The role of environmental optimization for storing bulls’ sperm cells

ABSTRACT

Artificial insemination has achieved a dynamic increase in genetic progress, and this is due to the improvement of sperm preservation technology. In recent years, a lot of attention has been paid to optimizing bull sperm storage environment and objectifying methods of sperm quality analysis. This review presents bull sperm preservation methods and ways to modify their storage environment. The main purpose of sperm preparation for artificial insemination is to obtain sperm with a high percentage of viable, motile sperm with normal morphology and low DNA fragmentation rates. Currently conducted experiments indicate the possibility of improving the quality of insemination doses produced using various components enriching common diluents. However, despite extensive research, no better results have been achieved than obtaining insemination doses with sperm viability that exceeds just over 60%. Obtaining a very good quality of frozen semen seems to be still unachievable today.

KEYWORDS:

• Bull
• environmental optimization
• storing
• sperm quality

Introduction

Artificial insemination (AI) remains the main driving force behind the rapid incorporation of desirable cattle genes around the world and the creation of their reserves. AI has achieved a dynamic increase in genetic progress, and this is due to the improvement of sperm preservation technology. In recent years, significant attention has been paid to optimizing bull sperm preservation environment (Kumar et al. 2013; Adami et al. 2018; Ugur et al. 2019) and objective methods of sperm quality analysis (Alvarez et al. 2005; Brito 2016).

Bull sperm preservation

Preservation in a liquid form

In the first half of the twentieth century, many reports were published about the dilution of livestock semen, and a large part of this work came from the former Soviet Union (Anderson 1945). The adopted principle of the technology of confectioning sperm in a liquid state was to keep sperm viable for a long time. For semen to be useful for AI, its minimum storage period had to be 2 to 4 days to allow its transport and use in remote farms. This forced the development of a semen management technique consisting of lowering the storage temperature to 5°C, which resulted in a decrease in the rate of sperm metabolism, and contributed to their better viability (Royere et al. 1996). The discovery of the positive effects of various substances on sperm survival contributed to even better storage effects. In the first half of the twentieth century, sodium citrate was found to have adequate buffering properties that allow extending the life span of sperm stored at 5°C outside the male body (White 1956). Many buffers (Tris, TES, MES, HEPES, PIPES, MOPS and BES) provided good buffering capacity (Good et al. 1966). However, the Tris-based diluent has become widely used for testing in combination with egg yolk and glycerol (Davis et al. 1963a, 1963b). Although the use of chicken egg yolk was more popular, it was shown that skimmed cow’s milk and coconut milk also helped maintain the fertilizing potential of sperm stored in a liquid state at a reduced temperature (5°C) (Norman et al. 1958; Melrose 1962). Methods for preparing dairy diluents and their various combinations have been reviewed (Norman et al. 1958; Foote 1978). The fertilization capacity of bull sperm stored in phosphates, egg yolk, and autoclaved milk dilutions was also compared (Tyrda et al., 2017), but the results were similar for the three diluents. An important aspect of storing semen in milk-based diluents was the need to inactivate lactenins (an antistreptococcal substance of milk, highly toxic to bovine spermatozoa) by pasteurization at 90°C.

Preservation temperature is an important factor, and its optimal range is considered to be 18°C – 24°C (Shah et al. 2017). Storage at higher temperatures is less effective compared to the Preservation at 5°C (Foote and Bratton 1960; Bartlett and Van Demark 1962), therefore attempts were made to use other methods of inhibiting sperm metabolism such as pH reduction (Niki 1987) or use of nitrogen (Shannon 1965) to obtain satisfactory results.

