In order to achieve intracellular vitrification, there are three main requirements i.e., high concentrations of permeating cryoprotectants, ultra-rapid cooling and low sample volume. Alteration in any one of these components can affect the success of the vitrification process. High concentrations of cryoprotectants are used to increase the viscosity of intra- and extra-cellular solutions, which increases the glass transition temperature . Ethylene glycol (EG) and DMSO are commonly used cryoprotectants in vitrification, but they are considered toxic due to their cell permeating nature and the high concentrations needed to induce vitrification . Ultra-rapid cooling allows the addition of permeating cryoprotectants at lower concentrations reducing toxicity to the cells . Decreasing the sample volume during the vitrification procedure increases the cooling rate which improves heat transfer .
In the present study, the most profound and consistent observation was that the high concentrations of cryoprotectants used (15% EG and DMSO) had no adverse effect on cleavage and blastocyst production rates. Similarly, exposure of vitrified oocytes to the warming solution containing sucrose for 5 min did not affect cleavage and blastocyst production, as compared to 1 min, regardless of group. On the contrary, vitrification of the GV stage oocytes affected cleavage rates and blastocyst rates profoundly following in vitro maturation and fertilization. In both experiments, the cleavage rates were significantly reduced in the Vitrified group and blastocyst rates were not only significantly reduced, but were exceptionally low. Although these experiments were not designed to determine the necessity of sucrose in the warming solution, it is difficult to imagine that the absence of sucrose in the warming solution could be anything but detrimental. Thus, we concluded that our vitrification procedure was deficient in one or more ways for the cryopreservation of germinal vesicle stage bovine oocytes.
In the current studies, cleavage and blastocyst production rates in the VS1 + VS2 group did not differ from that in the Control group (Table 1). This suggests that the 45–60 sec exposure time was not sufficient for permeating these cryoprotectants to penetrate inside the bovine oocytes , or if penetrated, DMSO and EG had no toxic effects on bovine oocytes. Although there is still considerable debate, published literature tends to confirm the likelihood that these cryoprotectants are not highly toxic. DMSO alone  or in combination with other cryoprotectants  has been used successfully for oocyte vitrification, and in at least one study , the exclusion of DMSO from vitrification solution resulted in lower survival rates of oocytes. However, it has also been reported that DMSO (used alone or in combination with EG) resulted in reduced developmental competence of oocytes [11, 18]. In other studies, DMSO-free vitrification systems e.g., glycerol and propanediol [35, 36], or ethylene glycol and sucrose [37–39], have provided promising results with oocytes .
An ultra-rapid cooling rate is generally considered as one of the requirements for successful vitrification. Recently, ultra-rapid warming has been reported to be more important than concentrations of cryoprotectants or cooling rate . The optimal warming time for vitrified bovine oocytes is still unknown. However, exposing oocytes to the warming solution for 5 min as opposed to 1 min in Experiment 2 appeared to have no obvious benefit on subsequent embryo development. Therefore, 1 min appears to be a sufficient time in the warming solution. It is also noteworthy that exposure to the warming solution containing sucrose for either 1 or 5 min did not have any adverse effect in nonvitrified oocytes in the VS1 and VS1 + VS2 groups.
Osmotic stress resulting from exposure to high concentrations of cryoprotectants in vitrification solutions could cause oocytes to undergo dramatic volume changes during warming. In this regard, one of the most important factors affecting cell survival during dilution is excessive cell swelling. Water moves through the cell membrane more rapidly than cryoprotectants, and because of the high concentration of cryoprotectants remaining in the cell, swelling occurs. Oocytes have biological limits in their tolerance to the osmotic stress associated with high concentrations of cryoprotectants . Volume fluctuations can affect the integrity of the plasma membrane as well as the cytoskeletal organization of oocytes . During this process, oocytes are rehydrated while cryoprotectants and water are exchanged due to their gradients across the cell membrane . For this reason, sucrose is commonly used in warming solution to counterbalance the osmotic shock during conventional freezing  and vitrification . Although these experiments were not designed to determine the benefits of sucrose in the warming solution, there was no evidence for any adverse effects.
Bovine oocytes at different nuclear stages (germinal vesicle, metaphase I and metaphase II) have been cryopreserved successfully, using both slow freezing or vitrification methods. Mature oocytes have meiotic spindles that are extremely sensitive to cryoprotectant additives and cooling rates which result in tubulin depolymerization [11, 41, 42]. Therefore, immature oocytes (GV stage) enclosed in cumulus cells (COCs) were used in the current study. The role of cumulus cells during oocyte cryopreservation is still not clear. Cumulus cells provide a rigid structure to oocytes and thus protect them against osmotic shock during cryopreservation . It has been reported that the survival, cleavage and blastocyst production rates of bovine oocytes were higher when vitrified with enclosed cumulus cells than partially denuded COCs , as cumulus cells support the oocyte during in vitro maturation and fertilization .
Cryopreservation of oocytes has been reported to result in several ultrastructural and morphological alterations including damage to the cell membrane and ooplasm, and abnormal distribution of chromosomes, microtubules and actin microfilaments [45–47]. Additionally, damage to gap junction integrity can result in disruption of the communication between cumulus cells and oocytes [11, 48]. These morphological changes have been linked to a failure in fertilization and embryo development [48, 49]. Consequently, the disruptions in protein synthesis, and the failure of cumulus cell expansion and further meiotic development may also be responsible for the failure of cleavage and subsequent embryo development following vitrification .
In a recent study, only 23% of vitrified COCs (GV stage) reached the MII stage following IVM as compared to 84% in nonvitrified control COCs . In another study using similar vitrification conditions (15% EG and DMSO in VS2, 45–60 sec exposure time and cryotop as a loading device), cleavage and blastocyst production rates were somewhat higher than in the present study, but still low . It is noteworthy that in the present study, cleavage and blastocyst rates were based on the original number of oocytes (COCs) that were vitrified. Thus, data have been presented and interpreted very conservatively. Based on these results and published literature, we must conclude that the optimum vitrification procedure for GV stage oocytes has yet not been developed.