Current perspectives on ruminant sperm freezability: Harnessing molecular changes related to semen quality through omics technologies

The recent advances in sperm cryopreservation transcend cryobanking and other assisted reproductive technologies. Since its discovery, cryopreservation has contributed positive impacts on animal breeding as well as in genetic exchange, improvement, and conservation efforts. However, cryoinjury and variabilities in cryopreservation outcomes remain as key challenges to sperm cryobiology. The present work explored the molecular bases for such freezability differences and freezing-thawing injuries in the ruminant sperm. Relevant biomarkers identified in the seminal plasma and the spermatozoa were highlighted, including lipids, proteins, metabolites, transcripts, and genes. Specific molecular mechanisms concerning sperm structures and functions were also examined relative to their association to cryotolerance, and spermiogram or seminogram modifications following cryopreservation procedures. Current conflicts and gaps in the knowledge base on ruminant spermatozoa were also emphasized. Further investigation of these areas using the available breakthrough molecular tools such as omics technologies is therefore proposed to improve, optimize, or even predict the overall quality of frozen-thawed ruminant semen towards reproductive efficiency.


INTRODUCTION
Over the years, substantial progress has been made toward the improvement of assisted reproductive technologies (ARTs) for domestic and wild animals. Sperm cryopreservation allowed for the long-term storage and utilization of male gametes from organisms with superior genetics (Ugur et al., 2019), declining conservation status, or even those at the brink of extinction (Pukazhenthi, 2016). Recently, the ruminant spermatozoa have been vitrified (Arando et al., 2017;Pradieé et al., 2018), and their freezing medium was supplemented with nanoparticles (Hozyen et al., 2020;Khalil et al., 2019) and other novel compounds (Batissaco et al., 2020;Fang et al., 2020). These approaches offer alternatives to conventional techniques and extender components to enhance cryosurvival and fertility in frozen-thawed semen.
Despite these developments, cryopreservation procedures compromise sperm quality and fertilizing ability. Spermatozoa are subjected to chemical, osmotic, mechanical, and thermal stresses during the dilution, cooling, equilibration, freezing, and thawing stages (Andrabi, 2009;Rasul et al., 2001). Even with optimized protocols, it is now generally accepted that only approximately half of the total sperm population (about 40%-50%) endure the freeze-thaw cycles (Grötter et al., 2019). Moreover, the cryosurvivability of spermatozoa differs within and between species and individuals. Cryopreservation outcomes still varied in different males with fundamentally similar spermiogram or seminogram results. Sperm from some animals with outstanding reproductive success in natural mating also exhibited poor freezing resilience (Gomes et al., 2020;Kumar et al., 2019;Rego et al., 2016).
The advent of functional genomics (lipidomics, metabolomics, proteomics, and transcriptomics) and epigenomics (chromatin dynamics and DNA methylation) (Khan et al., 2021;Peris-Frau et al., 2020;Ugur et al., 2019) revolutionized our current understanding of the complexities of reproductive biology, particularly that of sperm physiology. These have brought us closer than ever in elucidating the molecular changes in frozen-thawed spermatozoa relative to semen quality, as well as in unraveling the molecular factors involved in freezability differences, which were the main highlights of this review. Unless there is a compelling need to incorporate accounts from other species, discussions of cryoinjury and freezing resistance have primarily focused on ruminants. Moreover, the association of freezability with sperm structures and functions was also discussed in sections.

