Document Type : Original Article


1 Department of Theriogenology and Poultry Disease, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

2 Department of Clinical Sciences, Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran


Heat stress (HS) has caused relative hypoxia, oxidative stress and high level of homocysteine, which contributes significantly to fertility failures in bulls. The aim of present study was to evaluate the role of dietary betaine (BET) in improving dual purpose Simmental (Fleckvieh) post-thawed semen quality especially during the hottest summer days. A total number of 16 mature bulls were randomly assigned to three equal groups including: 1) Control condition (without betaine), 2) BET1: 57.00 mg of betaine kg-1 per day and 3) BET2: 114 mg of betaine kg-1 per day, through daily intakes for 90 days in summer. Plasma levels of homocysteine, seminal plasma antioxidants levels and sperm parameters such as DNA fragmentation, chromatin integrity, motility, viability, morphology and membrane integrity were evaluated. Under maximal HS, serum homocysteine concentrations were reached 16.67 ± 0.09 µmol L-1. Dietary betaine supplementation influenced DNA fragmentation of sperm and was higher in the control group compared to BET2 group. There were significant decreases in seminal plasma superoxide dismutase (SOD), glutathione peroxidase (GPx) activity and sperm viability and motility in bulls treated with betaine. The activity of GPx and SOD in the control group was increased up to 0.08 ± 0.00 U mg-1 protein and 0.52 ± 0.01 U mg-1 protein in seminal plasma. There were no significant differences between groups in the percentage of swollen spermatozoa, membrane integrity, sperm morphology, abnormal head morphology and percentage of spermatozoa stained with aniline blue. In conclusion, BET supplements improved semen parameters in sperm motility, sperm viability and influenced DNA fragmentation during HS with reduction in serum homocysteine concentrations.



    1. Armstrong DV. Heat stress interaction with shade and cooling. J Dairy Sci 1994; 77(7): 2044-2050.
    2. Brito LF, Silva AE, Barbosa RT, et al. Testicular thermoregulation in Bos indicus, crossbred and Bos taurus bulls: relationship with scrotal, testicular vascular cone and testicular morphology, and effects on semen quality and sperm production. Theriogenology. 2004 15; 61(2-3): 511-528.
    3. Fuerst-Waltl B, Schwarzenbacher H, Perner C, et al. Effects of age and environmental factors on semen production and semen quality of Austrian Simmental bulls. Anim Reprod Sci 2006; 95(1-2): 27-37.
    4. Taylor JF, Bean B, Marshall CE, et al. Genetic and environmental components of semen production traits of artificial insemination Holstein bulls. J Dairy Sci 1985; 68(10): 2703-2722.
    5. Collier RJ, Renquist BJ, Xiao Y. A 100-year review: stress physiology including heat stress. J Dairy Sci 2017; 100(12): 10367-10380.
    6. Lenzi A, Gandini L, Picardo M, et al. Lipoperoxidation damage of spermatozoa polyunsaturated fatty acids (PUFA): scavenger mechanisms and possible scavenger therapies. Front Biosci 2000; 5: E1-E15. doi: 10.2741/lenzi.
    7. Shadmehr S, Fatemi Tabatabaei SR, Hosseinifar S, et al. Attenuation of heat stress-induced spermatogenesis complications by betaine in mice. Theriogenology 2018; 106: 117-126.
    8. Rahman MB, Schellander K, Luceño NL, et al. Heat stress responses in spermatozoa: Mechanisms and consequences for cattle fertility. Theriogenology 2018; 113: 102-112.
    9. Lipiński K, Szramko E, Jeroch H, et al. Effects of betaine on energy utilization in growing pigs-A review. Ann Anim Sci 2012; 12(3): 291-300.
    10. Liu KS, Pan F, Chen YJ, et al. The influence of sperm DNA damage and semen homocysteine on male infertility. Reprod Dev Med 2017; 1(4): 228-232.
    11. Rizzo A, Sciorsci RL. Role of homocysteine metabolism in animal reproduction: A review. Res Vet Sci 2019; 122: 29-35.
