29 EFFECT OF EMBRYO CULTURE LENGTH ON PRODUCTION OF CLONED TRANSGENIC GOATS

The yield of blastocysts and hatched blastocysts using in vitro production (IVP) in goats are still low. The development of caprine embryos is frequently arrested at the 8- to 16-cell stage, indicating suboptimal culture conditions (Jimenez-Macedo et al. 2005 Theriogenology 64, 1249–1262). Our goal was to produce transgenic goats by somatic cell nuclear transfer (SCNT) and further determine whether the length of embryo culture has an effect on development to term. We compared the efficiency of transferring single-cell embryos 12 h post-activation to transferring 4- to 8-cell embryos cultured for 60 h post-activation. Nine transgenic goats from 2 cell lines were produced through SCNT. Somatic donor cells were obtained from 2 sources: adult fibroblasts and fetal fibroblasts. Adult fibroblasts were obtained from a transgenic doe skin biopsy. Fetal fibroblasts were isolated from a 25-day-old fetus and then electroporated with a pcDNA3.1DV5-MHC-TGF-β1cys33ser vector, followed by G418 selection, screening, and subsequent use for SCNT. Oocytes with >4 layers of cumulus cells were collected by slicing abattoir ovaries and matured in vitro for 21 to 23 h. After being denuded, oocytes presenting a first polar body were enucleated and received a donor cell from 1 of the 2 cell lines. Fused embryos were then activated for 5 min in 5 µM ionomycin, followed by 4 h in 2 mM DMAP with 5 µg of cycloheximide mL–1. Activated embryos were cultured in G1 medium with 5 mg of BSA mL–1 for either 12 or 60 h post-activation, followed by surgical transfer into the oviducts of recipients synchronized to show estrus within 12 h of SCNT. Overall, 376 embryos were transferred into 23 recipients. Pregnancy was examined by ultrasonography on Day 30 post-transfer. No pregnancy losses were observed after Day 30 of gestation. All kids were born live (42% of recipients receiving embryos cultured for 12 h gave birth, compared with only 9% when cultured for 60 h). The data (Table 1) suggest that a longer culture time in vitro significantly reduces viability of cloned embryos. Table 1.Twelve-hour versus 60-h embryo culture This work was supported by Utah Agricultural Experiment Station project no. 1100.