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

• Published in: Animals (Basel) Vol. 13; no. 7; p. 1200
• Main Authors: Málková, Anežka, Savvulidi, Filipp Georgijevič, Ptáček, Martin, Machová, Karolína, Janošíková, Martina, Nagy, Szabolcs, Stádník, Luděk
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 29.03.2023; MDPI
• Subjects: Animals; Balancing; Biotechnology; Chilling; Communication; Cryopreservation; cryopreservation capacity; Cryoprotectors; Flow cytometry; Freezing; Glycerin; Glycerol; Kinematics; Laboratories; Life sciences; Membrane potential; Membranes; Mitochondria; mitochondrial membrane potential; Motility; Parameters; plasma membrane and acrosome intactness; Review boards; Sheep; Sperm; sperm motility; Spermatozoa; Statistical analysis; Thawing; Vagina; Czech Republic; Germany; France; United States > US; plasma membrane and acrosome intactness; sperm motility; cryopreservation capacity; mitochondrial membrane potential
• ISSN: 2076-2615; 2076-2615
• DOI: 10.3390/ani13071200

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.