Effects of nonenzymatic and enzymatic antioxidants in cryopreserved canine semen extended with docosahexaenoic acid
Keywords:
Lipid peroxidation, oxidative stress, dog, sperm kinetic, polyunsaturated fatty acid
Abstract
Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), could be an alternative to improve cryopreserved canine sperm by increasing membrane fluidity. However, PUFAs also increase sperm susceptibility to lipid peroxidation. Supplementation of the extender with DHA and antioxidants could be an alternative. We evaluated supplementation of antioxidants (vitamin E and catalase) of cryopreserved canine semen extended with DHA. In Experiment 1, ejaculates from 8 dogs were divided into 4 aliquots, with increasing concentrations of DHA in the extender (0, 1, 5, and 10 µM) and cryopreserved. Sperm motility increased in 5 µM DHA and it was used for Experiment 2; ejaculates from 8 dogs were divided into 4 groups: control (5 µM DHA), vitamin E + DHA, catalase + DHA, and vitamin E + catalase + DHA. Samples were evaluated for susceptibility to lipid peroxidation, sperm kinetics, mitochondrial function, DNA integrity, and plasma and acrosomal membrane integrity. Vitamin E + DHA group had beneficial effects on the characteristics of sperm kinetics whereas the catalase + DHA group was harmful to sperm mitochondria. Vitamin E + catalase + DHA group decreased oxidative stress and damage to the plasma membrane and had low mitochondrial membrane activity. Addition of DHA + vitamin E in canine semen extender improved sperm quality after cryopreservation.
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References
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10. Lecewicz M, Strzezek R, Kordan W, et al: Effect of extender supplementation with low-molecular-weight antioxidants on selected quality parameters of cryopreserved canine spermatozoa. J Vet Res 2018;62:221-227. doi: 10.2478/jvetres-2018-0032
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18. Bendich A: Antioxidant micronutrients and immune responses. Ann NY Acad Sci 1990;587:168-180. doi: 10.1111/j.1749-6632.1990.tb00144.x
19. Perly B, Smith IC, Hughes L, et al: Estimation of the location of natural alpha-tocopherol in lipid bilayers by 13C-NMR spectroscopy. Biochim Biophys Acta 1985;819:131-135. doi: 10.1016/0005-2736(85)90203-2
20. Bradford A, Atkinson J, Fuller N, et al: The effect of vitamin E on the structure of membrane lipid assemblies. J Lipid Res 2003;44:1940-1945. doi: 10.1194/jlr.M300146-JLR200
21. Griveau JF, Le Lannou D: Reactive oxygen species and human spermatozoa: physiology and pathology. Int J Androl 1997;20:61-69. doi: 10.1046/j.1365-2605.1997.00044.x
22. Aitken RJ, Buckingham D, Harkiss D: Use of a xanthine-oxidase free-radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J Reprod Fertil 1993;97:441-450. doi: 10.1530/jrf.0.0970441
23. Griveau JF, Dumont E, Renard P, et al: Reactive oxygen species, lipid-peroxidation and enzymatic defense systems in human spermatozoa. J Reprod Fertil 1995;103:17-26. doi: 10.1530/jrf.0.1030017
24. Verstegen J, Iguer-Ouada M, Onclin K: Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 2002;57:149-179. doi: 10.1016/S0093-691X(01)00664-1
25. de Castro LS, de Assis PM, Siqueira AFP, et al: Sperm oxidative stress is detrimental to embryo development: A dose-dependent study model and a new and more sensitive oxidative status evaluation. Oxid Med Cell Longev 2016;1:8213071. doi: 10.1155/2016/8213071
