Effect of antioxidants ( taurine , cysteine , α-tocopherol ) on liquid preserved Kolbroek boar semen characteristics

Successful artificial insemination depends on maintaining longevity of semen in the reproductive tract, and semen characteristics among others. The study compared efficacy of Androhep extender supplemented with 75 mM taurine, 5 m M-cysteine, or 200 mg/l alpha-tocophero on liquid preservation of Kolbroek boar semen characteristics over 24 and 48 h after storage at 17°C. Total motility was evaluated using computer aided sperm analyzer (CASA), viability using SYBR/propidium iodidemembrane function using HOST test and lipid peroxidation using the MDA test. Statistical analysis was done using PROC GLM procedure of SAS (1996). Data were analyzed using analysis of variance (ANOVA). Untreated samples showed higher sperm quality in terms of membrane integrity, functionality and motility. All antioxidant treated spermatozoa did not show increased longevity (p>0.05) after 48 h of storage compared with untreated samples. Supplementation of Androhep extender with antioxidants did not improve sperm survivability of kolbroek liquid preserved semen. Therefore, the antioxidants were ineffective in improving longevity of Kolbroek liquid preserved semen after 48 h storage at 17°C. Further studies are required to find the effective antioxidant concentration to elicit sperm protection for Kolbroek boar semen liquid preservation.


INTRODUCTION
Kolbroek is a South African indigenous pig breed characterised by its unique gene pool.As such it is important to preserve the unique germplasm for conservation, research and future breeding purposes.
The breed is extremely hardy and can survive under harsh conditions; hence this breed could be enrolled for cross breeding to improve adaptability traits in the swine meat industry.However, the knowledge on Kolbroek *Corresponding author.E-mail: fungayi.chatiza@gmail.com.Tel: +263 (067) 22203, Ext: 268 or + 263 772 269 230.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License reproductive physiology is limited.
The effects of antioxidants are believed to have desirable effects in semen; however, the Kolbroek boar has shown some major differences in semen quality from the exotic landrace and large white boars therefore, there is need for further information on the sperm physiology of this pig breed.
Several extenders have been developed to preserve pig semen for long and short term storage.Kolbroek semen, has been extended with Beltsville thawing solution (BTS) and Kobidil used for short term storage; the latter performing better.Boar semen cooled to temperatures below 15°C, show an irreversible loss in sperm motility and metabolic activities (Orok et al., 2010).Thus for in vitro semen preservation, an extender should provide an environment that inhibits the formation of harmful reactive oxygen species (ROS) or lipid peroxide (Orok et al., 2010) and cold shock.Reactive oxygen species damages sperm membrane and DNA which in turn reduces the sperm's motility rate and ability to fuse with the oocyte which compromises the paternal genomic contribution to the embryo (Boonsorn et al., 2010).Preserving pig semen for short or prolonged period requires storage at low temperatures (15 to 18°C) however pig semen is known to be very sensitive to low temperature and is susceptible to lipid peroxidation during either liquid or cryopreserved (Basim et al., 2009) preservation forms.This is mainly due to the high continent of polyunsatured fatty acids (PUFAs) which is different from other species (Bresciani et al., 2012).
Some extenders have been shown to increase semen storage time for up to 3 days (Johnson et al., 2000), and even 5 to 7 days (Levis, 2000).Storing pig semen in cooled temperature around 5°C is a more cost effective alternative than liquid nitrogen preservation to help with increasing the use of artificial insemination in the pig industry.Therefore this study determines the efficacy of a long term pig semen extender supplemented with antioxidants (tocopherol, cysteine and taurine for liquid preservation of Kolbroek boar semen and possible increase in longevity during low temperature storage.The effectiveness of antioxidants in reducing the effect of reactive oxygen species damage is also evaluated at low temperature storage (17°C).

