Bovine Sperm Cryopreservation: A Review

http://amresearchreview.com/index.php/Journal/about Volume 3, Issue 12 (2025) Online ISSN Print ISSN . . http://amresearchreview.com/index.php/Journal/about Page 202 Bovine Sperm Cryopreservation: A Review Muhammad Faheem Sarwar Department of Zoology, University of Gujrat, Gujrat, Punjab, Pakistan Saima Ashraf Department of Zoology, University of Sialkot, Sialkot, Punjab, Pakistan Bovine semen cryopreservation is a pivotal technique in modern animal husbandry, enabling the efficient preservation and distribution of valuable genetic resources for breeding programs worldwide. Advances in the existing cryopreservation techniques have increased sperm survival and fertility rates, but challenges remain. The efficacy of cryopreservation is measured by post-thaw sperm motility, membrane integrity, and sperm reproductive potential, which are all influenced by parameters such as initial semen quality, type and concentration of the cryoprotectant, cooling rates, and storage duration. The process of cryopreservation causes oxidative stress, osmotic changes, cold shock and cryodamage that alters sperm plasma membrane structure and incurs deleterious changes at the molecular level, leading to compromised sperm integrity. Ongoing research focuses on optimizing procedures, investigating alternate CPAs, and improving cryoprotection with antioxidants and other additions. These enhancements aim to improve the effectiveness of bull sperm cryopreservation, boost genetic diversity and productivity in cattle breeding operations, and facilitate biotechnological applications such as genetic engineering. Keywords: Bovine, Cryopreservation, Spermatozoa, Cryodamage, Artificial Insemination Introduction There is an increased demand to enhance the efficiency and sustainability of animal food production to meet the growing global population (Medeiros et al., 2002). Global beef production in 2023 was estimated at 364 million tonnes, of which 76 million tonnes is contributed by bovine only (Statista, 2023). However, increasing the efficiency of animal product production requires both the development of new technology and an understanding of the reproductive dynamics of the various livestock species (Yánez-Ortiz et al., 2022). One way to increase global food production is to use cryopreserved semen from genetically superior breeds and artificial insemination (AI) to produce high-yield livestock. AI is a key reproductive technology that produces genetically superior breeds to enhance genetic advancement and selection (Bailey et al., 2000). Successful semen cryopreservation enhances the efficiency and success rate of AI, which leads to improvement in the global livestock industry (Bailey et al., 2000). The process of semen cryopreservation dates back to the 17th century; however, the first successful artificial insemination was reported by Spallanzani in dogs in 1784 http://amresearchreview.com/index.php/Journal/about Volume 3, Issue 12 (2025) Online ISSN Print ISSN . . http://amresearchreview.com/index.php/Journal/about Page 203 (Sharafi et al., 2022). A major improvement in the AI industry was witnessed after Polge reported that glycerol can facilitate cell preservation at low temperatures and could be used as a permeating cryoprotectant (Polge et al., 1949). Since the discovery of glycerol as a cryoprotective agent (CPA), significant progress has been made in developing protocols based on a variety of other CPAs for the optimal cryopreservation of bovine sperm (Foote, 2002). Sperm cryopreservation is crucial for livestock production since it allows for the rapid dissemination of genetic variety and the global dispersion of genetically superior animals; however, this process is very challenging for the sperm and affects its postthaw viability (Yánez-Ortiz et al., 2022). Sperm encounter physiological and structural obstacles such as osmotic imbalance, oxidative damage, and the development of intracellular ice crystals during cryopreservation (Ugur et al., 2019). These challenges lead to the death of approximately half of the sperm population (Sharafi, 2022), which is very concerning for the AI industry that aims to meet the global need for superior-quality genetics. Sperm cryopreservation treatments may be inefficient due to significant physiological damage suffered by a large number of sperm, resulting in reduced fertility after freezing and thawing. Therefore, new methods are being developed, and cryoprotectants are being explored to improve the survival of sperm during cryopreservation (Nijs et al., 2009). Cryopreservation of the sperm is still a very challenging task due to the complex physiological