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OPEN PULLED-STRAW TECHNOLOGY OF VITRIFICATION AND FIELD (WITHOUT-MICROSCOPE) TRANSPLANTATION OF THE SMALL RUMINANT EMBRYOS.
Isachenko, Vladimir1, Alabart, Jose luis1, Vajta, Gábor2, Cocero, Maria Jesus1, Olivera, Julio1, Roche, Alberto1, Folch, Jose1, 1 2
ABSTRACT- For testing of new technology, sixty in vivo-derived ovine morulae, compact morulae, blastocysts and expanded blastocysts were distributed into two analogous parts. Embryos of the control part (n=30) were frozen with programmable Biocool freezer (Minitube, Germany) using ethylene glycol (EG), and transferred to recipient using standard protocol. Embryos of second part (n=30) were vitrified using open pulled straws (OPS; Vajta et al., Mol Reprod Dev, 1998, 51:53-58) after exposure at room temperature either - for 5 min in 10% glycerol (G), then for 5 min in 10%G+20% EG, then for 30 sec in 25%G+25%EG (Group 1, n=20 [Donnay et al., Anim Reprod Sci., 1998, 52:93-104]); - for 3 min in 10% EG+10%DMSO, then for 30 sec in 20% EG+20%DMSO+0.3M trehalose (Group 2, n=10 [Vajta et al., Mol. Reprod. Dev., 1998, 51:53-58, with modification]). Both group embryos were then loaded to OPS (2 embryos per straw) and plunged into liquid nitrogen. Warming was performed by plunging of OPS into 15 ml tubes (Falcon, USA, 12x1.5 cm) containing 0.7ml (1.5 cm column) of 0.5M trehalose. Previous model experiments for testing of efficiency of this "in-straw dilution" mode, performed on 200 oocytes, proved that all oocytes remained in straws after thawing. Five to 10 min after thawing, direct transplantation of embryos was performed using the OPS as catheter for transplantation and an 1ml syringe. Eight weeks after transfer, the following attachment rates of embryos have been detected by echography: 46.7% (control part [4 recipients with 1 fetus and 5 recipients with 2 fetuses]), 25% (Group 1 [1 recipient with 1 fetus and 2 recipients with 2 fetuses]), and 60% (Group 2 [2 recipients with 1 fetus and 2 recipients with 2 fetus]), P>0.05. It is possible to use this technology with the following variations: 1) direct rehydration of embryos using tubes with culture medium for thawing; 2) stepping dilution of cryoprotectants (for example, plunging of OPS with embryos for 3 min into a tube with 0.2ml of 0.5M disaccharide, and then for 3 min into other tube with 0.7 ml of 0.2M disaccharide; 3) simultaneous transplantation of two embryos and additional biological objects, every of which was vitrified previously in different OPS and at different dates, using mode "OPS to OPS"; 5) use a conventional freezing in gas as well as in medium freezers. Supported by INIA.
KEY WORDS: embryo, vitrification, transplantation, technology
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