72 Methods of cryopreservation of sperm and oocytes of mammals

Increasing awareness on the reduction of breed diversity has prompted global efforts for conservation of threatened breeds through launching organizations such as Rare Breeds Canada, conferences like the Convention on Biological Diversity, and programs such as the Canadian Animal Genetic Resources. Efforts have focused on conservation of farm animal breeds for several reasons, for example, to keep potentially useful genes and gene combinations, to take advantage of heterosis, and to overcome selection plateaus, as well as for cultural reasons, research, and food security. Maintaining genetic diversity also provides insurance against climate change, disease, changing availability of feedstuffs, social change, selection errors, and unforeseen catastrophic events such as Chernobyl where many local breeds’ diversity became threatened.

The goals of conservation are to keep genetic variation as gene combinations in a reversible form and to keep specific genes of interest such as the Booroola fecundity gene in sheep [11]. In order to achieve these goals several steps are necessary. First an inventory must be taken to assess and monitor the risk status of a breed in an ongoing basis by examining the number of breeding males and females, the overall breed numbers, the number of subpopulations, and the trends in population size. The evaluation of stocks for phenotype and genotype must be done to determine the genetic distance of one group from another and the choice of breeds for conservation [6].

3.1. In Situ Conservation

Ideally, populations should be saved as live animals through in situ conservation programs; however this approach needs extensive infrastructure and management and thus is expensive [6]. Hence, ex situ in vitro strategies have been developed to cryopreserve animal genetic resources in a genome bank that creates a global gene pool to manage the exchange of genetic diversity or regenerate a population decades or centuries later [1, 2]. The Convention on Biological Diversity recommended that ex situ conservation be complementary to in situ conservation for farm animal genetic resources [12].

3.2. Ex Situ Conservation

One of the major issues surrounding genome banks is the amount and type of material that needs to be stored, which is a function of the intended future use of the material [12]. In order to avoid inbreeding, a gene bank of male and female genetics formed from the largest number of individuals would be ideal [13]. Ex situ in vitro conservation programs of livestock genetic resources have focused efforts on cryopreservation of gametes, embryos, and somatic cells as well as testis and ovarian tissues, effectively lengthening the genetic lifespan of individuals in a breeding program even after the death

Oocyte Cryopreservation

4.1. Principles

Cryopreservation involves cells or whole tissues preservation by exposure to subzero temperature in LN2(− 1 9 6∘C) [16]. At such a low temperature, biological activity is effectively stopped, and the cells functional status may be preserved for centuries [24]. 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 [25].

It is important to consider the nature and concentration of the cryoprotectant(s) for preservation of germplasm in any cryopreservation protocol. Dimethylsulfoxide (DMSO), ethylene glycol (EG), or glycerol alone or in combination protects the cells and tissues from freezing damage.

4.2. Cryoinjuries

During cryopreservation, the extent of injury incurred in cells largely depends on the size and shape of the cell, the permeability of the membranes, and the quality of the oocyte. However, these factors vary between species, developmental stage, and origin [26]. Although offspring have been born using frozen-thawed oocytes from various species, the ability to support embryo development following cryopreservation procedures is low. This may be attributed to the susceptibility of oocytes to damage during cooling and/or freezing and subsequent thawing because of their complex structure. Unfertilized mammalian oocytes are much larger therefore have small surface:volume ratio [21]. This makes dehydration and penetration of cryoprotectants difficult to achieve, which contributes to the complexity in cryopreservation

4.3. Freezing Procedures

Currently, two main methods for cryopreservation of oocytes are slow freezing and vitrification. With the exception of cryoprotectants concentration and cooling rate, these two methods differ slightly with regards to storage, warming, and rehydration [26]. Table 2 compares conventional freezing and vitrification methods for oocyte and embryo cryopreservation [3].