Several papers also concern the differentiation of the gaseous composition of the air in which the semen was stored. In 1940, carbon dioxide was found to be a very effective inhibitor of sperm motility (Sharma and Agarwal 1996). Initial experiments showed that sperm motility was reversibly inhibited while spermatozoa were exposed to CO₂ for a short period of time. Prolonged exposure to this gas was toxic to sperm. Van Demark and Sharma (1957) proposed to make sperm ‘dormant’ using CO₂ as a method effective in maintaining bull sperm viability and fertilization for 6–7 days at room temperature. The first diluent developed on the basis of immobilizing spermatozoa using CO₂ was IVT (Illini Variable Temperature) (Van Demark et al. 1957), which additionally contained a mixture of salt, sugar, antibacterial agents, and 10% egg yolk. Bull semen diluted with IVT and stored at room temperature, maintained sperm fertilization potential for more than 3 days (Bartlett and Van Demark 1962). Subsequently, several diluents were developed using this concept to optimize sperm survival and extend their shelf life in the liquid state. By optimizing the concentration of bicarbonate (0.1 M) and high glucose content (0.067 M) in IVT, sperm motility was maintained at high levels (> 45%) for over 90 days at 5°C (Bartlett and Van Demark 1962).

Preservation in a frozen state

Thanks to the discovery of glycerol as an effective cryoprotective agent and the development of an appropriate freezing method, it became possible to keep sperm frozen for a very long time without losing their fertilizing potential (Polge and Rowson 1952; Pickett and Berndtson 1974). Since then, a completely new semen preservation system has been introduced, which is now widely used in practice, and allows the creation of genetic reserves of outstanding individual bulls when compared to liquid preservation. The sperm freezing axiom is that even with the best preservation techniques and all changes that have taken place over the years in improving freezing procedures, the best cell recovery after thawing is just over 50% of viable sperm (Johnson et al. 2000; Bailey et al. 2008).

The basic principles of freezing, such as semen dilution, the use of a cryoprotective agent, and the appropriate rate of freezing and thawing were developed quite early, and several authors reviewed the physical effects of the freezing process (Mazur 1963, 1965; Watson 1979; Wang et al. 1997). Cooling and freezing a cell suspension causes several changes in their internal and external environments in terms of the flow of water and other substances. The rate at which water travels plays an important role in determining the rate of cooling that should be optimized based on cell survival after thawing (Mazur 1963, 1965). The essence of freezing sperm is balancing, which aims to enable translocation of water, and thus reduce the harmful effect of ice crystal formation during the freezing and thawing process. The optimal cooling rate is from 80°C/min to 120°C/min (Willett and Salisbury 1942; Watson 1979; Wang et al. 1997).

Diluents for freezing sperm are generally based on a similar composition to those used for storage at room temperature. Most of them contain ionic or nonionic substances which should maintain osmolarity and act as a buffer; a source of lipoprotein or high molecular weight material to prevent oxidative stress, for example, egg yolk or milk; glycerol, propanediol or dimethyl sulfoxide (DMSO) used as cryoprotectants; glucose or fructose used as an energy source; and other additives such as enzymes and antibiotics recommended as a protective agent against oxidative stress (Phillips 1939). Cryoprotectants protect sperm from ice crystal formation, osmotic, and chemical stress. Such components can be classified into permeating and non-permeating, and both types of cryoprotectants are usually included in the extenders. Glycerol is the permeating cryoprotectant most commonly used in ruminants during sperm cryopreservation, while egg yolk is the non-permeating cryoprotectant. The main task of cryoprotectants is to contribute to the extraction of intracellular water using a difference in osmotic pressure, without penetrating the cell.

Based on the results of breeding trials presented in 1940, frozen semen in an egg yolk and sodium citrate (EYC) diluent (Phillips and Lardy 1940) were used. Egg yolk in combination with TRIS and sodium citrate became the most common ingredient of diluents for bull sperm freezing. The reason why citrate became a better ionic substitute than phosphate was its ability to inhibit oxidation of lactic acid (phosphate caused its accumulation) (White 1956).

