Effect of vanillic acid in comparison with vitamin E+C on short- and long-term ‎preservation of ram semen

Document Type : Research Paper


1 M.Sc. Graduate,, Department of Animal Science, Sari Agricultural Sciences and Natural Resources ‎University, Sari, Iran

2 Associate Professor, Department of Animal Science, Sari Agricultural Sciences and Natural Resources University, Sari, Iran


In the present study, the effect of vanillic acid in comparison with vitamin E+C on the semen characteristics of Zel's ram under short and long-term preservation, was investigated. The experimental groups consisted of different concentrations of vanillic acid (0, 1, 10 and 100 μg/mL), a mixture of alpha-tocopherol and ascorbic acid (vitamin E+C), and dimethyl sulfoxide (DMSO) (as alpha-tocopherol solvent). The samples were evaluated at 0, 24, 48 and 72 hours after storage at 4 °C and following the freeze-thawing process. During the cold storage, sperm quality traits (motility, viability and membrane integrity) and mitochondrial activity increased significantly (P<0.05) at concentrations of 1 and 10 μg/ml vanillic acid in comparison with control and vitamin E+C groups. Our results revealed that the total motility and percentage of spermatozoids in the concentration of 1 μg/ml of vanillic acid were higher than control and vitamin E+C groups (P<0.05). Also, plasma membrane integrity and morphology of spermatozoid were higher in concentrations of 1 and 10 μg/ml of vanillic acid than control group. Concentration of 1 μg/ml of vanillic acid showed the highest mitochondrial activity than vitamin E+C treatment (P<0.05). Our findings showed that addition of vanillic acid at the concentrations of 1 and 10 μg/mL to the semen extender improved the quality of spermatozoid in the Zel breed sheep.


