A close association exists among testicular function, gut microbiota regulation, and organismal metabolism. In this study, serum and seminal plasma metabolomes, and the rumen microbiome of sheep with significant differences in sperm viability, were explored. Serum and seminal plasma metabolomes differed significantly between high-motility (HM) and low-motility (LM) groups of sheep, and 39 differential metabolites closely related to sperm motility in sheep were found in seminal plasma metabolomes, while 35 were found in serum samples. A 16S rRNA sequence analysis showed that the relative abundance of HM and LM rumen microorganisms, such as Ruminococcus and Quinella, was significantly higher in the HM group, whereas genera such as Rikenellaceae_RC9_gut_group and Lactobacillus were enriched in the mid-LM group. Serum hormone assays revealed that serum follicle-stimulating hormone (FSH) and MT levels were significantly lower in the LM group than in the HM group, whereas serum glucocorticoid (GC) levels were higher in the LM group than in the HM group, and they all affected sperm motility in sheep. Ruminococcus and other rumen microorganisms were positively correlated with sperm motility, whereas Lactobacillus was negatively correlated with FSH and GCs levels. Our findings suggest that rumen microbial activity can influence the host metabolism and hormone levels associated with fertility in sheep.

Ram sperm undergo a sequence of physiological and biochemical changes collectively termed as capacitation to perform oocyte fertilization. However, the protein changes induced by capacitation remain in need of further exploration. Thus, the present study investigated the comparative proteomic profiling in ram spermatozoa under non-capacitating (NC) and capacitating (CAP) conditions in vitro using a liquid chromatography–tandem mass spectrometry combined with tandem mass tag labeling strategy. As a results, 2050 proteins were identified and quantified; 348 of them were differentially abundant, with 280 of the proteins upregulated and 68 of the proteins downregulated between the CAP and NC spermatozoa, respectively. Functional enrichment analysis indicated that the differentially abundant proteins Prune Exopolyphosphatase 1, Galactose-1-Phosphate Uridylyltransferase, and ATP Citrate Lyase were strictly related to energy production and conversion, and Phosphoglycolate phosphatase, Glucosamine-6-Phosphate Deaminase 1 and 2 were related to metabolism, RNA processing, and vesicular transport pathways. Furthermore, the networks of protein–protein interaction indicated a strong interaction among these differential proteins in annotated pathways such as ubiquitin and transport metabolism. Our findings indicate that capacitation progress might be regulated through different pathways, providing insights into mechanisms involved in ram sperm capacitation and fertility.

The oviduct is a dynamic reproductive organ for mammalian reproduction and is required for gamete storage, maturation, fertilization, and early embryonic development, and it directly affects fecundity. However, the molecular regulation of prolificacy occurring in estrous periods remain poorly understood. This study aims to gain a better understanding of the genes involved in regulating goat fecundity in the proteome and transcriptome levels of the oviducts. Twenty female Yunshang black goats (between 2 and 3 years old, weight 52.22 ± 0.43 kg) were divided into high- and low-fecundity groups in the follicular (FH and FL, five individuals per group) and luteal (LH and LL, five individuals per group) phases, respectively. The DIA-based high-resolution mass spectrometry (MS) method was used to quantify proteins in twenty oviducts. A total of 5409 proteins were quantified, and Weighted gene co-expression network analysis (WGCNA) determined that the tan module was highly associated with the high-fecundity trait in the luteal phase, and identified NUP107, ANXA11, COX2, AKP13, and ITF140 as hub proteins. Subsequently, 98 and 167 differentially abundant proteins (DAPs) were identified in the FH vs. FL and LH vs. LL comparison groups, respectively. Parallel reaction monitoring (PRM) was used to validate the results of the proteomics data, and the hub proteins were analyzed with Western blot (WB). In addition, biological adhesion and transporter activity processes were associated with oviductal function, and several proteins that play roles in oviductal communication with gametes or embryos were identified, including CAMSAP3, ITGAM, SYVN1, EMG1, ND5, RING1, CBS, PES1, ELP3, SEC24C, SPP1, and HSPA8. Correlation analysis of proteomics and transcriptomic revealed that the DAPs and differentially expressed genes (DEGs) are commonly involved in the metabolic processes at the follicular phase; they may prepare the oviductal microenvironment for gamete reception; and the MAP kinase activity, estrogen receptor binding, and angiotensin receptor binding terms were enriched in the luteal phase, which may be actively involved in reproductive processes. By generating the proteome data of the oviduct at two critical phases and integrating transcriptome analysis, we uncovered novel aspects of oviductal gene regulation of fecundity and provided a reference for other mammals.

Sperm proteins play vital roles in improving sperm freezing resilience in domestic animals. However, it remains poorly defined which proteins regulate the freezing resilience of spermatozoa in rams (Ovis aries). Here, we compared the proteome of ram sperm with a high cryopreservation recovery ratio (HCR) with that of ram sperm with a low cryopreservation recovery ratio (LCR) using a tandem mass tag-based quantitative proteomics approach. Bioinformatic analysis was performed to evaluate differentially expressed proteins (DEPs). A total of 2464 proteins were identified, and 184 DEPs were screened. Seventy-two proteins were higher in the LCR group. One hundred and twelve proteins were more abundant in the HCR group, and they were mainly involved in the regulation of oxidative phosphorylation and thermogenesis pathways. Proteins in high abundance in the HCR group included the S100A family, such as S100A8, S100A9, S100A14, and S100A16, effectively controlling for CA²⁺ and maintaining flagella structure; HYOU1 and PRDX1, which participate in antioxidant protection and anti-apoptosis to prevent cell death; and HSP90B1, which maintains cell activity and immune response. Our results could help illuminate the molecular mechanisms underlying cryopreservation of ram semen and expand the potential direction of cryopreservation of high-quality semen.

The oviduct is associated with embryo development and transportation and regulates the pregnancy success of mammals. Previous studies have indicated a molecular mechanism of lncRNAs in gene regulation and reproduction. However, little is known about the function of lncRNAs in the oviduct in modulating goat kidding numbers. Therefore, we combined RNA sequencing (RNA-seq) to map the expression profiles of the oviduct at the luteal phase from high- and low-fecundity goats. The results showed that 2023 differentially expressed mRNAs (DEGs) and 377 differentially expressed lncRNAs (DELs) transcripts were screened, and 2109 regulated lncRNA-mRNA pairs were identified. Subsequently, the genes related to reproduction (IGF1, FGFRL1, and CREB1) and those associated with embryonic development and maturation (DHX34, LHX6) were identified. KEGG analysis of the DEGs revealed that the GnRH- and prolactin-signaling pathways, progesterone-mediated oocyte maturation, and oocyte meiosis were related to reproduction. GSEA and KEGG analyses of the target genes of DELs demonstrated that several biological processes and pathways might interact with oviduct functions and the prolificacy of goats. Furthermore, the co-expression network analysis showed that XLOC_029185, XLOC_040647, and XLOC_090025 were the cis-regulatory elements of the DEGs MUC1, PPP1R9A, and ALDOB, respectively; these factors might be associated with the success of pregnancy and glucolipid metabolism. In addition, the GATA4, LAMA2, SLC39A5, and S100G were trans-regulated by lncRNAs, predominantly mediating oviductal transport to the embryo and energy metabolism. Our findings could pave the way for a better understanding of the roles of mRNAs and lncRNAs in fecundity-related oviduct function in goats.