    12. Cronjé PB. Heat stress in livestock–the role of the gut in its aetiology and a potential role for betaine in its alleviation. Rec Adv Anim Nutr 2005; 15: 107-122.
    13. Morsy WA, Hassan RA, Abd El-Lateif AI. Effect of dietary ascorbic acid and betaine supplementation on semen characteristics of rabbit bucks under high ambient temperature. In proceedings: 10th World Rabbit Congress. Sharm El- Sheikh, Egypt 2012; 23-27.
    14. Lugar DW, Gellert T, Proctor J, et al. Effects of supplementation with betaine and superdosed phytase on semen characteristics of boars during and after mild heat stress. Prof Anim Sci 2018; 34(4): 326-338.
    15. Hall LW, Dunshea FR, Allen JD, et al. Evaluation of dietary betaine in lactating Holstein cows subjected to heat stress. J Dairy Sci 2016; 99(12): 9745-9753.
    16. Cabezón FA, Schinckel AP, Richert BT, et al. Effect of betaine supplementation during summer on sow lactation and subsequent farrowing performance. Prof Anim Sci 2016; 32(5): 698-706.
    17. National Research Council (U.S.). Committee on physio-logical effects of environmental factors on animals. A guide to environmental research on animals. Washing-ton, USA: National Academy of Sciences 1971; 217-228.
    18. Bohmanova J, Misztal I, Cole JB. Temperature-humidity indices as indicators of milk production losses due to heat stress. J Dairy Sci 2007; 90(4): 1947-56.
    19. Revell SG, Mrode RA. An osmotic resistance test for bovine semen. Anim Reprod Sci 1994; 36(1-2): 77-86.
    20. Tamuli MK, Watson PF. Use of a simple staining technique to distinguish acrosomal changes in the live sperm sub-population. Anim Reprod Sci 1994; 35(3-4): 247-254.
    21. Tvrdá E, Lukáč N, Schneidgenová M, et al. Impact of seminal chemical elements on the oxidative balance in bovine seminal plasma and spermatozoa. J Vet Med 2013; 2013: 125096. doi: 10.1155/2013/125096.
    22. Tejada RI, Mitchell JC, Norman A, et al. A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril 1984; 42(1): 87-91.
    23. Hofmann N, Hilscher B. Use of aniline blue to assess chromatin condensation in morphologically normal spermatozoa in normal and infertile men. Hum Reprod 1991; 6(7): 979-982.
    24. Aitken RJ, Roman SD. Antioxidant systems and oxidative stress in the testes. Oxid Med Cell Longev 2008; 1(1): 15-24.
    25. Reyes JG, Farias JG, Henríquez-Olavarrieta S, et al. The hypoxic testicle: physiology and pathophysiology. Oxid Med Cell Longev 2012; 2012: 929285. doi: 10.1155/ 2012/929285.
    26. Hou W, Dong Y, Zhang J, et al. Hypoxia‐induced deacetylation is required for tetraploid differentiation in response to testicular ischemia‐reperfusion (IR) injury. J Androl 2012; 33(6): 1379-1386.
    27. Hamilton TR, Mendes CM, de Castro LS, et al. Evaluation of lasting effects of heat stress on sperm profile and oxidative status of ram semen and epididymal sperm. Oxid Med Cell Longev 2016; 2016: 1687675. doi: 10.1155/2016/1687657.
    28. Rao M, Zhao XL, Yang J, et al. Effect of transient scrotal hyperthermia on sperm parameters, seminal plasma biochemical markers, and oxidative stress in men. Asian J Androl 2015; 17(4): 668-675.
    29. Koziorowska-Gilun M, Koziorowski M, Strzezek J, et al. Seasonal changes in antioxidant defence systems in seminal plasma and fluids of the boar reproductive tract. Reprod Biol 2011; 11(1): 37-47.
    30. Nichi M, Bols PEJ, Züge RM, et al. Seasonal variation in semen quality in Bos indicus and Bos taurus bulls raised under tropical conditions. Theriogenology. 2006; 66(4): 822-828.
    31. Agarwal A, Virk G, Ong C, et al. Effect of oxidative stress on male reproduction. World J Mens Health 2014; 32(1): 1-17.
