Storage temperature and sucrose concentrations affect ram sperm quality after vitrification

Abstract

The aim of this study was to analyse the characteristics of ram spermatozoa subjected to varying concentrations of sucrose, and the influence of storage temperature (22°C or 5°C) prior to vitrification. Ejaculated semen was diluted in TCFEY (tris-citric acid-fructose 20% egg yolk), and two aliquots were prepared at a final concentration of 100×106spz/ml, one maintained at room temperature (22°C) and the other at 5°C. In the first experiment, the toxicity of sucrose diluents on the sperm was analysed; sperm samples at different temperatures were diluted (1:2) in TCF-BSA 2% (control) or in the same extender supplemented with various sucrose concentrations (0.4M, 0.6M and 0.8M). The effects of vitrification were studied in the second experiment, where sperm samples were mixed with different concentrations of cryoprotectants (sucrose) and vitrified by being plunged directly into liquid nitrogen. In both experiments, the sperm quality was assessed by measuring motility, morphology, membrane functionality (HOST), viability, acrosome integrity and DNA fragmentation. The toxicity test revealed significant differences (p≤0.05) when different sucrose concentrations were used; lower total and progressive motility, normal morphology and membrane functionality were noted when sucrose concentration was higher, compared to the control treatment. Samples maintained at room temperature showed significantly (p≤0.05) higher viability than samples stored at 5°C. In contrast, although the quality of vitrified sperm was drastically decreased in comparison with fresh sperm, sucrose was associated with greater total motility, viability and membrane functionality. This improvement was closely linked to the temperature at which the sperm had been previously maintained, showing higher values when sperm was stored at 5°C. The main conclusions to be drawn from the study are therefore that sucrose shows promising potential as a cryoprotectant, and storing samples at 5°C is linked to improved sperm quality following vitrification.