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Many biotechnologies are currently used in livestock breeding with the aim of improving reproductive efficiency and increasing the rate of genetic progress in production animals. The term “cryopreservation” refers to methods that allow biological samples to be frozen and then warmed again without losing their vitality. Cryopreservation is a process that freezes and stores fertilized oocytes for later use, such as gametes, embryos, and primordial germ cells; it is a component of assisted reproductive technology. While some procedures still employ slow-freezing methods, the majority now use vitrification, or extremely rapid freezing, for both oocytes and embryos since it reduces the possibility of harm because there is not as much ice crystal formation as there is with slow-freezing methods. Vitrification has proven to be useful in a variety of applications, including the in vitro production (IVP) of embryos in agriculturally significant or endangered animal species, such as pigs, sheep, goats, cattle, etc., after in vitro fertilization (IVF) procedures in human embryology clinics.
Department of Agriculture and Animal Health, College of Agriculture and Environmental Sciences, University of South Africa, South Africa
Vhahangwele Makumbane
Department of Agriculture and Animal Health, College of Agriculture and Environmental Sciences, University of South Africa, South Africa
Lerato Deirdre Sehlabela
Department of Agriculture and Animal Health, College of Agriculture and Environmental Sciences, University of South Africa, South Africa
Zwivhuya Constance Raphalalani
Department of Agriculture and Animal Health, College of Agriculture and Environmental Sciences, University of South Africa, South Africa
North West Department of Agriculture and Rural Development, Animal Science, Potchefstroom, South Africa
Masindi Lottus Mphaphathi
Agricultural Research Council, Animal Production, Germplasm, Conservation and Reproduction Biotechnologies, Pretoria, South Africa
*Address all correspondence to: thivhiricha@gmail.com
1. Introduction
The genetic variety of many domestic livestock breeds is gradually declining; as a result, it is in the global community’s interest to conserve livestock genetics [1]. Ideally, preserving populations as live animals is the preferred method. However, this approach can be costly, and unless the breed serves a productive purpose, its success is unlikely [2]. Hence, ex situ in vitro cryopreservation methods have been devised to freeze animal genetic resources in genome/gene banks for the purpose of restoring a specific population later [3]. Substantial advancements have been achieved in the cryopreservation of semen and embryos in various domestic species. However, oocytes exhibit high sensitivity to chilling temperatures, and as of now, a universally accepted protocol has yet to be developed. The establishment of ova banks for the long-term storage of oocytes enables the preservation of female genetic material in an unfertilized state until a suitable male germplasm is chosen. Additionally, the successful cryopreservation of oocytes not only safeguards genetic material from deceased animals but also facilitates various assisted reproductive technologies [4, 5].
The success of animal oocyte cryopreservation was first reported in the 1980s [6]. Several issues were brought up in the literature regarding the typical development of embryos derived from frozen oocytes. Studies have shown that the development of embryos and fetuses was consistently poor in mice [7, 8, 9, 10, 11, 12], rabbits [13], and hamsters [14]. It is evident that cryopreservation procedures unavoidably cause cellular and molecular alterations, leading to a decrease in fertilization rate and embryo development [15]. Previous researchers indicated that two freezing methods, controlled-rate slow cryopreservation, and vitrification, are utilized in the clinical setting for oocyte cryopreservation in animals [16, 17]. The process of slow freezing mainly involves the use of a low concentration of cryoprotective agent (CPA) and slow cooling/freezing rates to control various factors, such as thermal shock, that can lead to cell damage [18]. By slowly reducing the speed of cooling from above zero to below zero, the CPA enables sufficient cellular dehydration, resulting in minimal intracellular ice formation [19].
Both forms of damage can be prevented, or at the very least lessened, by regulating the temperature reduction process and adjusting the cellular environment [20]. To illustrate, the piercing effect of ice crystals can be prevented by rapidly freezing the substance, while the substantial increase in solute concentration within cells due to ice crystal formation can be mitigated by using cryoprotectants [21]. The use of cryoprotectants such as glycerol, ethanediol, dimethyl sulfoxide, ethylene glycol, and propanediol [22]. Although most cells cannot endure the freezing procedure without the aid of a cryoprotectant, it should be emphasized that solely relying on these solutions is inadequate for cell survival post-freezing. The ability of cells to withstand different stresses induced by physical and physiochemical alterations during the process, along with the rates of cooling and warming, as well as the cell type, also plays a crucial role in their survival [23].
