PUFA-rich phospholipid classes and subclasses of ram spermatozoa are unevenly affected by cryopreservation with a soybean lecithin-based extender

Cryopreservation is known to affect spermatozoa structure and function. Ram sperm are among the most highly sensitive mammalian gametes to freezing, due to their lipid composition, which limit their effi-ciency in artificial insemination programs. The aim of this study was to investigate the effects of cryopreservation with a chemically defined soybean lecithin-based extender on ram spermatozoa functionality on the one hand, and quantifiable changes in lipid and fatty acid profile on the other. Freeze-thawing decreased sperm quality, as indicated by post-thaw parameters related to membrane integrity, mitochondrial viability and sperm motility. The most relevant lipid change after cryopreservation was a remarkable loss of all glycerophospholipids containing 22:6n-3. Species of sphingomyelin with very long chain polyunsaturated fatty acids (VLC-PUFA), that are exclusively located in the sperm head, where responsible of its reduction after cryostorage. Freezing caused a reduction in mitochondrial function, which was confirmed by significantly decreased of mitochondrial membrane potential and by the generation of 4-HNE. Mitochondria damage was accompanied by a loss in cardiolipin with 18:2n-6 and phosphatidylethanolamine with 20:4n-6, two well-known lipids that are critical components for mitochondrial membrane functionality. Loss of sterols after cryopreservation occurred along with a decrease in the order of sperm membrane lipids. Our research provides new insights on deleterious effects of cryopreservation on PUFA-rich phospholipids of ram sperm and highlight their importance as biomarkers of ultrastructural, biochemical and functional damage that ram spermatozoa undergo after freezing-thawing. (c) 2022 Elsevier Inc. All rights reserved.