125MORPHOLOGICAL CHARACTERIZATION OF DAY 14 AND DAY 21 IVP AND CLONED BOVINE EMBRYO DEVELOPMENT

Abstract

The establishment of viable embryonic development following in vitro production (IVP) or nuclear transfer by SUZI (subzonal injection) and handmade cloning (HMC) methods is pivotal for practical implementation of embryo technology. The development of cloned embryos is currently impeded by the high rate of pre- and postnatal losses. This project is aimed at developing screening methods using both ultra-structural and molecular markers of development of post-hatching/pre-implantation stage embryos to enable selection of embryos with greater developmental competence. Initially twenty embryos derived by IVP or nuclear transfer (SUZI or HMC) were transferred to six recipient cows on Day 7 (n=120). Embryos were collected on Days 14 and 21 by flushing the uterine tracts of slaughtered cows. Embryos were fixed in 4% paraformaldehyde and processed for paraffin embedding. Serial sections were stained with haematoxylin and eosin and evaluated by light microscopy. On each day of collection, certain characteristics were expected to be observed according to normal in vivo development (Maddox-Hyttel et al., 2003, Reproduction 125, 607–623). On Day 14, IVP embryos (n=6) were ovoid to filamentous in shape. Three embryos had extensive foldings of trophoblast in the process of forming an amniotic cavity. NT-SUZI embryos (n=4) had a mainly round uneven shape. No NT-HMC embryos were recovered. On Day 21, IVP embryos (n=4) more closely resembled in vivo embryos than any other group and formed either a neural groove or a neural groove with somites. NT-SUZI embryos (n=6) were mainly filamentous, with one embryo showing complete development of the neural tube and 5 somite pairs. NT-HMC (n=17) development ranged from ovoid to multi-protrusions of the yolk sac. One embryo displayed the formation of a neural tube. Embryos, irrespective of production method, which had an abnormal morphology, presented only an incomplete hypoblast and severe degeneration of the epiblast. Those embryos that were morphologically comparable to in vivo embryos also displayed developmental stages which ranged from presentation of a primitive streak and formation of a neural tube to having a neural tube, differentiation of mesoderm and somites. At present, the low number of embryos recovered as well as their abnormal development clearly reflect the low success rate to term of live calves. It is possible that no Day 14 NT-HMC embryos were recovered due to collection efficiency not being optimal. It is apparent that IVP and cloned embryos have different developmental time lines as compared to that of in vivo embryos.