50 FIRST EQUINE CLONE BORN IN ARGENTINA BY SOMATIC CELL NUCLEAR TRANSFER FROM A POLO ARGENTINO MARE

Abstract

Births of the first cloned equids were reported in 2003 (Woods et al. 2003 Science 301(5636), 1063; Galli et al. 2003 Nature 424 (6949), 635) but no equine clones have been reported in Latin America. The aim of this work was to inform the birth of the first equine clone in Argentina as a result of interdisciplinary teamwork. Equine oocytes were obtained from slaughterhouse ovaries and were transported at 38°C for 15 h in TCM-199 buffered with 20 mM HEPES and supplemented with 1 mM glutamine, 0.19 mM sodium pyruvate, 2 μg mL^–1 LH, 5 μg mL^–1 FSH, 100 ng mL^–1 EGF, 100 ng mL^–1 IGF-I, and 10% FCS. After 16 to 18 h of maturation, oocytes were denuded and enucleated. Frozen–thawed somatic cells from 1 of 2 aged (23 and 20 years old) Polo Argentino breed donor mares were inserted into the perivitelline space of each enucleated oocyte and fusion was induced. Activation was performed by exposure to Ionomycin (5 μM) for 4 min, followed by 4 h culture in a combination of 6-DMAP and cycloheximide in SOFaas culture media (Choi et al. 2001 Reproduction 122, 177–183). Embryos were cultured in 50 μL SOFm droplets for 7 to 8 days and then were transferred non-surgically to anovulatory recipient mares treated with 1.5 mg oestrogen, followed by long acting progesterone (BioRelease P4 300 LA, BET Pharm, KY). The production rates of the first cell line were: fusion 301/553 (54.4%), cleavage 163/218 (53.4%), blastocysts produced 20/163 (12.2%), blastocysts transferred 18/20 (90%), pregnancies 1/18 (5.5%) and no foal born. The production rates of the second cell line were: fusion 252/553 (45.6%), cleavage 55/252 (21.8%), blastocysts produced 8/55 (14.5%), blastocysts transferred 7/8 (87.5%), pregnancies 4/7 (57%) and 1 filly was born. DNA microsatellite analysis confirmed that the filly was a clone from the original donor cell line. The filly, born naturally on Day 329 of gestation, suffered premature placental separation and weighed 12 kg. Despite the intensive care provided, the filly’s respiratory, nervous, and cardiovascular functions deteriorated rapidly and she died 14 h postpartum. Fetal membranes weighed 5.7 kg (47.5% of the filly’s weight) and showed marked oedema of the chorioalantois and umbilical cord (which also showed a torsion of 9 twists). Histopathologically, the chorioalantois showed scant and poorly developed villi and also connective tissue oedema. Death could have been due to marked immaturity, as a consequence of placental insufficiency and umbilical cord torsion.