26. Hrudka F: Cytochemical and ultracytochemical demonstration of cytochrome c oxidase in spermatozoa and dynamics of its changes accompanying ageing or induced by stress. Int J Androl 1987;10:809-828. doi: 10.1111/j.1365-2605.1987.tb00385.x
27. Evenson D, Jost L: Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci 2000;22:169-189. doi: 10.1023/A:1009844109023
28. Lucio CF, Regazzi FM, Silva LCG, et al: Oxidative stress at different stages of two-step semen cryopreservation procedures in dogs. Theriogenology 2016;85:1568-1575. doi: 10.1016/j.theriogenology.2016.01.016
29. Nichi M, Goovaerts IGF, Cortada CNM, et al: Roles of lipid peroxidation and cytoplasmic droplets on in vitro fertilization capacity of sperm collected from bovine epididymides stored at 4 and 34 degrees C. Theriogenology 2007;67:334-340. doi: 10.1016/j.theriogenology.2006.08.002
30. Strzezek R, Koziorowska-Gilun M, Stawiszynska M: Cryopreservation of canine semen: the effect of two extender variants on the quality and antioxidant properties of spermatozoa. Pol J Vet Sci 2012;15:721-726. doi: 10.2478/v10181-012-0109-0
31. Mortimer ST: A critical review of the physiological importance and analysis of sperm movement in mammals. Hum Reprod Update 1997;3:403-439. doi: 10.1093/humupd/3.5.403
32. Martins MIM, Souza AK, Trautwein LGC: Subpopulações espermáticas. Rev Bras Reprod Anim 2017;41:243-247. Available from: http://www.cbra.org.br/portal/downloads/publicacoes/rbra/v41/n1/p243-247%20(RB653).pdf [cited 24 March 2024].
33. Kaka A, Haron W, Yusoff R, et al: Effect of docosahexanoic acid on quality of frozen-thawed bull semen in BioXcell extender. Reprod Fertil Dev 2017;29:490-495. doi: 10.1071/RD15089
34. Chanapiwat P, Kaeoket K, Tummaruk P: Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen. Asian J Androl 2009;11:600-608. doi: 10.1038/aja.2009.40
35. Aitken RJ, Wingate JK, De Iuliis GN, et al: Cis-unsaturated fatty acids stimulate reactive oxygen species generation and lipid peroxidation in human spermatozoa. J Clin Endocrinol Metab 2006;91:4154-4163. doi: 10.1210/jc.2006-1309
36. Kaeoket K, Sang-urai P, Thamniyom A, et al: Effect of docosahexaenoic acid on quality of cryopreserved boar semen in different breeds. Reprod Domest Anim 2010;45:458-463. doi: 10.1111/j.1439-0531.2008.01239.x
37. Wang N, Qian HY, Zhou XQ, et al: Mitochondrial energy metabolism dysfunction involved in reproductive toxicity of mice caused by endosulfan and protective effects of vitamin E. Ecotoxicol Environ Saf 2012;82:96-103. doi: 10.1016/j.ecoenv.2012.05.014
38. Kawakami E, Kobayashi M, Hori T, et al: Therapeutic effects of vitamin E supplementation in 4 dogs with poor semen quality and low superoxide dismutase activity in seminal plasma. J Vet Med Sci 2016;77:1711-1714. doi: 10.1292/jvms.15-0294
39. Beconi MT, Francia CR, Mora NG, et al: Effect of natural antioxidants on frozen bovine semen preservation. Theriogenology 1993;40:841-851. doi: 10.1016/0093-691X(93)90219-U
40. Pena FJ, Johannisson A, Wallgren M, et al: Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. Anim Reprod Sci 2003;78:85-98. doi: 10.1016/S0378-4320(03)00049-6
41. Angrimani DSR, Nichi M, Brito MM, et al: The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs. Arq Bras Med Vet Zootec 2018;70:419-428. doi: 10.1590/1678-4162-9684
