Identification of phospholipids associated with semen cooling ability of Mangalarga Marchador stallions

Cryopreservation exposes sperm to low non-physiological temperatures resulting in cryodamage to the plasma membrane. Sperm cryotolerance may be directly related to the inherent genetics of the animal, diet, and seminal plasma composition. There is some evidence of breed effects regarding cryotolerance; Mangalarga Marchador stallions appear particularly sensitive to cryopreservation and many stallions of this breed present poor cryotolerance when standard processing methods are used. The objectives of this study were to compare lipid composition of sperm of Mangalarga Marchador stallions classified either as bad (i.e., drop in ≥40% total motility, n=8) or good (i.e., drop in ≤30% total motility, n=12) coolers in response to standard cooling (5°C for 24h). One ejaculate was obtained from each stallion; gel-free semen was extended in skim-milk based extender at 50 million sperm/mL and stored in a passive semen container at 5°C for 24h. An aliquot of raw semen containing 50 million sperm was placed on a cryovial and stored in liquid nitrogen until analysis. A second aliquot of 50 million sperm was collected after cooling for 24h. The two aliquots were preserved in liquid nitrogen for lipidomic analyses by MALDI-MS. Analysis was performed with MetaboAnalyst 2.0 and the discriminating analyses of PLS-DA to demonstrate the variation during cryopreservation. Differentially expressed lipids were analyzed with ANOVA followed by Tukey test. Volcano plots were used to compare groups, using P<0.05and fold-change (FC≥1.5). Lipidomic analyses revealed that the fresh semen had 96 unique ion lipids, the cooled semen had 98 ion-lipids, and there were 82 lipids in common. Two phospholipids were more abundantly expressed in good cooler stallions (P<0.05-FC>1.5) with mass/charge (m/z) 772.5 ([PI 28:0 +NH4]+), phosphatidylinositol an essential lipid molecule involved in various cellular processes such as regulation of actin cytoskeleton and membrane traffic; and 774.6 that we were unable to identify the correct lipid, but that according to the base date it could be one of these 4: [PE 38:1 +H]+ phosphatidylethanolamine is a substrate for methylation to phosphatidylcholine, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins or [PC 35:1+H]+ phosphatidylcholines were associated with improved characteristics in thawed spermatozoa; or [PS O-37:0+H-H2O]+, phosphatidylserine is required for the targeting and function of various intracellular signaling, is a marker of apoptotic cells and can be synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine; or [PA 40:2 +NH4]+ phosphatide that plays a crucial role in lipid homeostasis. In conclusion, Mangalarga Marchador with good cooling ability differ from stallions with bad cooling ability with regard to the phospholipid composition of sperm.