27 BIRTH OF AFRICA’S FIRST NUCLEAR-TRANSFERRED ANIMAL PRODUCED WITH HANDMADE CLONING

Abstract

Cloning technology has the potential to stimulate the development of the animal biotechnology industry in southern Africa, as well as provide conservationists with an additional tool to possibly assist with conserving critically endangered wildlife species sometime in the future. The aim of this study was to determine whether cloning could produce blastocysts and possibly live progeny in a field-type laboratory without micromanipulators and CO₂ incubator. Approx. 1 × 1-cm ear skin notches were surgically removed from a physically immobilized 9-year-old Holstein cow, a former South African milk production record holder. The tissues were placed into physiological saline and transported to the laboratory at 4°C within 2 h, cleaned with chlorohexidine gluconate and sliced finely in Minimal Essential Medium supplemented with 10% fetal calf serum. The resultant tissue explants were treated as previously described (Bartels et al., 2003 Theriogenology 59, 387) and actively growing fibroblast cultures were made available for the nuclear transfer process. Bovine oocytes from slaughterhouse-derived ovaries were collected and matured for 21 h in modified TCM-199 medium supplemented with 15% cattle serum, 10 IU mL⁻¹ eCG and 15 IU mL⁻¹ hCG. Nuclear transfer was performed using the HMC technique (Vajta et al., 2003 Biol. Reprod. 68, 571–578). At 21 h after the start of maturation, cumulus cells and zonae pellucidae were removed and oocytes were randomly bisected by hand. Cytoplasts were selected using Hoechst staining and a fluorescent microscope. After a two-step fusion, reconstructed embryos were activated with calcium ionophore and dimethylaminopurine. Culture was performed in SOFaaci medium supplemented with 5% cattle serum using WOWs (Vajta et al., Mol. Reprod. Dev. 50, 185–191). All incubations including culture of donor cells were performed in the submarine incubator system (SIS; Vajta et al., 1997 Theriogenology 48, 1379–1385). In two consecutive experiments, 6 blastocysts were produced from 52 reconstructed embryos. On Day 7, 5 blastocysts were selected for transfer into 3 previously synchronized recipients. All three recipients became pregnant, but two of the recipients aborted at six and seven months, respectively. Post-mortem examination on the first aborted fetus did not reveal any identifiable etiology, but coincided with 6 abortions from natural pregnancies during a heat wave, while the organism Brucella abortis was isolated from the second aborted fetus. The third pregnancy went to term, and a healthy calf, weighing 27 kg, was delivered by Caesarean section. The three-month-old calf is being raised by a surrogate Jersey cow under standard dairy conditions and is expected to join the dairy in eighteen months’ time. The birth of ‘Futhi’, meaning ‘replicate’ in Zulu, is Africa’s first cloned animal and signifies an important milestone in the development of animal biotechnology in Africa.