Production of viable cloned miniature pigs by aggregation of handmade cloned embryos at the 4-cell stage
Chawalit Siriboon A , Ching-Fu Tu B , Michel Kere A , Ming-Sing Liu B , Hui-Jung Chang B , Lin-Lin Ho B , Miao-En Tai B , Wen-Der Fang B , Neng-Wen Lo C , Jung-Kai Tseng D and Jyh-Cherng Ju A E F
+ Author Affiliations
- Author Affiliations
A Department of Animal Science, National Chung Hsing University, 250 Kuokuang Road, Taichung 402, Taiwan, ROC.
B Animal Technology Institute Taiwan, 52 Kedung 2 Road, Ding-Pu LII, Chunan, Miaoli, Taiwan, ROC.
C Department of Animal Science and Biotechnology, Tunghai University, 181, Sec. 3, Taichung Harbor Road, Taichung 407, Taiwan, ROC.
D School of Optometry, Chung Shan Medical University, 110 Chien-Kuo North Road, Taichung 402, Taiwan, ROC.
E Agricultural Biotechnology Center, National Chung Hsing University, 250 Kuokuang Road, Taichung 402, Taiwan, ROC.
F Corresponding author. Email: jcju@dragon.nchu.edu.tw
Reproduction, Fertility and Development 26(3) 395-406 https://doi.org/10.1071/RD12243
Submitted: 25 July 2012 Accepted: 14 February 2013 Published: 2 April 2013
Abstract
The aim of the present study was to improve the quality of handmade cloned porcine embryos by multiple embryo aggregations. Embryos derived from aggregation of three cloned embryos (3×) had a better blastocyst rate than cloned control (1×) embryos (73.6% vs 35.1%, respectively; P < 0.05), but did not differ from those produced by aggregation of two cloned embryos (2×; 63.0%). Total cell numbers differed among treatments (P < 0.05), with the greatest cell numbers (126) in the 3× group and the lowest (55) in the control group. The ratio of inner cell mass : total cell number was comparable in the 2× and 3× groups (25.1% vs 26.1%, respectively) and was significantly better than that in the control group (15.3%). The proportion of apoptotic cells in 2× and 3× groups was lower than that in the control group (2.7% and 2.2% vs 4.7%, respectively; P < 0.05). Expression of Oct4 and Cdx2 was higher, whereas that of Bax was lower (P < 0.05), in the 3× compared with non-aggregate group. Seven piglets were born to two surrogate mothers after embryo transfer of 3× aggregated blastocysts. In conclusion, aggregated embryos had greater total cell numbers and better pluripotency gene expression, with reduced expression of the pro-apoptosis gene Bax. Collectively, these improvement may be associated with the development of cloned embryos to term.
Additional keywords: embryonic stem cells; oocyte bisection cloning technique.
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