nature of the sperm membrane and structure. There have been studies where rapid and slow cooling have been tried, but both methods have certain pros and cons. In both of these methods, cell membrane plays a crucial part, as the water permeability impacts intracellular events and is the primary site of injury (Mazur et al., 1972). Cells can be damaged during cryopreservation by exposure to excessive salt concentrations and the formation of ice crystals internally (Mazur, 1970). Rapid cooling results in ice crystal growth within a cell, which is damaging and potentially hazardous because it can physically disrupt the cell structure and cause it to cease its normal function (Mazur, 1970). Mazur states that reducing the cooling rate can help prevent ice development within the cell (Mazur et al., 1972). Slow cooling, on the other hand, can cause damage from prolonged exposure to high salt concentrations in the solutions surrounding the cell, causing cell dehydration and volume contraction (Ba-Awadh et al., 2023). Sperm cryopreservation is a challenging process because ice crystal formation poses a greater risk than sperm dehydration, affecting the success of sperm freezing. (Ba-Awadh et al., 2023) To reduce intracellular ice crystal formation and sperm dehydration, the optimal freezing process should be gradual and quick. These are the reasons that the cryopreservation of sperm is a challenging task, and there is still a need to optimize these protocols to take sperm cryopreservation to an optimal state (Ba-Awadh et al., 2023). Methods of cryopreservation Two traditional freezing techniques employed in sperm cryopreservation are slow freezing and vitrification (Behrman & Sawada, 1966). http://amresearchreview.com/index.php/Journal/about Volume 3, Issue 12 (2025) Online ISSN Print ISSN . . http://amresearchreview.com/index.php/Journal/about Page 204 Slow freezing Slow freezing is time-consuming and can span a duration of 2–4 hours, depending upon the specific species being targeted and necessitates the utilization of either a freezer or a programmable freezer (Silva et al., 2019). Although manual procedures have demonstrated successful sperm freezing, the reproducibility of this procedure may present certain challenges. Due to this reason, many researchers have conducted investigations on programmable freezers (Holt, 2000). The freezers employ a plate as a means of containing the semen straws, which are subsequently cooled by liquid nitrogen contained within a storage tank positioned beneath the plate. The semen straws are cooled gradually at different cooling rates to achieve a chilling process from 20°C to -80°C. Once the freezing is finished, the straws are extracted and stored in liquid nitrogen at -196°C (Santo et al., 2012). Several researchers contend that traditional slow freezing, whether performed manually or automatically, results in significant chemical-physical harm to the sperm, perhaps due to the formation of ice crystals (Santo et al., 2012). Vitrification Vitrification is a method that results in the solidification of living cells into a glasslike state without the creation of ice crystals during cooling (Kuleshova & Lopata, 2002). The traditional vitrification approach needs a rapid cooling rate and a high concentration of CPA (Vutyavanich et al., 2010). The conventional vitrification process involves the addition of CPA to the extender media cooling the straws on liquid nitrogen vapours at -80°C and then plunging them directly into liquid nitrogen at -196°C. As the process of vitrification involves the inclusion of a high concentration of CPA, it may lead to metabolic alterations and fatal osmotic damage (Vutyavanich et al., 2010). Vitrification of bovine semen has been documented to produce a significant number of spermatozoa that are non-viable and lack motility (Baiee et al., 2020). The dose of cryoprotectants that cells may tolerate during vitrification has a biological limit, and therefore, the goal of any vitrification strategy is to accelerate temperature change while maintaining the lowest feasible concentration of cryoprotectant (Liebermann et al., 2003). Cryopreservation damage in sperm The cryopreservation process incurs significant damage to sperm integrity due to the formation of intracellular ice crystals, oxidative stress, osmotic shock, and other factors. These factors lead to sperm plasma membrane damage, alterations in lipidome and proteome, DNA fragmentation, degradation of mRNA, mitochondrial dysfunction, etc. (Ugur et al., 2019). Cryopreservation also causes organelle damage, leading to spermatozoa with morphologic abnormalities, aberrant acrosomes, altered