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Assisted reproductive technologies are critical in the preservation of gametes from endangered species. As a result, cryobanking is critical in reproduction facilities for the gametes conservation of endangered species for future use. Furthermore, cryobanking allows for the preservation of genetic variability through biotechnological reproduction programs. If oocyte cryopreservation is successful, the timing of in vitro maturation and subsequent to in vitro fertilization (IVF) will be possible. Cattle oocytes are very sensitive to cryopreservation due to their complex structure, and they are also very sensitive to chilling, which can harm their viability. During the cryopreservation process, sperm membrane proteins and carbohydrate composition change, sperm membrane structure is disrupted, and sperm viability is reduced. Extenders are frequently required during cryopreservation, for improving sperm cryopreservation technologies and is therefore necessary to have a thorough understanding of the properties of the extenders. Extenders have been enriched with antioxidants such as Glutathione to protect sperm motility and integrity from oxidative damage and the reactive oxygen species produced during cryopreservation can be neutralized using antioxidants.
Agricultural Research Council, Animal Production, Germplasm, Conservation and Reproduction Biotechnologies, South Africa
Mahlatsana Ledwaba
Agricultural Research Council, Animal Production, Germplasm, Conservation and Reproduction Biotechnologies, South Africa
Faculty of Science, Department of Animal Sciences, Tshwane University of Technology, South Africa
Mamonene Thema
Agricultural Research Council, Animal Production, Germplasm, Conservation and Reproduction Biotechnologies, South Africa
Faculty of Science, Department of Animal Sciences, Tshwane University of Technology, South Africa
*Address all correspondence to: masindim@arc.agric.za
1. Introduction
Cryopreservation is the expertise of freezing and cryogenic storage of biological materials at extremely low temperatures, occasionally utilizing solid carbon dioxide at −80°C or more often liquid nitrogen at −196°C [1]. This procedure is essential for preserving gametes and genetic diversity in both known and endangered species. Cryopreservation of gametes and genetic diversity provides numerous advantages as it paves the way for the successful application of current biotechnologies like cloning, transgenesis, and long-term storage/conservation of animal genetic resources [2]. Oocyte cryopreservation is beneficial for the treatment of infertility and has broader clinical implications than embryo cryopreservation [3]. However, for the quality of the gametes or tissue not to deteriorate during long-term storage, a good and reliable methodology is required. Many reproductive centers have established cryopreservation methodologies for many species. However, the survival rate of the gametes or tissues has declined over time due to the lower temperature and metabolic reactions of the gametes which are impaired during cryopreservation. Cryopreservation causes the formation of intracellular ice crystals and osmotic stress, which causes cell damage, oocyte quality degradation due to their susceptibility to chilling, and a reduction in sperm survival rate [4]. Cryopreservation is one method of preserving sperm and oocytes ex-situ. Furthermore, the cryopreservation process has proven to can reduce the number of cases of extinction. Moreover, the use of semen extenders, medium, antioxidants and cryoprotectants (CPAs) proved beneficial in preserving the gametes. The semen extenders, mediums, antioxidants, and CPAs are nutrients and antibiotics that increase the quality and survival rate of the gametes. It was discovered that successful gametes cryopreservation, requires the use of semen extenders, mediums, antioxidants, and CPAs. Typically, the pace and susceptibility of sperm to subzero temperatures relate to the content of cryoprotective chemicals and membrane-stabilizing additives. Therefore, cryopreservation may be an effective method of preserving fertility as the frozen–thawed sperm may be utilized for intrauterine insemination, IVF, or intracytoplasmic sperm injection [5].
This chapter will discuss the current state of oocytes and sperm cryopreservation, as well as their prospects. Mammalian sperm cryopreservation has previously proven to be difficult due to the lack of methodologies that can help sperm and oocytes withstand extremely low temperatures. Whereas cryopreservation has been determined to be an ineffective reproductive medicine preservation treatment since 1970 [6]. To date, the sperm cryopreservation in ovine and bovine species has improved over time. Whereas sperm cryopreservation remains a challenge in mammalian species such as porcine and humans. Furthermore, cryopreservation of oocytes has proven to be difficult in all mammalian species. Several methodologies have been tested on mammalian oocytes, in the past, beginning with the first cryopreservation in the year and continuing to the present [7]. Cryopreservation of oocytes in many mammalian species, such as porcine and horses, remains a major challenge.
