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Abstract：
The in vitro maturation (IVM) rate of canine oocytes remains low compared to other mammals due to their unique reproductive characteristics. This study aimed to explore the effect of hormone supplementation during the IVM of canine immature oocytes on nuclear maturation and subsequently assess its potential application in canine somatic cell nuclear transfer (SCNT). Immature oocytes were collected and cultured in an IVM medium supplemented with hormones (follicle-stimulating hormone [FSH] and progesterone [P4]) or without hormones (control) for 24 hours. The maturation rates of oocytes in the hormone-treated group (94.92 ± 3.15%) were significantly higher than those in the control group (61.01 ± 4.23%). Both in vitro and in vivo matured oocytes underwent NT to evaluate their utility, and the fusion rates were higher in the in vitro matured group than those in the vivo matured group, not significant between in vivo and in vitro matured group (73.28% and 82.35%, respectively). As a result, 14 fused embryos from the in vitro matured group were transferred into two surrogates, with one surrogate achieving a successful pregnancy and delivering four puppies. Whereas in the in vivo matured group, 85 fused embryos were transferred to 8 surrogate mothers, leading to three surrogates becoming pregnant and delivering one, four, and two puppies. The pregnancy rates were not significant between both groups (50% and 37.50%), but the number of offspring exhibited a significant difference (28.57% and 8.23%). In conclusion, we achieved a remarkable milestone by successfully producing cloned puppies using in vitro matured oocytes, underscoring the feasibility of canine cloning from in vitro recovered oocytes. It is important to note that this study focused only on immature oocytes after ovulation and only during the estrus stage. Further research targeting other stages of the estrous cycle could potentially enhance canine cloning efficiency.
摘要：
与其他哺乳动物相比，犬卵母细胞的体外成熟（IVM）率由于其独特的生殖特征而仍然很低。本研究旨在探讨犬未成熟卵母细胞IVM期间补充激素对核成熟的影响，并随后评估其在犬体细胞核移植（SCNT）中的潜在应用。收集未成熟的卵母细胞并在补充有激素(促卵泡激素[FSH]和孕酮[P4])或无激素(对照)的IVM培养基中培养24小时。激素处理组卵母细胞成熟率(94.92±3.15%)明显高于对照组(61.01±4.23%)。体外和体内成熟的卵母细胞都接受了NT来评估它们的效用，体外成熟组的融合率高于体内成熟组，体内和体外成熟组之间不显著(73.28%和82.35%，分别)。因此,将来自体外成熟组的14个融合胚胎转移到两个替代品中，一个代孕者成功怀孕并分娩四只小狗。而在体内成熟组，85个融合胚胎被转移到8个代孕母亲身上,导致三个代理人怀孕并分娩一个，四,还有两只小狗.两组妊娠率无显著性差异(50%和37.50%),但后代数量差异显著(28.57%和8.23%)。总之,我们通过使用体外成熟的卵母细胞成功生产克隆的小狗，实现了一个非凡的里程碑，强调从体外回收的卵母细胞中克隆犬的可行性。重要的是要注意，这项研究仅关注排卵后的未成熟卵母细胞，并且仅在发情期。针对发情周期其他阶段的进一步研究可能会提高犬的克隆效率。
