Ferreira J C, Meira C, Papa F O, Landin e Alvarenga F C, Alvarenga M A, Buratini J
Department of Animal Reproduction and Veterinary Radiology, FMVZ-UNESP, Botucatu S.P. CEP, Brazil.
Equine Vet J Suppl. 1997 Dec(25):88-93. doi: 10.1111/j.2042-3306.1997.tb05109.x.
Six or 7-day-old equine embryos were divided into 4 groups; Group 1, n = 15, Day 7 embryos destined for immediate transfer; Group 2, n = 15, Day 6 embryos destined for deep-freezing with glycerol plus sucrose as cryoprotectant; Group 3, n = 10, Day 6 embryos destined for deep-freezing with glycerol plus 1,2-propanediol as cryoprotectant and Group 4, n = 3, fresh embryos destined for ultrastructural analysis. All the frozen/thawed embryos were transferred to recipient mares, except 3 embryos in Group 3 that were subjected to ultrastructural analysis. After thawing the cryoprotectants were removed by successive dilutions in PBS + 15% v:v fetal calf serum (FCS) containing decreasing concentrations of the cryoprotectants. Pregnancy was diagnosed ultrasonographically in 53.3%, 13.3% and 0% of the mares in Groups 1, 2 and 3 respectively. Ultrastructural analysis showed differences between frozen/thawed and fresh embryos. In the former, embryonic cells were deformed and showed dilation of the intercellular and perivitelline spaces, a decrease of desmosome number in the junctional complexes, few microvilli on the apical surface of the trophectoderm and an almost total absence of pinocytotic vesicles. Most of the mitochondria showed regions containing dilation and irregularities on the cristae, which appeared electron-dense. The results obtained with Groups 2 and 3 embryos showed that the cryoprotectants employed were not effective in protecting the embryos against damage during freezing and thawing. Indeed, the ultrastructural changes observed in the Group 3 embryos explained the absence of any established pregnancies in this group of mares.
将6或7日龄的马胚胎分为4组；第1组，n = 15，为第7天的胚胎，准备立即移植；第2组，n = 15，为第6天的胚胎，准备用甘油加蔗糖作为冷冻保护剂进行深度冷冻；第3组，n = 10，为第6天的胚胎，准备用甘油加1,2 - 丙二醇作为冷冻保护剂进行深度冷冻；第4组，n = 3，为新鲜胚胎，准备用于超微结构分析。除第3组中有3个胚胎用于超微结构分析外，所有冷冻/解冻后的胚胎均移植到受体母马体内。解冻后，通过在含有浓度逐渐降低的冷冻保护剂的PBS + 15%（体积比）胎牛血清（FCS）中连续稀释来去除冷冻保护剂。第1、2和3组母马的妊娠超声诊断率分别为53.3%、13.3%和0%。超微结构分析显示冷冻/解冻胚胎与新鲜胚胎之间存在差异。在冷冻/解冻胚胎中，胚胎细胞变形，细胞间和卵周间隙扩张，连接复合体中的桥粒数量减少，滋养外胚层顶端表面的微绒毛很少，几乎完全没有胞饮小泡。大多数线粒体显示出含有扩张和嵴不规则的区域，这些区域电子密度较高。第2组和第3组胚胎的结果表明，所使用的冷冻保护剂在冷冻和解冻过程中不能有效地保护胚胎免受损伤。实际上，在第3组胚胎中观察到的超微结构变化解释了该组母马中没有任何妊娠成功的原因。
