Sperm Cryopreservation in Farm Animals Using Nanotechnology

Abstract

Sperm cryopreservation is one premier biotechnology in assisted reproduction. In recent decades, there seemed to be an increasing trend in the usage of cryopreserved semen in the equine industry. Post-thaw semen quality and values are not the same, even in different equine species. Similarly, there are species-specific alterations in sperm physiology, i.e., sperm head, kinetic properties, plasma membrane integrity (PMI), and freezability. Albeit, the viability of sperm varies in the female reproductive tract in mares, jennies, and ponies. The absence of standardized methodology in various steps of sperm cryopreservation, i.e., male health examination, semen collection, dilution, semen centrifugation, and pre-and post-thaw semen quality analysis, results in variations in opinions. As compared to other farm animals, assisted reproductive technologies (A.R.T.) are not applied to the same extent in equines. This chapter aims to provide an update on sperm cryopreservation in equine.

1. Introduction

In 2022, the world population will elevate to 8 billion and rise to 9.7 billion by 2050—this massive surge in demands exploring sublime protein sources both from animals and plants [1]. Optimized equine production can be achieved by focusing on all aspects, including epidemiology, nutrition, management, and reproduction. To optimize equine production from farm animals, including horses, donkeys, ponies, etc., the application of biotechnology tools in copy is inevitable. For the better expansion of the equine industry, the semen quality of equine male stock and the causes of low conception rate in female equine stock needs dire attention.

Equine products (meat, milk, skin) are quite popular in European countries and China. Donkey skin and its byproducts are exported from Brazil to China [2]. In the past and even now, the donkey is considered a free-ranging animal, and less attention was given to its nutrition, welfare, and, most importantly, reproduction. Seasonality in reproduction makes equine more challenging for the application of reproductive biotechnologies. But the good news is that, with time and awareness, the current scenario is changing, and marvelous achievements have been made by focusing on assisted reproductive technologies, including artificial insemination (A.I.), sperm cryopreservation, and embryo transfer.

The exploration of cryoprotective agents proved to be a landmark in semen cryopreservation and, ultimately, farm animal reproduction [3, 4, 5]. Cryopreservation is a superb tool for conserving animal genetic resources. Endangered animal breeds can also be preserved by cryopreserving their semen and producing genetically improved animals. Female breeder stock in estrus can be inseminated anytime with cryopreserved semen. Semen quality and cryotolerance vary among different animal species [6]. There are several steps involved in semen cryopreservation, during which semen quality also lowers. Various studies elaborate on maintaining sperm structure and functionality during cryopreservation by enhancing the cryoprotectant’s concentration [7]. Sperm are sensitive to temperature fluctuation during cryopreservation [8]. In this scenario, the quality of post-thaw semen needs to be addressed. Cryopreservation alters acrosome integrity and mitochondrial activity and enhances reactive oxygen species (R.O.S) production.

As a consequence, the integrity of the nucleoprotein structure declines. So, different molecular alterations following sperm cryopreservation are inevitable to explore because one ejaculate is composed of millions of sperms. The fusion of sperm and egg is essential for fertilization [9]. Various breeds of donkeys are kept worldwide, and the trend of producing hybrid donkeys is increasing with time in many countries [10]. For example, a mating horse and donkey hybrid resulted in a highly desirable animal that combined the best features of both animals [11]. In the coming parts of the chapter, we will elaborate, step by step, on different structural and molecular changes in sperm during semen cryopreservation. Research involving donkey semen needs dire attention [12]. The fertility rates of frozen donkey semen are (0–36%) [13]. The optimum conception rate of 61.5% has been achieved till now for jennies artificially inseminated with donkey cryopreserved semen [14]. Reproductive biotechnologies applied in equine, especially donkeys, still need comprehensive research. In the following sections, we will discuss step by step various molecular alterations in semen cryopreservation across multiple farm animals including equine.

