Department of Obstetrics, Gynaecology and Reproduction, Chulalongkorn University, Faculty of Veterinary Science, Bangkok, Thailand.
Theriogenology. 2013 Jan 1;79(1):149-58. doi: 10.1016/j.theriogenology.2012.09.022. Epub 2012 Nov 6.
Cryopreservation of testicular tissue associated with intracytoplasmic sperm injection (ICSI) is a critical tool that still needs to be explored for preserving the fertility of endangered species. Using the domestic cat as a model for wild felids, the study aimed at determining the effect of different cryoprotectants and freezing techniques (two-step freezing vs. controlled slow freezing) on the sperm quality (membrane and DNA integrity). Then, spermatozoa were extracted from frozen-thawed testicular tissues and used for ICSI to assess early gamete activation or developmental competence in vitro. The percentage of spermatozoa with intact plasma membrane was not different (P > 0.05) among nonfrozen control, glycerol-, and ethylene glycol-frozen tissues (63.2 ± 2%, 58.2 ± 2.6%, 53.3 ± 2.3%, respectively). However, these percentages were significantly lower (P < 0.05) in groups of dimethyl sulfoxide (46.3 ± 3.3%) and 1,2 propanediol (44.3 ± 2.9%) when compared with control. Conventional freezing combined with 5% (vol/vol) glycerol best preserved sperm membrane integrity (55.0 ± 2.7%) when compared with other freezing techniques. The incidence of DNA fragmentation was found to be low (0.2%-1.1%) in all freezing protocols. After ICSI with frozen testicular spermatozoa, male and female gametes were asynchronously activated and the percentages of normal fertilization at 6, 12, and 18 hours were 11.2%, 20.6%, and 22.1%, respectively. Metaphase II-arrested oocytes containing or not a decondensed sperm head were predominantly found after ICSI with frozen testicular spermatozoa. Although two-pronucleus formation could be observed as soon as 6 hours post ICSI (10%), the rate increased dramatically after 12 and 18 hours post ICSI (17.2% and 19.5%, respectively). ICSI using frozen-thawed testicular spermatozoa yielded cleavage (32.7%), morula (6.5%), and blastocyst (4.4%) percentages similar to nonfrozen control (P > 0.05). It is concluded that conventional freezing technique with glycerol as a principle cryoprotectant is simplified and applicable for cat testicular tissue cryopreservation. We also demonstrate for the first time that feline spermatozoa derived from frozen-thawed testicular tissues retain their fertilizing ability and can be used to produce ICSI-derived embryos.
使用猫科动物作为野生猫科动物模型，本研究旨在确定不同的冷冻保护剂和冷冻技术（两步冷冻与控制性缓慢冷冻）对精子质量（膜和 DNA 完整性）的影响。然后，从冷冻-解冻的睾丸组织中提取精子，用于 ICSI 以评估体外早期配子激活或发育能力。未冷冻对照、甘油和乙二醇冷冻组织中完整质膜精子的百分比没有差异（P>0.05）（分别为 63.2±2%、58.2±2.6%和 53.3±2.3%）。然而，与对照组相比，二甲基亚砜（46.3±3.3%）和 1,2-丙二醇（44.3±2.9%）组的这些百分比明显较低（P<0.05）。与其他冷冻技术相比，常规冷冻结合 5%（体积/体积）甘油可最好地保持精子膜的完整性（55.0±2.7%）。在所有冷冻方案中，DNA 碎片化的发生率均较低（0.2%-1.1%）。用冷冻睾丸精子进行 ICSI 后，雌雄配子被异步激活，6、12 和 18 小时时正常受精的百分比分别为 11.2%、20.6%和 22.1%。在用冷冻睾丸精子进行 ICSI 后，主要发现中期 II 期阻滞卵母细胞中含有或不含有去浓缩精子头。尽管在 ICSI 后 6 小时即可观察到双核形成（10%），但在 ICSI 后 12 和 18 小时，双核形成的比例显著增加（分别为 17.2%和 19.5%）。使用冷冻-解冻睾丸精子进行 ICSI 的卵裂（32.7%）、桑葚胚（6.5%）和囊胚（4.4%）百分比与非冷冻对照相似（P>0.05）。结果表明，使用甘油作为主要冷冻保护剂的常规冷冻技术简化且适用于猫睾丸组织的冷冻保存。我们还首次证明，来自冷冻-解冻睾丸组织的猫精子保留了其受精能力，并且可以用于产生 ICSI 衍生的胚胎。