mitochondria, decreased ATP production, cellular integrity, viability, motility, and fertility (Gillan et al., 2005; Nishizono et al., 2004; Upadhyay et al., 2021). Efforts have been made to enhance freezing media and protocols, primarily with regard to the addition of cryoprotectants, it is important to remember that the quality of frozen-thawed sperm primarily depends on their ability to tolerate temperature changes without losing their primary functions (Sieme et al., 2008). http://amresearchreview.com/index.php/Journal/about Volume 3, Issue 12 (2025) Online ISSN Print ISSN . . http://amresearchreview.com/index.php/Journal/about Page 205 Figure 1: Cryopreservation-induced molecular and structural modifications in sperm Damage to sperm plasma membrane The majority of cryodamage in sperm is attributed to the structural integrity of the plasma membrane and is, therefore, connected to the composition of the plasma membrane (De Leeuw et al., 1993). Cryotolerance in bull spermatozoa is influenced by membrane structural characteristics such as cholesterol/phospholipid ratio, fatty acid mapping, hydrocarbon chain saturation, protein/phospholipid ratio, protein composition, and expression levels in seminal plasma (Esmaeili et al., 2015; Parks & Lynch, 1992; Ugur et al., 2019). For instance, certain phospholipids enhance the fluidity of the cell membrane, whereas cholesterol contributes to stability, which appears to enhance the ability of sperm to withstand freezing damage (Sharafi et al., 2022). Membranes having a lower cholesterol-to-phospholipid ratio, along with an asymmetrical distribution of cholesterol, appear to possess greater susceptibility to damage (Holt, 2000). The process of cryopreservation also causes elevated ROS production that result in increased lipid peroxidation disrupting the normal membrane function (Ugur et al., 2019). http://amresearchreview.com/index.php/Journal/about Volume 3, Issue 12 (2025) Online ISSN Print ISSN . . http://amresearchreview.com/index.php/Journal/about Page 206 Figure 2: Schematic representation of bovine sperm, sperm plasma membrane, phospholipid molecule and lipid class structures. Damage at molecular level Freezing and thawing during cryopreservation harm sperm DNA integrity, making it more susceptible to molecular and epigenetic alterations that influence embryo development (Lewis & Aitken, 2005). This detrimental process has been found to produce chromatin instability, resulting in DNA fragmentation for boar and avian sperm (Fraser & Strzezek, 2007). DNA damage is likely associated with many mechanisms that occur during cryopreservation, including double-strand breaks caused by high amounts of ROS generation (McCarthy et al., 2010), mechanical stress on the genomic regions of DNA molecules, where cell shrinkage causes chromatin compaction to increase (Kopeika et al., 2015). Epigenetic factors, including protamine, DNA methylation, and histone modifications, play key roles in spermatogenesis are also affected by the process of cryopreservation (Teperek et al., 2016; Ugur et al., 2019). Furthermore, epigenetic factors affect gene expression, which is dynamically regulated during cryopreservation (Zeng et al., 2014). The amount of sperm RNA is easily influenced by freezing-thawing cycles, and some degree of these RNAs is stable (Ugur et al., 2019). Cryodamage can also cause degradation of mRNAs (Giaretta et al., 2017), disrupting protein function and expression levels of fertility-related proteins (Kashir et al., 2011). To protect the sperm from these harms during cryopreservation, special chemical compounds known as cryoprotectants (CPAs) are added to the semen extender. Other than CPAs, the general recipe of semen extenders includes energy sources, antioxidants, antibiotics, etc. (Ugur et al., 2019). Cryoprotectants The crucial aspect for sperm is to retain its functionality in an environment that must offer optimal environment, including optimal pH, energy, temperature, and osmolarity to prevent physical harm. Therefore, cryopreservation media containing CPAs is added to ejaculated semen and helps reduce ice crystal formation and cold shock that cause damage to sperm during cryopreservation (Abdelhafez et al., 2009). Cryoprotectants are categorized into two types: permeating CPAs and non-permeating CPAs. Permeating CPAs possess high water solubility at low temperatures and have the ability to readily permeate biological membranes, and ideally, exhibit minimum toxicity (Wolkers, 2021). Non-permeable CPAs do not penetrate within the cell and thus exhibit their protective effects outside. They are usually bigger and covalently bonded as polymers, dimers, or trimers (Bartolac et