Canine-assisted reproductive techniques have been successful for several years; however, the lack of an oocyte in vitro maturation system has limited their application. The aim of this study was to evaluate the effect of canine oviduct epithelial cells (cOECs) on canine oocyte maturation in vitro. Specifically, the method used for isolation of cOECs did not affect the expression of epithelial markers, E-cadherin and cytokeratin, on fresh, cultured and cryopreserved cells. Moreover, BrdU analysis showed that cOECs cultured in Medium 171 supplemented with mammary epithelial growth supplement were more proliferative than counterparts in advanced Dulbecco's modified Eagle medium or Medium 199. Maturation rate of canine oocytes collected from bitches at diestrus was significantly increased when oocytes were co-cultured with either fresh, cultured or frozen/thawed cOECs (13.23 ± 1.15%, 10.38 ± 4.89%, or 10.54 ± 2.96%, respectively) than that of control oocytes cultured without cOECs (2.48 ± 2.16%, p < 0.05). Additionally, the number of oocytes collected from bitches at estrus the reached metaphase II was increased ∼4 fold in co-culture with fresh, cultured, or frozen/thawed cOECs (47.2 ± 3.82%, 45.4 ± 7.34%, and 46.9 ± 1.51%, respectively) as compared with oocytes cultured without cOECs (11.9 ± 3.18%, p < 0.05). Nuclear maturation was further confirmed by assessing the formation of normal metaphase-II spindles, whereas cytoplasmic maturation was confirmed by inducing parthenogenetic oocyte activation. Embryonic development to the 8-cell stage was similar between in vivo and in vitro matured oocytes. These results suggested that co-culturing immature canine oocytes with cOECs facilitated canine oocyte maturation and early stages of embryonic development.

Radioactive immunoassay (RIA) is a traditional serum hormone assay method, but the application of the method in reproductive studies is limited by the associated radioactivity. The aim of present study was to evaluate the reliability of RIA and to compare its canine serum progesterone concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). In vivo matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum progesterone levels were assessed to accurately determine ovulation and oocyte maturation. Canine serum progesterone concentrations during both proestrus and estrus were analyzed by RIA and ECLI to determine the ovulation day. Although both methods detected similar progesterone levels before ovulation, the mean progesterone concentration determined using ECLI was significantly higher than of RIA three days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of progesterone were used for determination of ovulation. A high percentage of mature oocytes was observed using ECLI when 6–15 ng/mL of progesterone was used for ovulation determination. To determine whether ECLI could be used for canine cloning, six canines were selected as oocyte donors, and two puppies were obtained after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.

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