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

E. Cabrita, V. Robles, R. Alvarez and M.P. Herráez-2001

Aquaculture, 201(3-4): 301-314
Abstract:

Traditional 0.25- and 0.5-ml straws have been successfully applied to the cryopreservation of rainbow trout sperm. In order to facilitate the fertilization of large egg batches, and with the purpose of transferring this technique to commercial hatcheries, the suitability of 1.8 and 5 ml straws was studied. Sperm was diluted at a 1:3 ratio in #6 Erdahl and Graham extender with 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. Freezing and thawing rates inside the straws were registered with a thermocouple. The success of sperm cryopreservation was checked in vitro using several 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- and 0.5-ml straws. However, lower freezing and thawing rates were achieved with 5-ml straws even at lower freezing and higher thawing temperatures. Motility analysis gave similar results in sperm cryopreserved in all the tested 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 benefits of 5-ml straws. Our results showed that cryopreservation of large sperm volumes could be useful for hatchery purposes.

(Department of Cell Biology, Faculty of Biology, University of Leon, 24071 Leon, Spain, Tel.: +34-987-291-492; fax: +34-987-291-487, e-mail of M.P. Herráez: dbcmho@unileon.es)

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