**2. Development of optimized methods for the cryopreservation of the ovarian tissue in domestic mammalian species**

The most common cryopreservation method is the slow freezing procedure, consisting of an initial slow, controlled-rate cooling to subzero temperatures followed by rapid cooling as the sample is plunged into liquid nitrogen for storage (−196°C). At such a low temperature, biological activity is effectively stopped, and the cells functional status may be preserved for centuries. However, several physical stresses damage the cells at these low temperatures. Intracellular ice formation is one the largest contributors to cell death; therefore, freezing protocols use a combination of dehydration, freezing point depression, supercooling, and intracellular vitrification in an attempt to avoid cell damage.

Currently used ovarian cortex cryopreservation protocols have been direct, or slight modifications of the methods developed for isolated oocytes and embryos. There were primarily developed by trial and error adjustments of cooling and warming rates, and choice of CPA and CPA concentrations. However, because there are a large number of protocol variables potentially affecting cell viability, an exhaustive experimental search for the optimal combination of these parameters has long been considered to be prohibitively expensive in terms of time and resources.

#### **2.1 Chemical and physical parameters affecting equilibration and freezing processes of ovarian tissue in mammalian species**

The result of a cryopreservation process is influenced by several chemophysical parameters affecting directly or not the functions and the integrity of the ovarian cells along the freezing process, from the equilibration to the thawing. Among these parameters, the method of equilibration, the freezing rate, the composition of the freezing solution and notably the nature of the permeating CPAs and the non-permeating CPAs, the concentration of each CPA, the use of serum, or the rate of thawing may be investigated to know the relative influence of each of them and the induced cell injuries.

In general, we can expect coupled flows of water and CPAs when CPAs are added, during freezing, thawing and when CPAs are removed from the cells, resulting in a series of

New Approaches of Ovarian Tissue Cryopreservation from Domestic Animal Species 211

Moreover, while the approach is sequential in nature, it is potentially increasing in complexity as the knowledge and understanding of the application and domain evolves. Design of experiments techniques provides a systematic, effective and efficient approach to the investigation of a phenomenon. The main advantages of this strategy were the saving in times and resources expended compared to other approaches and the resulting mathematical models that helps us to better understand the phenomena under investigation more fully. To analyze the response of ovarian cortices from different species to the freeze/thaw process, the authors decided to use fractional (2*n-p*) experimental designs

Using this statistical tool, the authors used only a fraction of the runs specified by the full factorial design; which runs to make and which to leave out was one of our subjects of picked. The authors used various strategies that ensure an appropriate choice of the runs. As for an example, fractional experimental designs 2 (5-2) presented in table 1 aim to evaluate the combined effect of five different factors according to two modalities for each of them. For each experimental design, eight combinations of factors were performed. For each of them, the ratio of morphological preservation and the ratio of viability of isolated preantral follicles were recorded. While the designs were similar for each of the species that were

evaluate, the parameters were chosen according to each species (Table 2 to Table 5).

Run I X1 X2 X3 X4 X5 X1.X2 1 +1 -1 -1 -1 +1 -1 +1 2 +1 +1 -1 -1 +1 +1 -1 3 +1 -1 +1 -1 -1 +1 -1 4 +1 +1 +1 -1 -1 -1 +1 5 +1 -1 -1 +1 -1 +1 +1 6 +1 +1 -1 +1 -1 -1 -1 7 +1 -1 +1 +1 +1 -1 -1 8 +1 +1 +1 +1 +1 +1 +1 Table 1. Fractional experimental design 2(5-2) used to evaluate the cryopreservation protocols

Variables Level -1 Level +1

X1: Permeating CPA DMSO PROH

X3: Non permeating CPA trehalose sucrose X4: Freezing rate 0.3°C/min 2°C/min X5: Equilibration 1 step 3 steps

X2: Concentration of permeating CPA 1.5M 2M

Variable

in the doe rabbit, in the queen and in the cow

Table 2. Dependent variable list evaluated in the rabbit doe

(Mechakra et al., 1999).

anisosmotic conditions. During freezing, the cells dehydrate and shrink and remain shrunken during storage, but return to their isosmotic volume upon thawing. Finally, the cells are subjected to potentially lethal swelling upon CPA dilution and removal. During the controlled slow cooling extracellular ice formation is induced (seeding) at a temperature just below the solutions' freezing point, and then the cooling continues at a given rate in the presence of a growing extracellular ice phase, which raises the extracellular solute concentration in the unfrozen fraction and results in water being removed from the cell via exosmosis.

