Li Ming W, Biggers John D, Elmoazzen Heidi Y, Toner Mehmet, McGinnis Lynda, Lloyd K C Kent
School of Veterinary Medicine, Center for Comparative Medicine, University of California, Davis, CA 95616, USA.
Reproduction. 2007 May;133(5):919-29. doi: 10.1530/REP-06-0096.
To determine if mouse spermatozoa could be preserved long-term without using liquid nitrogen, mouse spermatozoa in trehalose-EGTA solution were partially evaporatively dried under nitrogen gas (5 min at flow rate10 l/min) and stored for 1 week and 5 months at 4, -20, and -80 degrees C before intracytoplasmic sperm injection. Fertilization rates were neither different with spermatozoa stored at 4, -20, or -80 degrees C for 1 week or 1, 3, and 5 months respectively, nor blastocyst formation rates with spermatozoa stored for 1 week and 1 month. However, spermatozoa stored at 4 and -20 degrees C for 3 months resulted in fewer blastocysts (35.1 and 54.3% respectively) when compared with spermatozoa stored at -80 degrees C (74.4%). Blastocyst formation rates using spermatozoa stored for 5 months at -20 degrees C (57.4%) or -80 degrees C (74.5%) were not significantly different from those stored for 3 months at the same temperatures respectively, but were significantly better than those stored for 5 months at 4 degrees C (10.2%). Blastocysts derived from spermatozoa stored for 3 and 5 months at -20 and -80 degrees C respectively, were then transferred to pseudopregnant mothers to develop into healthy liveborn offspring. No significant differences were found in embryo transfer rates (number of pups born/number of embryos transferred), weaning rates, or sex ratios of resultant pups, which were healthy and reproductively sound. These results demonstrate for the first time that partially evaporatively dried mouse spermatozoa in trehalose-EGTA solution can be preserved for long term at -20 and -80 degrees C. The possibility that the storage temperature must be less than the glass transition temperature is discussed.
为了确定小鼠精子在不使用液氮的情况下能否长期保存，将处于海藻糖 - 乙二醇双乙酸酯（EGTA）溶液中的小鼠精子在氮气（流速10升/分钟，持续5分钟）下进行部分蒸发干燥，然后在4℃、-20℃和-80℃下保存1周和5个月，之后用于胞浆内精子注射。分别在4℃、-20℃或-80℃下保存1周以及1、3和5个月的精子，其受精率并无差异，在4℃、-20℃或-80℃下保存1周和1个月的精子，其囊胚形成率也无差异。然而，与在-80℃下保存的精子（74.4%）相比，在4℃和-20℃下保存3个月的精子形成的囊胚较少（分别为35.1%和54.3%）。使用在-20℃（57.4%）或-80℃（74.5%）下保存5个月的精子的囊胚形成率，与在相同温度下保存3个月的精子的囊胚形成率相比，差异不显著，但明显优于在4℃下保存5个月的精子的囊胚形成率（10.2%）。然后将分别在-20℃和-80℃下保存3个月和5个月的精子所形成的囊胚移植到假孕母鼠体内，使其发育成健康的活产后代。在胚胎移植率（出生幼崽数/移植胚胎数）、断奶率或所产幼崽的性别比例方面未发现显著差异，这些幼崽健康且生殖功能健全。这些结果首次证明，处于海藻糖 - EGTA溶液中的部分蒸发干燥的小鼠精子可以在-20℃和-80℃下长期保存。文中还讨论了储存温度必须低于玻璃化转变温度的可能性。