2. Sperm cryopreservation cause alterations in the cell membrane

There are several advantages of semen freezing, but certain disadvantages are also there. In equine, abrupt temperature alterations during semen freezing and thawing damage sperm cells resulting in cell membrane changes and even cell mortality up to 10–50% [15]. Cryoprotectants upgrade the ability of sperm to bear temperature fluctuation without affecting their functionality [16]. Thermal stress forms crystals inside sperm cells resulting in cryodamage [17]. In sperm cells, crystals are formed due to imbalanced extracellular and intracellular solute contents [18]. The extracellular medium upgrades sperm volume by passive diffusion [19]. During semen freezing and thawing, these factors affect lipid-protein complexes in Chinese hamsters [20]. They were lowering temperature results in protein adhesion. This process weakens sperm plasmalemma and enhances its permeability, ultimately decreasing sperm metabolism in poultry [21].

3. Molecular alterations after sperm cryopreservation

Molecular alterations after sperm cryopreservation in equine results in lowered spermatozoa longevity. In horses, such a situation is more problematic due to prolonged estrus, and the timing of ovulation requires more careful diagnoses for applying artificial insemination (AI). Regular ultrasound examinations are necessary for mares, and ovulation induction is unavoidable [22]. Significant differences in the reproductive biology of equine, bovine, poultry, and other animals affect sperm cryopreservation; e.g., the average ejaculate volume of poultry ranges from 0.1–0.3 mL as compared to 5–8 mL in the bull. In eutherian mammals, inside sperm chromatin, the type of protamines (P1 and P2) affects cryodamage [23]. Protamines (P1 and P2) alter in various animal species [24]. Inside chromatin, ROS production, and mechanical stress affect the DNA integrity of sperm [25, 26]. Cryopreservation lowers sperm messenger ribonucleic acid (mRNA), ultimately impairing its function [27]. Fertility of bull thawed bull semen is higher with elevated levels of mRNA’s AK1, IB5, TIMP, SNRPN2, and PLCz1 [28]. In oocytes, mRNA aids in the production of proteins during embryogenesis [29]. Sperms cannot replace the lost mRNA during cryopreservation [30]. Epigenetic factors affect freezing and thawing. All these factors collectively are involved in gene expression levels, post-translational histone alterations, chromatin remodeling, DNA methylation, and non-coding RNAs [31, 32]. The frozen, thawed bull semen had a differential abundance of 86 microRNAs [33]. In pig fresh and frozen semen, 135 miRNAs elaoborate metabolic pathways [34]. So, cryopreservation changes apoptotic genes, mitochondrial membrane, DNA fragmentation, phosphatidylserine externalization, and caspase activation [35, 36].

4. Impact of sperm cryopreservation on embryo

Application of assisted reproductive technologies (ART) in the horse is scarce and is only reported in vivo matured oocytes [37]. In vitro, matured oocytes were inseminated in mares, and embryos were generated [37]. Sperm DNA methylation is an inevitable aspect of embryo development, and DNA methylation is directly affected by freezing and thawing procedures, ultimately affecting embryo development [38]. This process involves connecting the methyl group to the cytosines of CpG regions [38]. In horse sperm, DNA methylation improves after cryopreservation (0.6% in fresh and 5.4% in thawed semen) [39]. Abnormal DNA methylation during cryopreservation is why fertilization fails during artificial insemination. Cryopreservation affects early embryonic development. Oocyte inherits epigenetics from nucleosomes and methylated DNA during fertilization [40]. Embryo development is affected by transcription factors [41]. After AI with frozen-thawed semen, downregulation of transcription factors was observed in horse embryos. Transcription factors were downregulated after AI in equine [42]. Hindrance during embryonic development is connected to lowered transcription factors, i.e., TCF7L1, BTEB3, CPBP, KLF3, and NF-1 [41]. In bovine, after cryopreservation, the appraisal of fresh and frozen-thawed semen showed variant mRNA and miRNA profiles [36]. mRNAs’ maternal functions can be controlled by sperm-born miRNAs [43]. The mRNA profiles of embryos can be changed by sperm cryopreservation [44]. Similar variations were noticed in frozen, thawed swine semen after adding glycerol as a cryoprotectant [45].

