Production of viable cloned miniature pigs by aggregation of handmade cloned embryos at the 4-cell stage

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.

Cheetah interspecific SCNT followed by embryo aggregation improves in vitro development but not pluripotent gene expression

“…Balbach et al (2010) reported that aggregation of mouse cloned embryos normalized the levels of CDX2 and this effect was attributed to higher cell numbers. In contrast to our findings, embryo aggregation in miniature pigs enhanced the expression of OCT4 and CDX2 (Siriboon et al 2014).…”

“…This mixture would compensate for epigenetic problems of one individual embryo. This approach, which is called embryo aggregation, results in higher blastocyst cell numbers, normalization of pluripotent gene expression and higher in vivo development in the mouse and miniature pigs (Boiani et al 2003, Balbach et al 2010, Siriboon et al 2014). Moreover, rates of blastocyst production improved for bovine and equine aggregated embryos as also did blastocyst cell numbers and pregnancy rates (Pedersen et al 2005, Zhou et al 2008, Ribeiro Ede et al 2009, Gambini et al 2012.…”

18 Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

“…It is believed that for a successful cloning, the pattern of epigenetic modifications in the donor cells must be remodeled to be similar to the pattern present in the fertilized embryos [35].As a result, we performed embryo aggregation and obtained a significant increase in in vitro embryo development and cell number, 20% cleavage and a three-fold increase in blastocyst rate. Similar observations were also reported following embryo aggregation in domestic animals such as the pig following aggregation of 2 cells [33], or of 4 cells [11,14,36,37] and in the bovine [17], the horse [8,9] and the feline [10]. Increased cleavage, development rates and cell number proved that the medium used was adequate for normal development.…”

“…Embryo aggregation consists of placing more than one zona free embryos in the same well; some of the reported benefits in mammals are: improvement in embryo development in pig [6], equine [8,9], feline [10] and mouse [11], increasing the density of cells in the inner cell mass on cattle [12], blastocyst diameter in equine and in bovine [8,13], reduction in apoptosis in bovine and pigs [13,14,15], normalization of gene expression in bovine [15]and in some cases, it has also improved in vivo embryo development and early pregnancy rates in mouse [11], equine [8] and in cattle [13,16,17]. As a single clone may have epigenetic defects [15], embryo aggregation could compensate the reprogramming deficiencies through the interactions between blastomeres [11] or by paracrine pathways [11].…”

Chimerism in piglets developed from aggregated cloned embryos

“…Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21: the EGFP gene was detected in the ear genomic DNA from piglets 2453, 2455, 2457, 2459, 2460, 2461, 2462, and 2463; EGFP expressing cells; tdTomato-transfected cells; and ddH 2 O (negative control), respectively; lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22: the tdTomato gene was detected in the ear genomic DNA from piglets 2453, 2455, 2457, 2459, 2460, 2461, 2462, and 2463; EGFP-expressing cells; tdTomato-transfected cells; and ddH 2 O (negative control), respectively. (C)The EGFP gene and the tdTomato gene were amplified from genomic DNA from different tissues of Rature.Lanes 1,3,5,7,9,11,13,15,17,…”