**3. Pig**

188 Current Frontiers in Cryopreservation

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

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

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

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

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.

stages are well described (Lavranos et al., 1994; Irving-Rodgers et al., 2006).

application of the vitrification procedure for ovarian cortex cryopreservation.

**2. Cow** 

up to a diameter > 100 μm.

investigated further.

The pig is a species that has been used in biomedical research, particularly regarding development and training of surgical techniques for later use in the human. The reproductive cycle of the pig lasts for 18-24 days and generally 8-15 oocytes are released from each ovary at ovulation (Soede et al., 2011). The size of the pig ovary is about 7.3 (+/- 2.2) cm3 (Gerritse et al., 2008), which is comparable to the human ovarian size of 6.5 (+/- 2.9) cm3 (Munn et al., 1986). The equal ovarian size of the sow and human female, as well as the possibility to get fresh pig ovaries from slaughterhouses, renders the pig as a good model for ovarian cryopreservation research. Nevertheless, a fairly low number of studies in this area with the pig ovary as an experimental model have been performed, as shown below.

One recent study on pig ovarian tissue evaluated whether the size of ovarian cortical pieces is important for the cryopreservation outcome (Jeremias et al., 2003). Cortical strips were all of 1 mm thickness but either 1x1 mm or 5x1 mm in surface area. The pieces were cryopreserved by slow freezing in 1.5 M DMSO and after rapid thawing the size of the surviving primordial follicle pool, was compared to fresh tissue (1x1 mm) (Jeremias et al., 2003). The freezing method was uncontrolled-rate freezing with the cryovials containing

Review on Ovarian Cryopreservation in Large Animals and Non-Human Primates 191

A variety of in vivo and in vitro sheep models have been used in research on both ovarian cortex cryopreservation and whole ovary cryopreservation, most probably due to the large knowledge about the physiology of the sheep ovary. In addition, the sheep ovary has, similarly to the human ovary, a collagen-dense outer stroma containing the pool of primordial follicles (Arav et al., 2005). Nevertheless, the size of the sheep ovary is only around 20% of the human premenopausal ovary (Munn et al., 1986; Gerritse et al., 2008) and this fact has to be taken into consideration, particularly in comparative studies concerning

The pioneering study in ovarian cryopreservation was published in 1994 by the Edinburgh group (Gosden et al., 1994), where live births (Table 2), after ovarian cortex cryopreservation and transplantation in a large experimental animal, were reported for the first time. In fact, this study opened up the field for future clinical fertility preservation and this is also accentuated in its conclusion by stating "that frozen storage and replacement of a patient´s own ovarian tissue might be practicable when fertility potential is threatened by chemotherapy/radiotherapy". The cryopreservation protocol applied in that study has subsequently been widely used in research and clinical practice, often with minor modifications. It should be emphasized that this slow freezing-thawing protocol was adapted from a study on cryopreservation of mouse primordial follicles (Carroll&Gosden, 1993) and the authors also wrote that they "have no evidence whether it is optimal". In the Gosden study, ovarian cortex pieces were positioned in cryovials with Leibowitz L-15 solution containing donor serum and 1.5 M DMSO and the cryovials were held on ice for 15 min. The ovarian cortex strips were then considered equilibrated in the CPA and cooled in a programmable freezer at a rate of 2ºC/min to -7ºC and maintained at -7ºC for 10 min before seeding. Freezing was performed further by reducing the temperature at a rate by 0.3ºC/min to -40ºC following by 10ºC/min until -140ºC before plunging into LN. Thawing was performed by exposure of frozen tissue to air temperature for 2 min and then placed in a water bath at room temperature for an unspecified time. The frozen-thawed slices were attached adjacent to the left ovarian pedicle at the second laparoscopy of the sheep. High progesterone level four months later indicated reestablished normal cyclicity. Eight months after grafting, one sheep gave birth and one more lamb was delivered by Cesarean section at

