Mitochondrial dysfunction, especially compromised respiratory metabolism, is a serious obstacle of sperm cryopreservation. This study aims to determine the effect of melatonin supplement on respiratory metabolism of frozen-thawed ram sperm. Semen was slowly frozen with or without melatonin supplement, while fresh semen was used as a control. The results showed that melatonin clearly improved ATP production, oxygen consumption and respiratory chain complex activities, while it decreased reactive oxygen species and nitrite concentrations in frozen-thawed sperm (p < 0.05). Consequently, the viability, motility and fertility of frozen-thawed sperm were also recovered by melatonin. Strikingly, promoter methylation levels of several key mitochondrial respiratory chain genes were dramatically increased along with decreased expression levels in frozen-thawed sperm, which can be partially repaired by melatonin supplement (p < 0.05). This might be attributed to the expression change of mtDNMT1 among three groups (p < 0.05). Furthermore, the declined expression of MTNR1A and MTNR1B were found in frozen-thawed sperm (p < 0.05). The treatment of melatonin receptor antagonist indicated that MTNR1A could have a key role in the regulation of melatonin on mitochondrial function of frozen-thawed sperm. Collectively, these findings provide a new perspective on the epigenetic regulation of sperm cryopreservation.

Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks during spermatogenesis. PRDM9 determines the localization of recombination hotspots by interacting with several protein complexes in mammals. The function of PRDM9 is not well understood during spermatogenesis in mice or yaks. In this study, we applied yeast two-hybrid assays combined with next-generation sequencing techniques to screen the complete set of PRDM9-interacting proteins and explore its novel functions in yak spermatogenesis. Our results showed that 267 PRDM9-interacting proteins were identified. The gene ontology (GO) analysis of the interacting proteins revealed that the GO terms were primarily associated with spermatogenesis, positive regulation of double-strand break repair via homologous recombination, RNA splicing, the ubiquitin-dependent ERAD pathway, and other biological processes. MKX and PDCD5 were verified to be strongly interacting with PRDM9 and expressed in prophase I of meiosis in both mouse and yak testes. The localizations of RNA splicing genes including THOC5, DDX5, and XRCC6 were expressed in spermatocytes. Cattleyak is the hybrid offspring of a yak and a domestic cow, and the male offspring are sterile. The gene expression of the interacting proteins was also examined in the sterile male hybrid of yak and cattle. Among the 58 detected genes, 55 were downregulated in cattleyak. In conclusion, we established a complete PRDM9 interaction network, and a novel function of PRDM9 was identified, which will further promote our understanding of spermatogenesis. It also provides new insights for the study of hybrid male sterility.

To investigate prenatal muscle satellite cell (MuSC) development and the associated epigenetic modifications in yak. Here, we conducted morphological and protein co-localization analyses of fetal longissimus dorsi muscle at various developmental stages using histology and immunofluorescence staining methods. Our study observed that primary muscle fibers began forming at 40 days of gestation, fully developed by 11 weeks, and secondary muscle fibers were predominantly formed by around 105 days. Throughout development, MuSCs were mainly located between the muscle fiber membrane and the basement membrane, acting as a reserve for the stem cell pool. MuSCs appeared within myotubes only during critical phases of primary and secondary muscle fiber formation. The proliferation of MuSCs gradually decreases until birth. MuSCs with 5mC modification show a trend of increasing first and then decreasing. MuSCs with 5hmC modification also present a dynamic change trend. The 41st day and 11th week are the critical periods for the changes of both. From the 11th week to around the 110th day of gestation, the modification effect of histone H3K4me3 is crucial for MuSCs during the development of the fetal longissimus dorsi muscle. Combined, our data identify key time points for yak fetal skeletal muscle growth and development and demonstrate that DNA methylation and histone modifications in MuSCs are closely related to this process, offering a valuable basis for future research into the molecular mechanisms underlying yak muscle development.

Yushu yaks face nutritional deficiency and poor production performance in the cold season on the Qinghai–Tibet Plateau, yet their metabolic responses to different feeding modes remain unclear. This study investigated the effects of traditional grazing, grazing with concentrate supplementation, and TMR stall-feeding on 90 four-year-old Yushu yaks (30 per group) during a 180-day cold-season trial, by determining body weight changes, serum biochemical parameters and plasma metabolic profiles. Stall-fed yaks had the highest average daily gain (259.05 ± 61.56 g/d), while grazing yaks showed negative gain (−279.50 ± 44.45 g/d) and supplementary-fed yaks had intermediate performance; grazing yaks had stronger antioxidant enzyme activity, and supplemented/stall-fed yaks had higher serum mineral and albumin levels. Metabolomic analysis identified 2024 plasma metabolites, with grazing yaks enriched in plateau adaptation and antioxidant pathways, stall-fed yaks in energy metabolism pathways, and supplementary-fed yaks in both growth and antioxidant pathways. Our findings confirm that gradient nutritional interventions regulate the balance between yak growth and plateau adaptability, with concentrate supplementation as the optimal cold-season feeding mode for local herders and stall-feeding suitable for intensive breeding to maximize growth efficiency.