140 PRODUCTION OF RECONSTRUCTED IN VITRO PRODUCED BOVINE EMBRYOS BY INNER CELL MASS AND TROPHECTODERM AGGREGATION IN VITRO

Abstract

The efficiency of embryonic chimerism tends to decrease when embryos in advanced stages of development, such as morulae and blastocysts, are used. To perform the inner cell mass (ICM) transfer to a trophectoderm (TE) receptor, it is essential to use embryos at an advanced stage and blastocoel presence. This method of embryo reconstruction has been performed only by the micromanipulator microinjection method (Zheng et al. 2005 Zygote 1, 73–71; Loi et al. 2007 Trends Biotechnol. 25, 195–200; Roth et al. 1989 Biol. Reprod. 41, 675–682; and Murakami et al. 2006 Cloning Stem Cells 8, 51–60). This study aimed to validate a manual procedure to reconstruct embryos using the method of ICM and TE approximation in the presence of phytohemagglutinin. Bos indicus ovaries from the abattoir were used to obtain 230 cumulus–oocyte complexes (COC; quality I and II). The COC were matured in 90-μL drops of TCM-199 bicarbonate supplemented with 10% fetal calf serum (FCS) and incubated in vitro for 22 to 24 h. Fertilization occurred in TALP-IVF medium, and the COC were incubated for 18 h. Presumptive zygotes were transferred to SOF medium to in vitro culture. Incubation conditions were 38.5°C and 5% CO₂ in air. In vitro produced (IVP) embryos 8.5 days after fertilization were used for the experiment. Ninety-three hatching or hatched blastocysts were put into 3-μL microdrops of protein-free HEPES-buffered SOF (HSOF) medium to hold the embryos on the dish bottom and to allow handmade sections of ICM and TE. The section was performed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under a stereomicroscope (35× magnification). Seventy half-structures from 35 different blastocysts were obtained to form pairs (ICM+TE). Each pair was transferred to drops with 500 μg mL^–1 of phytohemagglutinin-L (3 min) before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) to in vitro culture (38.5°C; 5% O₂ and 5% CO₂). The aggregation rate was 25.7% (9/35) and all the reconstructed blastocysts by aggregation (24 h) re-expanded after 48 h of culture. The technique of handmade ICM and TE section and posterior aggregation in the presence of an agglutinating agent was feasible for the structural and functional reconstruction and re-expansion of the blastocyst produced.