Successful cryopreservation of canine oocytes is a stepping stone towards applying assisted reproduction technology (ART) for conservation of endangered canids. To our knowledge, this is the first report conducted on vitrification of immature canine oocytes. Although the rate of meiotic resumption of vitrified-warmed oocytes was significantly lower than those of the control and exposure groups there was no statistically significant difference in rates of meiotic progression to the MII stage.
Various types of cryoprotectants (i.e., EG, DMSO, glycerol, propylene glycol, polyethylene glycol and 1,2-propanediol) have been used in different combinations for the vitrification of mammalian oocytes and embryos . EG has been found to be less toxic than glycerol and propylene glycol to mouse embryos . In addition, it was shown to allow much higher survival rates of bovine embryos . DMSO has been found to be an effective cryoprotectant for vitrification of mouse and hamster oocytes [19, 20]. In consequence, EG and DMSO were chosen as cryoprotectants in this present study to avoid ice formation based on the evidence of previous studies in which cryoprotection was useful for the successful vitrification of immature buffalo oocytes . The vitrification technique used high cryoprotectant concentrations, which have been described as toxic to cells . Contrarily, an appropriate phase composition of cryoprotectant mitigates the toxic and osmotic consequences of highly concentrated cryoprotectants . Thus, a mixture of cryoprotectants can decrease individual specific toxicity. In the present study, we vitrified canine oocytes in OPS using mixture of 20% EG and 20% DMSO as previously described .
Vitrification of immature bovine and equine oocytes using OPS resulting in subsequent cleavage and development to the blastocyst stage has been reported [25, 26]. The OPS method used for cryopreservation of oocytes offers many advantages over other methods. It is simple, inexpensive, achieves a great increase in the speed of cooling by reducing the volume to be vitrified and by thinning the isolating layer between the cooling agent (LN2) and the vitrification solution. A further advantage of using very small volumes of vitrified drops was a reduction of the amount of damage to the zona pellucida which occurred during cooling and warming .
Our results demonstrated that vitrified-warmed immature canine oocytes in OPS can successfully resume meiosis and develop to the MII stage following IVM. However, the efficiency of oocyte cryopreservation methods is still unsatisfactory. Vitrification procedure reduced oocyte competence to resume meiosis. The assessment of cryo-damaged processes and organelles is fundamental in the evaluation and refinement of current and future cryopreservation protocols. The ultrastructural alterations of vitrified-warmed oocytes revealed by TEM indicated that vitrification procedures affect the pericortical distribution and morphofunctional integrity of cortical granule, mitochondria, lipid droplet and SER.
Morphological examination demonstrated that cortical granules were reduced in numerical density and were damaged in vitrified-warmed oocytes, similar to results in previous studies [12, 28, 29]. Wessel et al.  reported that cortical granules are Golgi-derived, membrane-bound spherical or slightly ovoid organelles formed during the early stages of oocyte and at maturation. Cortical granules are believed to establish the block to polyspermy by preventing penetration of additional spermatozoa. Fuku et al.  showed that the numbers of cortical granules along the oolemma were substantially reduced, and fusion of cortical granules with the plasma membrane followed by exocytosis of granule core or intact cortical granule into the perivitelline space was seen in all vitrified bovine IVM oocytes. Ghetler et al.  found that the cryopreservation procedure resulted in the loss of cortical granules from the cortical area and in appearance of vesicles within the cytoplasm of both immature and mature human cryopreserved oocytes, which might indicate structural damage occurring from the freezing and warming process.
It has been suggested that the shape and intracellular distribution of mitochondria were related to the level of cell metabolism, proliferation and differentiation, and that these organelles generate the essentials required in a crucial period of the cell cycle . In addition, the present study demonstrated that mitochondria corresponding to the cortical zone were shown to increase in numerical density, to elongate, develop a coarse surface, broken and unclear, of decreased electron density. Similar findings were reported in the exposure of porcine oocytes to vitrification solutions . Roberto et al.  reported mitochondria of frozen-thawed human oocytes with decreased matrix electron density or with ruptures of the outer and inner membranes. Mitochondria were the most abundant organelles in mammalian oocytes and their dysfunction or abnormalities would critically determine oocyte and embryo developmental competence. Structural changes to lipid droplets in the present study were in agreement with previous reports in bovine and porcine oocytes [13, 31, 32]. Xiang et al.  suggested that the increased small lipid droplets came from broken larger lipid droplets and exist in the form of smaller drops during vitrification of porcine oocytes. Isachenko et al. also reported that lipid droplets in porcine oocytes changed morphologically during cooling; they changed into a spherical form with lucent streaks .
The present study showed the expression of stress genes (HSP70), Dnnt1 and SOD1 were similar in control and vitrified-warmed groups. The identical expression patterns of HSP70, Dnnt1 and SOD1 in the control and vitrified groups, indicating that the vitrification protocol did not alter the expression pattern of these stress genes. It is well known that members of Bcl2 gene family play a major role in regulation of apoptosis. In that regard, Bcl2 is anti-apoptosis and promotes cell survival, whereas BAX is pro-apoptosis and promotes cell death . Apoptosis is an underlying process in oocyte degeneration and embryo fragmentation . This experiment found that Bcl2 was strongly expressed in the vitrified-warmed group when compared to the control, whereas BAX wasn't expressed in both groups. Our results were similar to report in mouse embryos that the expression of BAX gene did not differ from control when morulae were vitrified . Contrarily, Dhali et al.  showed that Bcl2 displayed a greater decrease in the vitrified mouse embryos compared to control and expression of BAX gene was down-regulated in the treated group. The expression of Bcl2 was higher in normal than fragmented ones, and the expression did not vary significantly among embryos of varying quality . Moreover, the expression of Bcl-2 was comparatively lower in the vitrified embryos than the normal embryos . Therefore, the differential pattern of gene expression observed in the present study may be used to predict the quality and developmental ability of vitrified immature canine oocytes.
In conclusion, the present study demonstrated that it is possible to cryopreserve immature canine oocyte by vitrification using EG in combination with DMSO and modified OPS protocol. Our results showed that vitrified-warmed immature canine oocytes can resume meiosis and develop to the MII stage. Further studies are required to investigate the fertility and developmental ability of MII oocytes following fertilization.