Document Type : Original Article


1 Department of Theriogenology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

2 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Selcuk University, Konya, Türkiye


The reduction of spermatozoa survival time is a major problem of canine chilled sperm for artificial insemination. The current study looks at the possible advantages of chilling canine sperm to 4.00 ˚C for three days using Tribulus terrestris aqueous extract (TTAE). Three mixed-breed dogs were utilized to extract 24 ejaculates, which were then diluted in a Tris-based extender. The ejaculates were then divided into five groups including 20.00, 40.00 and 50.00 µg mL-1 of TTAE, sham (distilled water devoid of TTAE) and control (without TTAE) groups. During the three days of experiment, several parameters were measured every 24 hr. It was noticed that after 48 and 72 hr of liquid storage, total and progressive motilities were greater in the group with the 40.00 µg mL-1 TTAE concentration than the control group. Compared to the control group, the group with the 40.00 µg mL-1 TTAE concentration exhibited superior motility and viability. The percentages obtained from the hypo-osmotic swelling test were much greater. In contrast to the control group, DNA integrity was poorer in the 40.00 µg mL-1 TTAE concentration. After 72 hr of storage, the group with 40.00 µg mL-1 TTAE concentration had lower malondialdehyde levels but considerably greater total anti-oxidant capacity, superoxide dismutase, glutathione peroxidase and catalase levels than the control groups. The current study found that supplementing the semen extender with 40.00 µg mL-1 TTAE improves semen parameters after 72 hr of storage at 4.00 ˚C, and therefore can improve fertilization efficiency.