Throughout cryopreservation, cells encounter numerous unfamiliar surroundings, including chemical toxicity, osmotic fluctuations, and low temperatures, all of which have the potential to interfere with cell functions and lead to cell death [24]. Several variables, such as factors in species, the age and fertility of the oocyte donor, the stage of oocyte maturation, and the cryopreservation protocols, have been found to impact the efficacy of oocyte cryopreservation [25]. A large variation in oocyte parameters in particular livestock causes difficulties in obtaining a consensus on freezing protocols. Previous researchers reported that species, like pigs, have been suggested to exhibit a reduced ability to freeze oocytes due to elevated lipid levels [26, 27]. The freezing techniques and results in terms of fertilization rate, embryo development, and pregnancy rate after embryo transfer have shown variation among different species and laboratories. Nevertheless, the lack of significant achievements in oocyte cryopreservation hindered the widespread adoption of this method in routine clinical practice for an extended period [28]. During the 2000s, advancements in cryobiology through vitrification techniques paved the way for the successful cryopreservation of functional oocytes, resulting in significant advancements in oocyte cryopreservation programs within clinical settings [29]. A significant randomized trial on oocyte donation demonstrated that the quality of vitrified oocytes was comparable to fresh oocytes in relation to pregnancy outcomes [30]. This review aims to provide an overview of the recent advances in human oocyte and ovarian tissue banking and how these clinical reproductive technologies can be used to support fertility conservation strategies in animals. The focus of this chapter is the foundation of cryopreservation of oocytes and embryos in livestock animals.
There are two techniques that exist for cryopreserving oocytes and embryos: slow freezing and vitrification. The primary distinctions between these methods lie in the concentration of cryoprotective agent (CPA) employed and the speed at which the sample is cooled during preservation [31]. Nevertheless, a refined and altered iteration of the conventional vitrification process has since been introduced, which employs an exceptionally rapid cooling rate in contrast to the slow freezing or traditional vitrification techniques [32]. This enhanced vitrification technique is referred to as ultrarapid vitrification.
2.1 Slow freezing
Slow freezing was initially developed for the cryopreservation of embryos and oocytes [33]. Slow freezing is the process of gradually chilling a sample over the course of two to four hours, either manually or automatically with the use of a semi-programmable freezer [34]. Researchers reported that the technique was developed during the early 1970s [17, 35] and it has long been regarded as the benchmark for cryopreserving oocytes and embryos in animals [20]. In livestock animals, in this technique, specimens are adjusted to a concentration gradient of CPAs to reduce chemical and osmotic toxicity, and to establish an equilibrium between the factors affecting cell harm [36]. After reaching equilibrium, the specimens are placed into straws and gradually cooled at a rate of 1–2°C per minute until reaching temperatures between −5 and −7°C. Subsequently, they are seeded to start the process of extracellular freezing [37]. Subsequently, the samples are cooled slowly at a rate of 0.3–1°C/min until they reach a temperature ranging from −30 to −70°C [38], and then they are immersed in liquid nitrogen for preservation [39]. After being cooled down to an intense temperature ranging from −5 to −7°C, the cells undergo a gradual decrease in temperature at a rate of approximately 0.3–0.5°C per minute until they reach a temperature within the range of −30 to −65°C [40]. The straws are then placed into liquid nitrogen and left there for the necessary cryopreservation period [41]. This procedure stands out due to its gradual cooling rate, which, when combined with the appropriate concentration of CPAs, guarantees that the formation of ice crystals occurs exclusively outside the cell [42]. This mechanism occurs as a result of the solutes that inhibit the formation of ice crystals in the intracellular medium becoming more concentrated as the temperature decreases and the gradual and regulated loss of water [40]. Excessive solute concentrations that could result in cell death after prolonged exposure to such conditions can be caused by excessively slow cooling, which dehydrates the cell cytoplasm [36]. The optimal cooling procedure, therefore, is a balancing act between preventing the formation of ice crystals and the toxicity of high solute concentrations inside the cell is outlined in Figure 1.
Figure 1.
A diagram illustrating the process of controlled-rate slow freezing and the significance of the cooling rate in cell cryopreservation is presented. The formation of ice crystals commences near the cell as it undergoes a reduction in water volume after initial cooling to approximately −5°C. The relative value of the cooling rate in relation to the optimal rate and the potential harm resulting from the use of inappropriate cooling rates is depicted. A slow cooling rate leads to cell dehydration and osmotic damage due to solute concentration, resulting in chemical harm. Conversely, a rapid cooling rate elevates the likelihood of ice crystal formation within the cell, leading to structural damage [40].
