Influence of Different Combinations of Permeable and Nonpermeable Cryoprotectants on the Freezing Capacity of Equine Sperm

Abstract

This study was aimed to evaluate the effect of permeable cryoprotectants in combination with trehalose or sucrose on the freezing capacity of stallion sperm. For this purpose, the ejaculates (n = 24) were collected from four healthy mature Turkmen stallions. The ejaculates were pooled and diluted with one of the extenders containing a combination of 5% of permeating (dimethylacetamide [DMA]; dimethylformamide [DMF] or glycerol) and 50 mM of nonpermeating cryoprotectant agents (CPAs) (sucrose or trehalose) to a final concentration of 200 × 106 spermatozoa/mL. The extended samples were cryopreserved and thawed using a standard protocol. The samples were evaluated for motion kinetics, morphological abnormalities, plasma membrane functionality (PMF), viability, and lipid peroxidation. The results showed that the sperm cryopreserved in extender containing DMA produced higher (P ≤ .05) total motility, straightness, straight line velocity, curvilinear velocity, and lower (P ≤ .05) lipid peroxidation (malondialdehyde [MDA] concentration) compared with DMF and glycerol groups. Overall, both DMA and DMF have shown higher (P ≤ .05) sperm motion kinetics, viability, PMF, and lower (P ≤ .05) morphological abnormalities and MDA concentration compared with the glycerol. However, except morphological abnormalities, all of the other parameters did not differ between trehalose and sucrose. Likewise, there was no interaction between permeating and nonpermeating CPAs (P ≥ .05) except in terms of sperm abnormalities (P ≤ .05). In conclusion, the use of DMA or DMF as alternative CPAs of glycerol could be more effective for successful cryopreservation of stallion sperm. The nonsignificant interaction between permeating and nonpermeating CPAs for most of the post-thaw sperm parameters negates possible synergism among these compounds.