A Preliminary Report on Early Embryonic Development Following Oocyte Vitrification and In Vitro Fertilization in Sheep


J.T. Choi, D.A. Redmer, L.P. Reynolds, J.S. Luther, C. Navanukraw, D. Pant, R.M. Weigl,

J.D. Kirsch, K.C. Kraft, and A.T. Grazul-Bilska


Department of Animal and Range Sciences, North Dakota State University, Fargo




Interest in oocyte cryopreservation has recently increased with the growing importance of in vitro embryo production, nuclear transfer, and gene banking (Gordon, 1997; Andrus et al., 2000).  A relatively recent approach to achieve rapid freezing without the use of expensive freezing devices is called vitrification.  Vitrification is defined as the physical process by which a highly concentrated solution of cryoprotectant solidifies during cooling without formation of ice crystals (Rall, 1987). Oocyte vitrification has been developed using mouse embryos (Rall et al., 1985).  Recently, vitrification has also been attempted in the cow (Fuku et al., 1992; Vajta et al., 1998; Hurtt et al., 2000; Papis et al., 2000), pig (Isachenko et al., 1998), buffalo (Dhali, et al., 2000), and horse (Hurtt et al., 2000). The advantages of vitrification technology when compared with slow-rate freezing are the low price of equipment, the simplicity of the procedure, and the short time required (Palasz et al., 1996).


Despite the efforts of numerous research groups, successful cryopreservation of oocytes remains a difficult task (Niemann, 1991; Okatay et al., 1998). A limited number of experiments have reported blastocyst development and subsequent calf development from oocytes that have gone through the vitrification process (Fuku et al., 1992; Vajta et al., 1998). This lack of success presents an obstacle to planning and organizing of experiments (Ana et al., 2001).


The aim of this experiment was to evaluate early embryonic development following vitrification of oocytes at the germinal vesicle (GV) stage and in vitro fertilization (IVF) in sheep treated with follicle stimulating hormone (FSH).                                                                                                                                




Ewes of mixed breeds (n = 5) received exogenous FSH on days 13 and 14 of the estrous cycle (Stenbak et al., 2001).  Ovaries were surgically removed by ovariectomy (Reynolds et al., 1998) and placed in phosphate-buffer saline (PBS) containing penicillin/streptomycin (Gibco, Gaithesburg, MD) at 39° C (Stenbak et al., 2001). The number of visible follicles on each ovary was counted and oocytes were collected.  Oocytes were then evaluated based on morphology and categorized as healthy or atretic according to Thompson et al. (1995).


All oocytes were washed three times in maturation medium (Stenbak et al, 2001).  Oocytes were incubated in stabilized


maturation medium (incubated overnight under oil at 39° C in 5% CO2 and 95% air) containing 10 ng/ml of epidermal growth factor (EGF; Sigma). Dose of EGF was chosen on the basis of previously published experiments (Guler et al 2000; Grazul-Bilska et al., 2001).


After four hours, oocytes from each ewe were divided into two treatment groups: control (CON) and vitrification (VIT).  For CON group, oocytes were matured in vitro for additional 20 hours. Oocytes in the VIT group were vitrified, thawed, and then matured for additional 20 hours. There were two steps in the vitrification process.  First, oocytes were placed in holding medium [TCM-Hepes supplemented with 20 % fetal bovine serum (FBS) containing 10 % ethylene glycol (EG) and 10 % dimethyl sulfoxide (DMSO)] at 38°C for 45 seconds (Vajta et al., 1998).  Second, oocytes were placed to holding medium containing 20 % EG, 20 % DMSO and 0.5 M sucrose at 38 °C for 25 seconds.   Oocytes were then placed directly in liquid nitrogen (LN2, - 196 °C) for 1 hr followed by the thawing process. 


The thawing process consisted of three steps.  First, oocytes were placed in holding medium containing 0.25 M sucrose for 1 min.  Second, oocytes were placed in a holding medium containing 0.15 M sucrose for 5 min.  Third, oocytes were placed in holding medium for two, 5-minute intervals.  Subsequently, oocytes continued the maturation process and were then subjected to IVF and culture as described previously (Stenbak et al. 2001; Grazul-Bilska et al., 2001).