SPERM FREEZABILITY
During cryopreservation, the successive processes of temperature reduction, cell dehydration, freezing, storage, and finally thawing expose sperm to cryoinjuries, which affect cryosurvival (Ugur et al., 2019). These are generated, at least in part, by osmotic shock caused by solution effects on cell volume, mechanical disruptions from intracellular ice crystal formation, and thermal challenges from cold shock (Khan et al., 2021;Khalil et al., 2018). Deleterious changes to sperm architecture and functions (Figure 1)  Veterinary Integrative Sciences 489 such causes, including a breakdown of membrane permeability and fluidity, free radical production, cytoskeleton destabilization, ionic imbalance, DNA fragmentation, disturbance in macromolecular interactions, and protein, RNA, and epigenetic modifications. The ability of spermatozoa to maintain their structural integrity and functional competence following cryopreservation-induced stresses relates to their freezability, cryotolerance or cryoresistance (Martínez-Fresneda et al., 2021;Peris-Frau et al., 2020). In this context, males were frequently classified as either good or bad freezers. Semen samples were also phenotypically categorized into high and low freezing-resilient groups, using a variety of criteria, as shown in Table 1. These attributes utilized as sperm freezability determinants in ruminants include motility and velocity (Perumal et al., 2014;Rego et al., 2016;Ryu et al., 2019), viability (Gomes et al., 2020;Hitit et al., 2020), and morphometry (Esteso et al., 2006;Ramón et al., 2013;Ros-Santaella et al., 2014), with motility and viability being the most important indices for predicting cryoresistance and fertilizing ability.
A plethora of studies have identified candidate markers for semen freezability and fertility from genes, transcripts, proteins, and metabolites. Tables 2 and 3 listed some differentially represented proteins in the ruminant spermatozoa or semen with high and low freezability or motility, respectively. These proteins are involved with a wide array of cryosurvival functions, for instance, resistance to cold-shock as facilitated by the abundance of heat-shock protein (HSP90) (Wang et al., 2014), or tolerance to cryoprotective agents (CPAs) as enabled by the high expressions of cytosolic 5-nucleotidase 1B (NT5C1B) and fumarate hydratase (FH) (Song et al., 2020 Post-thaw sperm viability greater (high) or lesser (low) than the average of the sample population Holstein bull Gomes et al. (2020) Post-thaw motility of >60% (high) or <15% (low) Native Korean beef bull Ryu et al. (2019) Post-thaw motility of >50% (high) or <50% (low) Jersey crossbred bull Perumal et al. (2014) Post-thaw motility of >30% (high freezability) or <30% (low freezability) and sperm vigor of >3.0 (high) or <3.0 (low) Guzerat bull Rego et al. (2016) Pre-freeze and post-thaw motility difference, small ( Table 2 Proteins which were either found in greater abundance, or highly expressed in the spermatozoa or seminal plasma of ruminant species with high freezability (or motility) phenotypes

FREEZABILITY AND SPERM PLASMA MEMBRANE
The sperm's ability to survive in the female reproductive tract and subsequently bind and penetrate the outer oocytic investments is dependent on an intact and functional plasma membrane (Martí et al., 2003). However, its composition differs between animals, species, and even fertile and subfertile groups of similar species (Mandal et al., 2014), resulting in variations in sperm sensitivity to cryopreservation. The cholesterol to phospholipid and polyunsaturated to saturated phospholipid-bound fatty acid ratios in the plasma membrane impart for the spermatozoa's susceptibility or resistance to cold-shock through their contributions to membrane fluidity and stability at low temperatures (Mocé et al., 2010). As a result of their high unsaturated phospholipid and low cholesterol contents, the ruminant spermatozoa, therefore, are intrinsically very sensitive to cold shock compared to other animal species and humans (Bailey et al., 2000). Between domestic large ruminants, buffalo spermatozoa have been observed to be more vulnerable to freezing and thawing hazards than cattle spermatozoa (Andrabi, 2009). Contemporary lipidomics has established the possibility of predicting cryopreservation outcomes in the ruminant sperm. Quantities of saturated fatty acids like arachidic acid (22:0) and monounsaturated fatty acids such as oleic acid (18:1 cis9) differ between Holstein bulls of opposite freezabilities (Evans et al., 2020). This may also somewhat explain the higher plasma membrane integrity observed in good freezability phenotypes (Rego et al., 2016;Wang et al., 2014).
Greater degrees of cold shock suffered by the spermatozoa translate to greater damages to their plasma membrane (López Armengol et al., 2012). Table 3 Proteins which were either found in greater abundance, or highly expressed in the spermatozoa or seminal plasma of ruminant species with low freezability (or motility) phenotypes. (Cont.)
On the other hand, ROS, as redox signaling molecules, mediate reproductive processes such as sperm hyperactivation, capacitation, acrosome response, sperm-oocyte contact, and fertilization at appropriate physiological concentrations (Aitken, 1995;Gonçalves et al., 2010). Despite this, cryopreservation precipitates their elevated or even uncontrolled production in the spermatozoa (Chatterjee et al., 2001b;Gürler et al., 2016;Santiani et al., 2014). Kumaresan and co-workers (2017) employed the sperm ROS status, specifically that of hydrogen peroxide (H 2 O 2 ), in their development of a fertility prediction model in bull, taking advantage of the variations in values obtained from above-and below-average fertility animals. Moreover, during an experimental treatment with exogenous H 2 O 2 , motility was the major sperm function affected (Peris et al., 2007). Significant correlations were also demonstrated between the percentage of live H 2 O 2 -positive spermatozoa, and post-thaw viability and freezability in cattle ; however, more studies for other ruminant species are needed.
The stages of cryopreservation may also have an impact on sperm membrane or surface proteins. Dhanju et al. (2001) confirmed the gradual decrease in the sperm membrane protein content and quality, particularly in terms of number and molecular weight, throughout the duration of freezing. In addition, structural conformation, distribution, and abundance of sulf-hydryl-containing surface proteins in the bull sperm were also altered (Chatterjee et al., 2001b). Proteins associated with membrane stabilization and protection from cryostresses such as acidic seminal fluid protein (aSFP) (Jobim et al., 2004), binder of SPerm 1 protein (BSP1) (Magalhães et al., 2016;Singh et al., 2014), seminal plasma protein BSP-30 kDa (BSP5) (Gomes et al., 2020), 496 metalloproteinase domain-containing protein 2 (ADAM2) and metalloproteinase inhibitor 2 (TIMP-2) (Rego et al., 2016) varied between low-and high-freezing resilient ruminant spermatozoa, and thus may affect the membrane fusion events of fertilization.