Semen collection, processing and liquid storage
Semen was collected from three Kolbroek boars aged 9 to 12 months of proven good semen quality on four occasions using the hand gloved method (Roca et al., 2004).The sperm-rich fraction was collected using a thermo flask containing warm water (39°C) and a glass beaker covered with a gauze filter to separate the gel fraction from the sperm-rich fraction (Roca et al., 2004).Within an hour of collection, semen was transported to the laboratory for the evaluation.The semen was then diluted 1:1 (v/v) with isothermal Androhep extender supplemented with (i) 5 mM cysteine, (ii) 75 mM taurine, (iii) 200 µl α-tocopherol, and (iv) control/untreated to make four different treatments.After dilution, the four samples of the different treatments were then equilibrated at 17°C.Four replicates were used per treatment.Following equilibration at 0 h, then at 24 and 48 h the samples were evaluated using the swim-up (10 µL of semen was added to 500 µL of swim up media) method.Then 10 µL of each treated Kolbroek semen sample was placed in a 10 mL centrifuge tube containing 500 µL Bracket and Oliphant's spermwash media and co-incubated at 39°C for 5 min.After incubation, a drop (10 µL) of Kolbroek semen from each treatment was placed on a microscopic slide and evaluated microscopically for sperm progressive motility, viability and membrane permeability.Semen was analyzed just after collection, at 0 h, then at 24 and 48 h after collection in the different treatments of the antioxidants.

Sperm motility assessment using computer aided sperm analyser
Sperm motility characteristics were analyzed by the Sperm Class Analyzer (SCA, Masenya et al., 2012) (SCA ® V.4.001) as follows; a drop (5 µL) of Kolbroek semen from each different sample treatment was placed on a microscopic slide and evaluated microscopically for sperm motility rate, with the aid of sperm class analyzer.Progressive motility was assessed and four replicates were evaluated per treatments.

Membrane permeability
The hyper osmotic swelling test (HOST) was performed to evaluate membrane permeability from each of the four replicates per treatment by employing the technique developed by Maxwell and Johnson (1997).Briefly, a 50 µl semen sample from each treatment and replicate was added and mixed with 1 ml of 150 mOsm/kg HOS diluent (7.35 g Na-citrate, and 13.51 g fructose, in 1 L of distilled water) and then incubated for 30 min at 37°C in 5% CO2 incubator.The assessment of total sperm swelling and individual swelling patterns was recorded as coiled sperm tails which indicate good membrane functionality.A total of 200 spermatozoa were evaluated for coiled tails under a phase contrast microscope at 400x magnification.Four replicates were evaluated.

Viability
The percentages of viable sperm were evaluated from each of the four replicates per treatment using SYBR-14/propidium iodine (Fertilight, Sperm Viability Kit, Molecular Probes).Ten microliters (μl) of diluted spermatozoa from each treatment and replicate were mixed with 2.7 μl of the working solution containing SYBR-14 and 10 μl of propidium iodine.After incubation at 37°C for 20 min in the dark, a total of 200 spermatozoa were assessed (x400) using fluorescence microscopy (Carl Zeiss Inc., Axioskop 40, Oberkochen, Germany).The nuclei of spermatozoa with intact plasma membranes stained green with SYBR-14, while those with damaged membranes stained red with propidium iodine.The results were scored as the percentage of viable spermatozoa and non-viable (damaged and dead spermatozoa).Four replicates were evaluated.

Lipid peroxidation
Membrane lipid peroxidation was evaluated by the end point production of malondialdehyde (MDA) determined by the thiobarbituric acid (TBA) test (Esterbauer and Cheeseman, 1990).Briefly, diluted spermatozoa (250 × 10 6 cells in 1 mL of Androhep extender) from each treatment and replicate were mixed with 1.0 mL of cold 20% (wt/vol) trichloroacetic acid (TCA) to precipitate protein.The precipitate was pelleted by centrifugation (1500 × g for 10 min), and 1.0 mL of the supernatant was incubated with 1.0 mL of 0.67% (wt/vol) TBA in a boiling water bath at 100°C for 10 min.After cooling to room temperature, the absorbance was determined by a spectrophotometer (UNICAM PU 8610 Kinetics spectrophotometer; Philips, Eindhoven, Holland) at 534 nm.The results were expressed as a simple concentration of MDA (nmol/ml).Four replicates were evaluated.

Statistical analysis
Statistical analysis was done using PROC GLM procedure of SAS (1996).Analysis of variance (ANOVA) was applied to determine the effects of antioxidants on motility, viability and membrane permeability of liquid preserved semen.Data on motility and viability was analyzed by general linear-models (time series with repeated measures) procedure.Treatment means were separated using Fisher's protected t-test least significant difference (LSD) at 5% level of significance (Snedecor and Cochran, 1980).Results are presented as means ± SEM.