    32. Sahin K, Onderci M, Sahin N, et al. Dietary vitamin C and folic acid supplementation ameliorates the detrimental effects of heat stress in Japanese quail. J Nutr 2003; 133(6): 1882-1886.
    33. Kiliçkap A, Kozat S. Research of serum homocysteine levels in healthy cows. J Vet Sci Anim Husb 2017; 5(1): 103.
    34. Zalata A, El-Baz A, Othman G, et al. Seminal plasma S-adenosylmethionine and S-adenosylhomocysteine associations in infertile men. Hum Androl 2011; 1(3): 103-107.
    35. Montjean D, Benkhalifa M, Dessolle L, et al. Polymorphisms in MTHFR and MTRR genes associated with blood plasma homocysteine concentration and sperm counts. Fertil Steril 2011; 95(2): 635-640.
    36. Forges T, Monnier-Barbarino P, Alberto JM, et al. Impact of folate and homocysteine metabolism on human reproductive health. Hum Reprod Update 2007; 13(3): 225-238.
    37. Alirezaei M, Jelodar G, Ghayemi Z. Antioxidant defense of betaine against oxidative stress induced by ethanol in the rat testes. Int J Pept Res Ther 2012; 18(3): 239-247.
    38. Khaki A, Araghi A, Nourian A, et al. Evaluating the potential relationship between the semen quality parameters and serum catalase, superoxide dismutase, and glutathione peroxidase in Bulls. Caspian J Reprod Med 2018; 4(2): 1-9.
    39. Tapia JA, Macias‐Garcia B, Miro‐Moran A, et al. The membrane of the mammalian spermatozoa: much more than an inert envelope. Reprod Domest Anim 2012; 47(Suppl 3): 65-75.
    40. Go EK, Jung KJ, Kim JM, et al. Betaine modulates age-related NF-κB by thiol-enhancing action. Biol Pharm Bull 2007; 30(12): 2244-2249.
    41. Balkan J, Oztezcan S, Küçük M, et al. The effect of betaine treatment on triglyceride levels and oxidative stress in the liver of ethanol-treated guinea pigs. Exp Toxicol Pathol 2004; 55(6): 505-509.
    42. Erman F, Balkan J, Cevikbaş U, et al. Betaine or taurine administration prevents fibrosis and lipid peroxidation induced by rat liver by ethanol plus carbon tetrachloride intoxication. Amino Acids 2004; 27(2): 199-205.
    43. Shamsi MB, Venkatesh S, Kumar R, et al. Antioxidant levels in blood and seminal plasma and their impact on sperm parameters in infertile men. Indian J Biochem Biophys 2010; 47(1): 38-43.
    44. Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. Oxford, UK: Oxford University Press 2015; 100-113.
    45. Papas M, Catalán J, Fernandez-Fuertes B, et al. Specific activity of superoxide dismutase in stallion seminal plasma is related to sperm cryotolerance. Antioxidants (Basel) 2019; 8(11): 539. doi: 10.3390/antiox8110539.
    46. Del Prete C, Stout T, Montagnaro S, et al. Combined addition of superoxide dismutase, catalase and glutathione peroxidase improves quality of cooled stored stallion semen. Anim Reprod Sci 2019; 210: 106195. doi: 10.1016/j.anireprosci.2019.106195.
    47. Buffone MG, Calamera JC, Brugo-Olmedo S, et al. Super-oxide dismutase content in sperm correlates with motility recovery after thawing of cryopreserved human spermatozoa. Fertil Steril 2012; 97(2): 293-298.
    48. Kasimanickam R, Kasimanickam V, Thatcher C, et al. Relationships among lipid peroxidation, glutathione peroxidase, superoxide dismutase, sperm parameters, and competitive index in dairy bulls. Theriogenology 2007; 67(5): 1004-1012.
    49. Barranco I, Padilla L, Tvarijonaviciute A, et al. Levels of activity of superoxide dismutase in seminal plasma do not predict fertility of pig AI-semen doses. Theriogenology 2019; 140: 18-24.