Luizari Stábile Nicole A, Oliveira Frederico Rocha de, Furtado Ricardo Andrade, Felippe Carolina Barretto M L, Tavares Mariana Riboli, Martinelli Paulo E B, Fonseca-Alves Carlos Eduardo, Souza Fabiana Ferreira de, Colombo Martina, Luvoni Gaia Cecilia, Apparício Maricy
Universidade de Franca, Franca, SP, Brazil.
Universidade Estadual Paulista (UNESP), FCAV, Campus de Jaboticabal, Jaboticabal, SP, Brazil.
Theriogenology. 2024 Dec;230:8-14. doi: 10.1016/j.theriogenology.2024.08.032. Epub 2024 Aug 30.
In this study, we aimed to evaluate the efficacy of cryopreserving canine ovarian tissue using vitrification and slow freezing methods while investigating potential differences in cryotolerance based on follicular type and cryopreservation technique. Twenty-eight ovaries were collected from 14 anoestrus bitches of various breeds, aged between 2 and 5 years, and undergoing elective ovariohysterectomy. The ovaries were sectioned into small fragments and randomly assigned to three groups: vitrification, slow freezing, and a control group (fresh tissue). Vitrification was performed using cryotubes containing DAP 213 solution (2M DMSO, 1M acetamide, 3M propylene glycol) in two stages, while slow freezing involved cryotubes with 1.5M DMSO solution inserted into a programmable machine. The effects of cryopreservation were evaluated by histology and immunohistochemistry (cleaved caspase-3), to determine the percentage of cells undergoing apoptosis. Histological examination revealed that the slow freezing group exhibited a significantly higher percentage of intact follicles (45.75 %) compared to those subjected to vitrification (38.17 %; P = 0.01). Immunohistochemical evaluation further indicated that 84.21 % of the follicles in the slow freezing group did not express caspase-3, suggesting the absence of apoptosis. Conversely, vitrified samples exhibited significantly more apoptotic cells compared to other groups (P < 0.001). Furthermore, early antral follicles displayed a higher susceptibility to degeneration regardless of the cryopreservation method employed. Nevertheless, when comparing the cryopreserved groups, early antral follicles showed greater degeneration in slow freezing group, while preantral follicles were the most affected in the vitrification group. In conclusion, slow freezing demonstrated superior preservation of viable follicles compared to vitrification and emerged as the preferred technique for cryopreserving canine ovarian tissue. These findings contribute valuable insights into optimizing cryopreservation methods for canine ovarian tissue, potentially benefiting reproductive technologies and fertility preservation in canines.
在本研究中，我们旨在评估玻璃化冷冻和慢速冷冻方法保存犬卵巢组织的效果，同时研究基于卵泡类型和冷冻保存技术的耐冻性潜在差异。从14只年龄在2至5岁、处于乏情期、品种各异且接受择期卵巢子宫切除术的母犬身上采集了28个卵巢。将卵巢切成小碎片，随机分为三组：玻璃化冷冻组、慢速冷冻组和对照组（新鲜组织）。玻璃化冷冻分两个阶段使用含有DAP 213溶液（2M二甲基亚砜、1M乙酰胺、3M丙二醇）的冷冻管进行，而慢速冷冻则是将含有1.5M二甲基亚砜溶液的冷冻管放入程序降温仪中。通过组织学和免疫组织化学（裂解的半胱天冬酶-3）评估冷冻保存的效果，以确定凋亡细胞的百分比。组织学检查显示，与玻璃化冷冻组（38.17%；P = 0.01）相比，慢速冷冻组完整卵泡的百分比显著更高（45.75%）。免疫组织化学评估进一步表明，慢速冷冻组84.21%的卵泡不表达半胱天冬酶-3，表明不存在凋亡。相反，与其他组相比，玻璃化冷冻样本显示出明显更多的凋亡细胞（P < 0.001）。此外，无论采用何种冷冻保存方法，早期有腔卵泡对退化更敏感。然而，在比较冷冻保存组时，早期有腔卵泡在慢速冷冻组中退化更严重，而初级卵泡在玻璃化冷冻组中受影响最大。总之，与玻璃化冷冻相比，慢速冷冻在保存存活卵泡方面表现更优，成为冷冻保存犬卵巢组织的首选技术。这些发现为优化犬卵巢组织冷冻保存方法提供了有价值的见解，可能有益于犬类的生殖技术和生育力保存。
