and rumen metabolites by untargeted liquid chromatography-mass spectrometry (LC–MS) metabolomics techniques. The results showed that YC improved rumen papilla development and increased rumen papilla length (p < 0.05), while decreased cuticle thickness (p < 0.05). The 16S rDNA sequencing results showed that YC reduced the relative abundance of Prevotella_1 (p < 0.05), while significantly increased the relative abundance of Ruminococcaceae_UCG-005, uncultured_bacterium_f_Lachnospiraceae, and Ruminococcus_1 genus (p < 0.05). Metabolomics analysis showed that YC changed the abundance of metabolites related to amino acid metabolism, lipid metabolism and vitamin metabolism pathways in the rumen. In summary, YC might maintain rumen health under high-concentrate diet conditions by changing rumen microbiota structure and fermentation patterns, thereby affecting rumen metabolic profiles and repairing rumen epithelial injury.

Background: Copy number variation (CNV) is an important source of structural variation in the mammalian genome. CNV assays present a new method to explore the genomic diversity of environmental adaptations in animals and plants and genes associated with complex traits. In this study, the genome-wide CNV distribution characteristics of 20 Tibetan sheep from two breeds (10 Oula sheep and 10 Panou sheep) were analysed using whole-genome resequencing to investigate the variation in the genomic structure of Tibetan sheep during breeding. Results: CNVs were detected using CNVnator, and the overlapping regions of CNVs between individual sheep were combined. Among them, a total of 60,429 CNV events were detected between the indigenous sheep breed (Oula) and the synthetic sheep breed (Panou). After merging the overlapping CNVs, 4927 CNV regions (CNVRs) were finally obtained. Of these, 4559 CNVRs were shared by two breeds, and there were 368 differential CNVRs. Deletion events have a higher percentage of occurrences than duplication events. Functional enrichment analysis showed that the shared CNVRs were significantly enriched in 163 GO terms and 62 KEGG pathways, which were mainly associated with organ development, neural regulation, immune regulation, digestion and metabolism. In addition, 140 QTLs overlapped with some of the CNVRs at more than 1 kb, such as average daily gain QTL, body weight QTL, and total lambs born QTL. Many of the CNV-overlapping genes such as PPP3CA, SSTR1 and FASN, overlap with the average daily weight gain and carcass weight QTL regions. Moreover, V_ST analysis showed that XIRP2, ABCB1, CA1, ASPA and EEF2 differed significantly between the synthetic breed and local sheep breed. The duplication of the ABCB1 gene may be closely related to adaptation to the plateau environment in Panou sheep, which deserves further study. Additionally, cluster analysis, based on all individuals, showed that the CNV clustering could be divided into two origins, indicating that some Tibetan sheep CNVs are likely to arise independently in different populations and contribute to population differences. Conclusions: Collectively, we demonstrated the genome-wide distribution characteristics of CNVs in Panou sheep by whole genome resequencing. The results provides a valuable genetic variation resource and help to understand the genetic characteristics of Tibetan sheep. This study also provides useful information for the improvement and breeding of Tibetan sheep in the future.

Yeast products (YP) are commonly used as rumen regulators, but their mechanisms of action are still unclear. Based on our previous studies, we questioned whether yeast products would have an impact on rumen solid-associated (SA) and liquid-associated (LA) microorganisms and alter rumen fermentation patterns. Thirty 3-month-old male sheep weighing 19.27 ± 0.45 kg were selected and randomized into three groups for 60 days: (1) basal diet group (CON group), (2) basal diet add 20 g YP per day (low YP, LYP group) and (3) basal diet add 40 g YP per day (high YP, HYP group). The results demonstrated that the addition of YP increased rumen cellulase activity, butyrate and total volatile fatty acid (TVFA) concentrations (p < 0.05), while it decreased rumen amylase activity and abnormal metabolites, such as lactate, lipopolysaccharides (LPS) and histamine (HIS) (p < 0.05). Metagenomic analysis of rumen microorganisms in three groups revealed that YP mainly influenced the microbial profiles of the SA system. YP increased the relative abundance of R. flavefaciens and decreased methanogens in the SA system (p < 0.05). With the addition of YP, the abundance of only a few lactate-producing bacteria increased in the SA system, including Streptococcus and Lactobacillus (p < 0.05). However, almost all lactate-utilizing bacteria increased in the LA system, including Megasphaera, Selenomonas, Fusobacterium and Veillonella (p < 0.05). In addition, YP increased the abundance of certain GHs family members, including GH43 and GH98 (p < 0.05), but decreased the abundance of some KEGG metabolic pathways involved in starch and sucrose metabolism, biosynthesis of antibiotics and purine metabolism, among others. In conclusion, the addition of YP to high-concentrate diets can change the abundance of major functional microbiota in the rumen, especially in the solid fraction, which in turn affects rumen fermentation patterns and improves rumen digestibility.

