58 PRODUCTION OF CLONED BOVINE EMBRYOS DERIVED FROM AMNIOTIC CELLS OF PREGNANT COWS

Abstract

Progeny tests are widely used for selection of sires for beef and dairy cattle. A less costly method might be to clone the sire candidates at their earliest developmental stage possible. To produce cloned bulls, we obtained amniotic cells as donors for nuclear transfer by transvaginal aspiration of pregnant cows. However, the collected cells may include some maternal cells. In this study, we examined collection methods to obtain only fetal cells from the collected fluid. We also examined the developmental capacity of the embryos cloned from these cells. Amniotic fluids were aspirated from pregnant cows by ultrasound-guided aspiration. We collected amniotic fluids from 27 pregnant Japanese black beef cattle (between 58 and 132 days of gestation). In Method 1, cells were recovered from the whole amniotic fluid (approximately 15 mL). In Method 2, the initial 5 mL of aspirated fluid was discarded and then the next 10 mL sample was collected. Cells were recovered from the collected fluids. The cells in the fluids were washed twice by centrifugation and then cultured in AmnioMAX™-II medium (GIBCO, Grand Island, NY, USA). After 3–4 passages, the sex of the cell lines was determined by the loop-mediated isothermal amplification (LAMP) method (Eiken Chemical Co., Ltd., Tokyo, Japan). For the cell lines that were determined as 'male' by the LAMP method we further analyzed the sex of individual cells (137–620 cells of each cell line) by fluorescent in situ hybridization (FISH) using a bovineY chromosome-specific probe (Kobayashi et al. 1998 Mol. Reprod. Dev. 51, 390–394). The percentage of male cells obtained from Methods 1 and 2 were 0–0.4% (from 4 animals) and 93.7–99.5% (from 6 animals), respectively. Then, we used confluent amniotic cells from 3 cell lines obtained by Method 2 as donor cells for nuclear transfer and examined the developmental capacity of the cloned embryos. Bovine fibroblasts cultured under serum starvation were used as a control. The cells were electrically fused (2.7 kV cm–1, 11 µs, 2 times) with enucleated bovine oocytes, and activated with a calcium ionophore and cycloheximide. They were subsequently cultured in mSOF until 168 h post-activation. The data were analyzed with Fisher's protected least-squares difference (PLSD) test following ANOVA. The rates of fusion, cleavage, and development to the blastocyst stage of the cloned embryos were the same as those of the control embryos (78% v. 81%, 75% v. 75%, and 22% v. 27%, respectively; P > 0.05). Furthermore, the rate of male blastocysts derived from the cloned embryos with the three cell lines was 95% (19/20). These results indicate that the amniotic fluids collected from pregnant cows by Method 2 contained fewer maternal cells, and that the embryos cloned from the cells developed in a manner similar to that of embryos cloned from the fibroblasts. This work was supported byWakayama Prefecture CREATE, JST.