  1. de Fátima Lucio C, de Souza Ramos Angrimani D, Morales Brito M Oxidative stress challenges during the sperm cryopreservation in dogs. J Vet Androl 2017; 2(1): 1-7.
  2. Linde-Forsberg C. Artificial insemination with fresh, chilled extended, and frozen-thawed semen in the dog. Semin Vet Med Surg Small Anim 1995; 10(1): 48-58.
  3. Aitken RJ, Baker MA. Oxidative stress, sperm survival and fertility control. Mol Cell Endocrinol 2006; 250(1-2): 66-69.
  4. Allai L, Benmoula A, Marciane da Silva M, et al. Supplementation of ram semen extender to improve seminal quality and fertility rate. Anim Reprod Sci 2018; 192: 6-17.
  5. Michael A, Alexopoulos C, Pontiki E, et al. Effect of antioxidant supplementation on semen quality and reactive oxygen species of frozen-thawed canine spermatozoa. Theriogenology 2007; 68(2): 204-212.
  6. Gvozdjakova A, Kucharska J, Lipkova J, et al. Importance of the assessment of coenzyme Q10, alpha-tocopherol and oxidative stress for the diagnosis and therapy of infertility in men. Bratisl Lek Listy 2013; 114(11): 607-609.
  7. Liu JH, Li HY, Cao ZG, et al. Influence of several uropathogenic microorganisms on human sperm motility parameters in vitro. Asian J Androl 2002; 4(3): 179-182.
  8. Zheleva-Dimitrova D, Obreshkova D, Nedialkov P. Antioxidant activity of Tribulus terrestris - a natural product in infertility therapy. Int J Pharm Pharm Sci 2012; 4(4): 508-511.
  9. Gauthaman K, Ganesan AP, Prasad RN. Sexual effects of puncturevine (Tribulus terrestris) extract (protodioscin): an evaluation using a rat model. J Altern Complement Med 2003; 9(2): 257-265.
  10. Martino-Andrade AJ, Morais RN, Spercoski KM, et al. Effects of Tribulus terrestris on endocrine sensitive organs in male and female Wistar rats. J Ethnopharmacol 2010; 127(1): 165-170.
  11. Adaikan PG, Gauthaman K, Prasad RN, et al. Proerectile pharmacological effects of Tribulus terrestris extract on the rabbit corpus cavernosum. Ann Acad Med Singap 2000; 29(1): 22-26.
  12. Kistanova E, Zlatev H, Karcheva V, et al. Effect of plant Tribulus terrestris extract on reproductive performances of rams. Biotechnol Anim Husb 2005; 21(1-2): 55-63.
  13. MA, et al. Prospective analysis on the effect of botanical medicine (Tribulus terrestris) on serum testosterone level and semen parameters in males with unexplained infertility. J Diet Suppl 2017; 14(1):2 5-31.
  14. Sellandi TM, Thakar AB, Baghel MS. Clinical study of Tribulus terrestris in Oligozoospermia: A double blind study. Ayu 2012; 33(3): 356-364.
  15. Shalaby MA, Hammouda AA. Assessment of protective and anti-oxidant properties of Tribulus terrestris fruits against testicular toxicity in rats. J Intercult Ethnopharmacol 2014; 3(3): 113-118.
  16. Sharma P, Huq AU, Singh R. Cypermethrin induced reproductive toxicity in male Wistar rats: protective role of Tribulus terrestris. J Environ Biol 2013; 34(5): 857-862.
  17. Singh S, Nair V, Gupta YK. Evaluation of the aphrodisiac activity of Tribulus terrestris in sexually sluggish male albino rats. J Pharmacol Pharmacother 2012; 3(1): 43-47.
  18. Mattar AG, Adaay MH. Effect of aqueous and ethanolic extracts of Tribulus terrestris, Phoenix dactylifera and Nasturtium officinale mixture on some reproductive parameters in male mice. Baghdad Sci J 2012; 9(4): 640-650.
  19. Khaleghi S, Bakhtiari M, Asadmobini A, et al. Tribulus terrestris extract improves human sperm parameters in vitro. J Evid Based Complementary Altern Med. 2017; 22(3): 407-412.
  20. Dewanto V, Wu X, Adom KK, et al. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J Agric Food Chem 2002; 50(10): 3010-3014.
  21. Kutzler MA. Semen collection in the dog. Theriogenology 2005; 64(3): 747-754.
  22. Shakouri N, Soleimanzadeh A, Rakhshanpour A, et al. Antioxidant effects of supplementation of 3, 4‐dihydroxyphenyl glycol on sperm parameters and oxidative markers following cryopreservation in canine semen. Reprod Domest Anim 2021; 56(7): 1004-1014.
  23. World Health Organisation. WHO laboratory manual for the examination and processing of human semen. 6th Geneva, Switzerland: World Health Organization 2021; 26-29.
  24. Pinto CR, Kozink DM. Simplified hypoosmotic swelling testing (HOST) of fresh and frozen-thawed canine spermatozoa. Anim Reprod Sci 2008; 104(2-4): 450-455.
  25. Benzie IF, Strain JJ. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol 1999: 299: 15-27.
  26. Papas M, Arroyo L, Bassols A, et al. Activities of antioxidant seminal plasma enzymes (SOD, CAT, GPX and GSR) are higher in jackasses than in stallions and are correlated with sperm motility in jackasses. Theriogenology 2019; 140: 180-187.
  27. Maehly AC, Chance B. The assay of catalases and peroxidases. Methods Biochem Anal 1954; 1: 357-424.
  28. Bergmeyer HU. Methods of enzymatic analysis. 2nd New York, USA: Elsevier Science 2012; 201-568.
  29. Clément C, Witschi U, Kreuzer M. The potential influence of plant-based feed supplements on sperm quantity and quality in livestock: a review. Anim Reprod Sci 2012; 132(1-2): 1-10.
  30. Safavi Pour S, Pirestani A, Alirezai M, et al. The effect of adding different levels of aqueous extract of Tribulus terrestris in the extender on sperm motility Afshari rams at 5°C. J Chem Pharm Res 2015; 7(4): 957-959.
  31. Devaraj S, Mathur S, Basu A, et al. A dose-response study on the effects of purified lycopene supplementation on biomarkers of oxidative stress. J Am Coll Nutr 2008; 27(2): 267-273.
  32. Zini A, San Gabriel M, Libman J. Lycopene supplementation in vitro can protect human sperm deoxyribonucleic acid from oxidative damage. Fertil Steril 2010; 94(3): 1033-1036.