FREEZABILITY, CAPACITATION AND ACROSOME REACTION
Capacitation and acrosome reaction are fundamental events in fertilization. However, the freeze-thaw cycles can induce their precocious initiation (Khalil et al., 2018;Srivastava et al., 2013). Both partially and fully cryopreserved spermatozoa were observed to undergo cryocapacitation and premature acrosome reaction, thus, affecting their longevity, survivability, and fertility (Kumaresan et al., 2017;Talukdar et al., 2016;Watson, 2000). The post-thaw stability of the acrosomal membrane was also found to be correlated with freezability and motility phenotypes, with high freezing-tolerant and motile sperm samples having greater acrosomal integrity (Capra et al., 2017;Hitit et al., 2020;Singh et al., 2014;Wang et al., 2014).
In addition to its role in cryotolerance (Amorim et al., 2009;Rajoriya et al., 2014), cholesterol also plays a pivotal function in membrane stabilization to prevent capacitation-like changes (Longobardi et al., 2017;Rajoriya et al., 2020). Nonetheless, cryopreservation allows frozen-thawed spermatozoa to bypass the true capacitation cascade through the considerable removal of cholesterol from the sperm plasma membrane (Kadirvel et al., 2009b;Rajoriya et al., 2016;Yadav et al., 2017). Greater efflux of cholesterol was noted in non-freezable ejaculates compared to their freezable counterparts . As a consequence, this process enhances membrane fluidity and permeability and promotes calcium influx (Minervini et al., 2013;Watson, 2000). Because of the difficulty in maintaining and regulating concentration gradients over time, this uncontrolled intracellular calcium surge may be deleterious during cryoinjury, impacting sperm cryosurvival (Bailey et al., 1994;Bailey et al., 2000).

