**5.1 Chilling injury**

Chilling injuries refer to the irreversible changes that occur to the intracellular lipid droplets, lipid-containing membranes, and the cytoskeleton, during the cooling phase between +15 and −5°C [32]. Such injuries are commonly associated with the slow freezing technique. In contrast, the vitrification method substantially reduces the chances of chilling injuries to the frozen oocytes and embryos as they are exposed very briefly to the dangerous temperature zone due to high cooling rate [33]. Therefore, effective cryopreservation of porcine embryos containing extremely large amounts of chill-sensitive lipid droplets can be achieved only through vitrification [34]. Similarly, high survivability of cryopreserved oocytes of various other species such as cattle, sheep, and horse that are sensitive to chilling could be achieved through vitrification [35].

## **5.2 Formation of ice crystal**

The formation of ice crystals during cryopreservation is the major source of cryoinjury [36]. The slow freezing method induces ice crystal formation in the aqueous phase surrounding cells as well as inside the cells including the cytoplasm and nucleus at the temperature zone between −5 and −80°C. In contrast, high CPA concentration and rapid cooling rate of vitrification method allow solidification of intracellular and extracellular water into a glass-like state bypassing the formation of ice crystals.

#### **5.3 Fracture damage**

Fracture damages to the zona pellucida and blastomeres of oocytes and embryos are commonly observed following cryopreservation. Such damages usually occur during freezing because of the mechanical effect of the solidified solution at the temperature zone between −50 and −150°C [37].

#### **5.4 Formation of multiple asters**

 Aster formation is a newly discovered form of cryoinjury. It is frequently observed in the vitrified-warmed and fertilized oocytes [38]. This cryoinjury is likely accountable for the loss of ooplasmic function responsible for normal microtubule assembly. The exposure of oocytes to high CPA concentration and ultrarapid cooling during vitrification leads to the formation of multiple asters near the male pronucleus. The migration and development of pronuclei are disrupted by the asters resulting in delayed first cleavage and reduced blastocyst development [38].

#### **5.5 Osmotic stress**

 During the pre-freezing stage of cryopreservation, incubation of cells with high osmolar cryoprotectant solution causes cell shrinkage due to the outward movement of intracellular water in response to the difference in osmotic pressure between intracellular and extracellular solutions. Similarly, at the stage of thawing and CPA removal, the movement of water molecules occurs at the reverse direction that causes cell swelling. These phenomena are known as osmotic stress. The frozen cells are more permeable to water than cryoprotectants as compared to their fresh counterpart [39]. Therefore, the cryopreserved cells are more susceptible to osmotic stress as compared to the non-cryopreserved cells. The vitrification method employs considerably high concentration of CPAs and therefore induces greater osmotic stress as compared to the slow freezing technique. It may be noted that the required CPA concentration for vitrification is inversely related with the cooling rate. Therefore, a practical approach to reduce osmotic stress and CPA-mediated cell toxicity during vitrification is to increase the cooling rate and simultaneously reduce the concentration of CPAs.

#### **6. Deleterious effects of cryopreservation**

Cryopreservation of oocytes and embryos is associated with several deleterious consequences that in turn exert negative effects on their post freeze-thaw survivability and development.

Osmotic shock during cryopreservation and thawing may result in excessive shrinkage or swelling of cells that can damage the cellular cytoskeleton and in turn

#### *Cryopreservation of Oocytes and Embryos: Current Status and Opportunities DOI: http://dx.doi.org/10.5772/intechopen.81653*

the post freeze-thaw survivability and developmental ability of the cryopreserved cells. Similarly, the formation of intracellular ice crystals during freezing may damage the cellular cytoskeleton and cell organelles.

Mitochondria are the most abundant organelles in mammalian oocytes and embryos and they are the sole source of energy production. Mitochondrial dysfunction or abnormalities are critical for the development of oocytes and embryos. A reduction in the production of ATP by mitochondria is associated with the developmental failure of oocytes and embryos [40]. Cryopreservation may contribute to mitochondrial dysfunction, mitochondrial swelling [41, 42], abnormally shaped mitochondria, rupture of mitochondrial membranes [43, 44], and reduced cellular ATP content that might contribute to poor oocyte and embryo development following freeze-thawing [45, 46].

