**2.3.1 Morphology and ultrastructure**

212 Current Frontiers in Cryopreservation

These 2*(n-2)* experimental designs allowed discriminating between five factors influencing the cryopreservation process (variables X1 to X5) and the simultaneous interactions between two of them. The linearity (structural and estimated model) of the experimental model was evaluated by an ANOVA test. One randomly chosen assay was replicated three times to

So, eleven experiments were randomly performed. Multi-linear regression was performed using all the variables in order to evaluate experimental results according to this model:

Variables Level -1 Level +1

X5: Freezing rate after -40°C In the freezing chamber Direct immersion in LN2

Variables Level -1 Level +1

X4: Medium Euroflush® Medium with choline

X1: Permeating CPA PROH DMSO

permeating CPA 1.5M 2.5M X3: Non permeating CPA sucrose trehalose

X5: Cell surfactant Fetal calf serum Albumax®

Variables Level -1 Level +1

Results of experimental designs for each species were completed by at least one additional biological evaluation and one quantitative evaluation of normal and viable follicle rates after freezing, according to the best combination of factors chosen with experimental designs.

A survey of nearly all quality assays available to the preservation scientist reveals that they can be grouped into different categories. The following assay tier is not specific to

X1: Permeating CPA DMSO PROH X2: Non permeating CPA trehalose sucrose X3: Freezing rate 0.3°C/min 2°C/min X4: Equilibration 1 step 3 steps

X1: Permeating CPA PROH DMSO X2: add of sucrose no yes X3: Freezing rate 0.5°C/min 2°C/min X4: manual seeding no Yes

*1,2.*X1*.*X2 + E

estimate the experimental error (*E*).

ŷ = *0* + *1.*X1 + *2.*X2 + *3.*X3 + *4.*X4 + *5.*X5 + 

X2: Concentration of

Table 3. Dependent variable list evaluated in the queen

Table 4. Dependent variable list evaluated in the cow

Table 5. Dependent variable list evaluated in the bitch

**2.3 Criteria to assess the quality of frozen-thawed cortices** 

Assessment of the morphology of ovarian cells required the ovarian fragments were fixed in a preliminary defined fixative agent adapted to the species, before being processed for classical light microscopy. Primordial to primary follicles – from oocytes surrounded by flattened granulosa cells until oocytes surrounded by one layer of cuboidal granulosa cells (Gougeon and Chainy, 1987) – were usually classified into four types of morphological defects: *(Type I)* follicle without any morphological defect - follicle is regular, with joined follicular cells; cytoplasm of the oocyte is homogeneous and chromatin is diffused and regular; *(Type II*) follicle with cytoplasmic defect - cytoplasm of the oocyte is vacuolated or eosinophil; *(Type III)* follicles with nuclear defect - nucleus of the oocyte is picnotic, without apparent nuclear membrane or with an irregular nuclear membrane; *(Type IV)* degenerated follicle – oocyte with combined cytoplasmic and nuclear defects or follicle with irregular shape or with disjoined follicular cells or with swelled follicular cells.

The ultrastructure of ovarian follicles was also examined for the presence of apoptotic and para-apoptotic cell death. Ultrastructurally apoptosis is characterized by margination of condensed chromatin, nuclear fragmentation, and the formation of apoptotic bodies. Paraapoptosis, nonclassical apoptosis, is a specific morphologic type of non-necrotic cell death and is characterized by cytoplasmic vacuolization, condensed chromatin (but not early margination of the chromatin), and swollen mitochondria.

### **2.3.2 Cytolysis live/dead assay**

The cytolysis assays have both a very positive and a negative attribute to them. On the positive side, there are a variety of assays that can reveal cell membrane leakage that occurs as a final stage in most forms of cell death. Yet given that cytolysis is the last stage of preservation-induced cell death, these assays do not reveal early-stage mechanisms underlying preservation-induced cell death and thus have limited use in the future as a diagnostic means to develop improved preservation formulations and protocols. The LDH assay continues today to be useful for measuring preservation-induced cytotoxicity. The concept behind this cytolysis assay is simply that if the cell membrane is compromised, then LDH will leak into the extracellular milieu where it can be measured. The trypan blue assay is also one of the most commonly used cytolysis assays. A number of investigators has used the trypan blue exclusion assay in studying preservation efficacy. It does however share the same handicap as the LDH assay given that neither can be analyzed using fluorescence and/or bioluminescence. Currently, the best cytolysis live/dead assays are those that employ fluorescent indicator dyes. Available probes can be subdivided into two different subsets, one of which is trapped by the cell and leaks out only is a membrane rupture occurs. The other subtype, exemplified by ethidium homodimer or propidium iodide, is membrane insoluble and only stains the cell if it gains access through a compromised

New Approaches of Ovarian Tissue Cryopreservation from Domestic Animal Species 215

semen cryopreservation. Finally, the bitch was studied for preserving the reproductive