FREEZABILITY AND SPERM APOPTOSIS
The freeze-thaw cycles also incite apoptosis-like events in spermatozoa. Proportions of apoptotic sperm rose after cryopreservation, though they were already existent before the procedure (Anzar et al., 2002;Khan et al., 2009;Martin et al., 2007;Martin et al., 2004). Nakidkina et al. (2019) underscored the likely involvement of apoptosis in poor quality semen, as it might be an attribute related to low motility, plasma membrane integrity (Khan et al., 2009) and fertilizing ability (Anzar et al., 2002) in ruminants. Nevertheless, the ambiguous role of apoptosis in the ejaculated sperm (Martí et al., 2008b) has been the subject of recent andrological studies in several other domestic animals.
Externalization of phosphatidylserine is a well-known apoptosis-related characteristic. Rather than being sequestered in the inner cytosolic leaflet of the sperm plasma membrane by ATP-dependent aminophospholipid translocases or flippases (Dalal et al., 2016), the phospholipid is translocated and expressed to the outer surface during the dilution, cooling/ refrigeration, and freezing-thawing procedures (Ahmad et al., 2015;Del Valle et al., 2010;Mendoza et al., 2013). On the contrary, Januskauskas et al. (2003) and Müller et al. (1994) found that phosphatidylserine remained undisturbed in the cytoplasmic surface, and the stability of the plasma membrane bilayer asymmetry was maintained in intact ram and bull cryopreserved spermatozoa despite diminished aminophospholipid translocase activity, respectively.
Another key event in programmed cell death is the activation of caspases (Martí et al., 2008b). These members of the cysteinyl aspartate-specific protease family have also been found in cryopreserved ruminant spermatozoa, mostly in the post-acrosomal region and intermediate piece (Martin et al., 2004;Mendoza et al., 2013). Following refrigeration or cryopreservation of ram and bull spermatozoa, there was a remarkable increment in the quantity of active executioner caspases-3 and -7 (Mendoza et al., 2013) and initiator caspase-9 (Martin et al., 2007), respectively. On the other hand, caspase activity decreased in cold-shocked ram spermatozoa (Martí et al., 2008b). While the above texts highlight the dependence of biochemical and molecular apoptotic cascade to the structural and functional state of the mitochondria (Nakidkina et al., 2019), other cryopreservation-induced alterations to the sperm organelle and their effects on freezability and other semen quality parameters will be discussed in detail in a later section.

