Frozen Semen Quality of Kolbroek Boars Concerning Glutathione Concentrations

: Glutathione (GSH) is an antioxidant that is being studied for its ability to improve cellular responses to semen preservation challenges. The research goal aimed to evaluate the impact of introducing different glutathione concentrations to boar semen freezing extender (0, 1, 5, and 10 mM) on the Kolbroek boar sperm's quality after being frozen and thawed. For two hours, the collected sperm-rich fraction was chilled at 17°C. Following the 1 h of equilibration, fraction B: control (egg yolk 20% + BTS 72% + glycerol 8%), 1 mM (egg yolk 20% + BTS 70% + glycerol 8% + GSH 2%), 5 mM (egg yolk 20% + BTS 62 + 8% glycerol + GSH 10%) and 10 mM (egg yolk 20% + BTS 52% + glycerol 8% + GSH 20%) was added into the semen samples. Following that, 0.5 mL straws with the diluted, cooled semen samples were submerged in liquid nitrogen (LN 2 ) vapor and then stored inside the LN 2 tank (-196°C). After thawing (37°C), parameters of boar sperm motility, velocity, morphology and viability, membrane permeability, and Malondialdehyde content were assessed and recorded. Using the analysis of variance, the data were examined. The results revealed a difference in total motility (%) on fresh semen as compared to post-thawed control and GSH treatments. A significant difference was revealed in sperm progressive motility (%) in fresh semen (27.07±4.5). Significant differences were recorded in live sperm morphology and viability on fresh semen (81.8±2.8%) compared to control, 1, 5, and 10 mM. In the 10 mM diluted semen (78.5±6.8), the proportion of sperm with an intact plasma membrane integrity was highest ( P <0.05), while the percentage in the 1 mM diluted semen (73.0±2.7) was lower. Malondialdehyde levels were lowest ( P <0.05) in the group receiving 5 mM treatment. In conclusion, 5 mM of GSH is the required amount to be added to the freezing extender while cryopreserving semen from Kolbroek boars.


Introduction
An indigenous pig breed from South Africa known as Kolbroek is distinguished by its extraordinary adaptability features. This pig breed is popular amongst local South African pig farmers and their stock is protected as national treasures in order to preserve the genetic purity of the genotypes (Halimani et al., 2010;Netshirovha et al., 2020). In comparison to other pig genotypes, there has not been much research done on the ability of native Kolbroek boars to reproduce (Masenya et al., 2011). Semen conservation has been extensively studied and semen banking offers an effective method for maintaining higher male gametes for future use (Bucci et al., 2018). Regardless of these possible advantages, due to cell cryodamage that happens during the freezing and thawing processes, cryopreserved sperm is not frequently employed for artificial insemination in the swine sector (Giaretta et al., 2015). Such cryodamage eventually results in a decrease in sperm fertilizing ability (Didion et al., 2013). Furthermore, a sudden outbreak of an infectious disease or a natural disaster could have less of a negative impact if frozen-thawed boar semen could be used (Giaretta et al., 2015). Many studies have attempted to understand the mechanisms driving cryodamage to enhance cryopreservation technologies for boar sperm (Parrilla et al., 2019;Yeste et al., 2014). According to Crisóstomo et al. (2019), the most obvious effects of freeze-thawing techniques affect plasma and acrosome membranes, the mitochondrial midpiece, and the axoneme. Such sperm membrane injury, which is detrimental to the process of fertilization causes cryopreservation degradation in the quality of the boar semen . This membrane damage is most likely caused by the fact that boar sperm contains more unsaturated phospholipids and less cholesterol distribution is uneven and higher in some species than in others (Casas and Flores, 2013). According to Subczynski et al. (2017), Cellular resilience to heat shock and cholesterol go hand in one which has a substantial effect on the fluidity, membrane lipid, positioning of protein chains, and permeability which fall under the physical characteristics of membranes. The effectiveness of in vitro liquid perseveration for prolonged periods might be affected by the latter sperm cryo efficacy (Mphaphathi et al., 2016), the kind of semen extender employed, thawing temperature, storage temperature, and semen re-dilution (Sathe, 2012). Quantifying sperm characteristics using a computer-assisted sperm analyzer system (Neumann et al., 2017) has become critical to obtain objective data both before and after freezing. To achieve uniform results, artificial insemination centers (AI) and cryo biologist can make decisions (Alquézar-Baeta et al., 2019).
The cryopreservation technique is defined as a successful strategy for long-term gamete preservation (Yeste et al., 2016). Following AI, sperm viability, motility, and conception rates are routinely evaluated to determine the success of cryopreservation (Mphaphathi and Nedambale, 2021). After the cryopreservation process, frozen sperm can be thawed and, hopefully, biological activity can be resumed (Mphaphathi and Nedambale, 2021). One of the improvements of cryopreservation technology is the introduction of antioxidants in the boar's sperm cells (Giaretta et al., 2015). Different anti-oxidants, including a-tocopherol (Chatiza et al., 2018), decreased Glutathione (GSH) , superoxide dismutase and catalase (Bucci et al., 2018) have been included to freezing semen extenders to reduce sperm cryodamage. Impact of antioxidants are believed to have positive influences on boar semen in sperm motility, survivability, and capacity to fertilize both in vitro and in vivo after thawing . In this case, sperm is protected by GSH against oxidative damage. Silvestre et al. (2021), maintaining redox balance (Pacuła et al., 2017) and a defense mechanism against oxidative lipid peroxidation brought on by reactive oxygen species (ROS; Silvestre et al., 2021). The supplementation of various concentrations of glutathione is, however, not well understood in frozen-thawed Kolbroek boar's semen. The current study's goal was to find out how introducing glutathione to boar sperm freezing extenders affected the quality of frozen-thawed Kolbroek sperm.