The experimental variability expressed as the repetition of one single combination, showed that both the structural and the estimated models of the experimental design were valid when considering the morphological preservation ratio of the follicles. The concentration of the permeating CPA (*P* = 0.67) and the number of equilibration steps (*P* = 0.19) seemed to have no significant effect on the morphological preservation ratio of ovarian follicles. The nature of the permeating and non-permeating CPA seemed to influence the morphological preservation ratio of the follicles (*P* = 0.08 and *P* = 0.07 respectively) although the nonsignificant difference. DMSO tended to reduce the morphological preservation ratio, as compared with PROH. Morphological preservation ratio was increased in the presence of trehalose compared with sucrose. The freezing rate seemed to be the factor that had the greatest impact on the morphological preservation ratio of the doe rabbit follicles. At a freezing rate of 0.3°C/min we observed a significant increasing of the follicular morphological preservation ratio, as compared with 2°C/min (*P*<0.01). No significant interaction was observed between the nature of the permeating CPA and its concentration. According to the results of the experimental design, the precise evaluation of the best combination of factors influencing positively the morphological preservation ratio (3 steps equilibration protocol, 1.5M DMSO or 1.5M PROH, medium supplemented with either sucrose or trehalose) was performed. Ovarian pieces were treated according to the results obtained with experimental design. Ovarian cortices were equilibrated (3 steps) in the freezing media based on TCM 199 and 10% FCS, at room temperature. The freezing media was supplemented with 1.5 M DMSO or 1.5M PROH and 0.2 M sucrose or 0.2M trehalose. Freezing of ovarian fragments was slowly performed at 0.3°C/min from the temperature of seeding (-7°C/min) up to -35°C. Thawing, histology, viability tests and electron microscopy evaluation process were

potential of future guide dogs submitted to neutering surgery before training.

performed before and after cryopreservation as described previously.

and trehalose, with a post-seeding freezing rate at 0.3°C/min

A B Fig. 1. Rabbit follicular morphology before (A) and after (B) cryopreservation with PROH

**2.4.1 In the doe rabbit** 

plasma membrane. In this way, the authors did evaluate the viability of the frozen-thawed follicles using Calcein-AM and Ethidium homodimer I stains (Live/Dead Viability/Cytotoxicity Kit, Molecular Probes) on enzymatically isolated follicles (Fig. 2).

#### **2.3.3 In vivo functionality (autografting model) and in vivo growth potential (xenografting model)**

One of the key components of any preservation protocol must be a functional assay that matches the type of cells or tissue being analyzed. In some cases this determination is quite easy. As for example, the sperm motility is a well-accepted functional assay for this system, whereas the capacity to resume folliculogenesis sounds reasonable for the ovarian cortex.

Fresh and cryopreserved rabbit doe ovarian tissues were autografted into young females. Fresh ovarian tissue was grafted on the ipsilateral ovarian pedicle immediately after ovary resection (2 grafts per female), contrary to cryopreserved ovarian tissue grafted on the controlateral ovarian pedicle 24 hours after freezing (one graft per female). Control females were ovariectomised, according to the same surgery resection that used before graft. From height week after graft, grafted rabbit doe were inseminated every 3 weeks in case of negative pregnant diagnosis. Eleven months later, ovarian grafts were removed at necropsy to observe follicular structures by histology.

In the bitch, the growth ability of frozen-thawed ovarian tissue has also been assessed by implantation of small pieces of ovarian tissue into adult female SCID mice. After removing mice ovaries, the canine frozen thawed ovarian tissue was placed intramuscularly in the gluteal superficial muscle. To study the setting up of graft resumption, the graft was harvested at one, eight or 16 weeks and the resumption of the ovarian activity was controlled by vaginal cytology assessment. After harvesting, the grafts were processed for histological assessment.

#### **2.3.4 Vascularization**

In the bitch, the alpha smooth muscle actin (alpha-SMA), a marker of the mature blood vessel, was used to assess the vessel density within the ovarian tissue, using a primary antibody directed against alpha-SMA. For each slice, image analysis was performed under direct light microscope to determine the tissue areas. The stained vessels were counted in several fields of the same slice and in several slices per animal to deduce a blood vessel density.

#### **2.4 Experimental results**

To illustrate the interest of the use of fractional experimental design, the authors decided to present some of their results obtained in the doe rabbit, in the queen, in the bitch and in the cow during the last 6 years. The doe rabbit was used as a model for the human and the animal applications of the ovarian tissue cryopreservation, because of its biological and breeding characteristics. The cat was considered as a model for the ovarian tissue cryopreservation studies of endangered wild felids; from all the felids, the domestic cat is the only non endangered feline species. The cow was used as model for ruminants, with a special interest for preserving high valuable individuals in combination to embryos and to semen cryopreservation. Finally, the bitch was studied for preserving the reproductive potential of future guide dogs submitted to neutering surgery before training.