42. Panner Selvam MK, Agarwal A, Henkel R, et al: The effect of oxidative and reductive stress on semen parameters and functions of physiologically normal human spermatozoa. Free Radic Biol Med 2020;152:375-385. doi: 10.1016/j.freeradbiomed.2020.03.008
43. Arslan HO, Herrera C, Malama E, et al: Effect of the addition of different catalase concentrations to a TRIS-egg yolk extender on quality and in vitro fertilization rate of frozen-thawed bull sperm. Cryobiology 2019;91:40-52. doi: 10.1016/j.cryobiol.2019.10.200
44. Mohammadzadeh M, Ramazani V, Khalili MA, et al: Medium containing different concentrations of catalase as a strategy for optimising sperm parameters and chromatin in normospermic persons. Andrologia 2019;51:1-6. doi: 10.1111/and.13231
45. Kumar A, Prasad JK, Srivastava N, et al: Strategies to minimize various stress-related freeze–thaw damages during conventional cryopreservation of mammalian spermatozoa. Biopreserv Biobank 2019;17:603-612. doi: 10.1089/bio.2019.0037
2. Flesch FM, Gadella BM: Dynamics of the mammalian sperm plasma membrane in the process of fertilization. Biochim Biophys Acta 2000;1469:197-235. doi: 10.1016/S0304-4157(00)00018-6
3. Mandal R, Badyakar D, Chakrabarty J: Role of membrane lipid fatty acids in sperm cryopreservation. Adv Androl 2014;2014:1-9. doi: 10.1155/2014/190542
4. Rooke JA, Shao CC, Speake BK: Effects of feeding tuna oil on the lipid composition of pig spermatozoa and in vitro characteristics of semen. Reproduction 2001;121:315-322. doi: 10.1530/rep.0.1210315
5. Andrade ER, Melo-Sterza FA, Seneda MM, et al: Consequências da produção das espécies reativas de oxigênio na reprodução e principais mecanismos antioxidantes. Rev Bras Reprod Anim 2010;34:79-85. Available from: http://www.cbra.org.br/pages/publicacoes/rbra/v34n2/p79-86.pdf [cited 24 March 2024].
6. Parks JE, Lynch DV: Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes. Cryobiology 1992;29:255-266. doi: 10.1016/0011-2240(92)90024-V
7. 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;5:1-15. doi: 10.2741/lenzi
8. Agarwal A, Saleh RA, Bedaiwy MA: Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003;79:829-843. doi: 10.1016/S0015-0282(02)04948-8
9. Losano JDA, Angrimani DSR, Rui BR, et al: The addition of docosahexaenoic acid (DHA) and antioxidants (glutathione peroxidase and superoxide dismutase) in extenders to epididymal sperm cryopreservation in bulls. Zygote 2018b;26:199-206. doi: 10.1017/S0967199418000096
10. Lecewicz M, Strzezek R, Kordan W, et al: Effect of extender supplementation with low-molecular-weight antioxidants on selected quality parameters of cryopreserved canine spermatozoa. J Vet Res 2018;62:221-227. doi: 10.2478/jvetres-2018-0032
11. Aitken RJ, Krausz C: Oxidative stress, DNA damage and the Y chromosome. Reproduction 2001;122:497-506. doi: 10.1530/rep.0.1220497
12. Losano JDA, Angrimani DSR, Dalmazzo A, et al: Effect of vitamin E and polyunsaturated fatty acids on cryopreserved sperm quality in Bos taurus bulls under testicular heat stress. Anim Biotechnol 2018a;29:100-109. doi: 10.1080/10495398.2017.1322973
13. Vieira NMG, Losano JDA, Angrimani DSR, et al: Induced sperm oxidative stress in dogs: susceptibility against different reactive oxygen species and protective role of seminal plasma. Theriogenology 2018;108:39-45. doi: 10.1016/j.theriogenology.2017.11.020
14. Nordberg J, Arner ES: Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med 2001;31:1287-1312. doi: 10.1016/S0891-5849(01)00724-9
