Wang Yan-Ping, Zhao Xue-Ming, Zhou Guang-Bin, Hou Yun-Peng, Fan Zhi-Qiang, Yan Chang-Liang, Suo Lun, Fu Xiang-Wei, Zhu Shi-En
Laboratory of Animal Embryonic Biotechnology, College of Animal Science and Technology, China Agricultural University, Beijing, China.
Cryo Letters. 2008 Mar-Apr;29(2):111-9.
The present study was designed to investigate the optimized conditions for cryopreservation of Kunming (KM) mice spermatozoa (Experiment 1) and to compare the developmental potential of IVF embryos produced from fresh oocytes (Group 1), vitrified-warmed oocytes without (Group 2) or with partial zona pellucida incised by a piezo manipulator (ZIP) (Group 3) fertilized with frozen-thawed spermatozoa (Experiment 2). In experiment 1, spermatozoa were cryopreserved with the medium containing raffinose and egg yolk with different concentrations (0 to 60 percent) and then followed by fertilization with fresh oocytes after thawing. The highest cleavage (76.2 percent) and blastocysts formation rates (63.6 percent) were obtained when the egg yolk concentration was adjusted to 30 percent. To optimize the equilibration time, the spermatozoa were equilibrated in the optimized medium for 0, 10, 30, 50, 70, 90 min at 40 degree C before plunging into liquid nitrogen. After thawing, the highest cleavage rate (87.4 percent) of IVF embryos was observed when equilibrated for 30 min. In experiment 2, the cleavage and blastocyst rates in Group 1 (81.2 percent, 65.4 percent) and Group 3 (72.5 percent, 45.0 percent) were higher (P less then 0.05) than those in Group 2 (22.2 percent and 13.9 percent), respectively. When 2-cell embryos obtained in Group 1 and 3 were transferred, 32.1 percent and 22.7 percent of embryos in the pregnant receipts developed to term, respectively. In conclusion, the optimized protocol is highly efficient for the cryopreservation of KM mice spermatozoa; the ZIP technique is very useful for improvement of the fertilization efficiency using the cryopreserved gametes and normal offspring can be produced efficiently.
本研究旨在探究昆明（KM）小鼠精子冷冻保存的优化条件（实验1），并比较由新鲜卵母细胞（第1组）、未进行操作（第2组）或经压电操纵器部分切割透明带（ZIP）处理（第3组）的玻璃化-解冻卵母细胞与冷冻-解冻精子受精产生的体外受精（IVF）胚胎的发育潜能（实验2）。在实验1中，精子用含有不同浓度（0%至60%）棉子糖和蛋黄的培养基进行冷冻保存，解冻后再与新鲜卵母细胞受精。当蛋黄浓度调整为30%时，获得了最高的卵裂率（76.2%）和囊胚形成率（63.6%）。为优化平衡时间，精子在4℃下于优化培养基中平衡0、10、30、50、70、90分钟，然后投入液氮。解冻后，平衡30分钟时观察到IVF胚胎的最高卵裂率（87.4%）。在实验2中，第1组（81.2%，65.4%）和第3组（72.5%，45.0%）的卵裂率和囊胚率分别高于第2组（22.2%和13.9%）（P＜0.05）。当移植第1组和第3组获得的2细胞胚胎时，妊娠受体中分别有32.1%和22.7%的胚胎发育至足月。总之，优化方案对KM小鼠精子的冷冻保存非常有效；ZIP技术对于提高使用冷冻配子的受精效率非常有用，并且可以高效产生正常后代。