Experiments on the protective effect of egg yolk soon led to research on specific components that may be responsible for cryoprotective effects, namely phosphatidylcholine or lecithin, phospholipids, lipid extracts, lipoprotein fractions and specific lipoproteins (Mayer and Lesley 1945; Blackshaw 1954; Martin 1963; Lanz et al. 1965; Gebauer et al. 1970; Bollwein et al. 2008). Among these authors, there was consensus that lecithin provides some sperm protection during oxidative stress and the freezing process. One of the main experimental problems with the use of lecithin is its insolubility in aqueous solutions. It forms an unstable suspension, in which it partially precipitates without vigorous stirring, and may break down to form lysolecithins, which are toxic to spermatozoa (Jones 1976).

In addition to the egg yolk diluent, cryoprotective agents are important for protecting spermatozoa against thermal stress. Currently, glycerol is one of the most widely used cryoprotective agents for freezing bovine semen. Conventional freezing methods in 0.25 or 0.5 ml straws are based on approximately 7% glycerol in citrate-yolk and dairy diluents (Pickett and Berndtson 1974, 1978; Rodriguez et al. 1975; Watson 1979, 1995; De Leeuw et al. 1993).

Most semen producing companies prepare their own semen diluents with minor modifications, tailored to their needs and purposes. Over the past few years, several commercial diluents have been available, which have often been used in scientific research (most often cited). They include:

1. Biladyl® (Minitub, Germany),

2. Triladyl® (Minitub, Germany),

3. Biociphos (IMV, L’Aigle, Frace),

4. BIOXcell® (IMV, L’Aigle, France),

5. Laciphos (IMV, L’Aigle, France),

6. Tris – Gibco BRL (Holland Genetics, Holland), and

7. CAPROGEN® (Livestock Improvement, New Zealand).

Sperm change during cryopreservation

It is well known that freezing and thawing processes damage spermatozoa, due to changes in temperature, induction of osmotic stress and formation of ice crystals (Samper et al. 1991) and (Cerolini et al. 2001). It is noteworthy that these alterations affect acrosomes, plasma membrane, mitochondria, ROS, and DNA integrity, and in consequence reduce sperm motility and viability (Chatterjee et al. 2001; Ho et al. 2002; Gillan et al. 2004; Dziekonska et al. 2009).

Cryopreservation also causes significant changes in the distribution or abundance of these proteins that act as ROS scavengers. The defense of spermatozoa against antioxidants essentially depends on the antioxidative capacity of seminal plasma (Martin-Hidalgo et al. 2019), which contains enzymatic (glutathione peroxidase, superoxide dismutase, and catalase) and non-enzymatic antioxidants (glutathione, pyruvate, urate, ascorbic acid, α-tocopherol, taurine, and hypotaurine); however, its protective effect against oxidative stress is significantly reduced when semen is diluted in an extender before cryopreservation (Bilodeau et al. 2000). Excessive generation of ROS during cryopreservation leads to major protein, lipid and carbohydrate changes in the sperm membrane due to the reduction of disulfide bonds between membrane proteins (Chatterjee et al. 2001), peroxidation of membrane phospholipids and modifications of the sperm glycocalyx (Pini et al. 2018).

The first structure exposed to the cryopreservation process is the sperm cell membrane (Bailey et al. 2000). In ruminants, this membrane is characterized by high levels of unsaturated phospholipids and low cholesterol. In turn, this low level reduces sperm resistance to the freezing and thawing process (Darin-Bennett and White 1977). During freezing, phospholipids are redistributed across the membrane, and some of them change from liquid to gel. Such a change leads to the separation of lipid phases (Grötter et al. 2019) and then to a disorder of the lipid-protein interaction necessary for the proper functioning of the membrane (Lemma 2011). Furthermore, some sperm surface proteins, as well as membrane proteins, are lost or displaced, resulting in loss of their function.

In addition to cell membrane damage, oxidative stress disrupts mitochondrial activity, promotes the outflow of intracellular enzymes, and impairs several axonal proteins, which leads to loss of sperm motility (Aitken 1995).

The cryopreservation process also leads to the modification of mitochondrial and glycolytic proteins, which may be partly responsible for the reduced motility of frozen/thawed spermatozoa due to the lack of ATP (adenosine-5ʹ-triphosphate) production. Studies have shown that the loss of sperm motility during freezing and thawing was associated with impaired mitochondrial activity (Kadirvel et al. 2009; Yoon et al. 2015; Kumar et al. 2016).

