Accomplishment of canine cloning through in vitro matured oocytes: a pioneering milestone.

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.