It is evident that cryopreservation incurs negative effect on the expression of genes associated with oxidative stress, apoptosis, cell developmental process, and sperm-oocyte interaction [31, 47]. Such alteration in gene expression is one of the contributory factors of cryopreservation toward poor developmental ability of cryopreserved oocytes and embryos.

 Cryopreservation can be a potential cause of physical damage to DNA. The fragmentation of DNA increases in mouse and bovine oocytes following vitrification [48, 49]. It is suggested that slow freezing as well as vitrification affect the DNA integrity in embryos [50]. Further, cryoprotectants such as ethylene glycol and propanediol increase DNA fragmentation in porcine embryos, even without a cycle of freezing and thawing [51].

Cryopreservation may induce epigenetic changes in the genome of cryopreserved oocyte and embryos. Vitrification reduces or increases gene methylation in bovine and mouse oocytes and embryos [52–55]. Further, several reports indicate that vitrification significantly alters acetylation patterns in oocytes [56, 57]. It is suggested that the aberrant epigenetic modifications in response to cryopreservation are at least partially responsible for the reduced developmental competence of frozen oocytes and embryos [31].

### **7. Difficulties associated with oocyte cryopreservation**

 The cryopreservation of oocyte is more challenging than that of the embryos. As compared to an embryo, an oocyte has to maintain integrity of many of its unique structural features following freeze-thawing to undergo fertilization and further development. Oocyte being a single cell is more vulnerable to the steps of cryopreservation as compared to a multi-cellular preimplantation embryo. The larger volume of oocyte decreases the surface-to-volume ratio that makes it very sensitive to chilling and intracellular ice formation [58, 59]. The plasma membrane of matured oocytes has a low permeability coefficient, thus making the movement of cryoprotectants and water slower [60].

 In oocytes, the meiotic spindles play crucial roles in meiotic progression as well as chromosomal alignment and segregation [61]. Severe disorganization or disappearance of meiotic spindles is evident following slow freezing as well as vitrification with a more deleterious effect of the slow freezing procedure [31]. Cryopreservation exerts a negative influence on microfilament functions in oocytes that in turn can lead to abnormal distributions of mitochondria in the oolemma [6, 62, 63]. This consequently may result in reduced meiotic competence and fertilization ability of oocytes and developmental failure of early stage embryos.

During cryopreservation, CPA causes transient increase in the intracellular concentration of calcium in oocytes [64] that triggers exocytosis of cortical

granule [65] resulting in hardening of zona pellucida and in turn compromised sperm penetration and fertilization [66].

## **8. Future perspectives**

 The procedures of oocyte and embryo cryopreservation have evolved significantly since it was demonstrated for the first time five decades ago. Nevertheless, the success of oocyte cryopreservation is considerably poor as compared to that of the embryos at late developmental stage, even following the ultrarapid vitrification, which is considered as the best technique at present. Therefore, currently, the most important challenge in this field is to develop standardized protocols for effective cryopreservation of oocytes and early stage embryos. The theoretical target of success of such protocols should be comparable with that of their non-cryopreserved counterpart. It is evident from the current state of knowledge that the ability of oocytes and embryos to withstand cryopreservation process varies among the different species. It appears impossible to develop a single standardized protocol for all species. Therefore, future efforts should focus on developing species-specific optimized protocols for oocyte and embryo cryopreservation. Further, it will be fascinating to observe future efforts for the development of automated devices for oocyte and embryo vitrification. The implementation of an efficient and automated ultrarapid vitrification system for routine use in livestock can revolutionize the field worldwide. Conclusively, the most prominent future targets of cryopreservation are expected to focus on the development of protocols that would maintain as much as possible the structural and functional integrities of oocytes and embryos following freeze-thawing. The outcome of such protocols should be reproducible as well across the laboratories worldwide. Realization of such targets would definitely lead to the development of standardized and optimized methods for oocyte and embryo cryopreservation for routine use in livestock.
