Elsevier

Theriogenology

Volume 59, Issue 8, 15 April 2003, Pages 1839-1850
Theriogenology

Cryopreservation of goat oocytes and in vivo derived 2- to 4-cell embryos using the cryoloop (CLV) and solid-surface vitrification (SSV) methods

https://doi.org/10.1016/S0093-691X(02)01257-8Get rights and content

Abstract

This study evaluated the efficiency and toxicity of two cryopreservation methods, solid-surface vitrification (SSV) and cryoloop vitrification (CLV), on in vitro matured oocytes and in vivo derived early stage goat embryos. In the SSV method, oocytes were vitrified in a solution of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4% trehalose. Microdrops containing the oocytes were cryopreserved by dropping them on a cold metal surface that was partially immersed in liquid nitrogen. In the cryoloop method, oocytes were transferred onto a film of the CLV solution (20% DMSO, 20% EG, 10 mg/ml Ficoll and 0.65 M sucrose) suspended in the cryoloop. The cryoloop was then plunged into the liquid nitrogen. In vivo derived embryos were vitrified using the same procedures. The SSV microdrops were warmed in a solution of 0.3 M trehalose and those vitrified with CLV were warmed with incubation in 0.25 and 0.125 M sucrose. Oocytes and embryos vitrified by the SSV method had a significantly lower survival rate than the control (60 and 39% versus 100%, respectively; P<0.05), while the survival rate of CLV oocytes and embryos (89 and 88%, respectively) did not differ from controls. Cleavage and blastocyst rates of the surviving vitrified oocytes (parthenogenetically activated) and embryos (cultured for 9 days) were not significantly different (P>0.05) from the control nor did they differ between vitrification methods. Embryos vitrified with the CLV method gave rise to blastocysts (2/15). Our data demonstrated that the two vitrification methods employed resulted in acceptable levels of survival and cleavage of goat oocytes and embryos.

Introduction

Cryopreservation permits the long-term storage of cells and tissues such that acceptable numbers of cells are viable upon subsequent warming. The ability to apply cryopreservation techniques to oocytes and embryos is advantageous in the area of reproduction [1], [2], [3]. Cryopreservation could relieve dependency upon freshly collected oocytes, thereby simplifying the management of genetic resources (oocyte and embryo banking) in domestic and exotic species [3], [4].

Embryos can be cryopreserved using freezing or vitrification techniques. Vitrification is defined as glass-like solidification. It is achieved through use of a high concentration of cryoprotectant combined with a fast cooling rate, which avoids ice crystal formation during the cryopreservation process [5]. Rall et al. [6] were the first, in 1985, to report the vitrification of 8-celled mouse embryos, while Massip et al. [7] published the first report on successful vitrification of bovine embryos. Cryopreservation of embryos at the morula and blastocyst stage has become a routine procedure in several species; however, reliable methods of cryopreservation of oocytes and early cleavage stage embryos for most species are still under development.

Faster cooling rates reduce toxicity of the cryoprotectant and also diminish the length of time oocytes are exposed to temperatures toward which they are particularly sensitive [8], [9]. Based on survival rates of embryos cryopreserved by slow versus fast cooling methods it is evident that the rapid vitrification circumvents the chilling sensitivity of embryos [10], [11]. As a result of these advantages, vitrification is a promising tool for cryopreservation of mammalian oocytes and embryos. Furthermore, it is simpler, quicker, and less expensive than controlled freezing [12], [13].

To achieve the rapid cooling necessary for vitrification, several methods use small volumes of vitrification solution, which are plunged directly into liquid nitrogen (LN2) [14]. Electron microscopy grids [15], glass capillaries [16], open-pulled plastic straws [17], and cryoloops [2] have been used to reduce the volume of vitrification solution containing the oocytes. In an alternative technique called solid-surface vitrification (SSV), the oocytes in a droplet of solution are vitrified upon direct contact with a solid surface cooled to around −150 to −180 °C [4]. The cryoloop and SSV methods allow direct exposure of cryoprotectant containing embryos to the cooling environment, thus resulting in a rapid cooling. Both methods have been used to successfully cryopreserve bovine oocytes with survival rates in terms of development to blastocyst ranging from 15 to 41% [2], [4]. While either of the two methods could be readily used in field situations with minimal equipment requirements, the SSV method is lower in cost as no specialized supplies are needed.

The vitrification solutions used in our experiments contained a mixture of permeating and nonpermeating cryoprotectants. Permeating agents are organic solutes responsible for protecting the intracellular organelles of the cells during cooling and warming prior to and after storage in LN2 [1]. Ethylene glycol (EG), dimethylsulfoxide (DMSO) and polyethylene glycol are commonly used. The nonpermeating cryoprotectants are macromolecules and sugars, e.g. polyvinyl-pyrrolidone (PVP), sucrose and trehalose. Their role is to reduce ice formation during freezing [1], facilitate dehydration of cells prior to cooling and to protect the cellular membrane [18]. The optimal concentration of a given cryoprotectant, its permeating rates and toxicity depend upon the species and the developmental stage of the embryo [1], [8]. Toxicity and vitrification efficiencies for these solutions have not been previously reported for goat oocytes or cleavage-staged embryos. In this study, we compared the SSV and cryoloop methods in order to determine differences in toxicity of the vitrification solutions tested and the vitrification efficiency of the methods for goat oocytes and in vivo derived 2- to 4-cell embryos.

Section snippets

Materials and methods

Except where otherwise indicated, all chemicals were obtained from Sigma (St. Louis, MO, USA) and Canadian Life Technologies, Inc. (Burlington, Ontario, Canada).

Oocyte and embryo collection

A total of 447 oocytes were recovered in five sessions. Most (96%) were enclosed in cumulus cell layers, while only 4% were denuded at the time of recovery. All of the recovered oocytes were used in the experiment. Nuclear maturation was not assessed after oocyte maturation; however, consistent results with over 80% of matured oocytes reaching the metaphase II stage have been obtained in other experiments in our laboratory (data not shown).

A total of 194 zygotes were recovered in three

Discussion

In the present study, we compared the efficiency and toxicity of two vitrification methods developed for cattle oocytes and embryos [2], [4], for in vitro matured oocytes and in vivo derived embryos in the goat. This report demonstrated that goat oocytes or early stage embryos can develop to the morula or blastocyst stage following vitrification. While other groups have demonstrated goat embryo cryopreservation using morulae and blastocysts [23], [24], [25], [26], [27], to our knowledge, no

Acknowledgements

The authors gratefully acknowledge the support by technical and health staff on the Macdonald farm campus and from Nexia Biotechnologies, especially that of Denyse Laurin and Melanie Gauthier.

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