Study Site and Experimental Kolbroek Boars
The study was undertaken at the Agricultural Research Council (ARC) Irene's Germplasm Conservation and Reproductive Biotechnologies laboratory. Located 1525 meters above sea level, in Pretoria, South Africa's highveld at 25°53' 59.6 south latitudes and 28°12' 51.6 east longitudes. A total of 3 Kolbroek boars were adhere to the ARC's animal production ethical committee's standards of care (REF: APAEC2020/11) and the Tshwane University of Technology (REF: AREC-2021/11/006). The boars were ±1-2 years and kept at the ARC pig testing. The pen of each boar was cleaned before semen collection. Hygienic practices such as washed and drying hands were carefully applied, the collection glass was sterilized and the dummy sow was cleaned thoroughly to avoid semen contamination. The boars were housed in pens that included mechanical water nipples and self-feeders (Netshirovha et al., 2020). Feed was provided throughout the study and unlimited water was supplied during the experiment.

Chemicals and Reagents
Unless indicated otherwise, sigma-chemical Co, USA, and TOCRIS bioscience, UK were used to purchase all of the chemicals and reagents. Solutions were prepared with highly purified water (Adcock Ingram critical care, SA).

Beltsville
Thawing Solution (BTS) (IMV technologies, France) was the primary extender utilized in this study, which was composed of 1-liter deionized water and a 50 g BTS sachet. By moving the egg yolk from one half of the egg shell to the other and removing the albumin, the chicken egg yolk was separated from the albumen. An 18-gauge needle (Neoject, UK) was used to pierce the egg yolk, separating it from its membrane and transferring some albumin to a 50 mL (Plastro, SA) graduated tube. The egg yolk was then placed in a gauze swab (Johnson's, SA).

Kolbroek Boar's Semen Collection
Semen was collected twice weekly. The gloved-hand method was used to collect a total of ten ejaculates from each boar in a 500 mL glass bottle (10 ejaculates = 10 replicates/treatments). The filtered semen portion was contained inside an insulated thermo-flask that had been pre-warmed to 37C. For a microscopic semen analysis, the sperm was taken to the laboratory.

Evaluation of Kolbroek Boar's Sperm Motility and Velocity Traits
A Sperm Class Analyzer ® system (Microptic, Spain) was used to determine the parameters of sperm motility and velocity under a microscope (Nikon, Eclipse Ci-L, Japan) at the magnification of 10 . A swim-up (BTS) method was used to dilute semen before analysis. A drop of 10 µL was taken from the semen sample and diluted with 200 µL of BTS. Using a microscope with  10 magnification, a drop of 5 µL was deposited on a 37°C preheated microscopic glass slide (Labocare, UK) and mounted with a cover slip (Thermo Scientific, USA) (Nikon, Japan). Estimates of sperm motility and velocity traits were carried out on each semen sample in two separate microscopic fields. The movement of sperm traits was measured in percentages and velocity traits in micrometers per second and percentages.

Evaluation of Kolbroek Boar's Sperm Morphology and Viability
Boar sperm morphology and viability (live or dead) were analyzed using eosin nigrosine (university of Pretoria, SA) staining according to Brito et al. (2011). Smeared microscope glass slides were assessed on a phase microscope (Olympus, Japan) at 100  magnification, live sperm abnormalities were distinguished as unstained and dead sperm as stained. A smear was created at 37°C using a pipette tip after gently combining a drop of 7 µL of semen with a 20 µL stain using the tip of a hand pipette. On the clear end of a microscope slide, a drop of a mixed sample of 5 µL was placed. On the smeared microscope glass slide, an oil drop (UNILAB, SA) for the immersion lens was placed, and about 200 sperm cells per slide were counted.

