Dog cloning with in vivo matured oocytes obtained using electric chemiluminescence immunoassay-predicted ovulation method

Abstract

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.

Brachygnathia Inferior in Cloned Dogs Is Possibly Correlated with Variants of Wnt Signaling Pathway Initiators

“…Many animals have been cloned via nucleus transfer from somatic cells to mature oocytes in vitro [ 14 , 15 , 16 , 17 , 18 ]. However, owing to inadequate in vitro culture conditions and different estrus cycles, dogs are typically cloned using surgically recovered mature oocytes in vivo [ 5 , 6 ]. Nevertheless, the cloned dogs were also found to have many abnormalities as in other cloned animals [ 19 ].…”

“…For dog cloning, somatic cell nuclear transfer (SCNT) and embryo transfer were performed according to a previously described protocol [ 6 ] with minor modifications, as shown in Figure 1 . As donor cells, ear fibroblasts were collected from a 5-year-old male German shepherd dog (original donor) via ear skin biopsy after anesthesia.…”

“…Cells at the second to third passage were used for the production of cloned embryos. After SCNT, the embryos were immediately surgically transferred into the oviduct of a surrogate mother using a previously described method [ 6 ]. Pregnancy rates were determined by ultrasound diagnosis at ≈31 days after embryo transfer.…”

“…Dog cloning differs from cloning in other animals, such as sheep, cattle, and pigs, owing to the different reproductive processes, such as ovulation of oocytes at the metaphase I stage. Therefore, dog cloning is performed with in vivo-matured oocytes, after which cloned embryos are quickly transferred into the oviduct to overcome the inadequate in vitro culture systems [ 5 , 6 ]. The cloned offspring are expected to not only genetically but also phenotypically identical to the original donor dog [ 7 ].…”

Robotic Precisely Oocyte Blind Enucleation Method

“…In 1997, British scientist Wilmut et al used sheep mammary cells as donor cells for somatic cell nuclear transfer (SCNT) and obtained the world's first somatic cloned sheep, Dolly. Since the birth of Dolly, many other species, such as cloned mice, cloned dogs, cloned cows, cloned rabbits, cloned horses, etc., have also been born through nuclear transplantation technology [3][4][5][6][7][8]. The standard nuclear transplantation procedure is divided into two parts: the enucleation of oocytes and the injection of donor cells.…”