Conventional slow freezing was introduced first and is currently the gold standard for cryopreservation of embryos. This method typically involves the use of a single cryoprotectant in low concentrations (approximately 1 to 2 M) to minimize chemical and osmotic toxicity and attempts to maintain a balance between the various factors that influence cell damage [3].

4.4. Cryoprotectants

Cryopreservation strategies are based on two main principles: cryoprotectants and cooling-warming rates [26]. Because water is not very viscous, it can only be vitrified by extremely rapid cooling of a small sample or using high concentrations of cryoprotectants [53]. Cryoprotectants such as glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) are small molecules that penetrate cells and limit the amount of intracellular and extracellular water that converts into ice during cooling. Cryoprotective solutions are typically prepared in buffered media with a stable pH between 7.2 and 7.4 [54]. Cryoprotectants are compounds used in cryoprotective solutions to achieve cellular dehydration and to avoid intracellular ice crystal formation upon freezing. Typically combinations of cell permeating and nonpermeating cryoprotectants are used. Low molecular weight permeating cryoprotectants such as glycerol, EG, and DMSO are small molecules that enter the cell, form hydrogen bonds with intracellular water molecules, and lower the freezing temperature, preventing crystallization.

4.5. Cooling Rate

It has been reported that a cooling rate of approximately 2 5 0 0∘C/min can achieve a vitrified state [54]. However, using a suitable carrier system such as the open pulled straw, a cooling rate of 2 0 0 0 0∘C/min can be achieved [50]. The main reasons for increasing cooling and warming rates are to avoid chilling injury and to decrease the concentration of cryoprotectants in solution [53]. By passing cells through the critical temperature zone (15 to − 5∘C) quickly, water moves out of the cells and freezes extracellularly [20]. This prevents chilling injury to the intracellular lipid droplets, lipid containing membranes, and the cytoskeleton [30].

warming, and the small volume of solution prevents heterogeneous ice formation [30].

73 Basic properties of cryoprotectors and their components A cryoprotectant is a substance that is used to protect biological tissue from freezing damage (i.e. that due to ice formation). Without protection, cells will rupture when they freeze as a result of expanding water, causing severe injury or death to living organisms, and ruining tissue samples or frozen food products. The compound can work in a number of different ways. A common approach is to lower the freezing point, keeping the tissue flexible at temperatures that would normally result in freezing. Others bond to specific molecules to help tissue retain its structure under the intense pressures of cold temperatures. Cryoprotectants must (1) easily penetrate cells, and (2) not be toxic to the cell. Conventional cryoprotectants are glycols (alcohols containing at least two hydroxyl groups), such as ethylene glycol[citation needed], propylene glycol, and glycerol. Ethylene glycol is commonly used as automobile antifreeze, and propylene glycol has been used to reduce ice formation in ice cream. Dimethyl sulfoxide (DMSO) is also regarded as a conventional cryoprotectant. Glycerol and DMSO have been used for decades by cryobiologists to reduce ice formation in sperm and embryos that are cold-preserved in liquid nitrogen. Mixtures of cryoprotectants have less toxicity and are more effective than single-agent cryoprotectants. A mixture of formamide with DMSO (dimethyl sulfoxide), propylene glycol, and a colloid was for many years, the most effective of all artificially created cryoprotectants. Cryoprotectant mixtures have been used for vitrification (i.e. solidification without crystal ice formation). Vitrification has important applications in preserving embryos, biological tissues, and organs for transplant. Vitrification is also used in cryonics in an effort to eliminate freezing damage. Some cryoprotectants function by lowering the glass transition temperature of a solution or of a material. In this way, the cryoprotectant prevents actual freezing, and the solution maintains some flexibility in a glassy phase. Many cryoprotectants also function by forming hydrogen bonds with biological molecules as water molecules are displaced. Hydrogen bonding in aqueous solutions is important for proper protein and DNA function. Thus, as the cryoprotectant replaces the water molecules, the biological material retains its native physiological structure and function, although they are no longer immersed in an aqueous environment. This preservation strategy is most often utilized in anhydrobiosis.