2.2 Conventional vitrification
Vitrification serves as a substitute for the traditional slow-freezing technique. The controlled-rate slow-freezing approach of cryopreservation was made easier with the development of vitrification [43]. This technique enables the cell and the extracellular environment to solidify into a glass-like state without the formation of ice crystals [32]. The initial successful vitrification event was documented in 1985 by [44], while [45] showcased ice-free cryopreservation of mouse embryos at −196°C. The protocols rely on incubating embryos and oocytes in a solution containing various CPAs at elevated concentrations (up to 8 M) [40, 46]. Subsequently, bovine embryos or oocytes are separated into thin straws and exposed to cooling rates of −200°C per minute [47]. Typically, the straws are placed directly into liquid nitrogen at a temperature of −196°C, allowing them to cool quickly and become vitrified [40]. Samples can be preserved in liquid nitrogen for storage [48]. The primary functions of the high concentration of CPAs are to elevate the viscosity of the solution to desiccate the cells and prevent the crystallization of water [49]. This method requires a delicate balance between the harmful effects of CPA and the development of ice crystals within the substance (Figure 2). Bailey et al. [50] reported that the reason why this method is commonly used with oocytes and embryos, but not as much with spermatozoa, is due to its previous application. The susceptibility of spermatozoa to the toxicity induced by high concentrations of CPAs is greater than that of embryos and oocytes, primarily due to their heightened sensitivity to osmotic stress [51]. It is possible to reduce the CPA concentration by 50–75% through the use of small volumes (1–1.5 μL) of vitrifying solution [40]. Instant heat release homogeneity is improved after contacting a small amount of sample with liquid nitrogen. Vitrification maintains a liquid molecular structure despite being in a solid state; this prevents ice crystals from forming [52].
Figure 2.
A diagram illustrating the vitrification process and the significance of cryoprotectant agents (CPAs) in achieving cell cryopreservation is presented. Varying concentrations of CPAs result in different levels of cell damage post-vitrification. A low concentration of CPAs fails to facilitate vitrification, leading to the formation of ice crystals within the cell and subsequent structural harm. Conversely, a high concentration of CPAs elevates medium toxicity and osmotic damage, causing chemical injury to the cell [40].
The process of vitrification makes it possible to use this technique for livestock animals cryopreserving sperm cells [53]. According to [54], the protocols involve culturing ram spermatozoa in a 0.5 M sucrose solution for a minor shrinkage of the cells, followed by the direct discharge of small droplets of the sperm solution into liquid nitrogen (−196°C). The development of ice crystals, osmotic stress, and toxicity caused by a high concentration of CPAs, this procedure has demonstrated effectiveness in animals’ cryopreserving spermatozoa [55]. Despite its promising potential, this technique has not yet been established as a standard protocol for sperm cryopreservation [56].
However, it is unlikely that such a low concentration of CPAs in oocyte and embryo cryopreservation would be effective. This is because the lower surface/volume ratio of oocytes and embryos compared to sperm cells is associated with a higher likelihood of ice crystal formation [31]. However, for many years, oocytes and embryos have been successfully cryopreserved using vitrification techniques, which involve the use of high concentrations of cryoprotective agents (CPAs). This method has shown a continuous improvement in efficiency over the years [40, 57].
2.3 Ultrarapid vitrification
Ultrarapid vitrification represents an enhanced and updated approach to the conventional vitrification technique as reported by [58]. The previous researcher introduced the concept of ultrafast cooling for cryopreserving oocytes and embryos through their creation of the open-pulled straw [59, 60]. The relationship between the viscosity of the vitrification medium and the cooling rate is inversely proportional [35]. Consequently, a medium with reduced levels of cryoprotectants, with additional additives, can be effectively vitrified at an increased rate of cooling [36]. According to Barbosa et al. [53], achieving vitrification theoretically requires a 1.5 M concentration of any cryoprotectant, if a cooling rate of 15,000°C/min is utilized. The vitrification process enables the use of a low concentration of CPA solution with a very rapid cooling rate, effectively minimizing the toxicity and osmotic stress experienced by the vitrified cells [61]. The ultrarapid cooling rate is accomplished by decreasing the effective volume of the solution to be vitrified [62].
Successful vitrification of mammalian oocytes and embryos using ultrarapid methods is shown in Table 1. The findings indicate that ultrarapid vitrification is successful in protecting oocytes and embryos vulnerable to damage from cold temperatures [17]. The open-pulled straw and microdrop methods have also been reported to successfully vitrify bovine oocytes at the metaphase II (MII) stage, leading to the production of live offspring [58]. Booth et al. [67] indicated that full-term young were successfully developed from bovine embryos reconstructed using enucleated oocytes that had been vitrified through the open-pulled straw technique. Cameron et al. [64, 65] reported that the live offspring have been successfully achieved through vitrification using open-pulled straw in pig embryos, which are highly susceptible to chilling damage.