Evaluation of Kolbroek Boar's Sperm Plasma Membrane Permeability
The hyperosmotic swelling test (HOST) was performed to assess sperm membrane permeability. A drop of 100 μL HOST (distilled water, 1000 mL, 7.35 g sodium citrate, and 13.51 g fructose) (Zhang et al., 2016) and 10 μL of semen were placed in a centrifuge tube (Symport, Canada) at 37°C for 60 min. A drop of 5 μL of the semen mixture was placed on a microscope glass slide. Sperm swelling was observed as coiled sperm tails, indicating good membrane function. A total number of 200 sperm were analyzed from each microscope glass slide. The sperm cells were classified as positive or negative based on the presence or absence of a coiled tail.

Evaluation of Kolbroek Boar's Sperm Malondialdehyde Content
Malondialdehyde was based on the Thiobarbituric Acid (TBA) test used by Esterbauer and Cheeseman, (1990). Diluted sperm (1 mL of BTS extender) from each treatment and replicate was to precipitate protein, along with 1 mL of cold trichloracetic acid. A boiling water bath at 100°C was used to incubate 1 mL of the supernatant with 1 mL of 0.67% (wt/vol) TBA after the precipitate had been centrifuged (1500  g for 10 min). Once the semen samples were cooled to room temperature, a spectrophotometer (JENWAY ® 6310, UK) was used to calculate the absorbance at 534 nm. The results were expressed as nmol/mL.

Kolbroek Boar's Semen Thawing Process
Two straws per treatment were taken after at least 4 days of frozen semen straw storage at -196°C from the LN2 tank. For 10 sec on air, the straws were defrosted at 37°C and for 1 min in warm water. The semen samples were diluted with a BTS extender at a ratio of 1:2 after thawing. Kolbroek sperm motility traits, velocity traits, morphology traits, plasma membrane, and MDA content were evaluated.

Statistical Analysis
The data underwent analysis of variance. The generalized linear model procedure was used. At a 5% level of significance, Student's Least Significant Differences (t-LSDs) were computed to compare the means of significant source effects between treatments (Snedecor and Cochran, 1980). Statistical software SAS 9.4 (SAS, 1999) was used to carry out the stated analysis. Table 1 displays how varying glutathione concentrations affected sperm motility in frozenthawed semen from Kolbroek boars. The results showed that a difference was recorded in sperm total motility (%) on fresh semen compared to control, 1, 5, and 10 mM. There was a significant difference (P<0.05) in sperm progressive motility (%) in fresh semen compared to other treatments. Table 2 displays the impact of various glutathione concentrations on the sperm velocity rates of frozenthawed semen from Kolbroek boars. Sperm wobble (%) in 5 mM was significantly different from 1 mM (P<0.05). Sperm straight-line velocity (37.9±27.2 μm/sec) was lower at 10 mM compared to the control group, fresh semen, and 1 mM. In the control group, 1 mM and fresh semen on sperm straightness (%) indicated a higher percentage (P<0.05) in contrast to 10 mM.

Results
Sperm morphology and viability parameters in Kolbroek boars supplemented with glutathione following frozen-thawed are shown in Table 3. The findings demonstrated a distinction in the morphology and viability of live sperm on fresh semen compared to control, 1, 5, and 10 mM. Live sperm abnormalities are shown in Fig. 2 and dead sperm are shown in Fig. 1. However, in comparison to all other treatments, 10 mM had a decreased (P<0.05) level of live sperm viability and morphology. Table 4 displays the findings of the study on the impact of glutathione concentration on the integrity of the sperm plasma membrane in frozen-thawed boar semen from Kolbroek. On HOST+, 10 and 5 mM had the largest sperm membrane concentrations (%) without affecting the integrity of the plasma membrane, while 1 mM of glutathione had a lower (P<0.05) percentage of intact plasma membrane integrity. The control group was numerically lower on HOST+ than all other groups.
The impact of different glutathione concentrations on Kolbroek boar sperm MDA content during freezing is demonstrated in Fig. 3. The least improvement was shown in the 5 mM treated group MDA levels in all groups (P<0.05). The difference in 1 mM concentration was significant in contrast to the control group and 10 mM.

