78 ESTABLISHMENT OF PREGNANCIES IN LARGE WHITE SOW RECIPIENTS AFTER TRANSFER OF CLONED EMBRYOS OF DIFFERENT PIG BREEDS

Abstract

Production of cloned piglets, using normal or transgenic donor cells, has been of limited efficiency. One reason could be breed differences between recipient and embryos. The aim of this study was to compare pregnancies after transfer to Large White (LW) sow recipients of cloned LW embryos or cloned, transgenic minipig embryos. Large White donor cells were from LYxD, and minipig cells were transgenic with 1 of 5 genes related to different human diseases and from either Göttingen or Yucatan. The cells were used on Day 0 for handmade cloning (Du et al. 2005 Cloning Stem Cells 7, 199-205). The reconstructed embryos were cultured in vitro until transfer on Day 5 to 6. As recipients, 70 LW sows were weaned and anesthetized 4 days after natural heat. Through an abdominal incision the ovaries were controlled (CL formation, absence of cysts) and the embryos slowly introduced into the uterus via a catheter inserted 5 to 6 cm into the tip of the upper horn. To each of 33 recipients, 40 to 60 LW embryos were transferred, and 37 recipients each received 50 to 100 transgenic minipig embryos. Pregnancies were examined by ultrasound scanning every second week. Abortions were defined as absence of earlier confirmed scanning or delivery of aborted fetuses. Caesarean sections were performed on Day 114 (minipig) or Day 116 (LW) 24 h after injection of a prostaglandin analogue. At delivery, placental gross morphology was recorded with samples taken for later histology. The piglets were fed every 3 h with colostrum for the first 24 h and then by the recipient LW sow. Data were analyzed by Fisher’s Exact test with a significance level of P < 0.05. The overall pregnancy rate was 49% (34/70) with an abortion rate of 29% (8 aborted + 2 resorbed/34) from Day 30 to 45 giving 24/70 deliveries (34% of the transfers). There was no significant difference between minipig (54%, 20/37) and LW pregnancy results (42%, 14/33), although there tended to be more abortions with minipig pregnancies (8/20 v. 2/14; P = 0.14) resulting in 12 minipig and 12 LW litters of which 4 and 9, respectively, have grown up to adulthood. In almost every recipient the placenta and fetal membranes showed abnormal thick and edematous morphology. The total litter sizes ranged from 1 to 10 piglets (mean 4.4 ± 0.6), and in 13 of 24 litters there were 1 to 5 stillborn piglets. Except for one litter of 9 transgenic Yucatan piglets that all died within their first 2 weeks, the postnatal mortality of both LW- and mini-piglets seemed similar to that of farm piglets of the same age, and the piglets appeared normal with respect to weight gain, gross morphology, and behavior. These results demonstrate that, in spite of a rather high abortion rate and some fetal mortality, an acceptable birth rate can be achieved after transfer to LW recipients of cloned LW embryos (36%) as well as cloned, transgenic minipig embryos (32%). Therefore, a breed difference between the embryos and their recipient seems not to influence the pregnancy results.

The authors thank B. Synnestvedt, H. Kristiansen, S. Starsig, A. Pedersen, J. Adamsen, R. Kristiansen, and K. Villemoes for invaluable technical assistance.