Successful cryopreservation of livestock animals’ oocytes and embryos by ultrarapid vitrification, resulting in the production of offspring.
Oocytes and embryos have been effectively cryopreserved despite being susceptible to factors other than chilling injury. Certain categories of oocytes/embryos, such as pig oocytes, pig embryos prior to the peri-hatching stage, bovine oocytes, and bovine embryos during early cleavage stages, exhibit sensitivity to temperatures below +20°C [71]. Cells that possess a susceptibility to cold temperatures exhibit a dark appearance due to the presence of lipid droplets within their cytoplasm [72]; these lipid droplets are believed to be linked to the occurrence of chilling injury. Therefore, the successful cryopreservation of pig, cattle, and buffalo embryos has been achieved by using embryos at the peri-hatching stage, when there are fewer droplets [70, 73, 74].
Cryopreservation oocyte is a valuable technique for preserving germ cells for subsequent uses such as fertilization, as cytoplasts for somatic cells for use in fertilization, as cytoplasts for nuclear transfer from somatic cells to other cells, and for genome banking purposes for valuable animal species. However, Oocytes are highly sensitive to freezing, and the procedure is not as established as in semen or embryos, due to a low permeability to cryoprotectants [75], toxicity, temperature, and time of exposure to the cryoprotectants [76]. While cryopreservation does not affect some processes, it may impair others, like alterations in the oocyte plasma membrane structure and the number of lipids inside the cell in bovines [15] and the cytoskeleton [77]. These processes have been shown to be extremely sensitive to cryoinjury, which frequently results in cellular disruption and cell death [78]. However, the high concentration of cytotoxic CPAs and/or high cooling rates induce significant cytotoxicity, mechanical, and chemo-osmotic stresses to specimens, causing cellular viability and functional damage, and even cell death [79]. Pared [80] has demonstrated that a number of variables, including the stage of oocyte maturation at freezing, membrane composition, membrane permeability, the kinds of cryoprotectants employed, and the freezing methods, affect the result of oocyte cryopreservation. At prophase I, when the condensed chromatins are shielded by the nuclear membrane, immature oocytes are stopped [81]. Maturation oocytes complete nuclear and cytoplasmic maturation rapidly for fertilization and the ability to support further embryonic development [82].
The cryopreservation of oocytes and embryos involves four main steps, as illustrated in Figure 3. In the first step, cells are exposed to a CPA solution to remove water and dehydrate them. By replacing intracellular water with permeable CPA, the freezing point of the cell’s content is lowered, reducing the formation of intracellular ice crystals [42]. The second step involves cooling the equilibrated cells to a low temperature and storing them in liquid nitrogen (−196°C). Depending on the cooling method used, this process results in either the formation of small intracellular ice crystals (slow cooling) or the transformation of the intracellular content into a glass-like state without ice crystal formation (vitrification). In the third step, cryopreserved cells are thawed and warmed to reverse their frozen state. After completing Step 4, the cells that have been thawed and warmed are then balanced in the rehydration solution, which facilitates the substitution of intracellular CPA with water molecules. As a result of this crucial step, the preserved cells recover their vitality and resume their regular physiological functions. Steps involved in the cryopreservation of oocytes and embryos are shown in Figure 3 [33].
Figure 3.
Steps involved in cryopreservation of oocytes and embryos [33].
The cryopreservation of immature oocytes is superior to that of mature, as they lack a cold-sensitive meiotic spindle [83]. However, the cryopreservation processes itself disrupt the signals responsible for oocyte structure and maturation [84]. Therefore, frozen-thawed oocytes typically have lower rates of maturation and fertilization compared to oocytes that have not been cryopreserved [85]. Problems with oocyte freezing have been attributed to cryoinjury, which can result in issues such as excessive formation of harmful ice inside the cells and chromosome abnormalities [51], disturbance of hyperosmotic stress [86], disruption of actions and microtubules, and zona pellucida hardening [87]. In more recent studies, it has also been suggested that cryopreservation results in alterations in gene and protein expressions in boar [88, 89].