Cryopreservation of spermatozoa also results in damage to sperm DNA. Although the mechanisms associated with disruption of semen DNA integrity are still unclear, oxidative and mechanical stress appear to be the main causes during cryopreservation (Peris et al. 2007; Gürler et al. 2016). In addition, aside from DNA damage, changes in the relative abundance of RNAs, aberrant DNA methylation, abnormal histone modifications, or improper chromatin compaction in sperm due to alterations in the nucleoprotein structure could have a severe impact on fertilization or embryogenesis (Verma et al. 2014; Kumar et al. 2016; Ge et al. 2017).

Modification of diluent composition for bull sperm preservation

Over the last few decades of the dynamic development of cattle insemination, several diluents for bull sperm preservation have been tested (Pickett and Berndtson 1974, 1978; Dziekonska et al. 2009; Kaka et al. 2015; Singh and Sharma 2018). The diluents have various compositions, and their use in the production of insemination doses gives different results (Celeghini et al. 2008; Vera-Munoza et al. 2009). Insemination and breeding stations, which produce insemination doses, want to meet the market needs and satisfy the expectations of breeders, and that is why they are constantly examining new technologies and tools with which it will be possible to produce a straw with the semen of the highest quality (competitive to constantly emerging semen distribution stations).

This ‘pursuit of the ideal’ initiated the era of using various additives in bull semen diluents, which scientists from around the world are constantly testing for the possibility of using them in routine sperm production at insemination and breeding stations. These additives can be divided into 4 main groups: with antioxidant activity, improving sperm viability, positively affecting sperm motility and those that can potentially protect sperm DNA from damage.

Additives with antioxidant potential

The specific structure of the sperm cell membrane and the low level of antioxidants in the sperm cytoplasm makes them potentially susceptible to damage by free radicals (Bollwein et al. 2008). Antioxidants are the main defense against oxidative stress caused by free radicals (Shiva et al. 2011). There are two types of antioxidants: enzymatic and non-enzymatic (Kefer et al. 2009). During cryopreservation, osmotic stress is induced by changes in cell volumes due to the relocation of water and other substances passing through the sperm cell membrane, which generates reactive oxygen species (ROS) (Ball 2008). The generation of ROS, such as hydrogen peroxide (H₂O₂), superoxide anions (O₂⁻) and hydroxyl radicals (OH⁻) play a key role in sperm function during their capacitation, acrosome reaction and binding to pellucid zone (zona pellucida) (Agarwal et al. 2006).

Exposure to high concentrations of ROS causes the disruption of mitochondrial and plasma membranes, as well as the fragmentation of chromosomes and DNA, and leads to a decrease in sperm motility and viability (Taylor et al. 2009). One of the main symptoms of ROS-induced sperm damage is the oxidative attack of bis-allylic methylene groups of phospholipids of polyunsaturated fatty acids (PUFAs), which lead to lipid peroxidation (LPO) (Sharma and Agarwal 1996). Due to LPO, membrane permeability and fluidity are modified (Nagasaka et al. 2004), which results in irreversible loss of motility, leakage of intracellular enzymes, damage to sperm DNA, or difficulty in penetrating oocyte by sperm (Aitken et al. 2009). Under natural conditions, to neutralize the harmful effects of ROS, sperm and semen plasma with the help of many antioxidant systems function as ROS scavengers and prevent internal cell damage (Gadea et al. 2011). An imbalance between the presence of ROS and the antioxidant activity of sperm is one of the main causes of sperm damage (Wang et al. 1997; Ball 2008; Li et al. 2010).

