**1. Introduction**

The extent of metabolic processes and cellular functions of living cells are reduced dramatically in response to low temperature [1]. Further, at ultralow temperature, the biochemical and metabolic activities of living cells are virtually stopped and they enter into a suspended state of animation. Nevertheless, the exposure of living cells to ultralow temperature induces complex changes to the cells that are associated with its altered physical structure and biophysical processes. These facts indicate that living cells can be preserved at ultralow temperature for a long time. However, the preserved cells will be able to resume their normal physiological functions following recovery, if their physical structure and vitality are protected during the process and period of preservation. The methods for preserving living cells at ultralow temperature essentially employ these principles and the process is known as cryopreservation. It is the technique for preserving living cells or tissues at ultralow temperature, typically in liquid nitrogen (−196°C), at genetically and physiologically stabilized state.

#### *Infertility, Assisted Reproductive Technologies and Hormone Assays*


#### **Table 1.**

*First reported birth from the cryopreserved oocytes and embryos in different livestock species.* 

Preservation of oocytes and embryos is an integral part of the assisted reproductive technologies. It allows not only preserving the valuable female germplasm but also the rapid induction of genetic merits into population through in vitro fertilization and embryo transfer. The mammalian embryos could be cryopreserved successfully for the first time in 1972. It was shown that 50–70% of the early stage mouse embryos survived freezing to −196°C that required slow cooling and slow warming [2]. Subsequently, considerable efforts have been made until now to cryopreserve oocytes and embryos in different mammalian species including livestock (**Table 1**). The field of gamete cryobiology has undergone a tremendous advancement during the last five decades.

Cryopreservation of mammalian gamete has many potential applications. Cryobanking of oocytes and embryos derived from genetically superior animals is promising for enhancing the outcome of planned breeding programs. The technique is equally important for conserving biodiversity of endangered animal species and, valuable and genetically modified laboratory animals. It also ensures steady supply of oocytes and embryos for many downstream applications of assisted reproduction such as in vitro embryo production, embryo transfer, production of stem cells, and genetic engineering.

The underlying effects of cryopreservation on mammalian oocytes and embryos have been studied extensively by the scholars worldwide. Several methods have been demonstrated for cryopreserving oocytes and embryos in different species and some of these methods are real breakthroughs. Currently, devices and consumables required for cryopreservation are available commercially from many firms that transform the procedure into a routine practice in humans as well as in livestock. In this chapter, the fundamental aspects of oocyte and embryo cryopreservation are discussed in detail.

## **2. Principles of cryopreservation**

The process of cryopreservation exposes the cells to very low temperatures for preserving their structural and functional entity for a long period of time. As such, the freezing of cells results in the formation of both intracellular and extracellular sharp ice crystals that damage the cellular membranes and organelles and render the cells nonviable. Further, the formation of ice crystals causes osmotic stress to the cells that result from the altered concentration of intracellular solutes. Therefore, any cryopreservation protocol fundamentally includes steps that prevent and ameliorate such damages to cells during freezing. These damages are avoided by controlling the temperature during the freezing process and by incubating cells to cryoprotective solution [15]. A rapid freezing process helps avoiding the mechanical damage caused by the piercing action of ice crystals, and the rise in intracellular solute concentration

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

can be avoided by exposing cells to cryoprotectants [15]. Permeating cryoprotective agent (CPA) decreases ice formation by replacing the intracellular liquid [16].

 Irrespective of the methods, cryopreservation of oocytes and embryos basically includes four steps (**Figure 1**). Step 1: cells are equilibrated in a CPA solution that causes water egress from the cells and their dehydration. Replacement of intracellular water with permeable CPA lowers the freezing point of the intracellular content and reduces the extent of intracellular ice crystal formation. Step 2: equilibrated cells are cooled to low temperature and then stored in liquid nitrogen (−196°C). The cells are actually frozen during this step and, depending upon the cooling methods, either small intracellular ice crystals are formed (slow cooling) or the intracellular content is transformed into glass-like state bypassing ice crystal formation (vitrification). Step 3: cryopreserved cells are recovered by thawing and warming that reverse the frozen state of the cells. Step 4: finally, the thawed and warmed cells are

**Figure 1.** 

*Steps involved in cryopreservation of oocytes and embryos.* 

equilibrated in the rehydration solution that causes the replacement of intracellular CPA with water molecules. Following this step, the preserved cells regain their vitality and resume normal physiological processes.