3.1 Cryoprotective agents
Cryoprotective and cytoprotective agents (cytoprotective agents) are fundamental components of the cryopreservation process [24]. The action of CPAs can vary depending on the type of CPA and other factors, including their capability to interact with membrane phospholipids at low temperatures, helping to regulate membrane lipid-phase transition and maintain protein structure and cell integrity [90]. These changes involve alterations in interactions between lipids and lipids, lipids, and proteins, as well as in membrane receptor function, signal transduction, permeability, and transport mechanisms [91]. Therefore, CPAs can be divided into two primary groups: CPAs that can pass through membranes and CPAs that cannot pass through membranes [92]. These substances consist of glycerol, ethylene glycol, propylene glycol (1,2-propanediol), dimethyl sulfoxide, methanol, and butanediol according to [93]. After entering the intracellular fluid, the CPA takes the place of water and disrupts the hydrogen bonding among water molecules, which ultimately decreases the formation of ice inside the cell [94]. Additionally, penetrating CPA also helps increase cellular hydration status during cooling to prevent excessive accumulation of cellular electrolytes [95]. Their relatively small size and amphipathic nature allow them to easily penetrate cell membranes where they can exert their effects. The structure’s ability to hydrogen bond with water accounts for much of its protection [22]. The presence of high concentrations of penetrating CPAs in the molar range can induce cellular toxicity or apoptosis by causing osmotic stress or shock [40]. Mammalian cells should be able to tolerate changes in the volume, as they are exposed to being in contact with the hypertonic CPA solution [96]. This occurrence leads to different levels of water activity in various intra and extracellular compartments. If Mammalian cells can exceed the limit of volume expansion at sub-zero temperatures, resulting in cell destruction or apoptosis as a result of Osmotic Stress or Osmotic Shock [97, 98]. It is important to note that certain cell types will need a specific type and the right concentration of CPA to protect cryoinjury during cryopreservation and cooling [99]. Small molecules (such as sugars) arranged into polymers act to increase extracellular osmolality, resulting in cell dehydration, which serves as a stabilizing mechanism; for this reason, nonpenetrating agent cooling rates are slow [24]. CPAs with high amphiphilic properties and low molecular weight (typically less than 100 Dalton) are frequently organic compounds that can easily penetrate the plasma membrane due to their permeability in animals [40]. Nevertheless, alternative agents can be employed in cryopreservation to protect cells and tissue samples that do not possess cryoprotective properties. These agents function independently of their impact on ice crystal formation and osmotic effects, effectively preventing cellular damage [24]. The common nonpenetrating CPAs are typically less harmful than penetrating cryoprotectants, disaccharides, and polymers, such as ficoll, poly-ethylene glycol (PEG), hydroxyethyl starch (HES), polyvinyl alcohol (PVA), and polyvinyl pyrrolidone (PVP), are examples of such substances [92]. Utilizing a mix of penetrating and nonpenetrating CPAs offers the most effective approach to minimizing cryoinjury in the process of cryopreservation [36]. The regulation of molecular dynamics in cell membranes induced by CPAs is influenced by different mechanisms, which are determined by the specific types of CPAs used [36]. For instance, dimethyl sulfoxide exhibits a higher efficiency in diffusing through phospholipid bilayers when compared to other CPAs [100]. However, at higher concentrations (>7 mol %), DMSO causes the thinning of lipid bilayers, leading to membrane damage and cytotoxicity [40]. Additionally, an alternative suggested mechanism involves colligative properties, specifically the phase diagram alteration in solution, as demonstrated in glycerol [101]. This process could regulate the concentration of salt (NaCl) and modify the formation of crystalline solids that take place during the cryopreservation process [102].
3.2 Cryopreservation technique
The cryopreservation methods for oocytes, sperm, and embryos are typically categorized as controlled-rate slow freezing and vitrification, which is also known as “ice-free” freezing [15]. A programable freezer is necessary for conventional slow freezing to effectively regulate the optimal freezing rate [103]. Throughout the cooling process, the temperature is slowly lowered until it reaches a point below the freezing temperature, resulting in the formation of ice [21]. However, the survival condition of cells is associated with the status of the extracellular solution that results from ice formation [21, 104]. Excessive accumulation of ice inside cells, particularly intracellular ice formation, leads to the disruption of cell structure and function, ultimately leading to apoptosis or cell death [105]. The expansion of ice crystals depends on the rate at which freezing takes place [106]; however, the formation of ice crystals is commonly done to prevent the excessive accumulation of ice during supercooling [107]. By each ice crystal that is formed, the osmolarity of the extracellular solution increases, inducing the outflow of water from the intracellular compartment, due to the increased osmotic pressure [94]. Throughout the slow cooling process, embryonic blastomeres undergo dehydration due to the rising osmotic pressure, which escalates as extracellular ice crystals begin to form following ice seeding [58]. Due to the large size of oocytes and their low membrane permeability to cryoprotectants, most cryopreservation methods necessitate a slow freezing rate to ensure adequate CPA permeability [108]. The permeabilities of oocyte membranes to CPA and cryotolerant substances have been shown to vary between species [109]. Empirical study is often necessary to test freezing protocols before they can be used, despite the availability of theoretical models that can predict the optimal freezing rate. The reason for oocytes susceptibility to low temperatures is due to their sensitivity at different cellular levels, such as the zona pellucida, plasma membrane, meiotic spindles, and cytoskeleton [69]. On the contrary, if the freezing rate is too slow, oocytes will experience significant dehydration and a combination of relatively low CPA concentrations [16]. Hence, the ideal rate of freezing is a gradual process that strikes a harmonious equilibrium between sufficient cellular dehydration and limited formation of ice within the cells. This method typically necessitates low concentrations of CPA, effectively reducing osmotic shock and CPA toxicity. Vitrification, unlike slow freezing, enables a quick shift from a liquid state to a glassy phase or solidification of water [94]. Vitrification is simplified to develop the process of cryopreservation through the controlled-rate slow freezing method [40]. To obtain this outcome, living cells are first exposed to high concentrations of CPAs (including both permeable and nonpermeable CPAs) before being subjected to deep freezing in liquid nitrogen [18, 110]. Nevertheless, this method results in significant osmotic pressures and chemical harm [111, 112].
