**2. Cow**

Cow ovaries may be used in research on ovarian cryopreservation. They are much larger (14.3+/- 5.7 cm3) (Gerritse et al., 2008) as compared to the human, but on the other hand the tissue architecture is similar. The function (Yang&Fortune, 2006) and the structure (Rodgers&Irving-Rodgers, 2010) of the bovine ovary has been extensively studied. Also, the granulosa cells and the extracellular matrix of bovine follicles of various developmental stages are well described (Lavranos et al., 1994; Irving-Rodgers et al., 2006).

There exist one report of avascular ovarian transplantation in the cow after cryopreservation by vitrification using a solution consisting of 20% ethylene glycol (EG) and 20% dimethylsulphoxide (DMSO) in TCM-199 medium (Kagawa et al., 2009). In this study, vitrification was performed using the Cryotissue method (Kagawa et al., 2007), where the ovarian cortex was positioned on a thin metal strip that was plunged into liquid nitrogen (LN) and inserted into a protective container for storage in LN. Rapid post-thaw warming was done with immersion of the metal strip into TCM-199, supplemented with sucrose at 40ºC followed by washing in the identical solution but with decreasing sucrose concentration. After warming, the tissue was grafted subcutaneously to the neck or orthotopically to oophorectomized cows, and resumed cyclicity was seen in both groups within two months. Histological analysis of grafted tissue showed normal morphological appearance and about 80% viability among preantral follicles, as demonstrated by fluorescent staining. These results may be regarded as encouraging towards clinical application of the vitrification procedure for ovarian cortex cryopreservation.

In another study, aimed towards in vitro follicle maturation of bovine follicles, slow-frozen bovine ovarian cortical pieces were incubated after thawing for durations between 1 and 48 h (Paynter et al., 1999). The major finding was that the thawed tissue had a capacity to recover from damage during the subsequent incubation period. This idea was further utilized in later research, where bovine ovarian cortical pieces were cultured for six days followed by isolation of secondary follicles and culture in the presence of inhibin (McLaughlin&Telfer, 2010) showing significant estradiol (E2) secretion and oocytes growth up to a diameter > 100 μm.

The notion that antioxidants may enhance survival of frozen-thawed tissue was studied using bovine ovarian cortex (Kim et al., 2004). After cryopreservation by slow freezing with 1.5 M DMSO, bovine ovarian cortex was in vitro cultured for periods up to 48 h in minimal essential medium (MEM) with or without ascorbic acid. Interestingly, there was no difference between the two groups in apoptosis rate evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling test (TUNEL) or deoxyribonucleic acid laddering. Nevertheless, protective effects by ascorbic acid were seen in stromal cells that were cultured for 24 h. In addition, this study also demonstrated that stromal cells are more susceptible to damage mechanisms than primordial follicles, which is a finding also observed in frozen-thawed human ovarian tissue (Gook et al., 1999; Hreinsson et al., 2003). The model of supplementation of antioxidant agents to the cryoprotectant (CPA) should be investigated further.

The toxic effect of various concentrations and types of CPAs that are frequently used for slow freezing was compared using bovine ovarian cortical strips (Lucci et al., 2004; Celestino et al., 2008). Among other CPAs, DMSO at 1.5 M and 3 M were evaluated in both studies. While the study by Celestino and colleagues (Celestino et al., 2008) showed increased toxicity with rising concentration of DMSO, the other study (Lucci et al., 2004) showed slightly higher proportion of normal follicles in the 3 M DMSO group when assessed by conventional histology. However, ultrastructural analysis by transmission electron microscopy (TEM) revealed some irregularities in the cytoplasm of granulosa cells when 3 M DMSO was applied (Lucci et al., 2004).

There is one study on bovine ovarian cortical strips comparing slow freezing with vitrification and this study demonstrated higher efficiency of slow-freezing (Gandolfi et al., 2006). Furthermore, another study also demonstrated advantages of the slow-freezing method as compared to vitrification, when bovine ovarian cortical pieces were used, but on the other hand an advantage of the vitrification method was seen when whole ovaries with vasculature were used as the model system (Zhang et al., 2011). In the latter study, the effectiveness of the different cryo techniques was evaluated by Trypan blue test, histology as well as E2 and progesterone levels obtained from supernatant after in vitro culture of the tissue. The same research group (Zhang et al., 2011) performed controlled-rate slow freezing with DMSO of whole bovine ovary and compared different cooling rates and ice seeding temperatures. The cooling rate of 0.2ºC/min and ice seeding temperature of -5ºC showed superiority in comparison to different combinations of the cooling rates of 2ºC/min and 0.1ºC/min and the ice-seeding temperatures of -2ºC, -5ºC and -8ºC.

Additionally, one study on bovine ovarian tissue was designed to evaluate the effect of the thickness of the ovarian cortex strip on follicular morphology after incubation for 20 min and slow freezing with 1.5 M propylene glycol (PROH) (Ferreira et al., 2010). Ovarian cortex pieces of 10 x 3 mm, with a thickness of either 2 or 4 mm, were compared and considerably higher proportion of normal follicles were found in the 2 mm group compared to the 4 mm in both fresh and cryopreserved tissue. This result may be explained by superior tissue impregnation with CPA in the 2 mm group, but the exact mechanisms remain to be clarified.
