McClean Rhett, MacCallum Catriona, Blyde David, Holt William V, Johnston Stephen D
School of Animal Studies, University of Queensland, Gatton, Qld 4343, Australia.
Reprod Fertil Dev. 2006;18(4):469-76. doi: 10.1071/rd05148.
The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Tris-citrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P < 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa (P < 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol (P < 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.
本研究的目的是比较成熟和未成熟附睾袋鼠精子的冷冻保存、渗透耐受性和甘油毒性，以调查附睾尾部精子冷冻保存成功率低是否可能与附睾转运过程中精子超微结构复杂性增加有关。从赤颈袋鼠（RNW；Macropus rufogriseus）和东部灰袋鼠（EGK；M. giganteus）中回收附睾头部和尾部的精子。在实验1中，附睾头部和尾部的精子在含有或不含有20%甘油的柠檬酸盐缓冲液中，使用标准冷冻保存程序进行冷冻和解冻。尽管附睾头部精子的冷冻保存导致精子质膜损伤显著增加，但它们比从附睾尾部回收的精子对该程序的耐受性更强（P < 0.05）。在实验2中，将附睾头部和尾部的EGK精子稀释到不同渗透压的磷酸盐缓冲盐溶液中，并测定它们的渗透耐受性。附睾头部精子的质膜比附睾尾部精子对低渗介质的耐受性更强（P < 0.05）。在实验3中，将附睾头部和尾部的RNW精子在含有和不含有20%甘油的柠檬酸盐缓冲液中，于35℃和4℃下孵育，以检查甘油的细胞毒性作用。在两个温度下，当暴露于20%甘油时，附睾头部精子与附睾尾部精子相比，质膜损伤更少（P < 0.05）。这些实验清楚地表明，袋鼠精子的附睾成熟导致其承受与冷冻保存相关的生理应激的能力下降。