5. Redox imbalance and its effect on mitochondria

The balance between ROS production and the antioxidant defense mechanism is inevitable in cryopreservation. Otherwise, it leads to oxidative stress. Oxygen free radicals, i.e., hydrogen peroxide (H₂O₂), superoxide anion (O₂−), and hydroxyl radical (OH−), are part of ROS [46]. ROS are critical for sperm function. Sperm cryopreservation triggers apoptotic pathways, while ROS concentrations elevate the optimal value [47]. Increased ROS concentration structurally damages the sperm’s DNA, ultimately disrupting fertility [48]. During cryopreservation, ROS production modifies sperm’s mitochondrial membrane potential [35]. Static oxidative reducing potential (sORP) influences the redox balance in cryopreserved stallion semen [30]. The inclusion of Rosiglitazone can upgrade mitochondrial membrane activity in cryopreserved equine semen. It lowers caspase-3 activity and eventually halts the activation of apoptotic pathways. The addition of Rosiglitazone can aid to acquire redox balance in cryopreservation. In equine semen, rosiglitazone addition aids in AKT (kinase B) protein phosphorylation, which aids in maintaining a balance between apoptotic pathways and cell survival [30]. Compared to other farm animals, ROS production is well reported in equine. ROS production in the mitochondria is scarce in swine before freezing [49]. In swine, the effect of ROS on mitochondrial membrane integrity and lipid peroxidation is better in fresh semen compared to frozen-thawed semen [50]. In equine, lipid peroxidation (LPO) is a primary factor in sperm cryodamage [37]. Mitochondria is a significant source of oxidative stress to spermatozoa [51]. ROS production is elevated in sperm mitochondria during freezing-thawing, while the osmotic mechanism may upgrade mitochondrial membrane permeability, resulting in apoptosis [52].

6. Impact on sperm mobility

Sperm head, size, and movement are disrupted by cryopreservation. In humans, cryopreservation applies oxidative stress on sperm, resulting in cryodamage and reducing progressive motility [53]. Cryopreservation affects the lipids of sperm’s membrane, survival rate, and motility and harms DNA in humans [53]. After cryopreservation, sperm motility is notably affected [53]. Computer-assisted sperm analysis (CASA) aids in evaluating sperm kinetics [54]. Morphological changes in sperm can be analyzed with a phase contrast microscope camera attached to the computer screen. Sperms’ waggling movement can be recorded in sequential images [55]. CASA aids in analyzing different salient factors, e.g., oscillation, straightness, and linearity. It enunciates the frequency the sperm head movement [56]. Sperm can be classified based on their motility by using the CASA system [57]. The categorizing of sperm based on motility and morphology determined various aspects for elaborating sperm biology; sperm motility is influenced by thawing and freezing in bulls, stallions, rams, and boars [55]. In some previous research, sperm motility was connected with fertilizing capacity and viability [57]. Sperm cryopreservation affects sperm motility in equine also, and cryopreserved semen is less frequently used as compared to fresh semen [58]. Fertilizing capacity of frozen-thawed semen varies among various stallion species [59].

In bovine, sperm populations have quick and non-linearity in movements that are elevated in post-thaw sperm activity [60]. Cryopreservation can amend the plasma membrane of sperm [61, 62]. Table 1 enunciates the alterations in sperm cryopreservation in different farm animals and various factors.

Specie.	PM (%)	AI (%)	MF (%)	ROS (%)	Sperm M & V (%)	References
Bovine	40	10–19	15	48	50	[63]
Porcine	50	30	30	2	60	[64]
Ovine	80	50	30	1.5	30–40	[65]
Equine	70	12	35	1	30	[66]
Caprine	68–73	73–81	N/A	N/A	N/A	[67]

Table 1.

Sperm cryopreservation variations are specific to species.

Abbreviations: PM (%), progressive motility; A.I (%), acrosome integrity; M.F. (%) mitochondrial function; R.O.S. (%) reactive oxygen species; Sperm M&V (%), sperm motility and viability.

