Cryopreservation of dolphin spermatozoa

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In order to develop an effective and simple method for cryopreserving dolphin semen processed immediately after collection by the hand-manipulation method, we conducted some freezing and thawing experiments by straw method for fresh semen samples obtained from two bottlenose dolphins (Tursiops truncatus) and one Pacific white-sided dolphins (Lagenorhynchus obliquidens) in captivity. For both dolphin species, post-thaw sperm with total motility about 60% or more, percentage progressive motility over 95% and kinetic rating 4 to 5 (rapid forward progressive movement) were obtained by the straw method, with semen extender containing 2.9% trisodium citrate hydrate, 8% glycerol, 20% egg-yolk and antibiotics. Fresh semen was diluted twice with the semen extender to the final dilution 1:2 (semen : extender, v/v) and precryopreserved at 4 degrees C (refrigerator;1-1.5 hr) at the first dilution and then at 2 degree C (ice-water bath;0.25-1hr) at the second dilution. About 300-400 micro litters of extended semen put into 0.5mL-straw were frozen in the liquid nitrogen vapor for 15 min. and then straws were plunged into liquid nitrogen for complete freezing. Rapid thawing in the water bath at 35-36 degrees C led into better post-thaw semen characteristics. Post-thaw sperm frozen by the present straw method was used for the artificial insemination(AI) to a female bottlenose dolphin in September 2003,and a male calf was born as the second AI dolphin baby (the first with frozen-thawed sperm) in Japan in September 2004,