15. Halliwell B, Gutteridge JM: Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys 1986;246:501-514. doi: 10.1016/0003-9861(86)90305-X
16. Bjorneboe A, Bjorneboe GE, Drevon CA: Absorption, transport and distribution of vitamin E. J Nutr 1990;120:233-242. doi: 10.1093/jn/120.3.233
17. Mates JM: Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology. Toxicology 2000;153:83-104. doi: 10.1016/S0300-483X(00)00306-1
18. Bendich A: Antioxidant micronutrients and immune responses. Ann NY Acad Sci 1990;587:168-180. doi: 10.1111/j.1749-6632.1990.tb00144.x
19. Perly B, Smith IC, Hughes L, et al: Estimation of the location of natural alpha-tocopherol in lipid bilayers by 13C-NMR spectroscopy. Biochim Biophys Acta 1985;819:131-135. doi: 10.1016/0005-2736(85)90203-2
20. Bradford A, Atkinson J, Fuller N, et al: The effect of vitamin E on the structure of membrane lipid assemblies. J Lipid Res 2003;44:1940-1945. doi: 10.1194/jlr.M300146-JLR200
21. Griveau JF, Le Lannou D: Reactive oxygen species and human spermatozoa: physiology and pathology. Int J Androl 1997;20:61-69. doi: 10.1046/j.1365-2605.1997.00044.x
22. Aitken RJ, Buckingham D, Harkiss D: Use of a xanthine-oxidase free-radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J Reprod Fertil 1993;97:441-450. doi: 10.1530/jrf.0.0970441
23. Griveau JF, Dumont E, Renard P, et al: Reactive oxygen species, lipid-peroxidation and enzymatic defense systems in human spermatozoa. J Reprod Fertil 1995;103:17-26. doi: 10.1530/jrf.0.1030017
24. Verstegen J, Iguer-Ouada M, Onclin K: Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 2002;57:149-179. doi: 10.1016/S0093-691X(01)00664-1
25. de Castro LS, de Assis PM, Siqueira AFP, et al: Sperm oxidative stress is detrimental to embryo development: A dose-dependent study model and a new and more sensitive oxidative status evaluation. Oxid Med Cell Longev 2016;1:8213071. doi: 10.1155/2016/8213071
26. Hrudka F: Cytochemical and ultracytochemical demonstration of cytochrome c oxidase in spermatozoa and dynamics of its changes accompanying ageing or induced by stress. Int J Androl 1987;10:809-828. doi: 10.1111/j.1365-2605.1987.tb00385.x
27. Evenson D, Jost L: Sperm chromatin structure assay is useful for fertility assessment. Methods Cell Sci 2000;22:169-189. doi: 10.1023/A:1009844109023
28. Lucio CF, Regazzi FM, Silva LCG, et al: Oxidative stress at different stages of two-step semen cryopreservation procedures in dogs. Theriogenology 2016;85:1568-1575. doi: 10.1016/j.theriogenology.2016.01.016
29. Nichi M, Goovaerts IGF, Cortada CNM, et al: Roles of lipid peroxidation and cytoplasmic droplets on in vitro fertilization capacity of sperm collected from bovine epididymides stored at 4 and 34 degrees C. Theriogenology 2007;67:334-340. doi: 10.1016/j.theriogenology.2006.08.002
30. Strzezek R, Koziorowska-Gilun M, Stawiszynska M: Cryopreservation of canine semen: the effect of two extender variants on the quality and antioxidant properties of spermatozoa. Pol J Vet Sci 2012;15:721-726. doi: 10.2478/v10181-012-0109-0
31. Mortimer ST: A critical review of the physiological importance and analysis of sperm movement in mammals. Hum Reprod Update 1997;3:403-439. doi: 10.1093/humupd/3.5.403
32. Martins MIM, Souza AK, Trautwein LGC: Subpopulações espermáticas. Rev Bras Reprod Anim 2017;41:243-247. Available from: http://www.cbra.org.br/portal/downloads/publicacoes/rbra/v41/n1/p243-247%20(RB653).pdf [cited 24 March 2024].
