Cloning of adult cows by nuclear transfer of ovarian mural granulosa cells

Abstract

The objective was cloning three adult cows using their ovarian mural granulosa cells collected by transvaginal ovum pick up (OPU). One group (T1) of granulosa cells were matured together with their oocytes as part of the COC (Cumulus Oocyte Complex), a second group (T2) were cultured for 3 days independently of their oocytes and a third (T3) group was collected from the cells that are shed during the filtration of the OPU aspirations. The oocytes were denuded and then enucleated after previous treatment with pronase to digest the zona pellucida. The reconstructs were subjected to electro fusion before the chemical activation with ionomycin. A total of 324 embryo reconstructs were obtained; 158 from T1, 45 from T2 and 121 from T3. The overall response to activation of the embryo reconstructs was 91.35% and there were no statistical differences (P>0.05) between treatments. There were however significant differences (p<0.01) in the cleavage percent and in the percent of transferable embryos obtained with the different types of granulosa cells; the cleavage rate was 70 %, 61.7 % y 41.7 % for T2, T1 and T3 respectively and the rate of transferable embryo production was 30 %, 13.5 % and 0.86 %, for T2, T1 and T3, respectively. Two clone calves were obtained, one from T2 and one from T3. The calf from T2 is normal and healthy, the calf from T3 showed a congenital defect (cardiac teratology) and died 3 days after birth. Our results confirm previous work that show that ovarian mural granulosa cells obtained from adult live cows can be reprogrammed successfully after being transferred by the nuclear transfer procedure to matured enucleated oocytes and produce live normal cloned calves; also shows that there is no difference in the activation efficiency between the different groups of granulosa cells used.

Keywords

• clonation
• clonación
• células de granulosa
• vacas.
• Nuclear transfer
• granulosa cells
• cow