Cryopreservation of canine ovarian tissue by slow freezing and vitrification: Evaluation of follicular morphology and apoptosis rate

IF 2.4 2区 农林科学 Q3 REPRODUCTIVE BIOLOGY
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Abstract

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.

通过缓慢冷冻和玻璃化技术冷冻保存犬卵巢组织:卵泡形态和凋亡率评估
在这项研究中,我们旨在评估使用玻璃化和缓慢冷冻方法冷冻保存犬卵巢组织的效果,同时研究卵泡类型和冷冻保存技术在冷冻耐受性方面的潜在差异。从 14 只不同品种的发情母犬身上采集了 28 个卵巢,这些母犬的年龄在 2 到 5 岁之间,正在接受选择性卵巢切除术。卵巢被切成小块,随机分配到三组:玻璃化组、缓慢冷冻组和对照组(新鲜组织)。玻璃化是使用含有DAP 213溶液(2M二甲基亚砜、1M乙酰胺、3M丙二醇)的冷冻管分两个阶段进行的,而缓慢冷冻是将含有1.5M二甲基亚砜溶液的冷冻管插入可编程机器中进行的。冷冻效果通过组织学和免疫组织化学(裂解的 Caspase-3)进行评估,以确定细胞凋亡的百分比。组织学检查显示,慢冻组完整卵泡的比例(45.75%)明显高于玻璃化组(38.17%;P = 0.01)。免疫组化评估进一步表明,慢冻组84.21%的卵泡不表达caspase-3,表明没有卵泡凋亡。相反,玻璃化样本中的凋亡细胞明显多于其他组(P <0.001)。此外,无论采用哪种冷冻保存方法,早期前位卵泡都更容易发生变性。不过,在比较冷冻保存组时,慢冻组早期前位卵泡的退化程度更高,而玻璃化组前位卵泡受影响最大。总之,与玻璃化技术相比,慢冻技术能更好地保存存活卵泡,是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的冷冻保存方法提供了宝贵的见解,有可能对犬的生殖技术和生育力保存带来益处。
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来源期刊
Theriogenology
Theriogenology 农林科学-生殖生物学
CiteScore
5.50
自引率
14.30%
发文量
387
审稿时长
72 days
期刊介绍: Theriogenology provides an international forum for researchers, clinicians, and industry professionals in animal reproductive biology. This acclaimed journal publishes articles on a wide range of topics in reproductive and developmental biology, of domestic mammal, avian, and aquatic species as well as wild species which are the object of veterinary care in research or conservation programs.
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