Supplementation of a commercial extender (BTS) with melatonin to improve qualityof boar semen in cooling storage at 15 C Vibuntita Chankitisakul *

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1 แก นเกษตร 42 (4) : (2557) KHON KAEN AGR.J. 42 (4) : (2014) Supplementation of a commercial extender (BTS) with melatonin to improve qualityof boar semen in cooling storage at 15 C Vibuntita Chankitisakul * ABSTRACT: Sperm quality gradually deteriorates with declines in motility and viability because an excess of reactive oxygen species (ROS) occurred during cooling storage. The objective of this study was to determine the effects of adding melatonin (an enhancer of free radical scavenging) during storage on semen quality in terms of sperm motility, viability, and functional integrity of plasma membrane. Semen was extended in the absence (control) and presence (1 μm) of melatonin in the Beltsville Thawing Solution medium, and stored for 7 days. Semen quality was assessed every 12 hours (until day 7). The results demonstrated that the differences in semen quality between the presence and absence of melatonin were significant after the storage time had passed 72 hrs for sperm motility, and 96 hrs for sperm viability and functional integrity of sperms (P < 0.01). In conclusion, the quality of semen in the presence of melatonin improved as the incubation time increased when compared to that of the control. The presence of melatonin in the BTS medium could be used to preserve the quality of sperms, making them last longer while being stored in a refrigerator at 15 C. Keywords: host, melatonin, motility, ROS, viability Introduction Artificial insemination (AI) has been great importance to almost swine industry in Thailand. The use of AI had a major impact on genetic improvement, also reduced the risk of spreading sexually transmitted diseases. In accordance, for smallholder farms in rural areas, living of boars in each farm is restricted economically. AI service was better choice for inseminating as well, also the results from AI could improve those reproductive performances and cost of production (Am-In et al., 2010). Besides, another advantages of AI is that it s permit you to assess semen quality before insemination such as sperm motility, viability, morphology (Am-In et al., 2010), acrosome and DNA integrity, and in vitro fertilization rate (Boe-Hansen et al., 2008; Parrilla et al., 2012), resulting in using high semen quality for AI then. Normally fresh semen is extended with either commercial extender or not and then stored in the refrigerator at C (chilled semen). The limitation of using chilled semen is short storage time about 3-7 days, depended on extender types (Johnson et al., 2000; Waberski et al., 2011). Subsequently the fertility in terms of conception rate and litter sizes gradually decline with ongoing semen storage, probably due to oxidative stress during storage (Johnson et al., 2000). Oxidative stress is caused by the imbalance between production of reactive oxygen species (ROS) and the protective effect of the antioxidant system responsible for their neutralization and removal (Walczak-Jedrzejowska et al., 2013). 1 Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, * Corresponding author: vibuch@kku.ac.th

2 618 แก นเกษตร 42 (4) : (2557) In male reproductive tract, seminal plasma normally plays responsibility for produce antioxidants for instance vitamin E, vitamin C, glutathione. However, once ejaculated semen is collected, those are thoroughly diminished (Brezezinska- Slevbodzinska et al., 1995). Then, an excess of ROS causes a pathological response leading to damage of sperm membranes. The consequence of membrane damages is decreased sperm motility, viability, alternations in membrane permeability and fertility ability (Cummings et al., 1994). Boar sperm are particularly susceptible to peroxidative damage because their cell membrane contains large amounts of polyunsaturated fatty acids (PUFAs) which serve as preferred substrates for ROS in membranes (Awda et al., 2009; Kumaresan et al., 2009). Many attempts using antioxidants as semen additives in the extender have been proposed to improve the sperm function (Pena et al., 2003; Roca et al., 2004; Funahashi and Sano, 2005). Melatonin has an antioxidant and free radical scavenging abilities, reducing concentrations of reactive oxygen species at the mitochondrial level in somatic cells (Cruz et al., 2014). Recently, supplementation of melatonin as a potent antioxidant has been implied either during cryopreservation or not in sperm of deer (Domínguez-Rebolledo et al., 2010), horse (da Silva et al., 2011), buffalo (Li et al., 2012), and bull (Zhao et al., 2014) preservation. In pigs, it has only one report of melatonin supplemented in extender and storage for long term at 17 C (Martín-Hidalgo et al., 2011). However as temperature declines especially from at 15 C, there is an inevitable reduction in the normal sperm of boar (Johnson et al., 2000; Watson, 2000). In addition, the supplementation of melatonin in the Beltsville Thawing Solution (BTS) medium which is a well-known short term extenders in Thailand has never been examined. This study was therefore hypothesized that melatonin treated chilled BTS medium could extend the storage time and improve the semen quality while those are being stored in refrigerator at 15 C which is the standard storage temperature in Thailand. Therefore the present study was conducted to examine the effects of melatonin supplementation in the BTS medium for 7 days on semen quality in terms of sperm motility, viability, and functional integrity of the sperm plasma membrane. Materials and Methods The presented study was conducted at the swine unit and reproductive biotechnology laboratory of Animal Science, Faculty of Agriculture, Khon Kaen University. Semen collection and processing Nine ejaculated semen from three boars (Duroc, Large white, and Landrace; 2-4 years old) were collected using gloved-hand method. Those boars are used as inseminating to gilts and sows on a regular basis. Semen was immediately kept in an insulated thermo flask at 37 C during transporting to the laboratory within 15 min. At the laboratory, semen was firstly assessed the motility, less than 70 % motile sperm were excluded from the experiment. Besides, semen characteristics were evaluated and recorded with routinely analysis including sperm viability, and the functional integrity of the plasma membrane. The boar semen was diluted with BTS medium further to a final concentration of 3 x 10 7 sperm/ml. The samples were then stored in 15 ml centrifuge

