Trehalose is commonly used as an impermeable cryoprotectant for cryopreservation of cells, but its cryoprotective mechanism has now not but been determined. This study investigated the cryopreservation impact of trehalose on buck semen cryopreservation and finished metabolic profiling of freeze-thawed media by way of the GC–MS-based metabolomics for the first time. Metabolic pattern recognition and metabolite identification by means of principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and metabolic pathway topology analysis revealed the results of trehalose on buck sperm metabolism at some point of cryopreservation. The results confirmed that trehalose drastically progressed sperm motility parameters and structural integrity after thawing. PCA and PLS-DA analysis discovered that the metabolic patterns of the freezing-thawing media of buck semen cryopreserved with trehalose (T group) or without trehalose (G group, Control) were certainly separated. Using screening conditions of VIP >1.5 and p vaule <0.05, a total of 48 differential metabolites have been recognized, whithin l-isoleucine, L-leucine, L-threonine, and dihydroxyacetone were notably enriched in valine, leucine and isoleucine biosynthesis, glycerolipid metabolism, and aminoacyl-tRNA biosynthesis pathways. In brief, trehalose can efficiently improve membrane structural integrity and motion parameters in buck sperm after thawing, and it exerts a cryoprotective impact with the aid of changing sperm amino acid synthesis and the glycerol metabolism pathway.
Factors affecting sperm freezability in goat seminal plasma were investigated. Based on the total motility of thawed sperm, goats were divided into a high-freezability (HF) group with >60% total motility (n = 8) and a low-freezability (LF) group with <45% total motility (n = 8). Sperm and seminal plasma from the HF and LF groups were separated, HF seminal plasma was mixed with LF spermatozoa, LF seminal plasma was mixed with HF sperm, and the products were subjected to a freeze-thaw procedure. Semen from individual goats exhibited differences in freezability. HF semen had higher sperm motility parameters and plasma membrane and acrosome integrity after thawing; this difference could be related to the composition of seminal plasma. Seminal plasma from the HF and LF groups was evaluated using metabolomic analysis, and multivariate statistical analysis revealed a clear separation of metabolic patterns in the seminal plasma of goats with different freezability classifications. Forty-one differential metabolites were identified using the following screening conditions: variable importance in the projection > 1 and 0.05 < P-value < 0.1. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed significant enrichment of central carbon metabolism in cancer, protein digestion and absorption, aminoacyl-tRNA, and other pathways and significant differences in the abundance of seven differential metabolites, including L-glutamine, L-aspartate, L-arginine, phenylpyruvate, benzoic acid, ketoisocaproic acid, and choline between seminal plasma from the HF and LF groups (P-value < 0.05). These significantly differentially-expressed metabolites may be potential biomarkers for sperm freezability. L-glutamine, L-aspartate, and L-arginine may directly affect sperm freezability. Benzoic acid, ketoisocaproic acid, and choline may regulate sperm freezability by participating in anabolic processes involving phenylalanine, leucine, and phosphatidylcholine in sperm.
The current study aimed to detect the relationship between the spermatozoa cryotolerance and the post-thawed sperm lipidome. Ejaculates from 20 goats, and performed a uniform frozen-thawed procedure in this study. According to the total motility of thawed sperm of goats, semen samples were classified into HF group (High Freezers, n = 8) with >60% total motility and LF group (Low Freezers, n = 8) with < 45% total motility. The lipidomic analysis based on UHPLC-MS/MS was utilized to investigate the relationship between sperm cryotolerance and their lipid metabolites expression. The results showed that the cryotolerance of sperm from different individual goats were in great variation. The total motility of post-thawed sperm in HF group (60.93 ± 2.43%) is significantly higher than that in LF group (34.04 ± 3.41%, P < 0.01). And the post-thawed sperm in HF group exhibited significantly higher plasma membrane (59.06 ± 2.34%) and acrosome integrity (62.93 ± 1.15%) than that in LF group (34.06 ± 4.85%, 44.92 ± 2.19% respectively, P < 0.01). The total of 29 lipid subclasses and 1,133 lipid molecules in the post-thawed goat sperm were identified by lipidomics analysis. The lipid content of thawed sperm in HF group was higher than that in LF group, the lipid profile in HF group was significantly separated from LF group, which indicated that the difference in lipid composition and lipid metabolism mode of sperm between the two groups was existed, especially the expression of phosphatidylcholine and triglyceride molecules. In conclusion, the cryotolerance of sperm from different individual goats were in great variation. Sperm with high cryotolerance may be able to uptake more lipids during cryopreservation. The increase in phosphatidylcholine and triglyceride content of thawed. Sperm may relate to more active lipid anabolic processes.
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