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.

Objective: Skeletal muscle growth is an important economic trait for meat production, with notable differences between Tibetan pigs (TIBPs, a slow-growing breed) and Large White pigs (LWPs, a fast-growing breed). However, the genetic underpinnings of this disparity remain unclear.Methods: In the current study, we integrated differentially expressed genes (DEGs) and proteins (DEPs) from 60-day-old embryonic muscle tissue, along with whole-genome single nucleotide polymorphisms (SNPs) displaying absolute allele frequency differences (ΔAF) of 0.5 or more between the TIBP and LWP breeds, to unravel the genetic factors influencing skeletal muscle growth.Results: Our analysis revealed 3,499 DEGs and 628 DEPs with SNPs having a ΔAF equal to or greater than 0.5. Further functional analysis identified 145 DEGs and 23 DEPs involved in biological processes related to skeletal muscle development, and 22 DEGs and 3 DEPs implicated in the mechanistic target of rapamycin kinase signaling pathway, which is known for positively regulating protein synthesis. Among these genes, several DEGs and DEPs, enriched with TIPB-specific SNPs in regulatory or/and coding regions, showed marked ΔAF between the TIBP and LWP breeds, including MYF5, MYOF, ASB2, PDE9A, SDC1, PDGFRA, MYOM2, ACVR1, ZIC3, COL11A1, TGFBR1, EDNRA, TGFB2, PDE4D, PGAM2, GRK2, SCN4B, CACNA1S, MYL4, IGF1, and FOXO1. Additionally, genes such as CAPN3, MYOM2, and PGAM2, identified as both DEPs and DEGs related to skeletal muscle development, contained multiple TIBP-specific and LWP-predominant SNPs in regulatory and/or coding regions, underscoring significant ΔAF differences between the two breeds.Conclusion: This comprehensive investigation of SNPs in DEGs and DEPs identified a significant number of SNPs and genes related to skeletal muscle development during the prenatal stage. These findings not only shed light on potential causal genes for muscle divergence between the TIBP and LWP breeds but also offer valuable insights for pig breeding strategies aimed at enhancing meat production.

Spermatozoa are unique cells that carry a library of proteins that regulate the functions of molecules to achieve functional capabilities. Currently, large amounts of protein have been identified in spermatozoa from different species using proteomic approaches. However, the proteome characteristics and regulatory mechanisms of spermatozoa in bucks versus rams have not been fully unraveled. In this study, we performed a tandem mass tag (TMT)-labeled quantitative proteomic analysis to investigate the protein profiles in the spermatozoa of buck (Capra hircus) and ram (Ovis aries), two important economic livestock species with different fertility potentials. Overall, 2644 proteins were identified and quantified via this approach. Thus, 279 differentially abundant proteins (DAPs) were filtered with a p-value < 0.05, and a quantitative ratio of >2.0 or <0.5 (fold change, FC) in bucks versus rams, wherein 153 were upregulated and 126 were downregulated. Bioinformatics analysis revealed that these DAPs were mainly localized in the mitochondria, extracellular and in the nucleus, and were involved in sperm motility, membrane components, oxidoreductase activity, endopeptidase complex and proteasome-mediated ubiquitin-dependent protein catabolism. Specifically, partial DAPs, such as heat shock protein 90 α family class a member 1 (HSP90AA1), adenosine triphosphate citrate lyase (ACLY), proteasome 26S subunit and non-ATPase 4 (PSMD4), act as “cross-talk” nodes in protein–protein networks as key intermediates or enzymes, which are mainly involved in responses to stimuli, catalytic activity and molecular function regulator pathways that are strictly related to spermatozoa function. The results of our study offer valuable insights into the molecular mechanisms of ram spermatozoa function, and also promote an efficient spermatozoa utilization link to fertility or specific biotechnologies for bucks and rams.