3.3 Outcome following oocyte cryopreservation
Oocyte cryopreservation has proven to be a highly effective technique in laboratory animals, particularly in Zebrafish [113] and animals [62]. This success can be attributed to the remarkable tolerance of oocytes to cold stress in bovine [114]. The species has made significant advancements in the technology of producing embryos from frozen oocytes [115]. Cryopreservation of oocytes in Zebrafish and mice is a valuable technique for preserving the genetic material of breeds, including genetically modified animals. Understanding the process of oocyte maturation in Zebrafish and mice is more advanced compared to other animal species. While larger species’ oocytes are more susceptible to cold stress than those of Zebrafish and mice, viable offspring have been successfully produced from medium to large domestic animals, such as pigs [116], cows [117], and goat [118], as well as sheep [115].
3.3.1 Pigs
In pigs, successful cryopreservation of porcine gametes and embryos has been very challenging due to their sensitivity to cryoinjuries [119]. The high intracellular lipid content of porcine oocytes and embryos is one of the factors contributing to their low temperature sensitivity in buffalo [120]. Despite the negative impact on embryo development, the elimination of cytoplasmic lipids is harmful [121], improved freezing tolerance and embryo development were observed in porcine oocytes after undergoing partial dilapidation before vitrification [121, 122]. The of survival and embryo development, slow freezing has proven to be ineffective unlike in other species [123]. Due to the high sensitivity of porcine oocytes to cryopreservation, most of the research on porcine oocyte cryopreservation has used vitrification as the method [124, 125]. Vitrification has resulted in the successful birth of the initial piglets [126]. The effectiveness of this method was significantly improved during the latter part of the 2000s [125]. Therefore, experimentation involved testing various modifications to vitrification devices, cryoprotectant types, and procedures to minimize cryoinjuries and cellular alterations in vitrified-warmed oocytes. Significantly, the process of vitrification brings about various changes in porcine oocytes. These changes include disruption of the cytoskeleton [127], chromosomal abnormalities [128], dysfunction of organelles [129, 130, 131], oxidative stress [132], disturbance in calcium levels [133], apoptosis [134], and alteration in epigenetic patterns [17]. In recent, the vitrification of porcine oocytes has been linked to changes in transcriptomic and proteomic [135]. Both immature and mature-stage oocytes have been effectively preserved through either conventional slow freezing or vitrification methods [1, 127]. When immature oocytes are vitrified, there is an improvement in cell cytoskeleton rearrangement and embryo development compared to other methods [100]. Furthermore, research has demonstrated that vitrification can lead to parthenogenic activation at a significant rate of around 50% when oocytes are vitrified at the mature stage [125]. The vitrification process causes a disturbance in cellular functions, particularly in mitochondrial activities, which subsequently elevates the levels of reactive oxygen species. Consequently, this disruption leads to the apoptosis of cells [136]. Hence, the utilization of antioxidants like astaxanthin and caspase inhibitors resulted in a reduction of apoptosis and enhancement in the growth of vitrified warmed in pig oocytes [137, 138].
