P. Thananurak 1, C. Sittikasamkit 2, T. Vongpralub 2 and K. Sakwiwatkul 1* KHON KAEN AGR. J. 43 SUPPL. 2 : (2015).

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1 98 KHON KAEN AGR. J. 43 SUPPL. 2 : (2015). KHON KAEN AGR. J. 43 SUPPL. 2 : (2015). Effects of Addition of Reduced Glutathione to Thawing media on Motility Parameters, Lipid Peroxidation and Fertility Rate in Frozen-Thawed Chicken Spermatozoa P. Thananurak 1, C. Sittikasamkit 2, T. Vongpralub 2 and K. Sakwiwatkul 1* ABSTRACT: Sperm cryopreservation represents a useful tool in genetic conservation management of indigenous in chicken. However, freezing and thawing produce physical and chemical stress on the sperm membrane that reduces their viability and fertilizingability. The objective of the present study was, therefore, to evaluated the effects of reduced glutathione (GSH; 0.1, 0.5 and 1 mm) supplementation in the thawing diluent on post-thawed parameters of frozen chicken spermatozoa. Semen was collected from 24 Thai native chickens (Pradu Hang Dum). Semen sample were frozenby liquid nitrogen vapor method. After thawing, diluent supplementation with each of GSH concentrations. Sperm motility characteristics were assayed by CASA, ROS generation was assessed by TBARSand fertility test of frozen semen was carried on by inseminated to layer hens. The results showed that addition of 0.1, 0.5 and 1 mm GSH to the thawing media decreased the percentage of motile, progressively motile spermatozoa and fertility rate. Whereas, addition of GSH to the thawing media resulted in a reduction in ROS generation.our results do not support the use of GSH (0.1 to 1 mm) to improve the quality of post-thawed chicken semen. Keywords: glutathione, cryopreservation, native fowl Introduction Avian spermatozoa membranes are rich in polyunsaturated fatty acid (PUFAs) and can easily undergo lipid peroxidation (LPO) in the presence of reactive oxygen species (ROS) (Cerolini et al., 2006) ROS-induced damage to spermatozoa is mediated by oxidative attack of bis-allylic methylene group of sperm phospholipid bound PUFAs, leading to lipid peroxidation and reactive molecules produced during oxygen reduction that affect the deterioration of the function and viability of the spermatozoa, include irreversible loss of motility, inhibition of respiration, damage to sperm DNA and leakage of intracellular enzyme. To counteract the harmful effect of ROS, spermatozoa possess a number of antioxidant systems that scavenge ROS and prevent internal cellular damage (Jeulin et al., 1989) Glutatione (L-γ-glutamyl-L-cysteinylglycine) is a tripeptide ubiquitously distributed in living cells. It plays an important role in the intracellular defensive mechanism against oxidative stress. Glutathione peroxidase uses GSH to reduce hydrogen peroxidase to H 2 O and lipid peroxidase to alkly alcohols. GSH content has been reported in spermatozoa of different species, included goat (Gadea et al., 2013). A reduction in GSH content in spermatozoa induced by the freezing procedures has been previously described in human (Gadea et al., 2011) and in domestic animals such as boar (Gadea et al., 2004) or bull (Stradaioli et al., 2007) It is known that freezing induces a decrease in the antioxidant system and that increased ROS levels are present after cryopreservation, it is rea- 1 Faculty of Technology, Mahasarakham University, Mahasarakham 44150, Thailand 2 Faculty of Agricultural, Khonkean University, Khonkean 40002, Thailand * Corresponding author: sakwiwatkul4@hotmail.com