BOLL is implicated in mammalian testicular function maintenance and spermatogenesis. To understand the expression patterns and biological functions of sheep BOLL, we examined the expression and immunolocalization of BOLL in the developing testes of Small-Tail Han sheep aged 0 days (D0), 2 months (2M), 5 months (5M), 1 year (1Y), and 2 years (2Y), by qPCR, Western blot, and immunohistochemistry methods. Firstly, morphological studies revealed that, in addition to spermatogonia, ordered and clear spermatocytes, as well as round and elongated spermatids and sperm, were found in the 1Y and 2Y testicular seminiferous tubules of the sheep testes, compared with the D0, 2M, and 5M testes, as analyzed by hematoxylin and eosin (H&E) staining. The diameter and area of the seminiferous tubules, epithelial thickness, and the area and perimeter of the tubule lumens gradually increased with age. BOLL was specifically expressed in testes and upregulation of BOLL transcript expression was higher in the testes of the 1Y and 2Y groups than in those of the D0, 2M, and 5M groups. Similarly, BOLL protein was expressed mainly in the 1Y and 2Y testes, ranging from primary spermatocytes to round spermatids, as well as in the spermatozoa. This study is the first demonstration that sheep BOLL might serve as a key regulator of the spermiogenesis involved in sperm maturity, in addition to its role as a crucial meiotic regulator.

The double sex and mab-3-related transcription factors like family C2 (DMRTC2) gene is indispensable for mammalian testicular function and spermatogenesis. Despite its importance, what expression and roles of DMRTC2 possesses and how it regulates the testicular development and spermatogenesis in sheep, especially in Tibetan sheep, remains largely unknown. In this study, DMRTC2 cDNA from testes of Tibetan sheep was firstly cloned by the RT-PCR method, and its molecular characterization was identified. Subsequently, the expression and localization patterns of DMRTC2 were evaluated by quantitative real-time PCR (qPCR), Western blot, and immunofluorescence. The cloning and sequence analysis showed that the Tibetan sheep DMRTC2 cDNA fragment contained 1113 bp open reading frame (ORF) capable of encoding 370 amino acids, and displayed high identities with some other mammals, which shared an identical DM domain sequence of 47 amino acids ranged from residues 38 to 84. qPCR and Western blot results showed that DMRTC2 was expressed in testes throughout the development stages while not in epididymides (caput, corpus, and cauda), with higher mRNA and protein abundance in Tibetan sheep testes of one- and three-year-old (post-puberty) compared with that of three-month-old (pre-puberty). Immunofluorescence results revealed that immune staining for DMRTC2 protein was observed in spermatids and spermatogonia from post-puberty Tibetan sheep testes, and gonocytes from pre-puberty Tibetan sheep testes. Together, these results demonstrated, for the first time, in sheep, that DMRTC2, as a highly conserved gene in mammals, is essential for sheep spermatogenesis by regulating the proliferation or differentiation of gonocytes and development of spermatids in ram testes at different stages of maturity.

This research is designed to examine the genetic diversity and kinship among Hu sheep, as well as to discover genes associated with crucial economic traits. A selection of 50 unrelated adult male Hu sheep underwent genotyping with the SNP50K BeadChip. Seven indicators of genetic diversity were assessed based on high-quality SNP data: effective population size (Ne), polymorphic information content (PIC), polymorphic marker ratio (P_N), expected heterozygosity (He), observed heterozygosity (Ho), effective number of alleles, and minor allele frequency (MAF). Plink software was employed to compute the IBS genetic distance matrix and detect runs of homozygosity (ROHs), while the G matrix and principal component analysis were performed using GCTA software. Selective sweep analysis was carried out using ROH, Pi, and Tajima’s D methodologies. This study identified a total of 64,734 SNPs, of which 56,522 SNPs remained for downstream analysis after quality control. The population displayed relatively high genetic diversity. The 50 Hu sheep were ultimately grouped into 12 distinct families, with families 6, 8, and 10 having the highest numbers of individuals, each consisting of 6 sheep. Furthermore, a total of 294 ROHs were detected, with the majority having lengths between 1 and 5 Mb, and the inbreeding coefficient F_ROH was 0.01. In addition, 41, 440, and 994 candidate genes were identified by ROH, Pi, and Tajima’s D methods, respectively, with 3 genes overlapping (BMPR1B, KCNIP4, and FAM13A). These results offer valuable insights for future Hu sheep breeding, genetic assessment, and population management.