3.3.2 Bovine
Many reproductive biotechnologies have been utilized for the efficient production of large domestic animals, including pigs and cattle with a high significant economic importance value [58]. Cryopreservation of unfertilized oocytes can be combined with advanced reproductive techniques, along with its potential benefit as a means of preserving female genetic material. Oxidative stress can produce harmful effects on embryonic development, and among these effects, we highlight metabolic alterations such as the depletion of adenosine triphosphate (ATP) levels, changes in ion channels, deleterious effects on protein synthesis, lipid peroxidation, alterations in membrane permeability, and alterations in mitochondrial and ER functions [61, 62]. Therefore, it is important to use cryoprotective additives (CPA), such as dimethyl sulfoxide (DMSO), ethylene glycol (EG), or glycerol alone or in combination, when cryopreserving cells in any methods [58]. Due to both hydrophobic and hydrophilic characteristics, as well as the relatively small molecular weight, these CPAs are permeable to the plasma membrane. The application of vitrification as an alternative to conventional freezing can reduce the equipment required, but the technician-dependent performance of the vitrification process is a limited factor for its widespread use [58]. So far, successful vitrification-producing pregnancy and/or birth of live offspring has been reported with preimplantation embryos from various mammalian species including humans [63]. The method was found uniquely efficient for cryopreservation of pre-compaction stage in vitro-produced embryos, which so far are regarded as extremely sensitive to cryoinjuries [64]. Using identical parameters, embryos from the late stages of preimplantation development were also vitrified with high survival rates [65]. The higher lipid content in the in vitro-produced embryos has been related to its low cryotolerance [66]. However, the use of delipidant agents during in vitro culture does not necessarily result in increased cryotolerance [67]. In addition to the stress of in vitro production, it is relevant to mention that the cumulus-oocyte complexes (COC) have their own history when they arrive in the laboratory, which influences subsequent in vitro development [26]. Heat stress, for example, is a very common type of stress that affects oocyte and embryo development [70]. However, little is known about how lipid metabolism influences the embryonic development of oocytes under heat stress. However, it may be a good example to elucidate the importance of lipid reserves in oocytes and embryos under stress.
3.3.3 Goats
Oocyte maturation, parthenogenesis, cloning, and the creation of transgenic animals are among the biotechnological processes that demand a substantial quantity of oocytes [69]. Compared to the freezing of sperm or embryos, the cryopreservation of goat oocytes has proven to be much more challenging [68]. According to [35] oocytes suffer damage at various levels, impacting the cytoskeleton, meiotic spindle, zona pellucida, plasma membrane, and other structures, making them large and vulnerable cells for cryopreservation. Cooling and freezing matured or ovulated oocytes can cause spindle disorganization, loss or clumping of microtubules, chromosome scattering, increased polyploidy during fertilization, and decreased fertility [71]. The level of reactive oxygen species (ROS) during in vitro maturation significantly compromises the ability of oocytes to develop up to the blastocyst stage, as is well known [72] vitrification raises the ROS levels in oocytes of other species. Due to ROS playing a crucial role in the apoptotic process, there has been a close correlation between the increased rate of apoptosis and the increased ROS level of vitrified oocytes [73]. Oocytes in prepubertal females are less able to reduce reactive oxygen species (ROS), which damages the microfilament network and inhibits the development of blastocysts [74]. For this reason, the viability of the vitrification process depends critically on the oocyte’s quality and nuclear stage [68]. Kharche et al. [69] investigated the impact of propanediol-based GV-oocyte vitrification in goats, demonstrating a noteworthy decline in the rates of vitrification group zygote pronuclear formation and maturation following IVF. After parthenogenic activation, [75] assessed the impact of vitrifying GV and MII goat oocytes, but they discovered no variations in the two nuclear stage-oocyte groups’ blastocyst production. Nevertheless, cryotop [76] and open straw [77] were superior tools for vitrifying GV-oocytes in goats.