Motility
Progressive motility decreased significantly (p < 0.05) over time from 0 to 48 h for all treatments, however the rate of total motility decline was higher for samples supplemented with antioxidants than control samples.With the exception of cysteine, taurine and tocopherol, treated samples showed higher total motility than untreated samples after 24 h storage.Control samples performed better than treated samples after 48 h storage.
Taurine maintained the highest motility (95%) for 24 h with cysteine having the lowest motility of 71%.After 48 h, motility for all treatments declined below 50%, with taurine maintaining significantly (p < 0.05) higher motility of 49% and cysteine the lowest at 3% (Figure 1).

Membrane permeability
Membrane permeability decreased significantly (p< 0.05) with storage time.Cysteine was the least effective antioxidant in maintaining functional sperm membranes over 48 h.Taurine and tocopherol treated samples maintained membrane functionality better than untreated and cysteine treated samples.Rate of decline in membrane permeability was similar for untreated and treated samples from 24 to 48 h.Taurine (58%) treated samples showed the highest percentage of cells with functionaly active spermatozoa membrane and cysteine the least (30%) after 24 h, however there were no significant differences in efficacy of between taurine and tocopherol (Figure 2).

Viability
Viability was negatively affected by storage time.Untreated samples maintained viability better than treated samples over 48 h.Rate of decline in viable spermatozoa was higher for samples supplemented with antioxidants than untreated samples.Cysteine was the least effective antioxidant (Figure 3).

Lipid peroxidation
MDA concentration increased significantly with storage time indicating an increase in lipid peroxidation, with control samples showing the least antioxidant protection as expected addition of antioxidants to the extender had a positive impact on the decrease in the level of MDA during semen storage.
Cysteine was the most effective antioxidant in the maintenance of a constant MDA concentration from 0 to 48 h.Taurine had the highest concentration of MDA at 48 h, though it was not significantly different (p > 0.05) from tocopherol at 48 h (Figure 4).

