27 MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL CHARACTERIZATION OF DAY 21 IVP AND NT BOVINE EMBRYOS

Abstract

A major limitation of somatic cell nuclear transfer (NT) for the production of cloned calves is that only 1–5% of cloned embryos produce viable calves. The high rate of mortality is attributed to both pre- and post-natal losses and is primarily due to incomplete reprogramming of donor cells. Almost 50% of the pregnancy losses occur in the first trimester of pregnancy, indicating a major disruption in normal embryo development at NT. The aim of this study was to analyze germ layer formation by stereomicroscopy and immunohistochemical techniques for both NT embryos and their in vivo counterparts on Day 21 and to compare deviations from normal embryonic development as a measure of developmental capacity. Blastocyts derived by IVF (n = 20), conventional NT (n = 20), or hand made cloning (HMC; n = 20) were non surgically transferred to each synchronized recipient cow (n = 3). Each group of twenty embryos was transferred to one recipient. Cows were slaughtered on Day 21 and uterine tracts recovered and flushed with phosphate-buffer solution with 10% serum. Recovered Day 21 embryos were fixed in 4% paraformaldehyde, and embedded in paraffin; serial sections were stained with hematoxylin and eosin and evaluated by light microscopy. Immunohistochemical localization of cytokeratin 8 was used as a marker for potential ectoderm, alphafetoprotein for potential endoderm, and vimentin for potential mesoderm. Four IVF (20%; 4/20) embryos, six NT (30%; 6/20) embryos, and ten HMC (50%; 10/20) embryos were recovered following flushing. No obvious morphological differences were seen in the formation of a neural tube, differentiation of mesoderm, and number of somites among IVF (25%; 1/4), NT (33.3%; 2/6), and HMC (20%; 2/10) embryos. Delayed development with respect to the formation of neural groove and mesoderm differentiation was observed in 25% (1/4) of IVF, 16.7% (1/6) of NT, and 30% (3/10) of HMC embryos. In addition, 25% (1/4) of IVF, 16.7% (1/6) of NT, and 33.3% (2/6) of HMC embryos had not initiated gastrulation (i.e. displayed hypoblast and epiblast), suggesting a more substantial developmental delay. The remaining embryos showed gross abnormalities compared to their in vivo counterparts, including degenetration of epiblast and hypoblast cells. Cytokeratin was detected in the trophoblast, ectoderm, hypoblast, and endoderm. Alpha-fetoprotein was detected in the hypoblast while vimentin was seen in the mesoderm. In conclusion, although localization of staining in IVF and cloned embryos was consistent with that of in vivo embryos, the intensity was weaker, suggesting compromised or delayed development. It is also possible that the differences observed were due to the recipient and not to the treatment group. This work was supported by Genetics Australia Cooperative Limited, Victoria, Australia.