  1. Arora, A., Nair, MG. & Strasburg, GM. (1998). Structure–activity relationships for antioxidant activities of a series of flavonoids in a liposomal system. Free Radical Biology and Medicine, 24(9), 1355-1363.
  2. Benavente-García, O., Castillo, J., Marin, FR., Ortuño, A. & Del Río, JA. (1997). Uses and properties of citrus flavonoids. Journal of Agricultural and Food Chemistry, 45(12), 4505-4515.
  3. Blokhina, O., Virolainen, E. & Fagerstedt, KV. (2003). Antioxidants oxidative damage and oxygen deprivation stress- review. Annals of Botany, 91,179-194.
  4. Breininger, E., Beorlegui, NB., O’Flaherty, CM. & Beconi, MT. (2005). Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen. Theriogenology, 63(8),2126-2135.‏
  5. Bucak, MN., Tuncer, PB., Sarıozkan, S. & Ulutas, PA. (2009). Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters, lipid peroxidation and anti-oxidant activities. Small Ruminant Research, 81,13-17.
  6. Calixto-Campos, C., Carvalho, TT., Hohmann, MS., Pinho-Ribeiro, FA., Fattori, V., Manchope, MF., Zarpelon, AC., Baracat, MM., Georgetti, SR., Casagrande, R. & Verri, Jr WA. (2015). Vanillic acid inhibits inflammatory pain by inhibiting neutrophil recruitment, oxidative stress, cytokine production, and NFκB activation in mice. Journal of Natural Products, 78(8),1799-1808.
  7. Courot, M. (1976). Semen quality and quantity in the ram. Sheep Breeding: Studies in the Agricultural and Food Sciences, 495-504.
  8. ‏Delaquis, , Stanich, K.  & Toivonen, P. (2005). Effect of pH on the inhibition of Listeria spp. by vanillin and vanillic acid. Journal of Food Protection, 68(7),1472-1476.
  9. Dianat, M., Hamzavi, GR., Badavi, M. & Samarbafzadeh, A. (2014). Effects of Losartan and vanillic acid co-administration on ischemia-reperfusion-induced oxidative stress in isolated rat heart. Iranian Red Crescent Medical Journal, 16(7).‏
  10. Dragana, M., Stanisavljevic Sasa, S., Stojicevic Sofija, M., Dorcevic Branislav, P., Zlatkovic Dragan, T., Velickovic Ivana, T. & Karabegovic Miodrag, L. (2012). Antioxidant activity, the content of total phenols and flavonoids in the ethanol extracts of mentha longifolia L. Hudson dried by the use of different techniques. Available on line at Association of the Chemical Engineers of Serbia ACHE, 23,213-219.
  11. Evans, G., Hollinshead, FK. & MaxwellW, MC. (2004). Preservation and artificial insemination of sexed semen in sheep. Reproduction, Fertility and Development, 16,455-464.
  12. Fattah, A., Sharafi, M., Masoudi, R., Shahverdi, A., Esmaeili, V. & Najafi, A. (2017). l-Carnitine in rooster semen cryopreservation: Flow cytometric, biochemical and motion findings for frozen-thawed sperml. Cryobiology, 74,148-153.
  13. Fraser, L., Dziekońska, A., Strzeżek, R. & Strzeżek, J. (2007). Dialysis of boar semen prior to freezing–thawing: Its effects on post-thaw sperm characteristics. Theriogenology, 67(5), 994-1003.‏
  14. Itoh, A., Isoda, K., Kondoh, M., Kawase, M., Kobayashi, M., Tamesada, M. & Yagi, K. (2009). Hepatoprotective effect of syringic acid and vanillic acid on concanavalin a-induced liver injury. Biological and Pharmaceutical Bulletin, 32(7),1215-1219.
  15. Kim, MC., Kim, SJ., Kim, DS., Jeon, YD., Park, SJ., Lee, HS., Um, JY. & Hong, SH. (2011). Vanillic acid inhibits inflammatory mediators by suppressing NF-κB in lipopolysaccharide-stimulated mouse peritoneal macrophages. Immunopharmacology and Immunotoxicology, 33(3), 525-532.
  16. Lakshmanashetty, RH., Nagaraj, VB., Hiremath, MG. & Kumar, V. (2010). In vitro antioxidant activity of Vitex negundo L. leaf extracts. Chiang Mai Journal of Science, 37(3),489-497.
  17. Leal LKAM Pierdoná, TM., Góes, JGS., Fonsêca, KS., Canuto, KM., Silveira, ER., Bezerra, AME. & Viana, GSB. (2011). A comparative chemical and pharmacological study of standardized extracts and vanillic acid from wild and cultivated Amburana cearensis AC Smith. Phytomedicine, 18(2-3),230-233.
  18. Morucci, F., Lopez, P., Miño, J., Ferraro, G. & Gorzalczany, S. (2012). Antinociceptive activity of aqueous extract and isolated compounds of Lithrea molleoides. Journal of Ethnopharmacology, 142(2),401-406.
  19. Mustafa, S. & Esref, D. (2004). The effect of Ascorbic Acid on the freezability of ram semen diluted with extenders containing different proportions of glycerol Turk Journal, Vet. Animal Science Journal, 28,893-899.
  20. Najafi, A., Daghigh Kia, H., Dodaran, HV., Mehdipour, M. & Alvarez-Rodriguez, M. (2017). Ethylene glycol, but not DMSO, could replace glycerol inclusion in soybean lecithinbased extenders in ram sperm cryopreservation. Animal Reproduction Science, 177,35-4.
  21. Najafi, A., Kia, HD., Mohammadi, H., Najafi, MH., Zanganeh, Z., Sharafi, M. & MartinezPastor, F. (2014). Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender. Cryobiology, 69 (1),68-73.
  22. Prince, PSM., Dhanasekar, K. & Rajakumar, S. (2015). Vanillic acid prevents altered ion pumps, ions, inhibits Fas-receptor and caspase mediated apoptosis-signaling pathway and cardiomyocyte death in myocardial infarcted rats. Chemico-Biological Interactions, 232,68-76.‏
  23. Revell, SG. & Mrode, RA. (1994). An osmotic resistance test for bovine semen. Animal Reproduction Science, 36, 77-86.
  24. Rice-Evans, C., Miller, N. & Paganga, G. (1997). Antioxidant properties of phenolic compounds. Trends in Plant Science, 2(4), 152-159.
  25. Salamon, S. & MaxwellW, MC. (1995). Frozen storage of ram semen I. Processing, freezing, thawing and fertility after cervical insemination. Animal Reproduction Science, 37, 185-249.
  26. Salmani, H., Nabi, MM., Vaseghi-Dodaran, H., Rahman, MB., Mohammadi-Sangcheshmeh, A., Shakeri, M., Towhidi, A., Zare-Shahneh, A. & Zhandi, M. (2013). Effect of glutathione in soybean lecithin-based semen extender on goat semen quality after freeze–thawing. Small Ruminant Research, 112(1-3), 123-127.
  27. Sharma, RK. & Agarwal, A. (1996). Role of reactive oxygen species in male infertility. Urology, 48, 835-50.
  28. Sindhu, G., Nishanthi, E. & Sharmila, R. (2015). Nephroprotective effect of vanillic acid against cisplatin induced nephrotoxicity in wistar rats: a biochemical and molecular study. Environmental toxicology and pharmacology, 39(1), 392-404.
  29. Singh, JCH., Kakalij, RM., Kshirsagar, RP., Kumar, BH., Komakula, SSB. & Diwan, PV. (2015). Cognitive effects of vanillic acid against streptozotocin-induced neurodegeneration in mice. Pharmaceutical Biology, 53(5), 630-636.
  30. Smith, OB. & Akinbamijo, OO. (2000). Micronutrients and reproduction in farm animals. Animal Reproduction Science, 60, 549-560.
  31. Tai, A., Sawano, T. & Ito, H. (2012). Antioxidative properties of vanillic acid esters in multiple antioxidant assays. Bioscience, Biotechnology, and Biochemistry, 76(2), 314-318.
  32. Vinothiya, K. & Ashokkumar, N. (2017). Modulatory effect of vanillic acid on antioxidant status in high fat diet-induced changes in diabetic hypertensive rats. Biomedicine and Pharmacotherapy, 87, 640-652.
  33. Wang, G., Kang, N., Gong, H., Luo, Y., Bai, C. & Chen, Y. (2015). Upregulation of uncoupling protein Ucp2 through acute cold exposure increases post-thaw sperm quality in zebrafish. Cryobiology, 71(3), 464-471.