2 KHON KAEN AGR. J. 43 SUPPL. 2 : (2015). 99 sonable to explore the use of antioxidant strategies in an attempt to overcome cryodamage related to this oxidative stress (Bucak et al., 2010). No previous study was performed for investigating the effect of glutathione supplementation into postthawed chicken semen on subsequence sperm quality. The aim of this study was, therefore, to investigate the effect of addition of GSH to thawing media on motility parameters and ROS generation in frozen-thawed chicken spermatozoa. Material and methods Animal Twenty four mature (10 months), Thai native cocks (Pradu hang dam) were kept in individual cages. Cockerels were fed 130g/head/day and water was provided ad libitum. The animals were reared under natural environmental condition, where they received a natural light : dark photoperiod (11.14L : 12.46D to 13.01L : 11.59D) throughout the experiment. Semen collection The pooled semen from 24 individuals Thai native cocks was collected two time a week, by the dorso-abdominal massage method (Burrows and Quinn, 1937). Semen from an individual cock was collected in a 1.5 ml microtube containing 0.1 ml Schramm diluents (compose of 0.7 g magnesium acetate, 28.5 g sodium glutamate, 5 g glucose, 2.5 g inosital and 5 g potassium acetate dissolved in double-distilled water 1,000 ml). To maximize the semen quality and quantity, the collection was always performed by the same people, under the same conditions, time, and massage method. Ejaculates having good motility ( 85%) were used in this study. This research project was approved by Animal Ethics Committee of Khon Kean University (Approval No: /31) Semen extending, freezing, and thawing Irrespective of the treatment, the pooled ejaculates in every experiment were diluted using Schramm diluents, cooled to 5 ºC in 60 min. Then, diluted with DMF (N,N-Dimethylformamide; Sigma-Chemical Co., St.Louis, USA, D-4551) to the final concentration of 6% (v/v) in diluted semen. After 15 min of equilibration, 500- µl aliquots of treated semen were immediately loaded into 0.5 ml plastic strawswith sperm concentration of 500x106/straw (IMV ref ), sealed with polyvinylpyrrolidone (PVP) powder (IMV ref ). The straws cooled down to -35 ºC by exposure to liquid nitrogen vapor for 10 min at 11 cm above liquid nitrogen, located at -135 ºC for 5 min and then plunged into liquid nitrogen for storage at -196 ºC until analysis.(vongpralub et al., 2011). After storage, the straws were thawed individually in an iced water bath at 5 ºC for 5 min. To examine the effect of GSH supplementation on post-thawed semen, diluents with each of GSH at various concentrations (0.1, 0.5 and 1 mm GSH) was to each thawed semen sample and maintained at 5 ºCand then evaluated for sperm function. Analysis of the post-thaw sperm motion parameters and production of reactive oxygen species Analysis of post-thaw sperm motion parameters were determined using a computer assisted sperm analysis (CASA) (HTM-IVOS Model 10 Spermatozoa Analyzer; Hamilton Thorne Biosciences, Beverly, MA, USA)

3 100 KHON KAEN AGR. J. 43 SUPPL. 2 : (2015). Sperm oxidative levels were determined using the thiobarbituric acid (TBA) reaction. 250x106spz/ml. of each spermatozoa treatment was incubated in the presence of 2.78% ferrous sulfate.7h2o (Ajex, ), 0.1 ml 0.22% butylatedhydroxytoluene (Sigma, B1378) at 37 ºC for 60 min. The reaction was stopped by adding 1 ml 35% trichloroacetic acid (Sigma, T6399) and kept on ice for 15 min. Samples were centrifuged at 7,800xg for 15 min and the supernatant retained. The progress of endogenous peroxidation was followed by adding 1 ml 0.36% thiobarbituic acid (Sigma-T550-0) to 2 ml of supernatant. That mixture was boiled for 10 min, allowed to cool, thenproduction of reactive oxygen species was measured by a spectrophotometer Carry Conc. UV-Visible Spectrophotometer (Specord 250 plus, Analytikjena)and absorbance levels acquired by spectrometry at 532 nm(partyka et al., 2007) Fertilizing ability test: The fertilizing ability test was performed the frozen-thawed semen. Frozen-thawed semen (the experimental group) was inseminated once a week with a dose of 0.4 ml (48 layer hen were inseminated). All insemination were performed between Eggs were collected from day two after the first insemination. Fertility was determined by candling eggs on day seven of incubation. Statistical analysis The experiment was conducted as randomized complete block design (RCBD) and differences in number of particular categories of spermatozoa in frozen-thawed semen were analyzed with ANOVA and Duncan s multiple range tests. The results are presented as mean ± SE of measurements on sample from 6 replicate determinations (P<0.05). Results and Discussion Effects of addition of GSH to the thawing media on post-thaw sperm motility, ROS generation and fertility rate Addition of GSH to post-thawed semen at 0 hr (5 min) and 1 hr had a significant effect on the analyzed sperm motility parameter (Table 1, P<0.05). Total motility of spermatozoa in the control was significantly higher than 0.1, 0.5 and 1 mm GSH groups with no difference between GSH groups. However, for the progressive motility and other motility parameter, there were no statistically different. Table 1 Motility parameters of frozen-thawed chicken spermatozoa measured by the computer assisted semen analysis (CASA) system at 0 hr (5 min) and 1 hr. (mean ± SE; n=6) GSH (mm/ ml) Motility of parameter 0 hr (5min) 1 hr MOT PMOT VAP VSL VCL MOT PMOT VAP VSL VCL ±1.16 a 29.16± ± ± ± ±1.00 a 26.50± ± ± ± ±1.58 b 26.33± ± ± ± ±1.05 b 23.00± ± ± ± ±1.60 b 25.50± ± ± ± ±1.04 b 23.33± ± ± ± ±1.64 b 25.33± ± ± ± ±1.51 b 23.83± ± ± ±1.59 Different letters ( a,b ) within columns indicate significant differences (P<0.05). Total motile sperm (MOT), progressive motile sperm (PMOT), average path velocity (VAP), straight linear velocity (VSL) and curvilinear velocity (VCL)