In a follow up study the authors reported that the longevity of the transplants was at least 22 months, but at that time very few primordial follicles were found in the graft (Baird et al., 1999). The levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in animals which had received grafts were higher as compared to controls. However, all grafted animals demonstrated normal cyclical pattern. These findings were corroborated by the lower levels of inhibin, indicating reduced number of inhibin-producing small antral follicles. In the second part of the study Baird and colleagues grafted both fresh and frozenthawed ovarian pieces under the kidney capsule of nude mice to assess whether the freezing-thawing procedure per se or if the warm ischemic time post transplantation was the main reason for loss of the follicle pool already within two years. A follicular depletion of about 65% was seen after grafting of fresh tissue as compared to fresh non-grafted ovarian tissue. Additionally 7% was lost after transplantation of frozen-thawed pieces. Thus,

**4. Sheep**

whole ovary cryopreservation.

gestational age of about 144 days.

ovarian tissue were placed in a freezer at -20ºC for 30 min followed by plunging in LN vapor for 30 min and then stored in LN. The results of the experiment showed similar density of primordial follicles of the 5x1x1 mm group as compared to the fresh tissue, while lower number of primordial follicles was observed in the small (1x1 mm) frozen-thawed pieces. It was not further discussed why the larger pieces were more resistant to cryoinjury.

In one study, using porcine ovarian cortex samples, programmed slow-freezing was performed with four different CPAs (glycerol (GLY)-10%; DMSO-1.5 M, EG-1.5 M, PROH-1.5 M) (Borges et al., 2009). The ovarian cortex pieces were incubated after thawing, and histological analysis, including light microscopy and TEM, demonstrated that the follicular viability was decreased after freezing with better results obtained by DMSO and EG as compared to PROH and GLY. This result correlates to that pregnancies in the human so far only have been demonstrated after ovarian cryopreservation in either DMSO or EG as CPAs (Donnez et al., 2004; Andersen et al., 2008). However, it should be emphasized that species differences exist in this regard, as demonstrated in a comparative study between ovarian cortex of human, bovine and porcine ovarian tissue (Gandolfi et al., 2006). In that study, Gandolfi and colleagues study showed that DMSO and PROH were equally effective to protect primordial and primary follicles of the pig ovary to cryoinjury and the pig ovarian tissue was also more resistant to cryoinjury as compared to the bovine and the human ovarian tissue.

The pig model was also used to study vitrification procedures (Gandolfi et al., 2006; Moniruzzaman et al., 2009). In one study, ovarian strips from 15-day old pigs, were vitrified using 15% EG, 15% DMSO and 20% fetal calf serum with addition of either 0 M, 0.25 M or 0.5 M sucrose (Moniruzzaman et al., 2009). Histological evaluation after warming showed higher percentage of healthy primordial follicles when CPA solution with 0.25 M sucrose was applied as compared to 0 M and 0.5 M solution. Moreover, the higher oocyte shrinkage was observed with the sucrose supplementation of 0.5 M and these results can be explained by unsatisfactory cell dehydration without sucrose as well as to excessive dehydration at 0.5 M sucrose. In one study, xenografting of vitrified ovarian tissues under the kidney capsule of nude mice was performed (Moniruzzaman et al., 2009). Histological evaluation, two months after grafting, revealed decrease of the primordial follicle density by around 20% in both fresh and vitrified grafts, but in the vitrified grafts, the follicles did not developed beyond the secondary stage. Hence, it may be that follicular development is disturbed after vitrification or that a follicular stage dependant developmental blockage is incurred by cryoinjury. Another study using pig ovarian cortex demonstrated low survival rate of primordial follicles with either EG or a combination of EG and DMSO as CPA (Gandolfi et al., 2006)

The pig ovary has been used in one study of whole ovary cryopreservation (Imhof et al., 2004). The ovary was perfused through the ovarian artery with 1.5 M DMSO and cryopreserved with a slow freezing protocol. Light microscopy evaluation after thawing at 25ºC demonstrated a lower proportion of viable primordial follicles in the frozen-thawed ovaries (84%) as compared to fresh controls (98%). Furthermore, about 20% of healthy primordial follicles were seen when the ovaries were positioned directly into LN without previously perfusion with CPA. Noteworthy is that TEM did not demonstrate any major cellular difference between the fresh and frozen-thawed tissue.