al., 2018). Permeable cryoprotectants: Permeating cryoprotective agents like glycerol, ethylene glycol and dimethyl sulfoxide (Me2SO4) are recognized for their protective effect based on colligative characteristics (Mazur et al., 1972). Permeating cryoprotectants (Holt, 2000) have an amphiphilic nature and tiny size (usually fewer than 100 daltons), enabling them to easily pass through cell membranes and enhance the reorganization of membrane lipids and proteins, increase membrane fluidity, and promote higher dehydration at http://amresearchreview.com/index.php/Journal/about Volume 3, Issue 12 (2025) Online ISSN Print ISSN . . http://amresearchreview.com/index.php/Journal/about Page 207 lower temperatures, thus improving cryo-survival (Holt, 2000). Permeating cryoprotectants interact strongly with water through hydrogen bonding, the freezing point of water decreases, and fewer water molecules are present to form key nucleation sites necessary for crystal formation (Whaley et al., 2021). Glycerol largely enhances the colligative characteristics of a solution and decreases the salt content upon freezing (Lovelock, 1953). Glycerol molecules permeate lipid bilayers and induce modifications in the diffusion rates of electrolytes across the membrane, including Na+ and K+. These changes lead to the osmotic shrinkage of sperm cells, allowing them to withstand low temperatures. Despite the lower permeability of glycerol relative to other cryoprotectants, several effective cryopreservation methods nevertheless incorporate glycerol (Seki et al., 2007). In swine, glycerol and trehalose significantly increased acrosome integrity in comparison to glycerol (Gutiérrez-Pérez et al., 2009). Although glycerol has numerous advantages as a CPA, its toxicity in high concentrations is the subject of some debate. Glycerol in higher quantities is regarded to be damaging in numerous aspects of cell function (Hammerstedt & Graham, 1992). High concentrations of glycerol affect the polymerization and depolymerization of α and β tubulins, the main microtubule proteins in the sperm tail (Alvarenga et al., 2005). Increased glycerol content in thawed pig sperm reduces plasma membrane fluidity (Buhr et al., 2001). An increased concentration of glycerol alters the characteristics of F-actin, a globular protein in the cytoskeleton (García et al., 2012). One way to lower glycerol toxicity is to substitute glycerol with amides. Amides are a type of cryoprotectant; they cause less harm to sperm during cryopreservation in stallions because of their smaller molecular weight (Alvarenga et al., 2005). Substituting glycerol with dimethylformamide and methyl formamide improved the acrosome integrity, mitochondrial membrane potential, motility, and vitality of horse sperm (Wu et al., 2015). Moreover, a mixture of methyl formamide and glycerol enhanced the integrity and movement of the plasma membrane in cattle sperm (Akhtar et al., 2022). Among the other major permeating CPAs, ethylene glycol has been used as a substitute for glycerol in many species. Ethylene glycol has shown superior cryoprotectant properties compared to glycerol in bovine spermatozoa (Guthrie et al., 2002). A major advantage of this CPA is that it permeates the sperm membranes more rapidly, leading to decreased hydraulic conductivity and subsequently reduced osmotic stress on cells during cooling and freezing (Gilmore et al., 1995; Phelps et al., 1999). Research indicates that ethylene glycol has a less harmful impact on the survival and movement of sperm cells, offering superior protection to the acrosome compared to glycerol (Ball & Vo, 2001). Ethylene glycol has been successfully used as a cryoprotectant in the cryopreservation of dog (Swelum et al., 2011) and horse (Mantovani et al., 2002) spermatozoa. Dimethyl sulfoxide (Me2SO4) is another widely used CPA, which is an amphipathic molecule that dissolves in both aqueous and organic solutions (Santos et al., 2003). Dimethyl sulfoxide was used to freeze the first human oocytes, which led to a live birth after thawing (Chen, 1986). Dimethyl sulfoxide is believed to impact cellular permeability by influencing membrane dynamics in a concentration-dependent manner. Me2SO4 at low doses (5%) is believed to reduce membrane thickness, thereby http://amresearchreview.com/index.php/Journal/about Volume 3, Issue 12 (2025) Online ISSN Print ISSN . . http://amresearchreview.com/index.php/Journal/about Page 208 enhancing membrane permeability (He et al., 2012). 