Osmotic Tolerance of Equine Spermatozoa and the Effects of Soluble Cryoprotectants on Equine Sperm Motility, Viability, and Mitochondrial Membrane Potential

Abstract

Osmotic stress attributed to differences in the relative permeability of cryoprotectants, such as glycerol and water, appears to be an important factor in cryodamage. The objective of this study was to characterize the osmotic tolerance of equine spermatozoa, and to evaluate the effects of addition and removal of cryoprotectants from equine spermatozoa on their motility, and membrane and acrosomal integrity, as well as their mitochondrial membrane potential. Equine spermatozoa had a limited osmotic tolerance to anisosmotic conditions. Although the addition of increasing concentrations of glycerol decreased the motility and viability of equine spermatozoa, the rapid removal of glycerol by dilution in isosmotic media resulted in an even greater decline in motility and viability compared with spermatozoa maintained under anisosmotic conditions. Likewise, the addition and rapid removal of 1.0 M glycerol, ethylene glycol, dimethylsulfoxide, or propylene glycol resulted in a significant decline in sperm motility and viability. Among these cryoprotectants, ethylene glycol had the least detrimental effect on either viability or motility of spermatozoa following the rapid addition and removal of these cryoprotectants. These data demonstrate that equine spermatozoa have a limited osmotic tolerance compared with published reports for mouse or human spermatozoa, and appear to be more similar to boar spermatozoa in their osmotic tolerance. Of the 4 cryoprotectants evaluated in equine spermatozoa, the addition and removal of glycerol resulted in a more marked osmotic stress as indicated by alterations in motility, viability, and acrosomal integrity. These data suggest that alternative cryoprotectants should be considered for cryopreservation of equine spermatozoa in order to reduce osmotic stress associated with the addition of these agents during semen freezing.

Bovine spermatozoa react to in vitro heat stress by activating the mitogen-activated protein kinase 14 signalling pathway

“…normothermic temperatures in the presence of DMSO increased sperm DNA fragmentation. Although we do not know the mechanisms underlying the induction of DNA fragmentation by DMSO, the addition of DMSO as cryoprotectants for bull or stallion spermatozoa has been reported to reduce sperm motility and viability by inhibiting Na þ /K þ ion pumps (de la Cueva et al 1997;Ball and Vo 2001). Notably, incubation of spermatozoa at the hyperthermic temperature significantly augmented DNA fragmentation, which was prevented by SB203580 as a result of blockade of MAKP14 signalling transduction (Fig.…”

Toxicity of glycerol for the stallion spermatozoa: Effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential

“…To freeze mammalian sperm glycerol is the most widely used cryoprotectant [12], however beside the cryoprotective action, glycerol may have toxic effects on the spermatozoa on a dose dependent manner. In the equine species semen is frozen using glycerol at concentrations ranging from 2 to 5% [4,[13][14][15], however very few controlled studies have addressed the toxicity of different concentrations of glycerol [16][17][18]. The efficacy of cryopreservative agents is almost always based on comparing the prefreeze sperm motility with the immediate post thaw motility, however this approach do not take into account damage that may have been caused by the intrinsic toxicity of the cryoprotectant.…”

Evaluation of Different Cryoprotectants (CPAs) in Boar Semen Cryopreservation

“…Glycerol (at a concentration of 3%) is the most commonly used cryoprotectant (CPA) for boar sperm [3], but it has toxic effects as well as contraceptive effects on sperm [8]. Its detrimental effects have stimulated the study of alternative CPAs such as N,N-dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO) [2,10]. Due to its highly hydrophilic nature and low molecular weight, DMA reduces the formation of intracellular ice crystals and increases membrane permeability, thus decreasing osmotic damage [2].…”