Browne R K, Clulow J, Mahony M
Department of Biological Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.
Reproduction. 2001 Jan;121(1):167-73. doi: 10.1530/rep.0.1210167.
The responses of cane toad (Bufo marinus) gametes, used as a model for the development of assisted reproduction techniques for rare and endangered amphibians, to short-term storage at temperatures > 0 degrees C were studied. Whole excised testes were stored at 0 degrees or 4 degrees C for 15 days, and sperm motility was measured at excision and after storage for 2, 5, 7, 10, 12 and 15 days. Spermatozoa showed > 50% motility for 7 days at 0 degrees C and for 5 days at 4 degrees C. At 15 days, only spermatozoa stored at 0 degrees C still showed some motility (3%). Sperm suspensions were prepared at 5 day intervals over 30 days in simplified amphibian ringer (SAR) at dilutions of 1:1, 1:5 and 1:10 (w/v) testes:SAR. Aliquots from each dilution were stored at 0 degrees C in Eppendorf tubes opened at 5 day intervals of storage (aerated) or kept sealed (unaerated) (treatments: aerated or unaerated; 5, 10, 15, 20, 25 and 30 days storage). After 30 days, sperm motility and fertilizing capacity were determined. The optimal protocol for sperm storage up to 10 days, as assessed by the retention of fertilizing capacity, was as a 1:5 testis:SAR (w/v) suspension, whereas the longest absolute retention of both motility and fertilizing capacity was observed in concentrated (1:1 dilution), anaerobic suspensions (up to 25-30 days). Oviductal oocytes placed in SAR at 5, 10, 15, 20 and 25 degrees C immediately after ovulation lost viability when cooled rapidly to 5 degrees C and stored for 2 h. However, oocytes retained viability for up to 8 h at the optimum storage temperature of 15 degrees C. Thus, it is concluded that during short-term storage spermatozoa retain viability for longer than oocytes, and that spermatozoa in suspensions retain viability for longer than spermatozoa stored in situ in excised testes.
以甘蔗蟾蜍（海蟾蜍，Bufo marinus）配子作为珍稀濒危两栖动物辅助生殖技术开发的模型，研究了其配子在高于0摄氏度的温度下短期储存后的反应。将完整切除的睾丸在0摄氏度或4摄氏度下储存15天，并在切除时以及储存2、5、7、10、12和15天后测量精子活力。精子在0摄氏度下7天内活力大于50%，在4摄氏度下5天内活力大于50%。在15天时，只有储存在0摄氏度下的精子仍显示出一定活力（3%）。在30天内，每隔5天在简化两栖类林格氏液（SAR）中以1:1、1:5和1:10（重量/体积）睾丸:SAR的稀释度制备精子悬液。每个稀释度的等分试样在0摄氏度下储存在以5天为间隔打开（通气）或保持密封（不通气）的Eppendorf管中（处理：通气或不通气；储存5、10、15、20、25和30天）。30天后，测定精子活力和受精能力。根据受精能力的保留情况评估，精子储存长达10天的最佳方案是以1:5睾丸:SAR（重量/体积）悬液，而在浓缩（1:1稀释）、厌氧悬液中观察到活力和受精能力的最长绝对保留时间（长达25 - 30天）。排卵后立即置于5、10、15、20和25摄氏度的SAR中的输卵管卵母细胞，在迅速冷却至5摄氏度并储存2小时后失去活力。然而，在15摄氏度的最佳储存温度下，卵母细胞可保持活力长达8小时。因此，得出结论：在短期储存期间，精子保持活力的时间比卵母细胞长，并且悬液中的精子保持活力的时间比切除睾丸中原位储存的精子长。