33. Kaka A, Haron W, Yusoff R, et al: Effect of docosahexanoic acid on quality of frozen-thawed bull semen in BioXcell extender. Reprod Fertil Dev 2017;29:490-495. doi: 10.1071/RD15089
34. Chanapiwat P, Kaeoket K, Tummaruk P: Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen. Asian J Androl 2009;11:600-608. doi: 10.1038/aja.2009.40
35. Aitken RJ, Wingate JK, De Iuliis GN, et al: Cis-unsaturated fatty acids stimulate reactive oxygen species generation and lipid peroxidation in human spermatozoa. J Clin Endocrinol Metab 2006;91:4154-4163. doi: 10.1210/jc.2006-1309
36. Kaeoket K, Sang-urai P, Thamniyom A, et al: Effect of docosahexaenoic acid on quality of cryopreserved boar semen in different breeds. Reprod Domest Anim 2010;45:458-463. doi: 10.1111/j.1439-0531.2008.01239.x
37. Wang N, Qian HY, Zhou XQ, et al: Mitochondrial energy metabolism dysfunction involved in reproductive toxicity of mice caused by endosulfan and protective effects of vitamin E. Ecotoxicol Environ Saf 2012;82:96-103. doi: 10.1016/j.ecoenv.2012.05.014
38. Kawakami E, Kobayashi M, Hori T, et al: Therapeutic effects of vitamin E supplementation in 4 dogs with poor semen quality and low superoxide dismutase activity in seminal plasma. J Vet Med Sci 2016;77:1711-1714. doi: 10.1292/jvms.15-0294
39. Beconi MT, Francia CR, Mora NG, et al: Effect of natural antioxidants on frozen bovine semen preservation. Theriogenology 1993;40:841-851. doi: 10.1016/0093-691X(93)90219-U
40. Pena FJ, Johannisson A, Wallgren M, et al: Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. Anim Reprod Sci 2003;78:85-98. doi: 10.1016/S0378-4320(03)00049-6
41. Angrimani DSR, Nichi M, Brito MM, et al: The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs. Arq Bras Med Vet Zootec 2018;70:419-428. doi: 10.1590/1678-4162-9684
42. Panner Selvam MK, Agarwal A, Henkel R, et al: The effect of oxidative and reductive stress on semen parameters and functions of physiologically normal human spermatozoa. Free Radic Biol Med 2020;152:375-385. doi: 10.1016/j.freeradbiomed.2020.03.008
43. Arslan HO, Herrera C, Malama E, et al: Effect of the addition of different catalase concentrations to a TRIS-egg yolk extender on quality and in vitro fertilization rate of frozen-thawed bull sperm. Cryobiology 2019;91:40-52. doi: 10.1016/j.cryobiol.2019.10.200
44. Mohammadzadeh M, Ramazani V, Khalili MA, et al: Medium containing different concentrations of catalase as a strategy for optimising sperm parameters and chromatin in normospermic persons. Andrologia 2019;51:1-6. doi: 10.1111/and.13231
45. Kumar A, Prasad JK, Srivastava N, et al: Strategies to minimize various stress-related freeze–thaw damages during conventional cryopreservation of mammalian spermatozoa. Biopreserv Biobank 2019;17:603-612. doi: 10.1089/bio.2019.0037
Published
2025-08-01
How to Cite
Filho , R. R., Vieira , N., Brito , M., Losano , J., Kawai , G., Mendes , C., Rui , B., Nagai , K., Alves , Álvaro, Frias , H., Sousa , R., Sanches , F., Assumpção , M., & Nichi , M. (2025). Effects of nonenzymatic and enzymatic antioxidants in cryopreserved canine semen extended with docosahexaenoic acid. Clinical Theriogenology, 17. https://doi.org/10.58292/CT.v17.12690
Section
Research Reports
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