DISCUSSION
Efficacy of antioxidants was lower than for untreated samples in for long term (48 h) lipid preservation of Kolbroek semen at 17°C.Control samples showed higher motility, membrane functionality and integrity and general survivability over the 48 h storage.The study demonstrated that even though addition of antioxidants to extenders would decrease lipid peroxidation in general, the effective concentration and type of extenders is an important consideration when supplementing semen with antioxidants for protection against lipid peroxidation.Androhep extender (Vyt et al., 2004) which is a long term extender has been known to produce low survivability of semen compared to Beltsville thawing extender and Kobidil + which are short term extenders for liquid preservation of pig semen.Long term extenders are designed to preserve semen for longer periods of time thus contain sufficient amounts of antioxidants to minimise and reduce the occurrence of reactive oxygen species (ROS) during the holding period.It was well established that ROS can damage the sperm plasma membrane and hence reduced the sperm motility rate (Guthrie and Welch, 2005;Chanapiwat et al., 2009).However, addition of further amounts of antioxidants in the concentration used in this study has no added advantage for low temperature short term storage of Kolbroek semen using a long term extender such as Androhep, and does not improve longevity of liquid preserved semen.In order to counter the effects of ROS damage, generally semen contains a complex redox system that combines the antioxidant potential of seminal plasma and the pro-oxidant potential of sperm.Enzymatic antioxidant defence mechanisms in seminal plasma and spermatozoa include superoxide dismutase, glutathione reductase, gluthathione peroxidase and catalase.Nonenzymatic antioxidants include reduced glutathione (GSH), urate, ascorbic acid, a-tocopherol, taurine, hypotaurine, carotenoids, and ubiquinones.The interplay of antioxidant and pro-oxidant mechanisms in semen determines the overall rate of lipid peroxidation in sperm (Ochsendorf et al., 1998).
Contrary with other studies, addition of antioxidants in the present concentrations in this study was ineffective in improving the longevity and survivability of the Kolbroek boar semen while the addition of antioxidants such as a α-tocopherol, butylated hydroxytoluene, superoxide dismutase, catalase, cysteine or glutathione in extenders to both cooled and frozen-thawed semen have been reported to improve the semen quality in boar (Funahashi and Sano, 2005;Breininger et al., 2005;Satorre et al., 2007), bull (Bilodeau et al., 2001a), turkey (Donoghue and Donoghue, 1997), stallion (Ball et al., 2001b) and ram (Uysal and Bucak, 2007).
Lipid peroxidation which has been shown to also occur in Kolbroek spermatozoa is described as a process under which oxidants such as free radicals or non-radical species attack lipids containing carbon-carbon double bond(s).Glycolipids, phospholipids (PLs), and cholesterol (Ch) are well-known targets of damaging and potentially lethal peroxidative modifiation (Ayala et al., 2002).Lipids can be oxidized by enzymes, for instance lipoxygenases, cyclooxygenases, and cytochrome P450.In response to membrane lipid peroxidation, and according to specific cellular metabolic circumstances and repair capacities, the cells may promote cell survival or induce cell death.Under physiological or low lipid peroxidation rates (subtoxic conditions), the cells stimulate their maintenance and survival through constitutive antioxidants defence systems or signalling pathways activation that upregulate antioxidants proteins resulting in an adaptive stress response.In contrast, under medium or high lipid peroxidation rates (toxic conditions) h h h the extent of oxidative damage overwhelms repair capacity, and the cells induce apoptosis or necrosis programmed cell death.Either process eventually leads to molecular cell damage which may facilitate development of various pathological states and accelerated aging (Kohen and Nyska, 2002) which may be the case in this study.
The decline in sperm characteristics during liquid preservation at 15-17°C has adverse effects on the structure of sperm cell membrane, with consequential in the failure of membrane functions due to the effects of ROS (Mazur et al., 2008).Cerolini et al. (2000) concluded that liquid storage of boar semen decreases sperm viability, motility and progressive motility due to the susceptibility of sperm cells to thermal, mechanical and osmotic stress during cooling of boar semen particularly at low temperature storage because pig semen is known to be very sensitive to low temperature.
Sperm function in pigs is altered rapidly during in vitro storage at 17 to 19°C in pigs, of which one of the supreme factors of failure is reactive oxygen species (Jang et al., 2006).ROS have twofold effects on sperm function, at low concentrations inducing sperm capacitation (Leclerc et al., 1997;Ford, 2004), hyperactivation (de Lamirande and Gagnon, 1993), acrosome integrity, and syngamy, while, in contrast, extreme amounts of ROS cause DNA damage (Aitken and Bennetts, 2005), inhibit syngamy, and reduce equine and porcine sperm motility of (Guthrie and Welch, 2007).
Semen preservation disrupts interactions between plasma lipids and proteins, which weakens membrane permeability and plasticity.This has been correlated to the processes of apoptotic cell death.Apoptosis is defined as programmed cell death (Said, 2004).Apoptotic cells have been correlated to lower motility, in Stallions, as a result of cellular changes, and also have a reduced longevity in the female reproductive tract of sows.
Even though this study shows little effect of antioxidants, these compounds are known to suppress the formation of ROS and protect spermatozoa against ROS damage (Sikka et al., 1995).Studies have demonstrated that seminal plasma also contains a number of enzymatic antioxidants such as superoxide dismutase (SOD, glutathione peroxidase/glutathione reductase (GPX/GRD) and catalase which protect the spermatozoa against lipid peroxidation.Antioxidants are believed to counteract the negative effects of lipid peroxidation hence posing a very positive chance of increasing motility, survivability and the functional membrane permeability (Satorre et al., 2007;Bilodeau et al., 2001b).Antioxidants such as L-cysteine for example play a role in the intracellular protective mechanism against oxidative stress as membrane stabiliser and capacitation inhibitor (Johnson et al., 2000).