Permeating CPAs, such as glycerol, dimethyl sulfoxide, ethylene glycol or propylene glycol are typically included in the cryoprotective medium, to protect the cells against injury from the high concentrations of electrolytes that develop as water is removed from the solution as ice. During the equilibration step the inner cell water is partly replaced by the permeating CPAs. However, the CPAs can be damaging to the cells, especially when it is used at high concentrations. The toxicity can be reduced by decreasing the time or the temperature of the equilibration step (Karlsson and Toner, 1996). But equilibration at low temperatures requires increasing the exposition time to freezing solution. Furthermore, the CPAs may have dramatic osmotic effects upon the cells during their addition and their removal. Consequently, the use of several steps of increasing concentrations of CPAs during the equilibration allows reducing the osmotic gradient. The cells exposed to such permeating CPAs undergo initial dehydration, followed by rehydration, and potential gross swelling upon removal. This osmotic shock may generate membrane damages by mechanical means and predisposition of the cell to injuries during the other steps of cryopreservation, or even cell death (Mazur and Schneider, 1986). These kinds of damages could be reduced by using cells surfactant such as serum. During the freezing step, the follicular preservation depends on the nature and the concentration of the CPAs.

Control of the cooling and warming rates is also crucial, as the freezing/thawing rates and the temperature of seeding also influence the ice properties. If cells are cooled too rapidly during the controlled slow cooling process, water does not exit the cells fast enough to maintain equilibrium and, therefore, the oocytes and other ovarian cells freeze intracellularly, resulting in death in most cases. If cooling is too slow, the long duration can cause 'solution effects' injury resulting from the high concentration of extra- and intracellular solutes, probably due to the effects of the solutes on the cellular membrane or through osmotic dehydration. During warming the small intracellular ice crystals might subsequently undergo recrystallization, forming bigger ice crystals that rupture the cell membrane, thus leading to fatal damage. Finally, the thawing and the removal of the CPA depend on the temperature and on the presence of non-permeating CPA limiting the osmotic swelling during rinsing.

#### **2.2 Use of fractional experimental design**

The influence of the multiple chemical and physical parameters cannot be exhaustively performed as it would require too much time and resources. Even if the number of factors, *k*, in a design is small, the 2*k* runs specified for a full factorial can quickly become very large. For example, 2*5* = 32 runs is for a two-level, full factorial design with five factors. To this design we would need to add a good number of centerpoint runs and we could thus quickly run up a very large resource requirement for runs with only a modest number of factors.

anisosmotic conditions. During freezing, the cells dehydrate and shrink and remain shrunken during storage, but return to their isosmotic volume upon thawing. Finally, the cells are subjected to potentially lethal swelling upon CPA dilution and removal. During the controlled slow cooling extracellular ice formation is induced (seeding) at a temperature just below the solutions' freezing point, and then the cooling continues at a given rate in the presence of a growing extracellular ice phase, which raises the extracellular solute concentration in the unfrozen fraction and results in water being removed from the cell via

Permeating CPAs, such as glycerol, dimethyl sulfoxide, ethylene glycol or propylene glycol are typically included in the cryoprotective medium, to protect the cells against injury from the high concentrations of electrolytes that develop as water is removed from the solution as ice. During the equilibration step the inner cell water is partly replaced by the permeating CPAs. However, the CPAs can be damaging to the cells, especially when it is used at high concentrations. The toxicity can be reduced by decreasing the time or the temperature of the equilibration step (Karlsson and Toner, 1996). But equilibration at low temperatures requires increasing the exposition time to freezing solution. Furthermore, the CPAs may have dramatic osmotic effects upon the cells during their addition and their removal. Consequently, the use of several steps of increasing concentrations of CPAs during the equilibration allows reducing the osmotic gradient. The cells exposed to such permeating CPAs undergo initial dehydration, followed by rehydration, and potential gross swelling upon removal. This osmotic shock may generate membrane damages by mechanical means and predisposition of the cell to injuries during the other steps of cryopreservation, or even cell death (Mazur and Schneider, 1986). These kinds of damages could be reduced by using cells surfactant such as serum. During the freezing step, the follicular preservation depends

