Glycerol-Free Equilibration with the Addition of Glycerol Shortly before the Freezing Procedure: A Perspective Strategy for Cryopreservation of Wallachian Ram Sperm

SIMPLE SUMMARY: This study investigated the effect of cryoprotectant glycerol addition in different phases of sperm equilibration on sperm motility and viability parameters of thawed ram spermatozoa. The purpose of this study was to compare two strategies of glycerol addition for sperm cryo-preservation. The first strategy included the use of a glycerol-free extender for the procedure of glycerol-free equilibration and chilling, with the glycerolation of the extender by 6% glycerol shortly before sperm slow freezing. The second strategy included the use of a freezing extender already glycerolated by 6% glycerol before the equilibration and chilling of sperm and following slow freezing. The obtained results were statistically significant and demonstrated that the glycerol-free equilibration with the addition of glycerol at least 10 min before freezing is the best strategy for cryopreservation of Wallachian ram sperm. This represents a perspective strategy in the cryopreservation process for local sheep breeds and indicates a potential strategy to test for ram cryopreservation in a generalized manner. ABSTRACT: This study investigated the effect of glycerol added in different phases of sperm equilibration on CASA and flow cytometry parameters of thawed ram spermatozoa. Sperm was collected from adult Wallachian rams. The freezing extender was glycerol-free ANDROMED(®) (Minitub GmbH, Tiefenbach, Germany) supplied by 6% exogenous glycerol at different stages of the cryopreservation process. The purpose of this study was to compare two strategies of glycerol addition for sperm cryopreservation. The first strategy included the use of a glycerol-free extender for the procedure of glycerol-free equilibration and chilling, with the glycerolation of the extender by 6% glycerol shortly before sperm slow freezing (GFA). The second strategy included the use of a freezing extender already glycerolated by 6% glycerol before the equilibration and chilling of sperm and following slow freezing (GA). Sperm samples were analyzed after equilibration (but before freezing) and after thawing (at T0, T1 h, and T2 h time points). iSperm(®) mCASA (Aidmics Biotechnology Co., LTD., Taipei, Taiwan) was used for the evaluation of sperm kinematics. Flow cytometry was used to measure sperm viability (plasma membrane/acrosome intactness) and mitochondrial membrane potential. The obtained results significantly demonstrated that the glycerol-free equilibration with the addition of glycerol shortly before freezing is a perspective strategy for cryopreservation of Wallachian ram sperm.