4.2 Non-permeable cryoprotectants In the case of non-permeable CPAs, they are unable to pass through the cell membrane; instead, they cause water to move out of the cell and increase concentrations outside the cell (Sutton, 1992). Commonly utilized non-permeable CPAs include sugars like trehalose, glucose, and galactose (Whaley et al., 2021). Trehalose is synthesized by a diverse range of species, such as bacteria, fungi, yeast, insects, plants, and certain invertebrates (Elbein et al., 2003). It has been shown to help these organisms survive freezing, among its several roles. The structure of trehalose consists of two glucose units connected by an α-1,1-glycosidic bond. The acetal bond in each monomer avoids the decrease of C-1, enhancing stability at high temperatures and reducing vulnerability to acid hydrolysis in low-pH environments (Whaley et al., 2021). Trehalose as a CPA is expected to affect membrane fluidity by integrating into the phospholipid bilayer, making the membrane more stable during freezing (Aboagla & Terada, 2003). It was reported that trehalose can decrease cryocapacitation and sustain acrosomal integrity (Reddy et al., 2010). It has also been reported that trehalose reduces acrosomal damage and improves membrane fluidity during cryopreservation, consequently impacting fertility (Ahmad & Aksoy, 2012). Trehalose was found to improve membrane integrity in bulls when added to the semen extender (Hu et al., 2010). Glucose is another widely used non-permeating CPA that also serves as an energy source for gametes and embryos and is often present in culture media in millimolar quantities (Fuller et al., 2005). In bovine spermatozoa, the low concentration of nonmetabolizable glucose analogue, 3-O-methylglucose, improved the post-thaw motility (Bhat et al., 2020). In another study, glucose in the extender media, along with glycerol, improved the sperm quality parameters in bull spermatozoa (Awad, 2011). In boar spermatozoa, extender media containing a relatively higher concentration of glucose resulted in higher post-thaw motility, making glucose a good candidate as a CPA in boar sperm cryopreservation (Reyes et al., 2002). Table 1: Commonly used CPAs in sperm cryopreservation. Permeable CPAs Species Reference Glycerol Porcine (Gutiérrez-Pérez et al., 2009) Ethylene glycol Bovine (Guthrie et al., 2002) Dimethyl sulfate (Me2SO4) Human (Chen, 1986) Dimethyl sulfoxide (DMSO) Bovine (Taşdemir et al., 2013) Formamide Equine (Alvarenga et al., 2005) Acetamide Porcine (Yoshino et al., 1993) Methanol Equine (Bass et al., 2004) Butylene glycol Bovine (Suzuki et al., 1993) http://amresearchreview.com/index.php/Journal/about Volume 3, Issue 12 (2025) Online ISSN Print ISSN . . http://amresearchreview.com/index.php/Journal/about Page 209 Adonitol Human (Alvarez & Storey, 1993) Non-permeable CPAs Trehalose Bovine (Hu, Zan, et al., 2010) Glucose Bovine (Bhat et al., 2020) Galactose Equine (Swain & Smith, 2010) Polyethylene glycol Bovine (Ohboshi et al., 1997) Raffinose Equine (Swain & Smith, 2010) Hyaluronan Porcine (Peña et al., 2004) Arabinose Equine (Swain & Smith, 2010) Polyvinylpyrrolidone Bovine (Checura & Seidel, 2007) Lactose Bovine (Swain & Smith, 2010) Among sugars, galactose is also used as a non-permeating cryoprotectant(Swain & Smith, 2010). Galactose has been reported to hinder ice recrystallization during freezing/thawing, showing minimum toxicity at physiological temperatures, and can alleviate osmotic stress (Chaytor et al., 2012). Extender media As discussed earlier, sperm faces many challenges, including variable pH (Liu et al., 2016), elevated ROS production (Amin et al., 2018), media toxicity (Ghaniei et al., 2019), osmotic imbalance (Dorado et al., 2019), sperm membrane damage (Layek et al., 2016) etc., during cryopreservation. Researchers have developed a variety of extender media to ensure sperm resilience during chilling and freezing procedures (Baiee et al., 2018). Extenders preserve sperm maintain motility and fertility throughout time by stabilizing the membrane, providing energy substrates, preventing the negative effects of pH and osmolarity fluctuations (Foote & Leonard, 1963). Currently, multiple extenders use a variety of material sources, including animal sources, skimmed milk, egg yolk (Filho et al., 2009), and soybean lecithin (plant source) (Layek et al., 2016), which offer different characteristics and issues depending on the type of sperm extender and species (Bustani & Baiee, 2021). Some widely used extender media in bovine sperm cryopreservation are discussed here. Egg yolkolk-based extenders Egg yolk is an effective cryoprotectant for sperm cryopreservation at varying doses in different species (Abdel-Khalek, et al., 2018). The exact mechanism by which egg yolk protects sperm during freezing is still unknown (Moussa et al., 2002) however, it is