7. Markers of freezability

The composition of cryopreserved semen varies among the same species of sires and between the same ejaculates [18]. Different types of stress, like osmotic and thermal stress, dominate while thawing and freezing procedures and affect sperm response. Seminal plasma (SP) proteins affect sperm’s ability in cattle, pigs, equine, and sheep [68]. Sperm cryopreservation is affected by these SP proteins as they diversify from individual to individual [69]. During cryopreservation, these proteins go through carbonylation, which helps in oxidation; as a conclusion malfunctioning and alterations of protamine of SP proteins occur. These proteins withstand oxidative stress [49, 70]. AKAP4 and proAKAP4 proteins uncover cryodamage in ovine, equine, and porcine [71]. High quantities of HSP90AA1 and HSPA8 proteins enhance sperm cryopreservation chances in bovine [72, 73]. Upgraded post-thaw sperm viability and motility were observed after an elevated concentration of HSP90AA1 protein in swine. Likewise, inside the cell plasma membrane, aquaporins control the permeability of water and cryoprotectants during cryopreservation [74, 75]. In swine and bovine, AQP3 and AQP7 proteins affect sperm cry tolerance. Another crucial protein for sperm cryopreservation in pigs is VDAC2 [76]. Also, an upgraded amount of GSTM3 in swine sperm enunciates higher freezibility after cryopreservation [77]. The improved concentration of SP proteins aids in Cryotolerance. Fibronectin-1 (FN1) helps the thawing and freezing processes in boars [78]. The proteins TCP-1 and 26 S proteasome in swine promote sperm cryopreservation [79].

8. Impact of cryoprotectants

Cell metabolism is downregulated while storing various tissues and cells. The primary aim for sublime sperm functionality is to provide optimum pH, temperature, and osmolarity. In humans, cryoprotective media reduce ice crystal formation and cold shock, resulting in sperm functionality [80]. Permeable and non-permeable cryoprotectants have 4–5% glycerol and 20% egg yolk that guard against cryodamage. Sperm’s lipid bilayer is protected by a plasma membrane combined with egg yolk’s low-density lipoproteins (LDL) [81].

Addition of low-density lipoproteins (LDL) elevated sperm post-thaw quality in cattle [82], pigs [83], sheep [84], and horses [51]. During cryopreservation, a concentration of 9% LDL lowers sperm DNA damage in pigs [83]. Researchers and scientists are looking to substitute egg yolk as a cryoprotectant due to its bacterial contamination [85]. Soy lecithin is a vital substitute LDL having identical functionality [86]. Alternating egg yolk with soy lecithin upgraded post-thaw sperm motility (19%) in cattle [87], but some researchers observed a negative or no impact of soy lecithin on sperm motility [88]. Some researchers have enunciated the conclusive effects of soy lecithin (vegetable origin); egg yolk (animal origin) is still significantly used as a cryoprotectant agent. Liposomes are pure and are used as an alternative to freezing media in horses, pigs, and cattle while sperm cryopreservation [89, 90]. Liposome’s plasma membrane guards the permissibility of water and cryoprotectants [91]. Alcohol, glycerol, and ethylene glycerol are permeable cryoprotectants in cattle sperm cryopreservation [91]. After thawing, glycerol harms sperm quality, depending on the specie [92]. Plasma membrane fluidity is lowered by higher glycerol concentration in thawed swine semen [93]. Elevated concentration of glycerol >3.5%—alters the properties of F-actin (that belongs to globular proteins) in the cytoskeleton [94]. Much research is available exploring substitutes, but glycerol is still considered a sublime cryoprotectant. In research, lower glycerol concentrations were mixed with L-glutamine and trehalose in boar semen [95]. In Pigs, glycerol molecules come in contact with lipid bilayers and change membrane diffusion rates of electrolytes, which results in the osmotic contraction of sperm cells so that sperm can bear low temperatures [96]. Trehalose (60–100 mM) mixed with a low concentration of glycerol (1–3%) upgraded sheep-frozen sperm DNA quality [97]. Glycerol combined with trehalose did not affect cattle sperm quality [98]. In equine, amides cause less harm to sperm during cryopreservation [92]. Changing glycerol with dimethylformamide and methylformamamide increased sperm acrosome integrity, mitochondrial membrane potential, mobility, and capability in horses [99]. In sheep, a combination of 4.7% methyl formamide and 2.3% glycerol increased the integrity and mobility of the plasma membrane [100].

