Variation in semen lipids during cryopreservation by cooling with milk-based extender in donkeys

Donkey sperm cryopreservation is becoming increasingly necessary for the production of hybrids with horses and seed stock of endangered breeds. Therefore, this study aimed to assess lipid changes during the cryopreservation of donkey sperm. One ejaculate from each of seven donkeys was harvested, evaluated, and extended in a skim milk-based extender at 50 million sperm/mL and cooled for 24h at 5°C. Aliquots of fresh, not extended semen were preserved in liquid nitrogen for lipidomic analyses by MALDI-MS. The analyses were performed with MetaboAnalyst 2.0 and the discriminating analyses of PLS-DA to demonstrate lipid variation occurring during the cryopreservation process. The lipid types that were differentially expressed were analysed by T-test followed by Tukey. The Vulcano plot was used to compare groups, using the following criteria values of P<0.05 and fold-change (FC) ≥1.5. The identification of lipids was in the databases www.lipidmaps.org. Analysis of phospholipids between fresh and cooled semen showed 96 total lipids in fresh semen and 98 in cooled semen; 18 lipids are most abundant in the fresh semen, of which 6 are Glycerophospholipids, 2 Glycerolipids, 2 Sphingolipids, 2 are Fatty Acyls and 1 (728.6 m/z) can be Sphingolipids or Glycerolipids, 8 of which are known to be polyunsaturated and 4 saturated and the other 5 were not identified (733.9, 748, 764, 801, 826 m/z); 14 lipids are most abundant in refrigerated semen, 1 Sterol Lipids, 7 Glycerophospholipids, 1 Glycerolipids, 1 Fatty Acyls, 2 Sphingolipids, of these 11 are polyunsaturated and 1 saturated, another 2 (737, 749 m/z) were not identified. After refrigeration, 14 lipids were lost, 1 Sterol Lipids, 5 Glycerophospholipids 1, Sphingolipids and 4 Glycerolipids, 8 unsaturated and 4 saturated and 3 (736, 760, 762 m/z) were not identified, and even after refrigeration 16 lipids were incorporated on semen, 6 Glycerolipids, 1 Glycerophospholipids and 3 Fatty Acyls, of which 9 were unsaturated and 1 saturated, the remaining 6 (737, 755, 759, 798, 811, 859 m/z) lipids were not identified. Comparatively, refrigerated semen showed a greater presence of polyunsaturated lipids compared to fresh semen and the addition of the milk-based extender incorporated more unsaturated lipids into the sperm membrane. The presence of double bonds between carbons in polyunsaturated fatty acid molecules makes them very vulnerable to attack by free radicals and the initiation of the lipoperoxidation cascade. In conclusion, freezing donkey semen results in a change in phospholipids with loss of lipids, incorporation of new ones and change in relative abundance mainly of polyunsaturated lipids that can lead sperm to greater oxidative damage during the cryopreservation process.