Lipid profile differences in semen from “bad” and “good” freezer stallions

Lipids within the plasma membrane of sperm cells play a significant role in their resistance to cryopreservation. In equines, a considerable subset of stallions exhibits low sperm tolerance to freezing, which impacts fertility preservation efforts. This study aimed to elucidate and compare the lipid membrane profiles of sperm from stallions characterized as either cryo-sensitive or cryo-resistant. We analyzed two ejaculates from 39 stallions, all with established fertility and cryopreservation histories. Based on post-thaw seminal quality, stallions were categorized into two groups: “Bad Freezer” (n=19) with total motility <35% and/or membrane integrity <15%, and “Good Freezer” (n=20) with total motility >40% and/or membrane integrity >20%. Ejaculates were collected using an artificial vagina, filtered, and aliquots containing 50 × 10⁶ sperm cells were placed in cryotubes, without adding diluent medium, and stored at -196°C, for lipid extraction. Lipid profiles were obtained via MALDI-MS mass spectrometry. Data analysis was performed using MetaboAnalyst 2.0, partial least squares discriminant analysis (PLS-DA), showing the relationship between variance in the data and differences between groups. Lipid species were differentially expressed using ANOVA followed by Tukey's test, while vulcano plot test was used to compare the two groups of stallions, using the following criteria: P values≤0.05 and fold-change (FC) ≥1.5. The identification of lipids was in the databases www.lipidmaps.org. Five phospholipids were significantly more abundant in the “Bad Freezer” group: glycerophosphoethanolamines [PE 32:3 +NH4]+, glycerophosphoglycerols [PG 35:3 +Na]+, glycerophosphoinositols [PI 31:3 +NH4]+, glycerophosphoserines [PS 39:2 +Li]+, and simple Glc series [HexCer 36:1;O2 +H]+. Lipidomic analysis of spermatozoa in this study revealed that lipid patterns with different cryotolerances varied (PE, PG, PI, PS, HexCer) and these lipidomic differences were characterized by high carbon chain length (31-39) and polyunsaturation. These findings suggest that long-chain polyunsaturated fatty acids (PUFAs) in the plasma membrane may be critical determinants in sperm cryoresistance, offering insights for enhancing fertility preservation strategies in equine reproduction.