9. Addition of seminal plasma components and other supplements

During semen cryopreservation, seminal plasma is detached and changed with freezing media. In rams, complete or partial removal of seminal plasma downregulates sperm’s freezing capacity [101]. To improve sperm quality, detachment of seminal plasma components is not recommended. In swine frozen-thawed semen, the addition of 5% of seminal plasma before freezing elevated plasma membrane integrity and motility upto 10.5% and 9.2%, respectively [102]. In sheep sperm, the inclusion of 20% seminal plasma upgraded motility (14.7%), plasma membrane integrity (10.4%), and chromatin decondensation (13.9%) [103]. Adding seminal plasma proteins to freezing media has critical functions in sheep [104]. In boar sperm, sperm adhesins attach to glycoproteins in the oviduct, upgrading sperm membrane quality [105]. Furthermore, the task of sperm adhesins is elaborated in sperm cryopreservation. In swine sperm, including seminal plasma proteins AQN-1, AWN, and AQN-3 inhibited the capacitation of sperm at 5°C [106]. BSPs tend to upgrade plasma membrane integrity and halt capacitation [107]. In pigs, adding RSVP14 and RSVP20 in a freezing medium upgraded sperm viability [108]. It explains that sperm quality can be increased or at least sustained by these proteins [109, 110]. In cattle, seminal plasma proteins have a low molecular weight (14–16 kDa) [111]. Adding 1–1.5 mg of protein to MW (14–16 kDa) increased sperm viability by up to 20%. Seminal plasma (SP) proteins of one species can be used as sperm cryopreservation media for other species. SP proteins were added to the freezing medium in cattle [112, 113]. In rams, freezing capacity is elevated after altering bovine serum albumin (BSA) with egg yolk [114, 115]. Adding 4 mg/mL BSA to an excellent medium increased post-thaw sperm quality in bucks [116].

Sperm oxidation is lowered by enzymes inside seminal plasma [117, 118]. Antioxidant defensive enzymes in sperm are catalase, superoxide dismutase (SOD), glutathione reductase, and glutathione peroxidase. Superoxide dismutase is essential in sperm cry tolerance in equids [119]. In various breeds, the protective system of such enzymes is also related to seasonal breeding [120, 121]. In cattle, post-thaw sperm quality increased after adding antioxidants to the freezing medium. Cattle post-thaw sperm viability after inclusion of 100 IU/mL of superoxide dismutase (SOD) [122]. After adding SOD in rams, similar results were seen in post-thaw semen [123]. Post-thaw sperm quality increased after adding glutathione to freezing media in equine and swine, respectively [124, 125]. In rams, adding 2–5 mM glutathione in freezing media increased the acrosome integrity and motility of frozen-thawed sperm [123].

10. Conclusions

Animal biotechnology is now considered a pillar in research and animal production. Its wide application in all animals, including equine, still needs dire attention. Much work has been done in horses but lacks work in donkeys, especially pre- and post-thaw semen quality. Better research and collaborations between scientists, academia, and industry can aid in achieving milestones in equine biotechnology.

Funding

This research was funded by Shandong Province Modern Agricultural Industrial Technology System Project (SDAIT-27), Ministry of Agriculture and Rural Livestock Seed Industry Project “Donkey Camel Species Molecular ID Construction” (19211162), Key research and development project of Shandong Province “Innovation and Demonstration of Key Technologies for Integrated Development of Dong’e Black Donkey Industry” (2021TZXD012).

List of abbreviations

ROS	reactive oxygedefn species
mRNA	messenger Ribonucleic Acid
AKT	alpha serine/threonine-protein kinase
AWN	boar protein
BSPs	binder of sperm (BSP proteins)
miRNAs	micro–ribonucleic acid