**6. Conclusions**

198 Current Frontiers in Cryopreservation

in control group were frozen without CPA. Interestingly, steroid production after in vitro perfusion (Fig. 2) and cell culture was also demonstrated in control group which can be explained by adequate equilibration between cell dehydration and extracellular ice

As discussed above, cryopreservation injury may not only occur within the follicles but also in the vascular bed of the ovary or in the large vessels. It was hypothesized that perfusion with the anti-apoptopic agent sphingosine-1-phosphate (S-1-P) before cryopreservationperfusion by 1.5 M DMSO (Onions et al., 2008) may protect the tissue and diminish the cryoinjury. Histological examination after thawing showed arterial endothelial disruption of the vascular pedicle tissue in the cryopreserved ovaries, with the most extensive injury in the area where the cannula had been placed, followed by the hilus region and with less extensive injury on the venous side. No protective effect of the addition of sphingosine-1 phosphate could be demonstrated. In summary, this study was able to demonstrate the vulnerable state of the vasculature in whole ovary cryopreservation especially on the arterial side. It was also shown by proliferation and apoptosis markers that granulosa cells of antral

A number of non-human primate species have been used in research involving reproductive physiology and development of methods later used clinically in reproductive medicine. The benefit of these experimental animals is that the physiology and anatomy are similar to the human (Stevens, 1997; Weinbauer et al., 2008), although the ovarian size of the most studied cynomolgus monkey is considerably smaller (0.258+/- 0.159 cm3) (Jones et al., 2010) as compared to the human. Even if a lot of procedures are introduced in the human without

formation. However, a clear benefit of DMSO presence was seen.

Fig. 2. Photograph of the in vitro perfusion apparatus

follicles remain viable after cryopreservation.

**5. Non-human primates** 

Regardless pioneering success and reporting pregnancies after cryopreservation and avascular transplantation of ovarian tissue in human (Donnez et al., 2011) the future course regarding cryopreservation of ovarian tissue should be focused on further research using

Review on Ovarian Cryopreservation in Large Animals and Non-Human Primates 201

Celestino, J. J., R. R. Dos Santos, C. A. Lopes, F. S. Martins, M. H. Matos, M. A. Melo, S. N.

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Demirci, B., J. Lornage, B. Salle, L. Frappart, M. Franck&J. F. Guerin. (2001). Follicular

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on the histological appearance of human ovarian cortex following cryopreservation

animal models. The size of ovarian cortex pieces, whole ovary freezing or not, choice and exposure time to CPA, stepwise or direct addition of CPA are some of the unresolved questions. The difference between species, complexity of ovarian tissue and uneven distribution of primordial follicles should be taken into consideration when creating an optimal study design. Vitrification seems promising, but differences between the protocols used make the interpretation of the data difficult. However, further research on animal models should lead to better understanding and improvement of cryopreservation techniques and then to higher efficiency, when used as a clinical procedure.

#### **7. References**


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**11**

*France* 

**New Approaches of Ovarian** 

**Domestic Animal Species** 

Vanessa Neto1 et al.\*

**Tissue Cryopreservation from** 

*Université de Lyon, VetAgro Sup – Veterinary Campus of Lyon, UPSP ICE* 

Cryopreservation has been attempted for most of the developmental stages of both male and female reproductive cells, ranging from the immature gametes residing in ovarian or

However, each variant of the reproductive cells has introduced their own problems, and it has been realized that many aspects of the particular physiology of the cells will dictate how they respond to cryopreservation. Both male and female gametes have acquired highly specialized structural components (essential to fertilization and development) that may

Semen is one of the most practical means of storing germplasm due to its abundant availability and ease of application (Holt and Pickard, 1999; FAO, 2007). Stored spermatozoa could be introduced back into existing populations either immediately or decades or centuries afterwards. In this way, cryopreservation of spermatozoa associated with artificial insemination (AI) or *in vitro* fertilization (IVF) facilitates the management of domestic animals herds, especially cow dairy herds where it is now used since 60 years. Cryopreservation better allows the use of semen from genetically superior males of threatened livestock breeds and has the potential to protect existing diversity and maintain heterozygosity while minimizing the movement of living animals and the transmission of

Spermatozoon is a very small cell containing low amounts of cytoplasm and consequently low quantity of water. Furthermore sperm nuclear material is compacted and protected

respond to the freezing process in ways different from that of basic cell structures.

venereal diseases (Johnston and Lacy, 1995; Andrabi and Maxwell, 2007).

Thierry Joly1, Loris Commin1, Pierre Bruyère1, Anne Baudot2,

*1Université de Lyon, VetAgro Sup – Veterinary Campus of Lyon, UPSP ICE 'Interactions between Cells and their Environment', Team Cryobio, France 2Université Paris Descartes, Sorbonne Paris Cité, UFR Biomedical, France*

Gérard Louis2, Pierre Guérin1 and Samuel Buff1

testicular tissues through the mature oocytes and spermatozoa.

**1.1 Cryopreservation of spermatozoa** 

**1. Introduction** 

 \* *'Interactions between Cells and their Environment', Team Cryobio* 

