Evaluation of the morphological and functional state of cryopreserved sperm of bulls at different periods of long-term storage

Abstract

Objective. To study the morphological andfunctional state of cryopreserved sperm of bulls during storage for 6 - 27 years at a temperature of - 196 C in liquid nitrogen in a comparative aspect. Methods. The object of the study was cryopreserved semen of bulls stored for 6 - 10 or 20 - 27 years. In the study of the qualitative parameters offrozen- thawed spermatozoa, the method of visual assessment for motility and survival, the method ofers differential staining with trypan blue/Gymza dyes to determine the morphological and functional integrity of cell membranes and the test for the ability to bind to vesicular explants of bovine oviductal epithelial cells in vitro were used. Results. It was found that the extension of the storage period of spermatozoa to 20 - 27years had no significant effect on the functional characteristics o f spermatozoa in terms of motility, survival and absolute survival. The method of differential staining showed that the proportion of live sperm with an intact acrosome in the total distribution did not differ significantly in both studied storage periods and ranged from 61.6 - 63.9 %. The proportion of dead cells also did not differ significantly and was 26.4 % when stored for up to 10 years and 21.7 % when stored for more than 20 years. No significant difference was found in the overall assessment of the percentage of sperm with varying degrees of acrosomal membrane damage. This figure was 22.5 and 26.0 %, respectively. At the same time, when the shelf life of cryopreserved sperm was extended to 20 - 27 years, a significant increase in the percentage of live sperm with a missing acrosome was observed (p<0.01). It was determined that the index of sperm binding to explants of epithelial cells of the oviduct in vitro was significantly higher when storedfor 6-10 years and was 227.9±32.3 against 114.6±19.5 when stored for 20 - 27 years (p<0.05). Conclusions. The use of the traditional method of assessing sperm quality by indicators of sperm motility, survival and absolute sperm survival did not reveal any differences when storing cryopreserved sperm for 6 - 27 years. At the same time, the differential staining method showed a significant increase of 7.2% in the proportion of live sperm with a missing acrosome when the storage period was extended from 6 - 10 to 20 - 27 years. The in vitro test for sperm binding to oviduct epithelial cells showed a significant decrease of almost 2 times in the index of sperm binding to cellular explants during long-term storage. This may indicate a decrease in the ability of such sperm to fertilise. Keywords: bulls, sperm, cryopreservation, long-term storage, differential colouration, oviduct, sperm binding.