Semen cryopreservation results in the differential remodeling of the molecules presented in sperm, and these alterations related to reductions in sperm quality and its physiological function have not been fully understood. Given this, this study aimed to investigate the cryoinjury mechanism of goat sperm by analyzing changes of the metabolic characteristics in sperm during the cryopreservation process. The ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) technique was performed to explore metabolite profiles of fresh sperm (C group), equilibrated sperm (E group), and frozen–thawed sperm (F group). In total, 2570 metabolites in positive mode and 2306 metabolites in negative mode were identified, respectively. After comparative analyses among these three groups, 374 differentially abundant metabolites (DAMs) in C vs. E, 291 DAMs in C vs. F, and 189 DAMs in E vs. F were obtained in the positive mode; concurrently, 530 DAMs in C vs. E, 405 DAMs in C vs. F, and 193 DAMs in E vs. F were obtained in the negative mode, respectively. The DAMs were significantly enriched in various metabolic pathways, including 31 pathways in C vs. E, 25 pathways in C vs. F, and 28 pathways in E vs. F, respectively. Among them, 65 DAMs and 25 significantly enriched pathways across the three comparisons were discovered, which may be tightly associated with sperm characteristics and function. Particularly, the functional terms such as TCA cycle, biosynthesis of unsaturated fatty acids, sphingolipid metabolism, glycine, serine and threonine metabolism, alpha-linolenic acid metabolism, and pyruvate metabolism, as well as associated pivotal metabolites like ceramide, betaine, choline, fumaric acid, L-malic acid and L-lactic acid, were focused on. In conclusion, our research characterizes the composition of metabolites in goat sperm and their alterations induced by the cryopreservation process, offering a critical foundation for further exploring the molecular mechanisms of metabolism influencing the quality and freezing tolerance of goat sperm. Additionally, the impacts of equilibration at low temperature on sperm quality may need more attentions as compared to the freezing and thawing process.

Results of artificial insemination (AI) are affected by changes in sperm quality and the function throughout collection and preservation procedures. Proteome and metabolome alterations of sperm treated with the different procedures in goat, however, aren’t fully understood. To this end, we sought to investigate the impacts of rectal probe electrostimulation (EE) and artificial vagina (AV) semen collection methods on the quality and the cryotolerance of goat sperm, with additional focus on proteomic and metabolomic analyses. Semen samples were collected from Yunshang black goats and categorized into four groups: fresh sperm collected via AV (XAZ), fresh sperm collected via EE (XEZ), frozen sperm post-AV collection (DAZ) and frozen sperm post-EE collection (DEZ). Four comparisons (XAZ vs. XEZ, DAZ vs. XAZ, DEZ vs. XEZ, DAZ vs. DEZ) were performed, respectively. This study first evaluated sperm motility, acrosome integrity, plasma membrane integrity, mitochondrial activity, and reactive oxygen species (ROS) levels. The results indicated that there were no significant differences in fresh sperm quality parameters between the EE and AV methods. However, notable differences emerged post-cryopreservation. Specifically, the AV method proved more advantageous in preserving the motility, integrities of acrosome and plasma membrane, mitochondrial activity of frozen sperm compared to the EE method. Through the multi-omics approaches, a total of 210 differentially abundant proteins (DAPs) related to sperm characteristics and function were identified across the four comparations. Moreover, 32 differentially abundant metabolites (DAMs) were detected. Comprehensive bioinformatics analysis underscored significant molecular pathways in the co-enrichment of DAPs and DAMs, particularly focusing on the citrate cycle, ROS, oxidative phosphorylation, and glycine, serine, and threonine metabolism etc. We elucidated the differential impacts of AV and EE collection methods on the quality and cryotolerance of goat semen from omics perspectives, which offer a critical foundation for further exploration into optimizing semen collection and cryopreservation techniques in goat breeding program.