Abstract

The present study was undertaken to investigate the effects of PVP concentration and exposure temperature to vitrification solution on the post-thaw survival, in vitro maturation and development of immature bovine oocytes (germinal vesicle stage). The vitrification solution (VS) consisted of 40% ethylene glycol (EG)+0.5 M sucrose (S)+10% FBS. PVP was added to VS: 0%, 5% or 10%. The cumulus-oocyte complexes (COCs) were diluted in VS as one step, after 2 min the COCs were loaded in straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were plunged into 30°C water bath for 10s. After thawing, the oocytes were diluted in 0.5 M (in DPBS with 10% FBS) sucrose solution for 5 min. The survival rate (FDA-test and trypan blue) of immature bovine oocytes was measured. The survival rate was higher in 5% PVP (91.5%) than in 0% (64.2%) or in 10% PVP (79.7%). The proportion of metaphase II formation was 69.35% in control (no vitrified COCs), 9.3% in 40% EG+0.5 M S+0% PVP and 21.05% in 40% EG+0.5 M S+5% PVP (p 0.05). Two out of 151 oocytes (1.32%) developed to blastocyst stage after frozen-thawed with 5% PVP (p>0.05). Development of oocytes after frozen-thawing to the 2 cell were not significantly affected with or without PVP following IVF. However, the vitrification of immature bovine oocytes with PVP maintained the ability to develop to the blastocyst stage after IVM-IVF and IVC, while no blastocysts were obtained from oocytes vitrified without PVP. These results suggested that PVP has a protective role for vitrification of immature bovine oocytes as far as survival is concerned, however, the protection was not sufficient enough to support blastocyst formation. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 1 : 23- 28)

Highlights

  • The physical definition of vitrification is the solidification of a solution at low temperatures without ice crystal formation

  • These results suggested that PVP has a protective role for vitrification of immature bovine oocytes as far as survival is concerned, the protection was not sufficient enough to support blastocyst formation

  • The PVP is a macromolecular reagent that reduces the formation of ice crystal so that the cell membrane and zona of oocytes are protected during the freezing and warming (Fahy et al, 1994)

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Summary

INTRODUCTION

The physical definition of vitrification is the solidification of a solution (glass formation) at low temperatures without ice crystal formation. Successful vitrification requires the combination of a high concentration of permeable cryoprotectant and a very rapid cooling rate. The high concentrations of cryoprotectant cause cell damage due to the osmotic and cytotoxic effects (Rall and Fahy, 1985; Rall, 1987). Suzuki and Nishikata (1992) successfully cryopreserved immature bovine oocytes by a combined process of dehydration of the oocyte with sucrose and permeation with 1,2-propanediol before freezing.

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