Various compounds are added to the diluents to help the antioxidant enzyme system in the semen. One of them is vitamin E, which can break the covalent bonds that are formed between ROS and the side chains of fatty acids in cell membrane lipids (Jeong et al. 2009). Also, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), which is a water-soluble analog of vitamin E, has a great ability for scavenging free radicals (Mickle and Weisel 1992). Glutathione (GSH) (Lc-glutamyl-L-cysteinylglycine), which is the major non-protein thiol compound in mammalian cells (Atmaca 2004), participates directly in the neutralization of ROS (Gadea et al. 2011). Vitamin C (ascorbic acid) is a water-soluble antioxidant with low toxicity and high potency that can remove oxygen radicals (Niki 1987). Moreover, it neutralizes the effect of H₂O₂ on sperm DNA, processes inactive vitamin E and reduces lipid peroxidation (Sierens et al. 2002). Along with many others the following are often reported as antioxidant semen additives: L-cysteine (L-Cys) (Uysal and Bucak 2007b; Coyan et al. 2011), melatonin (N-acetyl-5-methoxytryptamine) (Jang et al. 2010; Ortiz 2011), selenium (Zhang et al. 2006; Thomson et al. 2009), herbal extracts of rosemary (Rosmarinus officinalis) (Daghigh-Kia et al. 2014), roseroot (Rhodiola rosea) (Yang et al. 2016).

Additives with a stabilizing effect on the plasma membrane

During cryopreservation, the formation of ice crystals changes the structure of the sperm cell membrane and impairs its function (Amann and Graham 1993; Lessard et al. 2000), resulting in a decrease in sperm viability (Wongtawan et al. 2006) and ultimately their death (Royere et al. 1996; Bailey et al. 2008). Cryopreservation affects the overall quality of sperm after thawing, causes particularly negative effects on motility and integrity of the sperm cell membrane (Royere et al. 1996; Lessard et al. 2000; Yoshida 2000). In order to increase the percentage of viable spermatozoa obtained after cryopreservation, it was proposed to supplement the diluents with additives showing a protective effect on the sperm cell membrane. These additives can be divided into natural and synthetic. The naturals are mainly planted extracts used both in animals and humans as semen additives.

One of the most popular is green tea extract (Camellia sinensis), whose use as an additive to a diluent for freezing sperm has a positive effect on maintaining the integrity of the sperm cell membrane (Mehdipour et al. 2016). The next additive of natural origin, widely used in research, is thyme extract (Thymus vulgaris). Vahedi et al. (2018) showed that the addition of 2 and 4 ml/dl thyme extract to the diluent improves sperm viability after thawing. Equally popular are Omega-3 acids, which are involved in regulating a cell membrane function, maintaining sperm viability and their fertilizing potential during cooling and freezing (Parks and Lynch 1992; Watson 2000; Medeiros et al. 2002). Kiernan et al. (2013) showed that palmitic acid (PA), alpha-linolenic acid (ALA) and oleic acid (OA) added to the diluent improve bull sperm motility and viability during cooling (for 7 days).

Synthetic additives with a positive effect on sperm viability are chloroquine diphosphate (Singh and Sharma 2018) and sodium pyruvate, which, in addition to its positive effect on the cell membrane, also has a protective effect on sperm DNA (Korkmaz et al. 2017). Soren et al. (2017) showed that iodixanol (2.5%) improves overall sperm viability and can be used as a cryoprotectant. Chuawongboon et al. (2017) found that antaxanthin (2 μM) reduces catalase and maintains sperm cell membrane integrity.

Additives improving the potential of sperm motility

Sperm motility is one of the most important parameters of sperm quality that determines successful fertilization. Spermatozoa must be highly motile to reach the fertilization site, and penetrate the pellucid zone (Hawk et al. 1978).