To achieve the best results during cryopreservation, a thorough grasp of sperm physiology is essential [8]. The fact that sperm are tiny cells with a vast surface area is a crucial aspect of sperm cryobiology [9]. Sperm are less vulnerable to potential harm because of these traits, which impact the intracellular cytosol’s viscosity and glass transition temperature [10]. Organelles in the sperm may be destroyed in the absence of cryoprotective substances due to cold shock and the stimulation of ice crystal formation [11]. More research is needed to determine the methodologies that can successfully improve the quality of oocytes after cryopreservation. Cryopreservation of semen and oocytes will aid in the preservation of domestic and wild species’ genetic diversity, as well as the dissemination of superior genetics, gene banking, and the extinction of superior and endangered species. Semen cryopreservation is also used in artificial insemination (AI) and IVF procedures. However, previous research has shown that using post-thawed semen for AI results in a lower pregnancy rate. Offspring have also been reported to be born from frozen–thawed oocytes in bovine, ovine, and horse.
Semen extenders are liquid diluents added to sperm to preserve its ability to fertilize, and they contain protective ingredients that allow sperm to survive outside the reproductive tract of the male animals [12, 13]. Furthermore, semen extenders protect sperm by stabilizing the plasma lemma, providing energy substrates, and preventing the harmful effects of pH and osmolarity changes over time during in vitro storage [14]. When a proper semen extender is added to the sample before evaluation, the accuracy of sperm motility determination may improve [15].
During the chilling and shipping processes, semen extenders act as a buffer to protect sperm cells from their own harmful byproducts as well as from cold shock and osmotic shock [16]. Semen extenders has the ability to prolong sperm storage and transportation, allowing it to be used during AI, IVF and other research studies. Currently, the semen extenders are categorized either as short (approximately 3 days; in vitro liquid storage) or long term (approximately 5 days; in vitro liquid storage or cryopreservation for years) [17]. For the past decades, some commercial vendors have made around 80% of the semen extenders for porcine sperm readily available [18].
3.2 Cryoprotectants
Cryopreservation methods aim to preserve the viability of tissues and cells by focusing on the mechanisms of harm and protection in living cells and tissues at low temperatures [19]. The impact of subzero temperatures on normally healthy tissue should be recognized to properly comprehend the role of cryoprotective agents. Since water makes up around 80% of tissue mass, both intracellularly and extracellularly, has the greatest impact on the detrimental biochemical and structural changes that are hypothesized to cause cryopreservation injury [20]. Due to the presence of salts and organic molecules in the cells, the freezing point of cell water is substantially lower (even −68°C) than the freezing point of pure water (about 0°C).
Cryoprotectants are divided into two groups: permeable and non-permeable CPAs. Non-permeable and permeable CPAs improve cell survival while decreasing cellular water content to help prevent intracellular ice crystal formation. Permeable CPAs are macromolecules that pass through the sperm plasma membrane. A cryopreservation diluent’s functions include providing the sperm with energy sources, shielding them from temperature related harm, and maintaining an environment that allows the sperm to survive for a while. Glycerol, Propylene glycol, and Ethylene glycol (EG) are three examples of permeable CPAs that are commonly used (Figure 1). To enhance post-thawed sperm viability and fertility, each of the several media components was studied alone and in combination [21]. The gametes exposed to those penetrating solutes undergo intense initial dehydration, then rehydration, resulting in a chance of gross cellular swelling [22]. Glycerol at 3% has shown to maintain the cryo survival rate of sperm from different species; thus, larger amounts of permeable CPAs concentrations have shown to lead to more cellular damage [23], while the higher concentrated CPAs are more toxic to oocytes. Glycerol reduces intracellular water freezing while adjusting sperm osmolality via invasive thermal protection [24]. The discovery of Glycerol’s effectiveness in preventing various phase transitions while freezing via increased water permeability and fluidity of the sperm membranes resulted from research to understand the mechanisms of CPAs [25]. Using minimum volume methods, a higher cooling rate can facilitate vitrification with less concentrated CPAs, and a higher warming rate will prevent it [26]. Non-permeable CPAs such as sucrose can facilitate dehydration and vitrification, which reduce the required concentration of permeable CPAs [27].
