Cryotop and development of vitrified immature bovine oocytes

H Hajarian,H Karamishabankareh,H Wahid,M Daliri,M Dashtizad,O Abas Mazni,Y Rosnina

doi:10.1590/s0102-09352011000100011

Abstract

The effectiveness of different cryodevices (open-pulled straw (OPS), electron microscopy grid (EMG), and Cryotop was evaluated for vitrification of immature bovine oocytes. Polar body, metaphase II stage (MII), survivability, and subsequent developmental rates were determined. Only oocytes with four or five layers of cumulus cells were used. Oocytes were equilibrated in two vitrification solutions - 1: 10% DMSO + 10% ethylene glycol (EG) for 30-45sec and 2: 20% DMSO + 20% EG +0.5M sucrose for 25sec -, mounted on one of the cryodevices and directly plunged into liquid nitrogen for 10 days. Immature vitrified oocytes using Cryotop showed the highest rates of polar body extrusion (PB) and nuclear maturity (MII); 41 and 58% respectively. Vitrified oocytes using OPS and EMG showed 26 and 32%; and 35 and 46% of PB and MII rates, respectively. The highest survivability resulted from Cryotop and EMG groups and no significant difference was found between them. Vitrified oocytes using Cryotop had the highest cleavage and blastocyst rates. All of the mean rates for vitrified immature oocytes were significantly lower than that of control group (P<0.05). The results of this study showed the superiority of Cryotop device for vitrification of immature bovine oocytes

Highlights

In 1985, vitrification emerged as another option for cryopreservation of cells and organs (Rall and Fahy, 1985)
Polar body assessment of denuded oocytes revealed that vitrification using Cryotop increased the percentage of oocytes with extruded polar body (P
There was no significant difference regarding the absence of polar body (PB-) between openpulled straw (OPS), Cryotop, and electron microscopy grid (EMG) groups (P>0.05), but control group represented the lowest percentage of PBcompared with other groups (P

Summary

Introduction

In 1985, vitrification emerged as another option for cryopreservation of cells and organs (Rall and Fahy, 1985). Formation of ice crystals is totally prevented in vitrification procedures using two main strategies: exceedingly high cooling/warming rates and highly viscose and concentrated vitrification solution (Smorag and Gajda, 1994; Vajta and Kuwayama, 2006). Some strategies such as reduction of vitrification solution volume (minimum volume, ≤1μL) and direct contact with liquid nitrogen or slush liquid nitrogen help to obtain necessary high cooling/warming rates (Yavin and Arav, 2007). The minimum volume-direct contact approach has been employed for cryopreservation of extremely chill sensitive Drosophila eggs by loading them on electron microscopy grids (Mazur et al, 1992). Cryotop have shown its superiority to OPS for vitrification of immature pig (Liu et al, 2008), human (Kuwayama et al, 2005b), and matured bovine oocytes (Morato et al, 2008), but there is still a gap of information for vitrification of immature bovine oocytes using different cryodevices

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Conclusion