L-cysteine can also improve survival time of semen and sperm chromatin structure in fresh chilled boar semen at 15°C (Szczesniak-Fabianczyk et al., 2003).Taurine is derived from cysteine, an amino acid which contains a thiol group.Taurine (2-aminoethanesulfonic acid) is the primary intracellular free-amino acid, which is normally present in most mammalian tissues (Chesney, 1985).Though it is not a component of any structural mammalian protein, taurine plays a variety of critical physiological roles including osmoregulation, cell propagation, viability and prevention of oxidant-induced injury in many tissues.The best known function of vitamin E or tocopherol as an antioxidant is the removal of free radicals that can initiate or propagate lipid oxidation.This action is due to its ability to interact faster with lipid peroxide radicals than they can do so with neighboring fatty acids or with membrane proteins.Tocopherol is anchored in the cell membrane through the isoprene chain, so that the chromatin ring that represents the active part of the molecule is on be on the surface of the structure of lipoproteins, in a position that would allow the interaction with the molecule and free radicals present in the cytoplasm.The tocopheroxil resulting radical reacts with another peroxyl radical, creating a stable product, tocopherol quinone.Thus, a single molecule of tocopherol is able to lead to the interruption of the two oxidation reactions (Ghiuru, 2013).
The current study concurs with that of Ball et al. (2001 b) that motility of boar spermatozoa was not significantly influenced by the addition of antioxidants especially αtocopherol in particular thus the decline over time.
Increased levels of ROS have been linked with reduced motility.Many theories have been proposed to explain this correlation.One hypothesis is that hydrogen peroxide can diffuse into the cytoplasm via the cell membrane and inhibit the activity of enzymes such as glucose-6phosphate dehydrogenase (G6PD) (Griveau et al., 1995).This enzyme controls the rate of flux of glucose via the hexose monophosphate shunt which then maintains the intracellular availability of nicotinamide adenine dinucleotide phosphate (NADPH).This molecule is used as a source of electrons by spermatozoa to fuel the generation of ROS by nicotinamide adenine dinucleotide phosphate oxidase, which is an enzyme system.Inhibition of G6PD leads to a decrease in the availability of NADPH and a concominant of oxidized glutathionine and reduced glutathionine.This reduces the defence mechanism of sperm antioxidants and can lead to membrane phospholipid peroxidation (Griveau et al., 1995) which results in a decline in viability as observed in the current study.
Contrary to this, Aguero et al. (1995) reported a positive effect of α-tocopherol on the motility of liquid preserved stallion semen.Andréa et al. (2008) also found a positive influence of cysteine on motility of refrigerated ram semen which is contrary to this study.According to this study, slight benefit of taurine was observed for less than 24 h of liquid storage.The valuable effects of taurine supplementation as an antioxidant in biological systems have been credited to its ability to stabilize biomembranes (Wright et al., 1986), utilize reactive oxygen species (Wright et al., 1985) and reduce the production of lipid peroxidation end products (Huxtable, 1992).
Cysteine showed the least protection of cells over 48 h of liquid preservation.Three concentrations of the antioxidants were used in a preliminary study (results not published).5 mM proved to produce better results, with cysteine though after 72 h all three concentrations had similar effect.This is in contrast to the results found by Aguero et al (1995).The probable reasons are not yet understood.Similar results were obtained with αtocopherol.
This study shows that Kolbroek boar spermatozoa have unique responses.Preliminary studies (unpublished data), indicate that instead of Androhep extender being a long term extender as with other breeds, only managed to preserve the semen for 3 days, and yet the short term extender, BTS maintain survival for up to 12 h.Further research should be done on the composition of the poly unsaturated fatty acids and seminal plasma constituents of this breed in order to understand the physiology Kolbroek boar spermatozoa physiology in relation to other breeds.Also the bacterial contamination of the Kolbroek semen should be checked during further studies, in fact, as reported from other authors the presence of bacteria could lead to detrimental effects during storage at 15 to 17°C (Bresciani et al., 2014) It is important to evaluate also the different concentrations of the antioxidants and different storage temperatures to establish a more efficient protocol for Kolbroek boar spermatozoa liquid preservation.Taurine was the most effective antioxidant for liquid preservation of Kolbroek spermatozoa at 17°C.The most effective time to elicit antioxidant protection for Kolbroek spermatozoa liquid preservation was 24 h.

Conclusion
Supplementation of androhep extender with antioxidants (cysteine, taurine and tocopherol) was not effective for increasing longevity and survivability of liquid preservation of Kolbroek boar semen at 17°C.Semen characteristics were higher in the untreated sample than samples supplemented with the antioxidants during low temperature; lipid preservation of Kolbroek semen at 17°C.Control samples showed higher motility, membrane functionality and integrity and general survivability over the 48 h storage.

Figure 1 .
Figure 1.Progressive motility of liquid preserved Kolbroek boar semen equilibrated at 17˚C in Androhep extender supplemented with 5 mM cysteine, 75 mM taurine, 200 µl αtocopherol, and control/untreated.Different letters between columns indicate significant differences between and across treatments (P<0.05).