23 IMPROVING EFFICIENCY IN WORK WITH TRANSFER OF CLONED PIG EMBRYOS
H. Callesen A , Y. Liu A , R. Li A and M. Schmidt BA Department of Animal Science, Aarhus University, Tjele, Denmark;
B Department of Large Animal Sciences, Faculty Life Sciences, University of Copenhagen, Frederiksberg C, Denmark
Reproduction, Fertility and Development 26(1) 126-126 https://doi.org/10.1071/RDv26n1Ab23
Published: 5 December 2013
Abstract
Cloning is a quite inefficient procedure with only around 10% of offspring born based on number of cloned embryos transferred. Every step to increase this level is therefore welcomed. Our group has worked with cloning of pig embryos since 2006, with the main purpose to establish a well-functioning cloning system to have transgenic piglets born as animal models for important human diseases. Here we report results from our attempts to improve efficiencies in several steps in the whole cloning procedure. Over 7.5 years, donor cells from 3 breeds were nontransgenic (50%, 4 types) or transgenic with 1 of 6 different types of gene. Oocytes from Large White (LW) sows or gilts were handmade cloned, so the zona-free cloned embryos were in vitro cultured until Day 5 to 6 to select 13 311 embryos (morulae or blastocysts) for transfer to 171 LW recipient sows or gilts. Of these, 126 were pregnant (74%; Day 35), but 20 aborted before term. A total of 704 offspring were delivered; half of the piglets were alive after 4 weeks and developed normally after that. Frequencies were compared using Chi-squared test; mean values by one-way ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Specific improvements were tested in 4 areas: (1) donor cells: stimulating reprogramming using Xenopus egg extract (Liu et al. 2013 Reprod. Fertil. Dev. http://dx.doi.org/10.1071/RD13147); (2) oocytes: preferably from sows, but also using larger gilt oocytes (Li et al. 2013 Zygote http://dx.doi.org/10.1017/S0967199412000676); (3) transfer: using both uterine sides (Theriogenology 74 : 1233); (4) number of embryos transferred: after cloning with same nontransgenic cells, embryo numbers per recipient were reduced from 90 to 30 (see Table 1). As a consequence of these different activities, overall results improved over the 7.5-year period [first 3.5 years v. last 4 years: 48% (32/67) v. 90% (94/104) recipients pregnant after transfer (P < 0.05); 5.6 ± 0.6 (n = 22) v. 6.9 ± 0.5 (n = 84) piglets/litter]. In our system, one good cloning person can now produce all embryos needed for one recipient in one good working day. Transfer of fewer cloned embryos results in fewer piglets, but it reduces the workload to produce cloned embryos and does not reduce efficiency. Further work is still needed to better understand the biological and technical challenges in work with cloning; 2 important areas are quality evaluation of the donor cells used for cloning and the recipient's reaction to transfer of many embryos. In conclusion, a reasonable increase in the overall efficiency in pig cloning work was achieved, which reduces the need for personnel, time, and material when working with this technology.
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