FREEZABILITY AND SPERM CHROMATIN, DNA AND RNA
Because of its direct role in fertilization and subsequent embryonic development via haploid paternal genetic contribution, the integrity of the sperm DNA cannot be undervalued in reproduction. Ultrastructural examination of cryopreserved sperm revealed irreversible nuclear and chromatin damages (Khalil et al., 2018). Spermatozoa exhibiting DNA fragmentation and/or chromatin overcondensation also increased after freezing and thawing (Erickson et al., 2015;Gürler et al., 2016;Kumar et al., 2011;Peris et al., 2007). Correspondingly, abnormal morphology and low motility were linked to DNA and chromatin damage in buffalo and bull sperm, with implications on the cryosurvival of the spermatozoa (Januskauskas et al., 2001;Mahmoud et al., 2015). A high correlation between DNA and chromatin stability and semen fertility has also been proposed (Dogan et al., 2015;Khalifa et al., 2013;Kumaresan et al., 2017;Waterhouse et al., 2006) since DNA-nicked sperm appears to result in poor cleavage and blastocyst turn-over rates (Erickson et al., 2015).
Other than the fact that the specific mechanisms governing sperm DNA injuries are not yet completely elucidated (Gürler et al., 2016;Peris et al., 2007), opposing results from some studies further confound the present knowledge on the effects of cryopreservation on ruminant sperm DNA and chromatin, and their relationships with semen quality. According to Martin et al. (2004), cryopreservation did not affect DNA fragmentation and nucleus condensation, but Kadirvel et al. (2009b) indicated that sperm DNA damage was only influenced by liquid storage rather than by the freezing and thawing procedures. Additionally, Dogan et al. (2013) claimed that the association between fertility and DNA damage in cryopreserved spermatozoa was lacking. Nevertheless, pieces of evidence point to lipid peroxidation, oxidative stress and ROS production as bases for DNA and chromatin integrity loss during cryopreservation Peris et al., 2007), as previously expounded.
With the emerging relevance of epigenetics on sperm functionality, the relationship of aberrant DNA methylation (formation of 5-methylcytosine from Veterinary Integrative Sciences 499 the covalent addition of a methyl group to carbon five of cytosine) with DNA fragmentation has recently been established in ram spermatozoa (Pool et al., 2020). However, when compared to humans and other animal species (Aurich et al., 2016;Khosravizadeh et al., 2020), there is a paucity of information on the role of cryopreservation in irregular DNA methylation in ruminants. Some researchers have associated global methylation to semen parameters such as motility, morphology (Capra et al., 2019;Pool et al., 2020) and fertility (Kropp et al., 2017;Verma et al., 2014), implicating its potential role in ruminant sperm cryosurvival and overall quality. Regarding freezability, Capra et al. (2019) determined several differentially methylated genes functioning in chromatin arrangement in both high and low motility bovine sperm populations. These consist of histone lysine demethylases 2A (KDM2A), histone lysine methyltransferases 2A (KMT2A), telomerase-associated protein 1 (TEP1), telomerase reverse transcriptase (TERT), nuclear receptor-binding SET Domain 2 (NSD2)/ multiple myeloma SET domain (MMSET)/ Wolf-Hirschhorn syndrome candidate-1(WHSC1), among others.
Protamination (the stepwise replacement of nuclear histones into transition proteins, and finally into much smaller and highly basic protamines) is one of the distinct epigenetic facets of sperm cells that act in the well-organized packaging of DNA through adequate chromatin compaction (Champroux et al., 2018;Gosálvez et al., 2011;Jenkins et al., 2012). Freezing and thawing affect sperm protamine levels, leading to reduced PRM2 and PRM3 transcripts and proteins in cryopreserved cattle spermatozoa (Lv et al., 2020). This deficiency could result in DNA fragmentation and low fertility in ruminants (Boe-Hansen et al., 2018;Dogan et al., 2015;Fortes et al., 2014;Pool et al., 2020), aside from its relationship to sperm concentration, mass activity and morphology. Surprisingly, despite comparable protamine deficits in spermatozoa of contrasting freezabilities, a notable association between the variables existed . Moreover, spermatozoa of lesser and greater motilities also showed insignificant differences in PRM1 and PRM2 differential expressions. However, PRM2 amino acid sequence changes that result from nucleotide base modifications (G35A, A49G and A64G) had a detrimental impact on sperm motility metrics (mass, initial progressive, and post-thaw) .
The chilling injury to the mitochondria causes mitochondrial permeability transition (MPT), as examined by Treulen and colleagues (2018) using bovine sperm as a model. Dramatic reductions in the sperm mitochondrial membrane potential (MMP) ensue from this change. These then cause detrimental repercussions to the other physiological aspects of the spermatozoa, including decreased motility, viability, and fertility, and thus, may impinge on cryoresistance and semen quality (Castro et al., 2016;Martin et al., 2007;Martin et al., 2004;Shah et al., 2017). The functional state of the mitochondria also differed considerably between high-and low-freezing tolerant spermatozoa, with the former having greater MMP than the latter (Ros-Santaella et al., 2014;Ryu et al., 2019). Moreover, high freezability spermatozoa were identified to be more viable, motile, and rapid than their low freezability counterparts (Rego et al., 2016;Ryu et al., 2019).
The metabolite profiles of the seminal plasma and spermatozoa were also explored to ascertain possible correlations with freezability and fertilizing ability. Such molecular elements may directly contribute to and reliably represent characteristic phenotypes as end-products of complex biochemical cascades (Kumar et al., 2015). The discovery of the varying levels of citrate, isoleucine, leucine, taurine, tryptamine (Kumar et al., 2015), fructose, 2-oxoglutaric acid (Velho et al., 2018), benzoic acid, lactic acid, palmitic acid, carbamate, GABA (Menezes et al., 2019), aspartic acid, iron, zinc (Narud et al., 2020), butyrylcarnitine, glycerophosphocholine, glycine betaine and l-carnitine (Longobardi et al., 2020) in fresh and cryopreserved semen of high-and low-fertility animals offered some promise in accurately predicting reproductive success. Moreover, good and poor freezability bovine groups presented distinct amino acid signatures. The former had more phenylalanine concentrations, which was linked to post-thaw viability and presumably contributed to antioxidant responses . While the effects of cryopreservation  (Longobardi et al., 2020), similar researches in other ruminant species are required to fill knowledge gaps and generate applications for conventional reproductive techniques.

CONCLUDING REMARKS
As particular molecules related to various sperm functions can be modified by the freezing-thawing procedures, there has been a growing interest in elucidating the molecular underpinnings of sperm biological response to cryopreservation. Sperm lipids, proteins, metabolites, transcripts, and genes can be harnessed as putative markers to classify and predict freezability and fertility phenotypes (Figure 2), or they can be incorporated as additives to freezing mediums to optimize cryopreserved semen quality. These pieces of information, along with modern molecular biological technologies, open new research horizons for the improvement of reproductive outcomes not only in ruminants but also in other domestic and wild animal species.

Figure 2
Putative biomarkers for sperm freezability identified by available omics technologies which can be used to improve semen quality.