36 EFFECT OF ACTIVATION METHODS ON DNA SYNTHESIS AND DEVELOPMENT OF CANINE PARTHENOGENETIC EMBRYOS

Abstract

Artificial activation is an important step for successful somatic cell nuclear transfer. In mammals, different methods of parthenogenesis have been studied to increase the developmental efficiency of cloned embryos. In an attempt to improve the techniques of nuclear transfer in canine species, this study investigated the timing of DNA synthesis and in vivo development of canine parthenotes produced by different activation treatments. For canine parthenotes, in vivo matured oocytes were obtained by flushing (~72 h after ovulation) the oviducts of mixed breed bitches. Denuded oocytes were cultured for 4 min in 10 μM calcium ionophore, and then they were divided into 2 groups: (1) 2DMAP group was cultured for 2 h in 6-DMAP; (2) 4DMAP group was cultured for 4 h in 6-DMAP. The first experiment determined DNA synthesis of parthenotes by 1 h treatment with incorporation and immunofluorescent detection of thymidine analogue 5-bromo-2′-deoxyuridine-5′-triphosphate (BrdU). The primary antibody was mouse anti-BrdU (Sigma, St. Louis, MO, USA), and the secondary antibody was fluorescein (FITC)-conjugated affinity purified goat anti-mouse IgG (Jackson). In order to examine the pronuclear formation and the onset of DNA synthesis in experimental groups, parthenotes derived from 2DMAP and 4DMAP groups were observed by BrdU incorporation at 2, 4, and 12 h post-activation (hpa). Data were analysed using Graph Prism software (GraphPad, San Diego, CA, USa). The next experiment observed in vivo development as follows: parthenotes were surgically transferred to synchronized recipient female dogs. The implantation rate of parthenogenetic fetuses was observed in uterus of recipients on Day 26 of pregnancy. All parthenotes of the 2DMAP group showed BrdU incorporation at 2 hpa, whereas 4DMAP parthenotes showed 96% BrdU incorporation at 2 hpa. Incorporation of BrdU was detected in all parthenotes of both experimental groups after 4 hpa. A total of 98 parthenotes were transferred to 9 surrogate mothers (53 parthenotes into 5 recipients in 2DMAP group and 45 parthenotes into 4 recipients in 4DMAP group). There was no significant difference in pregnancy rate between the 2 groups (2DMAP: 60% v. 4DMAP: 50%), whereas the implantation rates were significantly higher in 2DMAP (24.5%) compared with 4DMAP (4.4%; P < 0.001). The recovered parthenotes were able to develop to the stages of limb-bud formation, but few parthenotes showed the small and degenerating formation. Regardless of treatment group, the implantation site of the fetuses indicated either one side or both of the uterus. In conclusion, the present results demonstrated that the protocols using combined treatment with 10 μM of calcium ionophore (4 min) followed by a 2-h culture with 1.9 mM of DMAP resulted in completing pronuclear formation and enhancing fetal formation. This result could be useful method for improving canine cloned embryos production. This study was supported by IPET (#311062–04–2-SB010), RNL Bio (#550–20130013), RDA (PJ008975022013), and Research Institute for Veterinary Science and Natural Balance.