85 PREGNANCIES AND VIABILITY OF THE OFFSPRING AFTER TRANSFER OF HANDMADE CLONED PORCINE EMBRYOS

Abstract

In several species, nuclear transfer causes a high frequency of fetal loss, high birth weight, malformations, and neonatal death. This study investigates the fetal development of porcine embryos produced by handmade cloning (HMC) and compares the piglets with littermates produced by traditional cloning (TC). HMC embryos were produced from one fetal cell line, LYxD (Landrace/Yorkshire x Duroc) and TC from another (LYDxLYD). For transfer, 11 sows were weaned, and at cycle stage Day 4 after heat, they were anesthetized in lateral recumbence. Through an abdominal incision 40–60 Days 5–7 embryos (69% HMC + 31% TC) were transferred to the upper uterine horn. The tip of the uterine horn was punctured with a blunt 18G needle and a temporary catheter introduced to absorb the blood before inserting an insemination catheter (J-IUIC-351341 Insemi-Cath; Cook Ireland Ltd., Limerick, Ireland) 5–6 cm into the horn and releasing the embryos. Pregnancy was examined by ultrasonography every second week to Day 100. Caesarian sections were performed at Day 115, 24 h after injection of PGF₂ (175 µg Estrumate^®; Pitman-Moore Ltd., Harefield, UK). The piglets were fed with 15 mL kg^-1/3 h colostrum through a stomach tube for the first 12–24 h, until they were left with their dam. Body weight, blood values (oxygen tension, and glucose and hemoglobin concentrations), and body temperature were recorded at birth, 24 h, and 3 weeks of age. The data were analyzed by Fisher's Exact test and are given as LS means ± SEM values with a significance level of P < 0.05. The pregnancy rate was 6/11 (55%), with 2 pregnancies developing to term, 2 ongoing (Day 100 and Day 60), and 2 lost (about Day 35). So far, the litter sizes were 3 (1 TC, 2 HMC) and 10 (2 TC, 8 HMC) live piglets, respectively. The mean birth weight of the TC offspring (1045 ± 135 g) was lower than that of the HMC piglets (1614 ± 177 g) and that of normal piglets on the farm (1520 ± 240 g; P < 0.05). One of the HMC piglets of the first litter was rather oversized (2690 g) and another showed rigid flexure of a carpal joint. All other piglets appeared normal on gross morphology and behavior. Blood values were similar for HMC and TC piglets, but the body temperature at Day 0 (36.3 ± 0.2 vs. 35.1 ± 1.1°C; P < 0.05) and the daily weight gain during the first week (129 ± 21 vs. 74 ± 13 g; P = 0.06) tended to be higher for the HMC piglets. After 24 hrs, the mean blood glucose concentration was higher in the HMC than in the TC piglets (6.09 ± 0.74 vs. 3.67 ± 0.19 mmol L^-1) and similar to farm piglets of the same age (6.93 ± 0.44 mmol L^-1), but the difference was not significant after 3 weeks. These preliminary results demonstrate for the first time that HMC in pigs can result in an acceptable pregnancy rate, birth rate, and litter size. The minor differences in birth weight, weight gain, and blood glucose observed between HMC and TC offspring may be explained by the different cell lines used for the 2 methods, and are under current investigation.