3 KHON KAEN AGR.J. 42 (4) : (2014) 619 tube. To reduce the effects of cold shock on the sperm during cooling, the tubes were placed in a room temperature (25 to 27 C) for min. Then they were placed in the refrigerator at 15 C and stored for 7 days. The temperature was monitored constantly by digital temperature through the experiment. Every 12 hours, an aliquot of each sample was evaluated for sperm characteristics as described below. Experimental design Base on the preliminary studies in swine (Martín-Hidalgo et al., 2011) and horse (da Silva et al., 2011), the concentration of melatonin was 1 mm (34 mm stock solution in ethanol). To examine the effects of melatonin supplementation in the cooling extender on sperm quality (in terms of sperm motility, viability, and functional integrity of the plasma membrane), fresh boar semen was extended with BTS medium supplemented with or without 1 mm melatonin and stored for 7 days. Then the semen samples were assessed every 12 hours (hour 0 = time of storage). Sperm quality assessments A 500 ml aliquot of each chilled semen sample was taken on each experimental hour. The sample was warmed at 37 C for 15 min before assessment. Sperm motility Semen samples were dropped on the slide, motility then was subjectively evaluated by visual estimation by the same personal throughout the study under a phase-contrast microscopy at x 400 magnification. The motility was expressed as percentage of progressively motile sperm. Sperm viability The percentage of sperm viability was determined by eosin-nigrosin stain. Total or partly red stained cells were considered dead while viable sperm remained unstained. Two hundred sperm per smear were counted under a phase-contrast microscopy at x 400 magnification. At least two smears per sample were assessed. The functional integrity of the sperm plasma membrane The functional integrity of the sperm plasma membrane was assessed using a short hypo-osmotic swelling test (shost) (Chanapiwat et al., 2012). Sperm were incubated at 37 C for 30 min, with 75 mosm/kg a hypo-osmotic solution [0.368% (w/v) Na-citrate, 0.675% (w/v) fructose (Merck, Darmstadt, Germany)]. Then they were fixed in hypo-osmotic solution plus 5% formaldehyde (Merck, Darmstadt, Germany), for later assessment. A small drop from suspension was placed on a clean slide and covered with a cover slip. At least two hundreds sperm were assessed under a phase contrast microscope at 400 x magnification. Two slides per sample were assessed. The coil-tailed sperm were determined as functional intact plasma membrane. Statistical analysis Before analyzing, the boar effect was tested by analysis of variance method. It was found that the effect of different breeds on semen quality was not significantly both in melatonin and control groups (P>0.05). Then data on motility, viability and plasma membrane integrity were analyzed by repeated measurement in complete randomized design. Testing of significant of treatments used

4 620 แก นเกษตร 42 (4) : (2557) least significant different (LSD) by SAS version 9.0. Statistical significant difference was defined as P < 0.05 and Results After semen samples were extended in BTS medium supplemented with or without (control) 1 mm melatonin, the following parameters were initially evaluated: sperm motility, viability and plasma membrane integrity before stored in the refrigerator (hour 0). The results were demonstrated that they were no significant difference between the melatonin group and control group in the percentages of sperm quality in terms of sperm motility, viability, and plasma membrane integrity (85.5±5.2 vs 85.5±5.2, 92.3±1.8 vs 92.3±2.2, and 85.7±7.4 vs 88.5±7.8, respectively; P>0.01). Figure 1 shows changes in sperm quality with storage time between the presence and absence of melatonin as follows: Effects of melatonin supplementation in the chilling extender on sperm motility Sperm motility decreased dramatically throughout the storage time in both groups. However before 72 hours of storage time, no statistical difference on sperm motility was observed between the melatonin and the control group. The sperm motility in the melatonin group were better than the control group since the storage time was 72 hours (P<0.01; Figure 1a). Effects of melatonin supplementation in the chilling extender on sperm viability Melatonin maintained the percentage of sperm viability not less than 80 through the whole period. The percentage of sperm viability was decreased significantly in the control group than in melatonin group since the storage time was 96 hours (P<0.01; Figure 1b). Effects of melatonin supplementation in the chilling extender on the HOST The percentage of the functional integrity of the plasma membrane (HOST) also decreased with storage time. The percentage of incidence of coiled tail sperm reacting positively to the test was significantly lower in the absence of melatonin after 96 hours of preservation (P<0.01; Figure 1c).