Cyclodextrins (5 mM) (Ezz et al. 2017) is one of the additives that support the maintenance of the normal lipid profile of the spermatozoa cell membrane and has a positive effect on the total motility of cryopreserved spermatozoa. These molecules have a high affinity for sterols in vitro, and if they are pre-loaded with cholesterol can insert cholesterol into cell membranes (Navratil et al. 2003). The sperm membrane cholesterol to phospholipid ratio has an important role in sperm resistance to cold shock-induced damage (Watson 1979), moreover, the cholesterol amount of the sperm membrane can be regulated by the use of cyclodextrins (Purdy 2006). The sperm from species characterized by lesser ratio of cholesterol to phospholipid are more prone to cold shock-induced damage than the sperm with greater cholesterol to phospholipid ratio (Parks and Lynch 1992; Watson 1995). Furthermore, the efflux of cholesterol from the membrane of sperm impairs signaling mechanisms that regulate the capacitation of sperm (Watson 1995, 2000), and sperm longevity is reduced. Soy lecithin is another commonly used additive with an effect on improving sperm motility (Mehdipour et al. 2016; Tarig et al. 2017; Singh and Sharma 2018). Positive effects of coconut water (Vale et al. 1997), curcumin (Shah et al. 2017), and sodium pyruvate (Korkmaz et al. 2017) on sperm motility have also been observed.

Additives preventing sperm DNA defragmentation

The main purpose of sperm preparation for artificial insemination is to obtain sperm with a high percentage of viable, motile sperm with normal morphology and low DNA fragmentation rates. These criteria affect the subsequent development of embryos and their high quality. Sperm DNA integrity affects the fertilization percentage (Tavalaee et al. 2009), embryo development and pregnancy (Aitken 1995; Collins et al. 2008), miscarriage rates (Zini et al. 2008) and abnormalities in offspring (Aitken et al. 2009). In animals, the development of tumors and the shorter life expectancy of their offspring are associated with the use of spermatozoa with high rates of DNA fragmentation (Fernández-Gonzalez et al. 2008; Pérez-Crespo et al. 2008).

Tvrda et al. (2017) reported the protective potential of lycopene against sperm DNA fragmentation. Other authors indicated that supplementation of the diluent with hyaluronic acid (Sarıözkan et al. 2015) and vitamin E (alpha-Tocopherol) (Adami et al. 2018) improved the overall percentage of sperm with intact DNA.

The effect of various additives on sperm functions

Many of the semen diluent additions listed in this review are characterized by multidirectional effects on sperm function/quality parameters. Table 1 presents some of them in relation to their impact on semen quality parameters.

Table 1. Selected agents used in cryopreservation of sperm and their impact on sperm quality.

Function	Agent	Effect on sperm cells quality parameters	References
Plasma membrane stabilizer	Egg yolk, skimmed cow’s milk coconut milk, plant extracts (green tee, thyme), Omega-3 acids, chloroquine diphosphate, sodium pyruvate, iodixanol, antaxanthin	Better viability, motility, and fertilizing potential, reduction of effects of oxidative stress	(Chuawongboon et al. 2017; Davis et al. 1963a; Davis et al. 1963b; Kiernan et al. 2013; Korkmaz et al. 2017; Medeiros et al. 2002; Mehdipour et al. 2016 Melrose 1962; Norman et al. 1958; Parks and Lynch 1992; Singh and Sharma 2018; Soren et al. 2017; Vahedi et al. 2018; Watson 2000)
Cryoprotectant	Glycerol, propanediol dimethyl sulfoxide	Better viability, motility, and fertilizing potential, reduction of effects of oxidative stress, reduction of morphological defects	(Watson 1995; Watson 1995; Rodriguez et al. 1975; Pickett and Berndtson 1978; Pickett and Berndtson 1974; De Leeuw et al. 1993)
Preventing DNA damage	Lycopene, hyaluronic acid, vitamin E	Preservation of DNA structure, better viability, motility, and fertilizing potential	(Adami et al. 2018; Sarıözkan et al. 2015; Tyrda et al., 2017)
Antioxidant	Vitamin E, vitamin C, L-cysteine, melatonin, selenium, herbal extracts of rosemary, roseroot	Protection from protein, lipid and carbohydrate changes in the sperm membrane, peroxidation of membrane phospholipids and modifications of the sperm glycocalyx, better viability, motility, and fertilizing potential, reduction of effects of oxidative stress	(Thomson et al. 2009; Uysal and Bucak 2007b; Yang et al. 2016; Zhang et al. 2006; Pini et al. 2018; Ortiz 2011; Niki 1987; Jeong et al. 2009; Jang et al. 2010; Daghigh-Kia et al. 2014; Coyan et al. 2011; Chatterjee et al. 2001)

