Apparicio M, Ruggeri E, Luvoni G C
Departmento de Medicina Veterinária Preventiva e Reprodução Animal, UNESP - Univ Estadual Paulista, Jaboticabal Campus, Jaboticabal, SP, Brazil.
Reprod Domest Anim. 2013 Apr;48(2):240-4. doi: 10.1111/j.1439-0531.2012.02138.x. Epub 2012 Jul 7.
The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n = 72) using a vitrification kit for bovine embryo or slow frozen (n = 69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48 h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n = 92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p < 0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p < 0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p < 0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48 h of culture.
本研究的目的是评估一种用于牛胚胎玻璃化的商业试剂盒对猫卵母细胞冷冻保存的适用性，并比较其与慢速冷冻程序在形态学和卵母细胞减数分裂恢复方面对冷冻耐受性的影响。从猫卵巢分离的生发泡期卵母细胞，一部分（n = 72）使用牛胚胎玻璃化试剂盒进行玻璃化处理，另一部分（n = 69）在转移至程序控制胚胎冷冻仪之前，先将卵母细胞暴露于乙二醇溶液中进行慢速冷冻。解冻和复温后，将卵母细胞培养48小时，然后使用双苯甲酰亚胺荧光染色（Hoechst 33342）检查减数分裂恢复情况。新鲜未成熟卵母细胞（n = 92）用作对照组。解冻/复温后形态正常的卵母细胞比例，冷冻组（94.5%）显著高于玻璃化组（75%，p < 0.01）。培养后的形态完整性在玻璃化组（73.6%）和慢速冷冻组（76.8%）中相似；然而，玻璃化处理后形态正常的卵母细胞只有37.5%恢复减数分裂，而慢速冷冻组为60.9%（p < 0.01）。无论采用何种程序，新鲜卵母细胞的形态完整性（91.3%）和减数分裂恢复率（82.6%，p < 0.002）均高于冷冻保存的卵母细胞。这些结果表明，用牛胚胎试剂盒玻璃化的未成熟猫卵母细胞在复温和培养后仍保留减数分裂恢复能力，尽管恢复率低于慢速冷冻的卵母细胞。玻璃化和慢速冷冻方法在培养后形态正常的卵母细胞比例相似，这表明抵抗冷冻损伤的解冻和复温卵母细胞在培养48小时后保持完整性的机会相同。