suggested that low-density lipoproteins (LDL) in egg yolk can protect spermatozoa against cold shock and improve sperm motility (Graham & Foote., 1987). Egg yolk is one of the most commonly used extenders for preserving bull sperm because it can protect against membrane damage (Purdy, 2006; Qureshi et al., 2014). Poultry egg yolk can be utilized in sperm extenders; however, some research suggests that other animal egg yolks, such as quail (Bustani & Baiee, 2021), turkey and pigeon (Akhter et http://amresearchreview.com/index.php/Journal/about Volume 3, Issue 12 (2025) Online ISSN Print ISSN . . http://amresearchreview.com/index.php/Journal/about Page 210 al., 2018), and ostrich (Naz et al., 2019), can also be used. Furthermore, researchers discovered that quail egg yolk improved sperm motility and membrane integrity in bull semen better than other bird egg yolks (Akhter et al., 2018). The predominant component of yolk plasma is LDL, followed by livetins (Alexandra, 2013). Livetins are serum proteins composed of albumin, α-2-glycoprotein, and immunoglobulin Y (IgY) (Schade & Chacana, 2007). Egg yolk also contains phosvitin, which is a highly phosphorylated protein with antibacterial and antioxidant effects (Anton et al., 2007). Generally, the percentage of egg yolk in egg yolk-based extenders is 20% (AmiratBriand et al., 2010; Layek et al., 2016). Tris-buffered egg yolk extenders with fructose and glycerol maintain animal sperm fertility at high extension rates (Bustani & Baiee, 2021). However, the use of egg yolk comes with a number of drawbacks. As the egg yolk is an animal-derived ingredient, it poses a substantial danger of microbial contamination of the semen, necessitating the use of antibiotics in egg yolk-based semen extenders. Egg yolk contains granular components that threaten the maintenance of the quality of spermatozoa and interfere with a variety of other metabolic processes (Layek et al., 2016). Egg yolk is an incredibly complex substance whose composition varies across batches and changes with diet, and these variables make it impossible to have constant quality and composition in egg yolk (Moreno et al., 2013). Thus, complete egg yolk has some sperm toxicity and interferes with laboratory testing. For these reasons, two significant plant-derived replacements for entire egg yolk have shown potential: soy lecithin and crude soymilk (Singh et al., 2012). In addition, some researchers have shown that egg yolk toxicity can be minimized by lowering the content of granular and high-density material in the media using clarification-repeated centrifugation at low temperatures (Amirat et al., 2005). Milk-based extenders Milk has been frequently used to cryopreserve mammalian sperm, usually mixed with arabinose, fructose, or egg yolk (Barbas & Mascarenhas, 2009). Skim milk proteins can buffer semen pH and may also bind heavy metal ions (Perea et al., 2017). Lactose, an important milk ingredient, is hydrophilic and cannot disperse the sperm cell membrane, hence protect the cell wall and prevent freezing shock (Namdeo Tukaram et al., 2010; Perea et al., 2017; Rahman et al., 2018). The protective component of milk is most likely micelles of caseins, the main proteins in milk. Caseins (α, β, and κ) are found in milk proteins as heterogeneous colloidal particles called casein micelles (Bergeron et al., 2007). Casein micelles are composed of a hydrophobic core of α and β caseins, surrounded by κ caseins (Dalgleish, 1998). It has been demonstrated that casein micelles extracted from milk can protect bull sperm while stored at 4-5°C (O’shea & Wales, 1966). Furthermore, casein micelles can protect bull sperm during freezing in the presence of glycerol (Bergeron et al., 2007). Plant-based extenders Plant-based semen extenders, which are usually produced from soybean lecithin, are easier to modify and are more stable in composition than animal-derived extenders (Mousavi et al., 2023). Soybean lecithin, an alternative to egg yolk, has been created and commercially used for sperm preservation (El-Sisy et al., 2016). Keeping in view http://amresearchreview.com/index.php/Journal/about Volume 3, Issue 12 (2025) Online ISSN Print ISSN . . http://amresearchreview.com/index.php/Journal/about Page 217 (2016). The role of antioxidants in sperm freezing: a review. Cell and Tissue Banking, 17(4), 745–756. https://doi.org/10.1007/s10561-016-9566-5 Amin, B. Y., Prasad, J. K., Ghosh, S. K., Lone, S. A., Kumar, A., Mustapha, A. R., Din, O., & Kumar, A. (2018). Effect of various levels of dissolved oxygen on reactive oxygen species and cryocapacitation-like changes in bull sperm. 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