5 KHON KAEN AGR.J. 42 (4) : (2014) 621 Figure 1 Mean±SE of individual motility, viability, and integrity of plasma membrane (HOST) of chilled boar sperm after storage every 12 hours. Nine replicates were performed and analyzed. a,b values with different are statistically different within each storage hour (P<0.01)

6 622 แก นเกษตร 42 (4) : (2557) Discussion In the present study, melatonin supplementation in commercial extender effectively improved the quality of semen in terms of sperm motility, viability and functional of sperm integrity during storage for 7 days in refrigerator at 15 C. Changes in the quality of sperm in relation to storage time between the presence and absence of melatonin were determined after 72 hours for sperm motility, after 96 hours for sperm viability and functional of sperm integrity. Even the sperm quality in the melatonin group was better than the control group; subsequently leading to possibly extend the storage time of semen in BTS extender from three days as manufacturers recommendation to be more. However the percentage of motility and plasma membrane integrity were less than 70% after Day 5 of storage. This circumstance alerted the awareness whether it perhaps impossible to use semen even if the sperm quality kept in BTS with melatonin was better than control. As practically, after ejaculated sperm had been collected, it was preserved as liquid for a short while either in the storage box or in the refrigerator in which the temperature was controlled constantly. It commonly hypothesized that sperm quality was gradually deteriorated with decline in motility and viability as sperm cells exposed to a number of potential sources of stress including dilution and cooling incubation (Cummings et al., 1994; Waberski et al., 2011) resulted in damaging to sperm plasma membrane through lipid peroxidation from increased level of ROS (Walczak-Jedrzejowska et al., 2013). Boar sperm are particularly wellknown regarded as susceptible to peroxidative damage because of their high content of PUFAs that serve as preferred substrates for reactive oxygen species in membranes (Brouwers et al., 2005). Besides, another organelle involves with ROS generation in the sperm is mitochondria in which more sensitive whether it confronts cooling or freezing temperatures (Kopper et al., 2008). In animal cells, the main function of melatonin is facilitating the activities of several enzymes (superoxide dismutase, glutathione peroxidase, and catalase) involved in metabolizing ROS and thereby preserves cell membrane fluidity (García et al., 1997; Okatani et al., 2000) as well as protects the mitochondria (da Silva et al., 2011). In the present study, the presence of melatonin in extender medium improved the quality of semen in terms of sperm motility, viability and functional of sperm integrity during storage for 7 days when compared with control. A similar improvement of sperm quality which enhances sperm motility, viability, and survival rate as well as preserving membrane integrity and lowering lipid peroxidation was addressed in several studies (Jang et al., 2010; Martín-Hidalgo et al., 2011; Cruz et al., 2014; Domínguez-Rebolledo et a., 2014). It was important to clarify whether how long the BTS medium supplemented with melatonin could be stored and still effectively maintained sperm quality for insemination. It was noted that the difference of semen quality between the presence and absence of melatonin was significantly since the storage time passed to 72 hours for sperm

7 KHON KAEN AGR.J. 42 (4) : (2014) 623 motility and 96 hours for sperm viability and functional of sperm integrity. However the motility and plasma membrane integrity were lower than 70% since the storage was 120 hours (Day 5) meanwhile the percentage of sperm viability was higher than 80% through the storage time. BTS medium is the most widely used semen extender throughout the world; it is suitable for short term storage without effect on sperm quality and fertility (Gadea, 2003; Mapeka et al., 2012). The good extenders normally could act as an energy source for sperm metabolism, could provide ph buffering from sperm cell waste, ions for membrane and cell balance (Knox, 2011). However, the properties of BTS medium were limited by time. Hofmo (1991) showed the evident that fertility and litter size assessed after insemination with semen preserved in BTS for hours were significantly decreased. Similar results were obtained by Alexopoulos et al. (1996) who reported reduced fertility when semen was stored for more than 72 hours in BTS medium. In accordance, Guthrie et al. (2008) has recently inferred that ROS formation and lipid peroxidation seemed to be low during the first five days of cooling storage. It would be due to the fact that viable sperm may have a substantial amount of intracellular superoxide dismutase (SOD) for scavenging ROS produce after cooling (Kim et al., 2011; Walczak-Jedrzejowska et al., 2013). It was therefore a time-dependent, a significant increase in the percentage of ROS releasing. Thus it was inferred that the quality of sperm either by the presence of melatonin or not had been maintained for a short while during the first few days. Later the quality of extended semen was decreasingly by a rapid loss of motility and membrane integrity because of the reduction of both antioxidants and medium quality. Interestingly, those did not affect viable sperm function. In this study, the viability of sperm was always higher than the motility and HOST irrespective of the storage time. It would be due to the fact that some sperm are still living even they are immotile. A similar finding was found in the previous study, the proportion of live sperm with an intact acrosome was extended after storage for 7 days (Martín-Hidalgo et al., 2011). The other word, boar sperm motility and plasma membrane integrity were more sensitive damaged of oxidative stress than sperm viability. Therefore there is the answer that even the extender was supplemented with melatonin and the sperm motility and membrane integrity were significantly improved, but their functions could be still more damaged than sperm viability. The motility is an indication of the active metabolism and integrity of membrane (Johnson et al., 2000). The percentages of motility and plasma membrane integrity would be therefore consideration for practical using in case that the storage time will be postponed. Otherwise those sub-optimal sperm quality could not be able move into the uterus, could not bind or react with the oocytes, and could not fertilize the mature oocytes, eventually. It would be kept in mind in making a decision of an optimal sperm quality which perhaps results in the production then.

