Cryopreservation of dolphin spermatozoa

为了探索一种简单有效的海豚精液冷冻保存方法，对人工饲养的两条宽吻海豚(Tursioptruncatus)和一条太平洋白边海豚(Lagnowhynchus Obliquidens)的新鲜精液进行了吸管法冷冻和解冻试验。两种海豚精液解冻后，用含2.9%柠檬酸三钠、8%甘油、20%蛋黄和抗生素的吸管法精液解冻后，精子的总活动率在60%左右，前向运动百分率在95%以上，运动能力分级为4~5级(快速前进运动)。新鲜精液用精液稀释液稀释两次，最终稀释度为1：2(精液：稀释液，v/v)，第一次稀释液在4℃(冰箱；1-1.5小时)预冻，第二次稀释液在2℃(冰水浴；0.25-1小时)预冻。将大约300-400微升的延伸精液放入0.5ml的吸管中，在液氮蒸气中冷冻15分钟。然后将稻草放入液氮中完全冷冻。在35-36摄氏度的水浴中快速解冻可以获得更好的解冻后精液特性。2003年9月，采用目前管道法冷冻的解冻精子对雌性宽吻海豚进行人工授精，2004年9月日本出生了第二只雄性海豚幼崽(第一只冷冻解冻精子)，

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,