Temerario Letizia, Martino Nicola Antonio, Bennink Monika, de Wit Agnes, Hiemstra Sipke Joost, Dell'Aquila Maria Elena, Lamy Julie
Department of Biosciences, Biotechnology & Environment, University of Bari Aldo Moro, Strada per Casamassima km 3, 70010 Valenzano, Italy.
Animal Breeding and Genomics, Wageningen University & Research, 6700 AH Wageningen, The Netherlands.
Animals (Basel). 2024 Aug 14;14(16):2351. doi: 10.3390/ani14162351.
Oocyte vitrification allows for the storing of endangered breed female gametes. Cryoprotectant (CPA) concentration and exposure time should ensure cell protection with minimal toxicity. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating CPAs, were evaluated on meiotic competence and bioenergetic-oxidative status of pre-pubertal lamb immature COCs after in vitro maturation (IVM). For each protocol, COCs vitrified through a traditional protocol and fresh ones were used as controls. Both protocols allowed COC morphology preservation after vitrification-warming (V-W) and cumulus expansion after IVM. The maturation rate (7% and 14%) was comparable to the vitrified control (13% and 21%) but not satisfactory compared to fresh ones (58% and 64%; < 0.001). The rate of mature oocytes displaying a perinuclear/subcortical (P/S) mitochondrial distribution pattern, an index of cytoplasmic maturity, was comparable between vitrified and fresh oocytes. The LC-SE vitrification protocol did not affect quantitative bioenergetic-oxidative parameters compared to both controls whereas HC-RE protocol significantly reduced intracellular reactive oxygen species (ROS) levels, indicating cell viability loss. In conclusion, to improve pre-pubertal lamb immature COC vitrification, the combination of low CPA concentrations with prolonged exposure time could be more promising to investigate further.
卵母细胞玻璃化冷冻可用于储存濒危品种的雌性配子。冷冻保护剂（CPA）的浓度和暴露时间应确保在毒性最小的情况下保护细胞。在本研究中，使用二甲亚砜（DMSO）和乙二醇（EG）作为渗透性CPA，对青春期前羔羊未成熟卵丘-卵母细胞复合体（COCs）体外成熟（IVM）后的减数分裂能力和生物能量-氧化状态进行了高浓度-快速暴露（HC-RE）和低浓度-缓慢暴露（LC-SE）玻璃化冷冻方案的评估。对于每个方案，将通过传统方案玻璃化冷冻的COCs和新鲜的COCs用作对照。两种方案在玻璃化-复温（V-W）后均能保持COCs的形态，并且在IVM后能使卵丘扩展。成熟率（7%和14%）与玻璃化冷冻对照组（13%和21%）相当，但与新鲜组相比并不理想（58%和64%；P<0.001）。显示核周/皮质下（P/S）线粒体分布模式（细胞质成熟的指标）的成熟卵母细胞比例在玻璃化冷冻和新鲜卵母细胞之间相当。与两个对照组相比，LC-SE玻璃化冷冻方案不影响生物能量-氧化的定量参数，而HC-RE方案显著降低了细胞内活性氧（ROS）水平，表明细胞活力丧失。总之，为了改进青春期前羔羊未成熟COCs的玻璃化冷冻，低CPA浓度与延长暴露时间的组合可能更有前景，值得进一步研究。