Implications and cryopreservation of sperm cells

As shown in this review, there is currently a wide range of extenders that can be used during cryopreservation of semen in various species of ruminants (Purdy 2006; Barbas and Mascarenhas 2009; Layek et al. 2016; Lv et al. 2018; Allai et al. 2018; Ugur et al. 2019); however, they do not provide the same protection during freezing. That is why sperm susceptibility to freezing and their quality after thawing can be influenced by the type of cryoprotectants, antioxidants, and other ingredients contained in the diluent, as well as their concentration (Fernández-Santos et al. 2006; Mata-Campuzano et al. 2015; Ugur et al. 2019). Extenders usually have different compositions in terms of the buffer, antibiotics, fatty acids, sugars, cryoprotectants, antioxidants, and other substances to effectively protect the fertilizing potential of sperm during the freezing process (Allai et al. 2018).

Particular attention should be paid to the interaction of cryoprotectants with spermatozoa, as this interaction may affect the results of cryopreservation of sperm and the effectiveness of its fertilizing potential. Some of them, such as commonly used glycerol, have cytotoxic effects (McClean et al. 2006). In turn, egg yolk changes the sperm proteome before cryopreservation (Ramírez-Vasquez et al. 2019). The concentration of these substances in the extender seems to be key. Supplementing the diluent with antioxidants reduces the negative effects generated by excessive ROS production during cryopreservation (Uysal and Bucak 2007b; Reddy et al. 2010), however, as in the above case, the concentration of antioxidant additives should be taken into account, because as demonstrated by Mizera et al. (2019), higher doses of various substances may be toxic to sperm.

Understanding the molecular damage caused by this process and looking for methods to prevent or reduce changes in the structure or function of spermatozoa that negatively affect their fertilization potential is essential to protect sperm during cryopreservation. This seems to be particularly important in ruminants since most of these species are more susceptible to sperm damage during freezing and require significant improvements in the freezing and thawing process in order to obtain fertilization rates comparable to fresh semen.

On one hand, advances in proteomics, transcriptomic, and epigenomic technologies are constantly providing new information on the mechanisms underlying sperm cryocells and factors affecting cryotolerance of these cells. Nevertheless, despite extensive multidirectional research, several problems remain to be solved. To contribute to their resolution, future research should combine many technologies to simultaneously study changes in the level and location of sperm protein and their effect on sperm function. The focus of research around the combination of high-throughput mass spectrometry with multi-parameter, computational and imaging flow cytometry seems to be particularly interesting, which will provide a deeper insight into the molecular and cellular changes induced by the freezing and thawing process, facilitating the interpretation of data to improve sperm functionality and fertility of frozen semen.

On the other hand, extenders require further optimization to achieve better efficiency than before, mainly in breeding centers (according to the idea of science for practice). Contiunued research is needed with a broader range of concentrations or combinations of various new cryoprotectants, antioxidants and proteins, and improvement of the technological process itself, which is cryopreservation.

Conclusion

Current experiments indicate the possibility of improving the quality of insemination doses produced using various components enriching common diluents. However, despite extensive research, no better results have been achieved than obtaining insemination doses with sperm viability that exceeds 60%. It probably results from still little knowledge about the mechanisms and dynamics of changes in cell structures subjected to the cryopreservation process in this species. Obtaining a very good quality of frozen semen seems to be still unachievable today.

Author contributions

We declare that all authors made substantial contributions to this manuscript. Conception of the review and preparation of manuscript, text translation into English, editorial preparation: AK; collection of literature and preparation of the manuscript: EC-P; correction of the scientific coherence of the manuscript and preparation of the manuscript: MK.

Disclosure statement

The authors declare no conflict of interest.