8 624 แก นเกษตร 42 (4) : (2557) Conclusions Accordingly, in the present results, melatonin treatment did not affect quality of sperm whether semen were stored in the commercial extender (BTS) before Day 3. However the presence of melatonin could extend the storage time and improve the semen quality while those are being stored in refrigerator at 15 C. The sperm quality kept in BTS with melatonin in this study could be used for insemination until Day 5 of storage. Acknowledgments The author is grateful to Department of Animal Science, Faculty of Agriculture, Khon Kaen University for supporting the financial and providing facilities. Thank you to Assistant Professor Wuttigrai Boonkum for his assistance in statistical analysis. References Alexopoulos, C., C. Boscos, P. H. Saratsis, C. Saoulidis, and S. Kyriakis The effect of storage time and number of spermatozoa per insemination dose on semen characteristics and fertilizing capacity of boar semen diluted with Beltsville Thaw Solution (BTS) extender. Anim. Sci. 62: Am-in, N., W. Tantasuparuk, and M. Techakumphu Comparison of artificial insemination with natural mating on smallholder farms in Thailand, and the effects of boar stimulation and distance of semen delivery on sow reproductive performance. Trop. Anim. Health. Prod. 42: Awda, B. J., M. Mackenzie-Bell, and M. M. Buhr Reactive oxygen species and boar sperm function. Biol. Reprod. 81: Boe-Hansen, G. B., P. Christensen, D. Vibjerg, M. B. Nielsen, and A. M. Hedeboe Sperm chromatin structure integrity in liquid stored boar semen and its relationships with field fertility. Theriogenology. 69: Brezezinska-Slevbodzinska, E., A. B. Slebodzinski, B. Prietas, and G. Wieezorek Antioxidant effect of vitamin E and glutathione on lipid peroxidation in boar seminal plasma. Biol. Trace. Elem. Res. 47: Brouwers, J. F., P. F. Silva, and B. M. Gadella New assays for detection and localization of endogenous lipid peroxidation products in living boar sperm after BTS dilution or after freeze-thawing. Theriogenology. 63: Chanapiwat, P., K. Kaeoket, and P. Tummaruk Cryopreservation of boar semen by egg yolk-based extenders containing lactose or fructose is better than sorbitol. J. Vet. Med. Sci. 74: Cruz, M. H., C. L. Leal, J. F. da Cruz., D. X. Tan, and R. J. Reiter Role of melatonin on production and preservation of gametes and embryos: a brief review. Anim. Reprod. Sci. 145: Cummings, J. M., A. M. Jequier, and R. Kan Molecular biology of the human male infertility: links with ageing, mitochondrial genetics and oxidative stress. Mol. Reprod. Dev. 37: da Silva, C. M., B. Macías-García, A. Miró-Morán, L. González-Fernández, A. Morillo-Rodriguez, C. Ortega-Ferrusola, J. M. Gallardo-Bolaños, G. Stilwell, J. A. Tapia, and F. J. Peña Melatonin reduces lipid peroxidation and apoptotic-like changes in stallion spermatozoa. J. Pineal. Res. 51: Domínguez-Rebolledo, A. E., M. R. Fernández-Santos, A. Bisbal, J. L. Ros-Santaella, M. Ramón, M. Carmona, F. Martínez-Pastor, and J. J. Garde Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments. Reprod. Fertil. Dev. 22: Funahashi, H., and T. Sano Select antioxidants improve the function of extended boar semen stored at 10 C. Theriogenology. 63: Gadea, J Review: Semen extenders used in the artificial insemination of swine. Span. J. Agric. Res. 1:

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