Optimizing the freezing rate for ovine semen cryopreservation: Phospholipid profiles and functions of the plasma membrane and quality and fertilization of spermatozoa

• Freezing changed spermatozoa plasma membrane phospholipid profiles and functions.
• A freezing rate of −40 °C/min resulted in better plasma membrane structure and function, as well as fertilization rate.
• The −40 °C/min freezing rate minimized cryoinjury to phospholipids.

The objective of this study was to investigate the effects of freezing rate on plasma membrane (PM) phospholipids and the function and fertilization of ram spermatozoa during freezing. Semen was obtained from 10 Merino rams using an artificial vagina, diluted with glucose-egg yolk buffer, and frozen in a programmable freezer at rates of −1, −20, −40, −60, and −80 °C/min. After thawing, we examined PM phospholipid content and distribution, fluidity, permeability, potential, spermatozoa quality, and in vivo and in vitro fertilization rates. The results showed that phospholipid, phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) contents of the PM decreased significantly (P < 0.05), whereas diphosphatidylglycerol (DPG) content and the cholesterol: phospholipid (CH: PL) ratio increased significantly compared to those of fresh semen after freezing (P < 0.05). PE, phosphatidylinositol (PI), and DPG were transferred to the outer leaflet, whereas PS and PG were transferred to the inner leaflet after freezing. Spermatozoa cryopreserved at −40 °C/min resulted in distributions of PS, PE, and DPG, as well as increased PM fluidity, permeability, and potential that was greater than those of the other freezing-rate groups (P < 0.05). No differences in vitality, PM and acrosome integrity, in vitro fertilization hatching rate, or artificial insemination conception rate were observed in any of the freezing-rate groups (P > 0.05), whereas cleavage rate (69.67 ± 5.11%) at −40 °C/min increased significantly (P < 0.05). Therefore, the optimal freezing rate to cryopreserve ram spermatozoa based on cryoinjury of phospholipids, membrane functions, and fertilization rate was −40 °C/min.