Control of the cooling and warming rates is also crucial, as the freezing/thawing rates and the temperature of seeding also influence the ice properties. If cells are cooled too rapidly during the controlled slow cooling process, water does not exit the cells fast enough to maintain equilibrium and, therefore, the oocytes and other ovarian cells freeze intracellularly, resulting in death in most cases. If cooling is too slow, the long duration can cause 'solution effects' injury resulting from the high concentration of extra- and intracellular solutes, probably due to the effects of the solutes on the cellular membrane or through osmotic dehydration. During warming the small intracellular ice crystals might subsequently undergo recrystallization, forming bigger ice crystals that rupture the cell membrane, thus leading to fatal damage. Finally, the thawing and the removal of the CPA depend on the temperature and on the presence of non-permeating CPA limiting the

The influence of the multiple chemical and physical parameters cannot be exhaustively performed as it would require too much time and resources. Even if the number of factors, *k*, in a design is small, the 2*k* runs specified for a full factorial can quickly become very large. For example, 2*5* = 32 runs is for a two-level, full factorial design with five factors. To this design we would need to add a good number of centerpoint runs and we could thus quickly run up a very large resource requirement for runs with only a modest number of factors.

exosmosis.

on the nature and the concentration of the CPAs.

osmotic swelling during rinsing.

**2.2 Use of fractional experimental design** 

Moreover, while the approach is sequential in nature, it is potentially increasing in complexity as the knowledge and understanding of the application and domain evolves. Design of experiments techniques provides a systematic, effective and efficient approach to the investigation of a phenomenon. The main advantages of this strategy were the saving in times and resources expended compared to other approaches and the resulting mathematical models that helps us to better understand the phenomena under investigation more fully. To analyze the response of ovarian cortices from different species to the freeze/thaw process, the authors decided to use fractional (2*n-p*) experimental designs (Mechakra et al., 1999).

Using this statistical tool, the authors used only a fraction of the runs specified by the full factorial design; which runs to make and which to leave out was one of our subjects of picked. The authors used various strategies that ensure an appropriate choice of the runs. As for an example, fractional experimental designs 2 (5-2) presented in table 1 aim to evaluate the combined effect of five different factors according to two modalities for each of them. For each experimental design, eight combinations of factors were performed. For each of them, the ratio of morphological preservation and the ratio of viability of isolated preantral follicles were recorded. While the designs were similar for each of the species that were evaluate, the parameters were chosen according to each species (Table 2 to Table 5).


Table 1. Fractional experimental design 2(5-2) used to evaluate the cryopreservation protocols in the doe rabbit, in the queen and in the cow


Table 2. Dependent variable list evaluated in the rabbit doe

New Approaches of Ovarian Tissue Cryopreservation from Domestic Animal Species 213

cryopreservation, but is presented below as a support that can guide those who work with preserved tissues. The authors decided to assess the quality of the protocols developed in different species using all or part of alternate test presented below. The morphology and the viability of the ovarian follicles were systematically assessed, in combination with the investigation of the ultrastructure of the follicles, and their capacity to resume

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

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

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

shape or with disjoined follicular cells or with swelled follicular cells.

margination of the chromatin), and swollen mitochondria.

**2.3.2 Cytolysis live/dead assay** 

folliculogenesis after graft.

**2.3.1 Morphology and ultrastructure** 

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 estimate the experimental error (*E*).

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:


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

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.

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

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 cryopreservation, but is presented below as a support that can guide those who work with preserved tissues. The authors decided to assess the quality of the protocols developed in different species using all or part of alternate test presented below. The morphology and the viability of the ovarian follicles were systematically assessed, in combination with the investigation of the ultrastructure of the follicles, and their capacity to resume folliculogenesis after graft.
