The effects of different freezing techniques monosaccharide sugars in freezing extender on post-thawed quality of stallion semen

Abstract

The objectives of this study were to compare the quality of stallion spermatozoa frozen with either conventional or controlled-rate freezing technique (EXP 1), and the effects of monosaccharide sugars on post-thawed semen quality (EXP 2). EXP. 1. Semen from 3 stallions (3 ejaculates per stallion; >70% motility, viability and normal morphology) were used in this study. Cryopreservation reduced significantly the motility and plasma membrane integrity of stallion sperm (51.7±9.9% and 61.3±7.4%) compared unfavorably to postequilibrated/ non-frozen sperm (67.8±4.4% and 74.0±4.8%). However, sperm viability of stallion no.1 and no.2 frozen with controlled rate freezer was significantly greater than conventional method (p<0.05) when examined at 2 h (39.6±1.1% vs. 49.4±4.2%) and 4 h (32.0±1.9% vs. 40.4±2.8%) post-thawing. In contrast, there was no difference in sperm viability of stallion No.3 between the two freezing techniques. EXP. 2. Semen from six fertility proven stallions (four ejaculates per stallion) was cryopreserved. After freezing and thawing, the quality of the cryopreserved semen was reduced significantly, in terms of the physical and functional characteristics of stallion sperm. All sugars (glucose, fructose and sorbitol) had a strong influence on motility and viability of stallion semen post-thawing. Sorbitol-based extender significantly yielded in higher percentages of motile (50.2%) and viable sperm (54.3%) than glucose extender (40.6% and 48.6%) or fructose extender (40.0% and 46.9%). Using sorbitol and glucose in freezing extender also resulted in higher percentage of acrosome and plasma membrane integrity of sperm than fructose (P<0.05). Cryopreserved stallion spermatozoa using sorbitol-based extender were inseminated into estrus mare (n=4), two (50%) mares were pregnant. These results demonstrated convincingly that freezing techniques and sugar types in the freezing extenders play a central role in protecting sperm against cryoinjury that occurs during freezing and thawing. Sorbitol-based semen extender improves cryopreservability of stallion spermatozoa.