Metabolomics Reveals Betaine as a Biomarker for Better Boar Spermatozoa Freezability

Artificial insemination in the pig industry requires frozen semen of better freezability and post-thaw quality. Several recent metabolomics studies were carried out to discover sperm freezability-related biomarkers. However, only a limited number of pig breeds were examined and results remain obscure. Here, boar semen samples were collected and quality evaluated for 3 Western breeds (Duroc, Landrace and Yorkshire), originating from a nucleus farm and a boar station. Post-thaw sperm from the nucleus farm exhibited significantly higher motility and vitality rates (p < 0.01), and also better acrosome integrity and plasma membrane integrity (p < 0.05). For the high and low freezability groups (36 samples in total), ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) on spermatozoa identified significantly differential metabolites (p < 0.05, including betaine), with 258 metabolites in Duroc, 126 in Landrace, and 215 in Yorkshire, which were significantly enriched in 11, 8 and 13 metabolic pathways (KEGG) (p < 0.05), respectively. Besides breed-specific pathways (Duroc: cysteine and methionine metabolism; Landrace: arginine and proline metabolism, tryptophan metabolism, lysine degradation, galactose metabolism and pyruvate metabolism; Yorkshire: steroid hormone biosynthesis, riboflavin metabolism and linoleic acid metabolism), two pathways common to 3 breeds (alanine, aspartate and glutamate metabolism, and pentose phosphate pathway) were found. Betaine was confirmed to be at a significantly higher level in the semen of high freezability for all three pig breeds (p < 0.05). Taken together, metabolites and metabolic pathways common and specific to commercial Western breeds were identified. Betaine was related to better spermatozoa freezability. Our findings provide the basis and insights into better understanding the role of metabolic molecules and pathways important to boar spermatozoa freezability.