Figure 1.
The penetration CPAs that are widely used: Glycerol (GLY), dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propylene glycol (PG). Adapted from Whaley et al. [20].
3.3 Antioxidant
During semen cryopreservation and thawing, increased reactive oxygen species (ROS) generation and decreased antioxidant levels were observed. As a result, oxidative stress may have a role in sperm injury during cryopreservation. Oxidative stress is the imbalance between the formation of ROS and antioxidant defenses, resulting in considerable loss of sperm function. Therefore, the ROS generated during oxidative stress can be neutralized by the use of antioxidants. Antioxidants are chemicals and reactions that dispose of scavenging, suppressing, or resisting the creation of ROS. Antioxidants have been shown to inhibit or reduce the lipid peroxidation reaction, resulting in less oxidative stress and damage [28]. Antioxidants and antioxidant enzymes that are found in the seminal plasma of the semen protect sperm from oxidative damage. These antioxidant mechanisms protect the sperm, including enzymatic antioxidants [Catalase, Superoxide Dismutase (SOD), reduced Glutathione & Glutathione Peroxidase (GPx)], [29, 30] and non-enzymatic antioxidant systems [GPx, Vitamin C, E, Cysteine & Glutathione (GSH)]. These antioxidants protect sperm from cryo injuries caused by reactive oxygen species [31, 32]. However, due to the addition of extenders to the seminal plasma during cryopreservation, the quantities of these antioxidants decrease [33]. Therefore, finding low cytotoxic antioxidants at a suitable concentration is very important in improving the frozen–thawed sperm quality [34].
Recent studies suggest that supplementing cryopreservation extenders with antioxidants improves sperm quality in bovine, ovine, porcine, canine, and human, enhancing sperm motility and membrane integrity following thawing [35]. A range of antioxidants is active in the body including enzymatic (endogenous) and non-enzymatic (mainly brought by food) antioxidants. All of them can be intracellular or extracellular antioxidants and can be used during the cryopreservation of semen. To reduce cryodamage, numerous exogenous, non-enzymatic antioxidants were introduced to maturation, vitrification media, and extenders for mammalian sperm, oocytes, and embryos.
There are numerous cryopreservation methods for cryopreserving semen and oocytes of different species; methods include slow freezing (programmable freezer), rapid freezing, and ultra-rapid freezing (also known as kinetic vitrification). These methods represent a particular drawback in determining the most appropriate method for cryopreservation [36]. Various processes have been developed for semen and oocytes cryopreservation technology in recent years [37].
4.1 Conventional slow freezing
Slow cryopreservation of semen has been a useful method and is still used during cryopreserving semen and oocytes [38, 39]. A programmable freezer with significant control over the ideal freezing rate is necessary for conventional slow freezing. The temperature progressively drops below the freezing point during cooling, whereas both extracellular and intracellular spaces can generate ice [40]. In order to ensure fine control over numerous elements (such as thermal shock) that lead to cell damage, slow freezing primarily calls for a relatively low concentration of CPA agent, combined with sufficiently slow cooling/freezing rates [41]. In a summary, slow cooling is mixing low concentrations of a penetrating agent like DMSO (usually ≤1.5 M) as well as a non-permeating agent (often sucrose or trehalose, ≤ 0.3 M) with controlled slow chilling rates to gradually dehydrate sperm and oocytes. The sperm and oocytes are kept in liquid nitrogen at −196°C till they are required for usage after cooling to about ≤150°C [20]. One disadvantage of this procedure is the inability to freeze extremely small amounts of semen, as in the case of surgical testicular sperm retrieval [37]. Slow freezing methods using a programmed freezer is traditionally used for oocyte cryopreservation, and these procedures typically take several hours [3], oocytes are progressively chilled over 2 to 3 hours in two or more steps, either manually or automatically with the aid of a programmable freezer [42]. Semen straws can be frozen in huge quantities with the use of programmable freezers, which also allow for regulating the freezing pace. By cryopreserving the semen straws at −80°C for 7 to 15 minutes and then submerging them in liquid nitrogen, these freezers can be used to stimulate pellet freezing [43]. The advantage of several programmable freezers is the ability to personalize the freezing curve, for example, 4 to −5°C at 4°C/min, −5 to 110°C at 25°C/min, and − 110 to 140°C at 35°C/min, before submerging the semen straws in liquid nitrogen [44]. According to some theories, the formation of ice crystals during slow freezing raises the electrolyte concentrations inside cells and could harm the sperm and oocytes chemically and physically. Therefore, slow freezing appears to have a lower survival rate than vitrification [3].
