52 ESTABLISHMENT OF A PREGNANCY WITH IDENTICAL TWINS AFTER THE TRANSFER OF A VITRIFIED CLONED BOVINE EMBRYO

Abstract

Identical twin pregnancies occur naturally in some species because of spontaneous embryo division during initial stages in development. Bovine embryo splitting usually does not compromise subsequent in vivo development, being a method commonly used in embryo transfer programs to enhance overall pregnancy and parity rates. The goal of this study was to establish pregnancies from vitrified bovine cloned embryos. A spontaneous twin pregnancy was obtained after the transfer of a vitrified cloned hatching blastocyst produced by nuclear transfer procedures. Briefly, selected COCs obtained via slicing from bovine ovaries collected in a local slaughterhouse, and in vitro-matured for 17 h, were denuded by pipetting in HEPES-SOF (HSOF)-BSA and screened for the presence of the first polar body. Selected oocytes were enucleated by micromanipulation in HSOF + cytochalasin B and Hoechst 33 342 under UV fluorescence control. Frozen-thawed cumulus cells obtained by ovum pickup (OPU) from a Holstein cow were used as nucleus donors at passage 2 and high confluence (>90%). A single cell was inserted into the perivitelline space by micromanipulation. Fusion was induced electrically between two 200-μm-wide electrodes, 150 μm apart, using a single 20-V electrical DC pulse for 45 μs. Fusion outcome was observed 30 min after the electrical stimulus, with the immediate activation of the reconstructed embryos in ionomycin for 5 min, followed by incubation in cycloheximide and cytochalasin D for 5 h. Then, activated embryos were cultured in vitro for 7 days in SOF with amino acids (SOFaa) +10% estrous cow serum (ECS), at 39°C, high humidity, and 5% CO₂, 5% O₂ and 90% N₂. Day-7 blastocysts were vitrified in hand-pulled glass micropipettes (GMP) using super-cooled LN₂ (Vieira et al. 2007 Anim. Reprod. Sci. 99, 377-383), with the subsequent transfer of the GMP content into a straw upon warming, for the direct transfer of 4 embryos to female recipients, 1 per recipient, without the embryo evaluation under a stereomicroscope (Vieira et al. 2008 Reprod. Domest. Anim. 43, 314-318). A pregnancy was diagnosed on Day 35; on Day 260 of pregnancy, the female recipient aborted identical 20-kg twin fetuses showing no macro- or microscopic alterations following the necropsy. Samples of donor cells and tissue samples from the female recipient and cloned twins subjected to microsatellite DNA analysis confirmed that the twins were indeed identical clones and derived from the donor cells. The embryo from which the twin pregnancy derived was a hatching blastocyst, vitrified when about half of the embryo, including half of the inner cell mass, was projected outside the zona pellucida. Most likely, the hatching blastocyst was physically and unintentionally bisected during the process of vitrification, warming, or embryo transfer, producing hemi-embryos that later developed into identical twins.

This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq).