Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, Milan, Italy
Cryobanking of female gametes has important potential applications in carnivores. The availability of cryostored oocytes or gonadal tissue, as reservoir of a large number of oocytes, would greatly improve assisted reproductive techniques aimed at ensuring the future survival of high genetic value individuals.
Long-term storage of gametes would allow the planning of in vitro embryo production and transfer at proper time, that is, whenever male gametes and embryo recipients are available. It would also maintain genetic diversity that would otherwise be lost when an animal dies or is gonadectomized.
This is particularly crucial in wild and rare species threatened by extinction or in domestic valuable breeds.
Oocytes can be either cryopreserved at GV stage and then matured and fertilized in vitro after thawing, or at the Metaphase II (MII) stage (mature oocytes) and then fertilized in vitro after thawing.
Ovarian tissue, retrieved from isolated ovaries by sectioning ovarian cortex into small fragments, represents a large source of oocytes for in vitro maturation (IVM) and fertilization (IVF). It is rich in primordial and primary follicles that can be transplanted to obtain follicular development and matured oocytes for IVF procedures. Yet, primordial and primary follicles can be isolated from cryopreserved ovarian tissue and cultured in vitro, although in vitro development of ovarian follicles from primordial to pre-ovulatory stage is still challenging (for review, Santos et al. 2010). A population of antral follicles already exists in the ovarian tissue and healthy immature oocytes can be retrieved after cryopreservation and matured in vitro for IVF. This approach might increase the number of competent oocytes for in vitro embryo production other than being an alternative to oocyte cryopreservation.
Despite oocytes and tissue are very sensitive to chilling and optimal cryopreservation procedures are not yet been established, significant advances have recently been achieved, particularly in feline species.
Technical Aspects of Oocyte and Ovarian Tissue Cryopreservation
Cryopreservation procedures must be defined on the basis of physical characteristics of the cells: "large cells," as oocytes, have different cryotolerance than "small cells," as spermatozoa that are successfully frozen. Oocytes have a lower surface to volume ratio compared to spermatozoa and require a longer time to reach osmotic balance with the cryoprotectant (CPA) solution. They are surrounded by a proteinaceous coat (zona pellucida); thus, their permeability is very different from that of spermatozoa in which a plasmatic membrane only is present around the cell.
Studies on mammalian oocytes revealed that they are particularly vulnerable to cold injuries (for review, Saragusty and Arav 2011), but cryopreservation of ovarian tissue is even more demanding than that of oocytes for the presence of several different cellular types with a different cryotolerance.
Two main methods for cryopreserving oocytes and ovarian tissue have been proposed: slow freezing and vitrification.
This method has been derived from protocols used for embryo freezing and it is based on water crystallization. Samples are exposed to hypertonic solutions of cryoprotectants (CPAs) that prevent intracellular ice crystal formation through dehydration. Among permeating CPAs, dimethylsulphoxide (DMSO), 1,2-propanediol (PROH) and ethylene glycol (EG) are the most widely used for cryopreservation. Oocytes loaded into straws (0.25 ml) and tissue fragments in cryovials are slow cooled with the aid of a programmable freezing unit to an intermediate temperature before they are plunged into liquid nitrogen for storage.
Frozen samples must be warmed rapidly to allow a very rapid dispersion of intracellular ice crystals. Then, the cryoprotectant is removed by washing the samples in medium with a decreasing concentration of CPAs. The presence in the medium of non-permeating CPAs, as sucrose, reduces the osmotic shock and controls the water afflux inside the cell reducing the risk of swelling.
Increasing application of vitrification to mammalian gametes is due to the simplicity of the procedure and the satisfying results obtained in cell survival.
It consists in a formation of a glassy vitrified state of the samples without ice crystallization. To obtain this amorphous state, highly viscous CPA solutions in small volumes are rapidly cooled by direct immersion into liquid nitrogen. Viscosity of the vitrification solution is obtained with high CPA concentrations or a combination of relatively low concentrations of different CPAs. The small sample volume has been achieved with different carrier systems (Saragusty and Arav 2011). Among these, Cryoloop consists in a nylon loop mounted on a stainless steel pipe; samples are placed on the loop that had been coated with a thin film of CPA solution. Cryotop is a fine polypropylene strip attached to a plastic handle; samples are placed on the end of the strip in a minimal volume of medium droplet. Open Pulled Straws (OPS) have a diameter of approximately half size than that of regular straws and their end is open. The capillary effect exerted by touching with the open end a very small droplet of CPA solution containing the oocytes allows the loading of the cells into the OPS. All these carriers are then directly immersed into liquid nitrogen. Before use, samples are rapidly warmed and washed in decreasing concentration of CPAs.
Cryopreservation of Feline and Canine Oocytes
Our first report showed that immature feline oocytes were able to survive as, after thawing and culture, they matured in vitro. However, development in vitro after fertilization was enhanced when mature oocytes were slow frozen. Following these demonstrations, cryosurvival of feline oocytes has been further reported (Luvoni, 2006).
Vitrification in straws, OPS and Cryoloop resulted in embryo development after warming and IVF of mature (Murakami et al. 2004; Merlo et al. 2008) and immature oocytes (Comizzoli et al. 2009; Cocchia et al. 2010). The first pregnancy was established in 2011 in one recipient receiving embryos derived from vitrified immature oocytes (Tharasanit et al. 2011). The first kittens were born last year after Cryotop vitrification of matured oocytes, ICSI (Intracytoplasmic Sperm Injection) and transfer of derived-embryos into recipients (Pope et al. 2012).
The low efficiency of in vitro embryo production in canine species has limited the studies on oocyte cryopreservation. However, vitrification with Cryotop or OPS of dog oocytes has been recently reported. Results showed that immature canine oocytes maintained their integrity (Abe et al. 2010) and resumed meiosis in vitro (Turathum et al. 2010) after warming. Dog and Mexican wolves (endangered canid) oocytes have also been vitrified with Cryotop method. The results are promising since proportions of viable oocytes after warming ranged between 57% and 61% (Boutelle et al. 2011).
Cryopreservation of Feline and Canine Ovarian Tissue
Cryopreservation of cat ovarian tissue followed by xenotransplantation to immunodeficient (SCID) mice has been documented (Bosch et al. 2004). Although only few follicles survived after freezing and transplantation, some retained the ability to resume growth from early to more advanced stages. We firstly demonstrated that feline immature oocytes retrieved from cryopreserved ovarian tissue maintain the capability of resuming meiosis after warming (Luvoni et al. 2012). Recent findings on comparative cryosurvival of oocytes vitrified as isolated or enclosed in the ovarian tissue demonstrated that different procedures of vitrification preserve the viability of feline oocytes at high extent (Alves et al. 2012).
Vitrification of canine ovarian fragments resulted in the preservation of tissue normal histology, although after xenotransplantation into mice ovarian bursa, antral follicular development did not occur (Ishijima et al. 2006). Further research demonstrated the beneficial effect of erythropoietin administered to the host mice in enhancing the survival of the follicles of transplanted cryopreserved ovaries (Suzuki et al. 2008). Recent results showed that canine ovarian tissue can be successfully preserved by slow freezing protocol as follicular growth after xenotransplantation in SCID mice has been obtained (Commin et al. 2012).
Cryobanking of oocytes and ovarian tissue in cats and dogs would allow the long-term preservation of precious germplasm. The establishment of cryobanks would be very helpful for improving assisted reproductive programs aimed to the preservation of biodiversity. Cryopreservation protocols must be adequate to each different type of cell in order to maintain their viability and limit damages occurring with the exposure to non-physiological conditions, as sub-zero temperatures. Protocols of cryopreservation are still far from being standardized and results, although encouraging, are still variable. Nowadays it is still premature to purpose to owners and breeders this technique, but recent findings demonstrated that this possibility is not remote.
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