Discussion
The primary conclusion reached by this investigation was that there was a decrease in boar sperm motility after cryopreservation. The results obtained in sperm motility showed differences in TM (%) of GSH between the treatments. However, no difference was observed in the control group, 1, 5, and 10 mM. These differences could be related to differences in the fertility potential of boars used. According to Ansari et al. (2010); Câmara et al. (2011), GSH at high doses of 3, 5, and 10 mM showed no positive impact on semen quality indicators, which is similar to the results of the present investigation. Nonetheless, comparisons between studies are typically challenging to understand because of variations in research techniques and sperm processing regimens. This study describes a reduction in GSH content in swine sperm brought on by freezing methods, however, it is not the first to do so.
Human GSH content has decreased by a certain proportion of 64% (Gadea et al., 2011), while in boar it was 32% (Silvestre et al., 2021) and in bull, it was 58% or 78% (Estrada et al., 2017). Glutathione levels in mouse sperm have been found to be substantially greater than other mammalian species (90 mM GSH/10 8 cells) (Chanapiwat et al., 2010). Furthermore, excessive glutathione and glutathione peroxidase, concentrations may compromise the functional integrity of the sperm axosome, which is linked to the motility of sperm cells (Salmani et al., 2013).
Glutathione deficiency may lead to mitochondrial DNA degradation (Gadella, 2008). An essential component of sperm, mitochondria provide the energy needed for sperm motility (Hu et al., 2010). Hence, damage to the mitochondria and axosomes may contribute to the deleterious effects of high GSH levels on progressive motility (%) and other motion characteristics. (Allai et al., 2018). Our findings contrast with previous research by Vozaf et al. (2021) who, in contrast to the control group, demonstrated enhanced sperm motility. This variance could be due to various extenders used or supplemented with various animal breeds.
The results of the current investigation demonstrated that greatly improving sperm motility by adding 5 mM GSH to freezing media diminishes viable sperm.
According to the data, sperm movement patterns are altered by increases in values that drop along the straightline and average route for curved trajectory wobbling, this was controversial to what (Gadea et al., 2011) obtained. Sperm speed on the average path and speed on the curve line, both expressed in percentages remained constant and steady between the treatment groups. The swine genetic industry relies on the cryobanking of boar sperm hence the cryopreservation process needs to be enhanced. In addition to its function, superior male sperm cryobanking is an important backup for proper insemination dosages in the event of sickness, infertility, or death (Vozaf et al., 2021). Cryopreservation has been shown to damage some structural and ultrastructural sperm components.
In comparison to dead sperm (%) and in fresh sperm, there was a high percentage of live normal sperm morphology (%). However, a high percentage of dead sperm was recorded in all treatments. Although the dead sperm (%) was significantly higher, the results were still satisfactory. There are several different types of defective sperm and some of them are hereditary (Morrell et al., 2018). These include primary sperm head abnormalities, such as an acrosomal cap deficiency (Hirohashi and Yanagimachi, 2018), and secondary sperm tail abnormalities (Bó and Mapletoft, 2014). In this study, these primary sperm abnormalities variables were taken into account.
In this study, in the presence of various glutathione concentrations upon cryopreservation, MDA levels were measured and examined. The MDA concentration was higher in the treatments than that of the control group, except for 5 mM. According to Chatiza et al. (2018), cells stimulate their maintenance and survival under low lipid peroxidation rates. This was due to activated antioxidant defense systems (Chatiza et al., 2018). The rate of lipid peroxidation when high, oxidative damage exceeds the potential for repair, and cells trigger apoptosis (Chatiza et al., 2018). Whichever process ultimately causes cell damage, which can lead to the development of various accelerated aging and pathological states (Halliwell and Gutteridg, 2015).
According to (Silvestre et al., 2021), plasma membrane functional integrity is related to sperm motility. These findings suggested that supplementation with glutathione in a freezing extender could enhance sperm motility by shielding the plasma membrane from ROS. The sperm membrane's capacity for function can be damaged when introduced to high concentrations of antioxidants (El-Seadawy et al., 2022).
Little is known about the mechanisms underlying cryodamage to boar sperm and the major contributors, which include ROS (Vaughan et al., 2020). Human sperm cells are assumed to be multifactorial (Vaughan et al., 2020). Significant contributions have previously been identified as increased ROS production caused by freezing and thawing, as well as oxidative stress (Vaughan et al., 2020).
In mammalian cells, GSH is the most prevalent nonprotein thiol molecule (Gadea et al., 2011). Multiple cellular activities rely on glutathione, including the transfer of amino acids, DNA, protein synthesis, and disulfide bond depletion (Gadea et al., 2011). Glutathione has been demonstrated to keep certain domestic animals' sperm moving (Masoudia et al., 2020) and to guard sperm against oxidative damage (Silvestre et al., 2021) it is anticipated that adding this antioxidant to extenders can improve the sperm's quality and ability to fertilize eggs through the freezing and thawing processes. The GSH's sulphydryl groups have been demonstrated to protect cells from oxidants, electrophiles, and free radicals (Perumal et al., 2011).

Conclusion
In comparison to fresh semen, the freezing extender combined with various GSH concentrations lowered sperm motility parameters. The amount of 5 mM of GSH, was however found to be the most effective concentration to be added to the sperm-freezing extender.