Cryopreservation of rainbow trout sperm in large volume straws: application to large scale fertilization

Traditional 0.25- and 0.5-ml straws have been successfully applied to the c ryopreservation of rainbow trout sperm. In order to facilitate the fertiliz ation of large egg batches, and with the purpose of transferring this techn ique to commercial hatcheries, the suitability of 1.8 and 5 ml straws was s tudied. Sperm was diluted at a 1:3 ratio in #6 Erdahl and Graham extender w ith 7% DMSO, 10% egg yolk and 7.5 mg/ml Promine. Freezing was performed at different levels above the surface of liquid nitrogen using a styrofoam box and thawing was carried out in a water bath at different temperatures. Fre ezing and thawing rates inside the straws were registered with a thermocoup le. The success of sperm cryopreservation was checked in vitro using severa l sperm quality parameters: motility, viability and membrane functionality. Fertility tests were also performed on small batches using a ratio of 200 eggs/ml of diluted sperm. Freezing and thawing rates were similar in 1.8- a nd 0.5-ml straws. However, lower freezing and thawing rates were achieved w ith 5-ml straws even at lower freezing and higher thawing temperatures. Mot ility analysis gave similar results in sperm cryopreserved in all the teste d straws (0.5, 1.8 and 5 ml). However, cell viability (61.9%) and fertility (73.2%) were slightly lower in large volumes than in 0.5-ml straws (77.4% and 84%, respectively). Fertilization of larger egg batches (1600-2000 eggs ) with sperm frozen in 5-ml straws provided similar fertilisation rates (73 %), demonstrating that the slight loss in fertility is compensated by the b enefits of 5-ml straws. Our results showed that cryopreservation of large s perm volumes could be useful for hatchery purposes. (C) 2001 Elsevier Scien ce B.V. All rights reserved.