4.2 Conventional straw vitrification
Vitrification is another important method for improving the survival rate of cryopreserved oocytes that is both time saving and does not require any special equipment [45]. The most typical process for vitrification includes adding CPAs step by step in cryomedia [20]. Although vitrification’s rapid freezing defends cells from the majority of chilling-related harm, including membrane damage, it necessitates the use of hazardous CPAs solutions in higher concentrations [46]. Oocytes are introduced to a solution that contains 7.5% v/v EG and 7.5% v/v DMSO for 5 to 15 minutes during the initial equilibrium phase. The oocytes are subsequently subjected to a vitrification solution containing 0.5 M sucrose, 15% v/v DMSO, and 15% v/v EG. The oocytes are then kept in liquid nitrogen at −196°C after a brief incubation (≤ 1 minute). After gradually removing the CPA, the oocytes are promptly warmed to prevent the development of ice crystals, and then cultured in a culture medium until use [4, 47, 48]. Vitrification solution for embryos must be treated at a low temperature of 4°C [47]. One method for increasing the cooling rate and vitrification is to use liquid nitrogen vapor instead of liquid nitrogen only [49]. The study discovered that the vitrification method involving the use of only non-permeable CPAs for cryopreservation of abnormal sperm samples was an effective alternative to the vitrification method [44]. The differences between the vitrification and slow freezing method are shown in Table 1.
Characteristics
Vitrification
Slow freezing
Direct contact with liquid nitrogen
Yes
No
Ice formation
No
Yes
Time
Fast (minutes)
Slow (hours)
CPA equilibration
Yes
Yes
CPA concentration
High (over 40%)
Low (10–15%)
Cooling rates (°C/min)
15,000-30,000
0.15–0.30
Cost
Protocol-dependent (usually inexpensive)
Equipment-dependent (usually expensive)
Special equipment
No
Yes
Technical expertise
Risky
Simple
Routinely applied for cryopreservation of human ovarian tissue
No
Yes
Table 1.
Comparison of the characteristics between vitrification and slow freezing methods.
Semen is poured into 0.25 or 0.5 ml straws, placed on a rack, and frozen in liquid nitrogen vapor. The temperature of which should be determined by the height above the liquid nitrogen after dilution and cooling of the semen samples [21]. When utilizing a styrofoam box, the samples are placed on a rack that is suspended 3 to 4 cm above the liquid for 7 to 8 minutes before the straws are submerged into liquid nitrogen for storage [43]. Alternatively, the freezing height above the liquid nitrogen should be determined by the reported straw size [50]. For storage, it was recommended that 0.5 ml straws be frozen 4 cm over liquid nitrogen for 5 minutes, whereas 0.25 ml straws must be placed 16 cm over liquid nitrogen for 2 minutes, lowered to 4 cm for 3 minutes, and then submerged in liquid nitrogen [21].
4.4 Solid surface vitrification
Solid surface vitrification (SSV) is a cryopreservation technique applied to the preservation of embryos and oocytes. In this technique, which combines many others, oocytes are combined with CPAs and partially submerging a metal surface in liquid nitrogen to pre-cool it to −180°C, SSV employs the metal surface as a cooling template for microdrops of vitrification solution containing oocytes or embryos [51]. The SSV maximizes cooling rates, leaves ample room for tissue, and prevents the formation of gas phase liquid nitrogen bubbles [52]. This technique was initially created for use with mammalian oocytes [51]. To store the vitrified droplets in liquid nitrogen, they are put into a cryovial. Sperm can be stuffed into tiny capillaries in this experiment. The capillaries can be permitted to be exposed to the cryo-chilly chamber’s (−180°C) surface. The benefits of this approach include lessened DNA damage and lessened sperm tail damage [53].
