Dog cloning with in vivo matured oocytes obtaining using serum estradiol levels for predicting time of ovulation

Abstract

Dog cloning using in vivo-matured oocytes has been carried out for a decade. To obtain mature oocytes, serum progesterone (P₄) levels are used to evaluate ovulation. However, the accuracy of these methods is not sufficient. Thus, the aim of the present study was to verify the feasibility of serum estradiol (E₂) on canine ovulation determination as assessed by the percentage of dogs yielding mature oocytes. In vivo-matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum P₄ and E₂ levels were assessed to determine ovulation and oocyte maturation. Canine serum P₄ and E₂ concentrations during both pro-estrus and estrus were analyzed by electrochemiluminescence immunoassay. The percentage of dogs yielding mature oocytes using each of the two ovulation prediction methods were compared, and correlations between the percentage of each method and temperature were analyzed. Following evaluation, oocytes were collected surgically, and a significantly higher percentage (P < 0.05) of dogs yielding mature oocytes was observed using E₂ (56.43%) for ovulation detection as compared with that using P₄ (39.60%). The percentage of dogs yielding mature oocytes using P₄ significantly lower (P < 0.05) than E₂ in autumn (P₄, 37.50% vs. E₂, 52.00%) and winter (P₄, 29.17% vs. E₂, 59.09%). Using E₂, the percentage was maintained at about 52.00-66.67% regardless of the season and temperature. Correlation analysis showed that the dynamic of percentage of dogs yielding mature oocyte using P₄ was highly correlated with environmental temperature (R_P4 = 0.862), whereas E₂ was not affected by temperature (R_E2 = 0.199). To determine whether serum E₂ could be used for ovulation prediction for canine cloning, ovulation of 25 and 19 dogs (P < 0.05) were predicted using P₄ or E₂ methods, respectively and two puppies, one from each ovulation prediction method, were obtained after SCNT and embryo transfer. Thus, compared with the P₄ method, E₂ was an accurate and reliable method for canine cloning.

Keywords: Estrus cycle prediction; Oocyte maturation; Somatic cell nuclear transfer.