4 KHON KAEN AGR. J. 43 SUPPL. 2 : (2015). 101 The generation of ROS was significantly reduced following the addition of GSH to the thawing media compared to control group during the 0 hr (5 min) and 1 hr of incubation period. Differences were found between 1 mm GSH was reduced to remain 20% of the values compare in the control group. Results showed that addition of GSH to the thawing media decrease fertility rate (Table 2, P<0.05). Table2 Malondialdehyde concentration as level of lipid peroxidation (MDA) and fertility rate in frozen-thawed chicken semen. (mean ± SE; n=6) maloaldehyde (MDA) Fertility rate (µm/ml/50x10 6 spz) 0 hr (5 min) 1 hr Number of set egg Fertility (%) ±0.36 a 1.13±0.07 a ±1.61 a Glutathione ±0.26 b 0.87±0.65 b ±1.92 b (mm/ml) ±0.35 b 0.74±0.09 b ±1.23 b ±0.67 c 0.18±0.06 c ±1.30 b Different letters ( a,b ) within columns indicate significant differences (P<0.05) In this studies, evaluated the effects of GSH supplementation of the thawing media on sperm function. The main findings emerging from this study indicate that addition of GSH to the thawing media resulted in a lower number of reduction in ROS generation. GSH supplementation to thawing media could compensate for the decreased GSH content observed during sperm freezing and thawing. When add to the thawing media,this protective effect on sperm function was more pronounced with 1 mm GSH. This could be related to the lower membrane stability caused by a high concentration of GSH when GSH levels are within normal limit in semen (Baty et al., 2002). Addition of GSH to the thawing media would be expected to improve motility and fertilizing ability of frozenthawed boarand human spermatozoa, as addition of GSH has been shown to help to maintain sperm motility and improves sperm s in vitro oocyte penetration ability (Gadea et al., 2004; 2005; 2011). In contrast, to this study, motility and fertility rate was declined after the higher concentration of GSH was added to thawing media.nevertheless, the efficiency in the supplementation of antioxidants is increased when it is done after the thawing process as previously our research group showed in human (Martinez-Sato et al., 2010, Gadea et al., 2011), boar (Gadea et al., 2004;2005) and goat frozen-thawed spermatozoa (Gadea et al., 2013). In conclusion, the addition of GSH (0.1 to 1 mm) are negative effect on post-thawed chicken spermatozoa quality. However, the results show that GSH significant inhibits ROS generation. To our knowledge, this study is the first report studying the effect of GSH supplementation into postthawed semen on the quality and fertility in chicken. Acknowledgment The authors would like to thank the Research and Development Network Center for Animal Breeding (Native Chicken) and Department of

5 102 KHON KAEN AGR. J. 43 SUPPL. 2 : (2015). Animal Science agriculture, Khon Kaen University for supported animal experiment. We would like to thanks to Institute of agricultural technology, Suranaree University of Technology for semen quality technical assistant. Moreover we would like to thanks research grand from Mahasarakham University. References Buty, B.A., M.B. Hampton and C. C. Winterbourn Detection of oxidant sensitive and thiol proteins by fluorescence labeling and two-dimensional electrophoresis. Proteomics. 2: Bucak, M.N., S. Sarlozkan, P. B. Tuncer, F. Sakin, A. Atessahin and R. Kulakslz The effect of antioxidants on post-thawed Angora goat (Capra hircusancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small Rumin Res. 89: Gadea, J., E. Selles, M.A. Marco, P. Coy, C. Matas, R. Romar and S. Ruiz Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders. Theriogenology. 62: Gadea, J., F. Garcia-Vazquez, C. Matas, J.C. Gardon, S. Canovas and D. Gumbao Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function. J Androl.26: Gadea, J., M. Molla, E. Selles, M.A. Marco, F.A. Garcia- Vazquez and J.C. Gardon Redued glutathione content in human sperm is decreased after cryopreservation: effect of addition of reducedglutathione to the freezing and thawing extenders. Cryobiology. 62: Gadea, J., D. Gumbao, B. Gomez-Gimenez and J.C. Gardon Supplematation of the thawing medium with reduced glutathione improves function of frozen-thawed goat. Reprod Biol. 13: Jeulin, C, J. C. Soufir, P. Weber, D. Laval-Martin and R. Calvayrac Catalase activity in human spermatozoa and seminal plasma. Gam Res. 24: Martinez-Soto, J. C., J. de DiosHourcade, A. Gutierrez- Aden, J. L. Landeras and J. Gadea Effect of genistein supplementation of thawing medium on characteristics of frozen human spermatozoa. J Androl. 12: Partyka, A., A. Jerysz and P. Pokorny Lipid peroxidation in fresh and storage semen of Greenlegged partridge. EJPAU 10(2), [Online]. Available: /art-08.html Stradaiolo, G., T. Noro, L. Sylla and M. Monaci Decrease in glutathione (GSH) content in bovine sperm after cryopreservation: comparison between two extenders. Theriogenology.67: Vongpralub T., A. Utha, J. Pongpeng, P. Sonseeda and W. Yindee A comparison of simple vapour method and programmable freezer on motility and fertility of frozen Thai native chicken semen. The 3 rd International conference on sustainable animal agriculture for developing countries (SAA- DC2011)