The sperm quality after thawing is believed to be influenced by its pre-freezing qualities. The cryo survival rate of post-thawed sperm can also be impacted by pre-freezing semen quality factors, such as sperm motility and the abstinence time of sperm donors [54]. Moreover, sperm with aberrant motility characteristics (asthenozoospermia and oligoasthenozoospermia) are especially vulnerable to cryo-damage, which could lower their fertility [55]. The sperm plasma membrane’s lipid content has a significant impact on the sperm’s cryotolerance and cold sensitivity. The size, shape, and lipid composition of sperm from various species may vary, which could have an impact on how resistant they are to cryo-injuries [56, 57, 58].
Two competing theories attempt to explain why cryopreserving the gametes is harmful. (i) The creation of intracellular ice crystals during cryopreservation causes severe osmotic and physical damage to the sperm, which later reduced sperm functioning [34], and (ii) during chilling, ice crystals form inside the cells, and fatal increases in solute concentration occur in the remaining liquid phase. The cryopreservation and thawing procedure during cryopreservation creates oxidative, osmotic, and thermal pressures, which might compromise sperm quality and lead to a low fertility rate [33]. Despite advancements in cryopreservation technology, the rate of functional post-thawed sperm recovery remains low [59].
The cryo survival rate of oocytes cryopreserve with the use of the slow freezing method ranged from 74–90% [60]. Oocytes are prone to ice recrystallization episodes during storage and thawing [54, 61, 62]. During the thawing process, sperm and oocytes are vulnerable to metabolic damage caused by oxidative stress [63]. The cytoskeleton, lipid droplets, membrane system, and microtubules are the parts of the cell that are most impacted [49]. Oocyte cell membranes resemble female mammalian gametes visually and are much more vulnerable to the effects of cooling than embryonic and even zygotic membranes [63]. Oocyte membranes have a high melting point, making it easier for the lipids to be damaged by a drop in temperature and lose their ability to function as a membrane.
Transzonal processes, tiny microfilaments that keep the meiotic spindle in the proper position during maturation, are damaged by cryopreservation, which has an impact on communication between oocytes and the cumulus cells around them [64]. It was recently discovered that the gamete’s lipid and adenosine triphosphate content is influenced by the number of cumulus cells adhering to the oocyte [65]. The decreased survival and development rates of cryopreserved immature and in vitro matured oocytes are believed to be mostly caused by this [64].
The maintenance of fertility through semen/sperm and oocytes cryopreservation is a crucial component of assisted reproductive techniques, although sperm and oocytes functions may be negatively impacted by cryodamage to cellular constituents. However, the effectiveness of freezing sperm and oocytes can be improved by comprehending the cellular and molecular alterations. To optimize cryopreservation and thawing methodologies, increasing pregnancy rates in IVF cycles, better understand the role of oxidative stress in the lower developmental competency of cryopreserved gametes, and additional studies are required.
The extenders, mediums, kind of molecules, and concentration utilized for each species samples are all related to the varied effects of each antioxidant supplementation, which improves distinct measures of sperm quality. Antioxidants are increasingly common when semen is being cryopreserved because they may lessen oxidation. Certain antioxidants have been shown to have superior efficacy, while others have less encouraging outcomes. Antioxidant combination, extender concentration, and quality are just a few of the variables that might make adding antioxidants to semen extender during cryopreservation successful or unsuccessful. The development of more effective methods for cryopreservation of cells and the expansion of their clinical applications may be made possible by understanding the underlying chemistry and biology of freezing and thawing processes.
The Agricultural Research Council (ARC) is acknowledged for funding the running costs and providing the facility for the project, the Department of Agriculture Land Reform and Rural Development (DALRRD), South Africa is acknowledged for funding the running costs of this research. Agricultural Research Council, Animal Production, Germplasm Conservation and Reproductive Biotechnologies colleagues for their support.
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Written By
Masindi Mphaphathi, Mahlatsana Ledwaba and Mamonene Thema
Submitted: 23 July 2022Reviewed: 26 August 2022Published: 08 February 2023
Open access peer-reviewed chapter
