系统低温生物学技术在实验小鼠资源保存中的建立与应用

目的建立小鼠胚胎与配子冷冻库，以安全、有效地保存小鼠资源。方法选择不同遗传背景（近交系、远交群、免疫缺陷、疾病模型和基因改变等）的实验小鼠，系统进行了超数排卵、体外受精（IVF）率、胚胎与精子的冷冻复苏效果、卵巢冷冻与移植、辅助体外受精等比较研究。结果①小鼠年龄和遗传背景的不同，其超数排卵的结果也不同（P〈0．01）。三个日龄段中，28日龄最好，其次为112日龄，56日龄最差；不同遗传背景小鼠的超数排卵结果显示，封闭群和大部分近交系小鼠优于转基因小鼠（P〈0．05），自发性疾病小鼠和基因剔除小鼠的结果最差；②不同品系小鼠的新鲜精子和冻融精子的体外受精率差异有显著性（P〈0．05），特别是C57BL／6J小鼠冻融精子的IVF率（10．3±4．2％）与新鲜精子（89．8±4.8％）相比，差异极显著（P〈0．01）；③不同品系小鼠的胚胎复苏率，除MRL／mp小鼠的复苏率略低外，其他小鼠品系均有较高的冷冻胚胎复苏率（58．2％～83．9％），表明，不同遗传背景小鼠之间差异有显著性（P〈0．05），但均可以达到有效保存小鼠资源的目的。④小鼠的遗传背景、年龄等对小鼠精子的冷冻效果都有影响，采用改良的FERTIUP冷冻保护剂和细胞质内单精注射（ICSI）技术可有效提高以C57BL／6J为背景的基因改变小鼠的精子冷冻复苏率。⑤卵巢冷冻保存可以改善雌性小鼠的繁殖困难或不孕。结论小鼠资源的安全保存，除了长期连续繁殖保种外，最好的或最保险的方法是低温保存。通过将胚胎、配子、卵巢等长期保存在液氮（-196℃）中避免遗传性状的改变，并在将来复苏后获得正常的小鼠后代，以用于生物学和医学等研究。

Establishment and Application of Systemic Cryobiological Techniques in Laboratory Mouse Resource Preservation

Objective To establish the banking of mouse embryos and gametes for preserving mouse resources safely and efficaciously. Methods To select diverse genetic background mouse strains of inbred, closed colony, immunodeficiency, disease model and mutagenesis, and make comparative analysis based on superovulation, the rate of in vitro fertilization （IVF）, thethawed rate of frozen embryos and sperms, ovary cryopreservation and subsequent transplantation, and intracytoplasmic sperm injection （ICSI） .Results ①the age and genetic background of mice were major factor influencing the superovulation in mice. The donor mouse age of 28 days showed significant different effect compared with that of 56 and 112 day-old mice （P 〈 0.01 ）, and the oocytes superovulation of closed colonies and mostly inbred strains were higher than that in genetic manipulated mice and spontaneous diseased mice（ P 〈 0.05）. ②There were distinct differences in the rate of fertilization and in fresh vs. post-thawed sperms in mice with different genetic background （P 〈 0.05）, especially in C57BL/6J mice （89.8 ±4.8 % vs. 10.3 ±4.2% ）. ③There were higher thawed rates （58.2% ～83.9% ） except for MRL/mp strain in all mouse strains used in this experiment. ④ The survival of thawed sperms were influenced by genetic background and age of mice, and the thawed rate of genetic manipulated mice with C57BL/6J background were remarkably improved by using FERTIUP cryopmteetant solution and ICSI. ⑤The results confirmed previous findings that grafting ovaries frozen in DMSO can restore fertility in female mice. Conclusions These results suggest that besides the method of long-term continuous breeding, the best and most safe method is cryopreservation and banking of mice embryos, gametes and ovaries. The preservation of gametes or ovaries in liquid nitrogen at - 196℃for extensive periods without genetic changes and subsequent recovery of normal offspring from the stored material has important implications in animal breeding, biomedical research and medicine.