**4. Acknowledgment**

The results presented in this chapter were partially supported financially by Région Rhône-Alpes and the breeding center (CESECAH) of the French Guide Dog Federation (FFAC). The authors also thank the LLC Hycole (Marcoing, France) for their technical support.

The authors want to thank equally regards the Electronic Microscopy Center of the University of Lyon, the Laboratory of Pathological Anatomy of the Veterinary Campus of Lyon and the Institute Claude Bourgelat for access to their facilities. The authors are also grateful to all veterinarians which allowed the ovary collection.

### **5. References**

228 Current Frontiers in Cryopreservation

Bitch 36.81 Doe rabbit 45.79 Cow 37.64

Then, our team decided to explore two research areas. On the one hand, DSC can compare the quantity of ice formed in two different cryopreservation solutions. Thus, it seems possible to select the most suitable cryopreservation solutions for slow freezing methods. The first results obtained in doe rabbit seem to confirm this hypothesis. In fact between two cryopreservation solutions tested, those for which the quantity of ice formed was the lowest, was also the one with the best biological results. On the other hand, for a given solution, DSC allows the measure of the quantity of ice formed for different freezing kinetics. Consequently, it seems also possible to select the most suitable kinetics according to the cryopreservation solutions.

Another approach to optimize cryopreservation process should be to control the ice crystal growth and shape in order to promote the less deleterious crystallization. It is already assumed that intracellular ice formation is lethal. Nonetheless, the recent observations of rabbit ovarian tissue by cryoscanning electronic microscopy and cryofracture reveal that depending on the cooling rate, the extracellular ice shape is modified. Moreover, according to these results the seeding temperature influences the shape and regularity of the ice crystals resulting in large uniform crystals when seeding was induced close to the solution

Otherwise, a better understanding of intracellular ice formation can also be advantageous to improve cryopreservation processes. Indeed, Han *et al* (2009) investigated the size of intracellular ice crystals in mouse oocytes by cryomicroscopy. They conclude that increasing the concentration of macromolecules in the cells by increasing the extracellular non permeating solute concentration significantly reduced the required permeating CPA concentration for intracellular vitrification. Moreover they also observed that intracellular ice melting point was always lower than extracellular ice. Taken together, this information

Regarding DSC, a recent study showed that it is possible to differentiate the crystallization of intra and extracellular ice depending on freezing kinetics (Seki et al., 2009). Consequently, in addition to the measure of the quantity of crystallized ice, DSC can provide a better

The results presented in this chapter were partially supported financially by Région Rhône-Alpes and the breeding center (CESECAH) of the French Guide Dog Federation (FFAC). The

The authors want to thank equally regards the Electronic Microscopy Center of the University of Lyon, the Laboratory of Pathological Anatomy of the Veterinary Campus of

authors also thank the LLC Hycole (Marcoing, France) for their technical support.

Table 6. Maximal quantity of ice crystallized (Qmax) in solutions used for the

cryopreservation of ovarian tissue of bitch, doe rabbit and cow.

**3.3 Promote the formation of a non-vulnerable extracellular ice** 

can be helpful to optimize the cryopreservation protocols.

melting point (Gosden et al., 2010).

control of ice formation.

**4. Acknowledgment** 

Species Qmax in percentage (w/w) of solution


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**Part 4** 

**Cryopreservation of Aquatic Species** 

