The lipidomic profiles of donkey semen present greater diversity and abundance than horse semen

While donkeys and horses belong to the same family and are able to cross-mate to produce hybrids, there are many unique features of either species regarding their sexual behavior, semen characteristics, and tolerance to cryopreservation; therefore, a better understanding of the physiology of either species can enhance semen processing and fertility. Lipids play a paramount role in the fluidity and flexibility of the sperm plasma membrane and are intrinsically related to sperm motility and plasma membrane integrity. This study aimed to determine the lipid profile of donkey and horse semen. Semen from 19 jacks and 25 stallions was collected by artificial vagina, and gel-free semen was assessed for motility, morphology, concentration, and volume. An aliquot of gel-free semen (50 million sperm/mL) was placed in cryovials and then plunged into liquid nitrogen until lipidomic analysis. The lipidomic profile of the gel-free semen was performed with MALDI-MS with no addition of an extender. The analyses were performed with MetaboAnalyst 2.0 and the discriminating analyses of PLS-DA to demonstrate differences between horses and donkeys. The lipid types that were differentially expressed were analyzed by ANOVA followed by Tukey test. The Volcano plot was used to compare species, using the criteria values of P<0.05 andfold-change (FC) ≥1.5. A total of 101 ion lipids were found in horse semen and 105 in donkey semen. Five ion-lipids with mass charge (mz) 762, 822, 865, 867, and 879 were uniquely present in horse semen, but not identified in the databases (www.lipidmaps.org), whereas 9-ion-lipid were present in donkey semen (m/z 709.4, 731, 753, 756, 778, 796, 798, 811 and 880) with only 709.4 being identified (Phosphatidylglycerol). The donkeys showed 18 ion-lipid with greater relative abundance (P<0.05 and fold-change>1.5), 12 identified in the database (4 triacylglycerols, 2 phosphatidylglycerols, 2 phosphatidylcholine, 2 phosphatidylinositols and 2 sphingolipids); whereas the horse had ten ion-lipid with greater abundance (P<0.05 and fold-change >1.5) but only 4 identified (2 diacylglycerols, 1 triacylglycerol and 1 phosphatidylglycerol). Among these identified lipids, 10 have one or more double bonds, making them mono- or polyunsaturated, while in horses only 1 was polyunsaturated. This may be one of the underlying reasons for lower tolerance to cryopreservation/cooling of donkey semen. In conclusion, the lipidomic profile of donkey and stallion semen differ with regard to relative abundance and diversity.