All data are reported as means ± standard error (SEM). Data were analyzed by using the general linear models (GLM) procedure of the Statistical Analysis System (SAS, 1985) with the vitrification process as the main effect. In addition, data for the number of oocytes cleaved, morula, and blastocysts were analyzed by Chi-square (Spiegel, 1961).        





The number of visible follicles was 30.4 ± 1.8/ewe, the number of recovered oocytes was 31.2 ± 2/ewe, and the number of healthy oocytes was 22.4 ± 2.3/ewe.


The total number of oocytes used for IVM was 150, the CON group consisted of 71 oocytes, and the VIT group consisted of 79 oocytes.  Following maturation, cumulus cells were much more expanded in the CON than VIT group.  Some oocytes were damaged in the VIT group.


Table 1 shows the number of oocytes used for IVF, oocytes fertilized, and the number of morula and blastocysts in the CON and VIT group.


Table 1. Number of oocytes used for IVF, oocytes fertilized, morulas and blastocysts of CON

and VIT groups.



No.of oocytes used for IVF

No. of fertilized oocytes (%)

No. of non-fertilized oocytes (%)

No. of morula (%) on day 6

No. of blastocyst (%) on day 8


Per ewe


Per ewe


Per ewe


Per ewe


Per  ewe

Control (CON)


17.8 ±4
































* Values (means + SEM) differ within a column; P<0.05


Control oocytes achieved a greater (P<0.05) fertilization rate than oocytes from the VIT group. Total number of fertilized oocytes was also greater (P<0.05) in CON than VIT group.  However, no differences (P>0.1) were observed between CON and VIT groups in morula and blastocyst development following fertilization.




Data of this experiment demonstrated that the vitrification procedure could be applied to cryopreserve oocytes before fertilization since several embryos developed after vitrification and IVF.  However, fertilization rates of vitrified oocytes were significantly lower than control oocytes.  Therefore, future studies will need to focus on improving the process of vitrification for subsequent fertilization and early production procedures in sheep.  


Vitrification has been used to cryopreserve oocytes in several species, including mice (Rall and Fahy, 1985; Ko and Threlfall, 1988), cow (Fuku et al., 1992; Martino et al., 1996; Vajta et al., 1998; Hurtt et al., 2000), pig (Isachenko et al., 1998), horse (Hurtt et al., 2000) and buffalo (Dhali et al., 2000).  However, the efficiency of this procedure is very low.  Vitrification diminishes the rates of maturation, fertilization and blastocyst formation, when compared to slow-rate freezing techniques and/or no freezing conditions (Fuku et al., 1992, 1995; 30% Martino et al., 1996; Isachenko et al., 1998;Vajta et al., 1998; Dhali et al., 2000; Hurtt et al., 2000).


In our experiment, the fertilization rate of non-vitrified oocytes was 38% and vitrified oocytes 7.5%.  Additional data for vitrified ovine oocytes are not available at present, but for non-vitrified oocytes the rates of fertilization are usually in the range 60-85% (see Grazul-Bilska et al., 2001 and Stenbak et al., 2001).  Relatively high fertilization rates (45-75%) of vitrified oocytes were reported for cows (Vajta et al., 1998; Papis et al., 2000).  However,  these fertilization rates were lower than achieved for non-vitrified oocytes  (72-91%; Vajta et al., 1998; Papis et al., 2000).  Others reported much lower rates of fertilization for bovine vitrified oocytes (0.8-7%, Fuku et al., 1992, 1995; 30%, Martino et al., 1996).  Bovine vitrified and then fertilized oocytes were able to develop to blastocyst stage (Papis et al., 2000; Vajta et al., 1998; Martino et al., 1996).  Interestingly, healthy calves were born following transfer of embryos developed after vitrification of oocytes (Fuku et al., 1992; Vajta et al., 1998). 


High fertilization rates for bovine vitrified oocytes were achieved by introducing several modifications to the simple vitrification procedure (Papis et al., 2000).  Therefore, these modified protocols will be used in future experiments to improve the vitrification procedure for ovine oocytes.


In summary, the present data demonstrated that vitrification procedures have a potential to be used to cryopreserve ovine oocytes.  Future experiments have the potential to establish an efficient vitrification method for ovine oocytes.  Vitrification can be applied to make oocyte banks for storage of gametes from genetically superior ewes.




Supported in part by Hatch Project ND 01705 and ND SBARE.




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