3.3.4 Sheep
Cryopreservation of sheep oocytes is significant for preserving the genetic potential of female sheep. The procedure involves freezing eggs, and this maintains their viability and fertility. It is crucial for the long-term preservation of sheep’s genetic resources. This method advances sheep embryo biotechnology supporting ewe genetic resources [111]. There are two techniques for oocyte cryopreservation which are vitrification and slow cooling. Both have been tested for cryopreservation of sheep oocytes. The slow-cooling method is more susceptible to cold shock than the vitrification method and as such, cold shock must be thoroughly looked out for in slow cooling. During vitrification, oocytes pass through the dangerous temperature zone at extremely high speed [111]. The status of cryopreservation of sheep oocytes is limited due to the lower cryotolerance of sheep embryos compared to other species like cattle. Despite efforts to improve the process, actual results are not as promising as expected and it is mainly due to the cellular and biochemical changes that occur during freezing and heating. Ultrastructural evaluation of vitrified ovine embryos shows significant damage. Fresh embryos exhibit fewer microvilli and less extensive intercellular junctions and there are also more lipid droplets and cell debris [111, 139]. These challenges call for new approaches to achieving cryopreservation goals, with new tools being developed for vitrification and different ultrafast vitrification protocols. These result in higher efficiency and better tolerance of the samples to temperature during the cryopreservation process. Further, methods like manipulation of lipid content in embryos as a potential way to enhance cryopreservation effects are indeed under consideration [139, 140]. The latest technological advancements in sheep oocyte cryopreservation involve the use of alternative methods such as vitrification and ultrarapid vitrification protocols, which provide a more efficient and temperature-permissive cryopreservation process. Additionally, strategies like manipulating the lipid content of the embryo are being investigated as a promising approach to improve cryopreservation efficiency [141]. Dilapidation i.e., studies aimed at understanding the correlation between oocyte lipid content and the success rate of vitrified-warmed ovine oocytes have suggested that lowering oocyte lipid content may be a feasible strategy for enhancing the survival of cryopreserved sheep oocytes. Originally, the concept of this strategy lies in the fact that ovine oocytes contain a high level of lipids, and these lipids contribute to low cryotolerance and poor cryosurvival rates. A couple of studies by [111, 142] support it to some extent. Thus, while dilapidation may be helpful to enhance the survival rate of cryopreserved ovine oocytes the outcomes are not significantly different because intracellular lipid content is only one factor among many variables that can lead to reduced tolerance to cryopreservation [111, 142]. Cryopreservation of sheep oocytes is a valuable tool for preserving the genetic potential of female sheep and advancing sheep embryonic biotechnology. While there are challenges associated with sheep oocyte cryopreservation, researchers are actively exploring alternative methods and strategies to improve the efficiency and effectiveness of the process. Ongoing research in this field is expected to further enhance our ability to preserve and utilize sheep genetic resources.
Progress in oocyte cryopreservation has advanced steadily over the years, yielding positive results. However, enhancing outcomes for cryopreserved oocytes through the study of cryobiology remains a complex task. However, the efficacy of oocyte cryopreservation remains significantly lower in comparison to embryos at a later developmental stage, despite utilizing ultrarapid vitrification, which is currently regarded as the most effective technique available. Therefore, the primary obstacle in this domain is to establish uniform procedures for efficient cryopreservation of oocytes and embryos at their early stages. The aim of achieving success in such protocols theoretically should be like that of their non-cryopreserved equivalent. It is clear from the existing knowledge that the capacity of oocytes and embryos to endure the cryopreservation process differs among various species. Developing a universal standardized protocol for all species seems unattainable. Therefore, it is imperative to prioritize the advancement of species-specific optimized procedures for the cryopreservation of oocytes and embryos. Additionally, it will be captivating to witness forthcoming endeavors in the creation of automated devices for the vitrification of oocytes and embryos. The worldwide field of livestock can be revolutionized by the implementation of an efficient and automated ultrarapid vitrification system. In the future, the focus of cryopreservation will be on developing protocols that preserve the structural and functional integrities of oocytes and embryos after freeze-thawing. These protocols should be reproducible across laboratories worldwide. Achieving these targets will lead to the development of standardized and optimized methods for routine oocyte and embryo cryopreservation in livestock.
In conclusion, cryopreservation is the process of preserving living cells at an extremely low temperature, typically in liquid nitrogen. It is essential for maintaining and conserving genetically superior germplasm by preserving oocytes and embryos. The cryopreservation procedure involves three main stages: equilibration of cells with a concentrated cryoprotective agent solution, cooling and storing cells at ultra-low temperatures, and recovering frozen cells after thawing and warming. Cryoprotective substances are crucial for ensuring the viability of cryopreserved cells during processing and storage at very low temperatures. There are two primary techniques for preserving oocytes and embryos: slow freezing and vitrification. Slow freezing involves gradually cooling samples to form ice crystals inside and outside cells, while vitrification rapidly cools samples to create a glass-like state without ice crystal formation. Ultrarapid vitrification is an advanced technique with rapid cooling and warming rates, considered superior to slow freezing and conventional vitrification. Cryopreservation often results in cryoinjuries such as chilling injury, ice crystal formation, fracture damage, osmotic stress, and the development of multiple asters. The extent of cryoinjuries depends on various factors and significantly impacts the viability of cryopreserved cells. The cryopreservation process has adverse effects that affect the survival of frozen cells after thawing, especially when preserving oocytes.
The University of South Africa, Department of Agriculture and Animal Health, College of Agriculture and Environmental Sciences, South Africa colleagues are acknowledged for their support.
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Written By
Thivhilaheli Richard Netshirovha, Vhahangwele Makumbane, Lerato Deirdre Sehlabela, Zwivhuya Constance Raphalalani and Masindi Lottus Mphaphathi
Submitted: 16 June 2024Reviewed: 26 June 2024Published: 06 September 2024
Open access peer-reviewed chapter - ONLINE FIRST
