Comparison of different freezing techniques, extenders, and cryoprotectants on quality and fertility of cryopreserved Salmo trutta f. fario sperm

Abstract

There is still a lack of standardized methodology in the cryopreservation of Salmo trutta f. fario sperm. Thus, the present study was designed to compare current freezing protocols to improve and as well as to examine the post-thaw quality and fertilizing ability of cryopreserved sperm in Salmo trutta f. fario. Sperm samples were diluted at a 1:10 ratio in one of three extenders (Alsever´s solution, glucose-based, and ionic-based) containing four types of cryoprotectant (DMSO, DMA, MeOH, glycerol) at 10% concentration and frozen in 0.1 mL pellets on surface of the dry ice (solid carbon dioxide, 79°C) or in 0.25 mL straws 2 cm above of the liquid nitrogen (LN₂) surface at a rate of ~30°C for 10 min.¹ before storage in a mL cryotank (-196°C). The frozen sperm cells in straws and pellets were thawed in a water bath at 25°C for 30 s and at 20°C for 6 s respectively. Fertilization was carried out using a ratio of 5x10⁵ sperm/egg in both freezing methods. The glucose-based solution including glycerol produced the highest post-thaw progressive motility (62.5 ± 1.24%), motility duration (57.2 ± 0.46 s), and viability (56.4 ± 1.57%) (p < 005) in the straw method. In breeding trials, similarly, sperm frozen-thawed with the glucose-based solution including glycerol produced the highest fertilization (54.2 ± 0.36%) and hatching (30.6 ± 0.28%) in the straw method. Fresh sperm used as control produced 82.6 ± 0.45% and 78.4 ± 1.27% fertilization and hatching respectively. It was concluded that sperm frozen in straws produced higher post-thaw sperm motility and fertility of eggs than those frozen in pellets. Also, the results suggest using of glycerol-supplemented glucose solution because of producing better results in both freezing techniques.