Cryopreservation of spermatozoa is part of the assisted reproductive biotechnology to enhance reproductive capacity in livestock. Conventional cryopreservation applies slow-gradual freezing method permitted ice crystallization and causes cryodamage resulting in poor post-thawed semen quality. Hence, vitrification is introduced by solidifying the solution into glassy state without causing any crystallization in fast and inexpensive manner. This ultra-rapid cooling method requires high concentration of cryoprotectants that potentially toxic the spermatozoa. Therefore, this study was conducted to determine the effect of vitrification on the quality of bull semen. A total of 24 bull semen samples were collected using an electro-ejaculation technique. Tris-based extender was compared with vitrification using solution of different concentration of cryoprotectants at 10% (Vitrification Solution 1; VS-1) and 20% (VS-2) containing dimethyl sulfoxide (DMSO) and ethylene glycol respectively. The result revealed that high mortality and nearly zero motility in all post-warmed vitrified spermatozoa, but the general and progressive motilities parameters in VS-1 at initial evaluation was 22.45% and 24.87% respectively better than Tris-based extender and was statistically significant. In conclusion, vitrification has potential as an alternative for cryopreservation. Therefore, further research on spermatozoa vitrification technique on enhancement in cooling and warming should be conducted and investigated.


  • Agha-Rahimi, A., Khalili, M. A., Nabi, A., & Ashourzadeh, S. (2014). Vitrification is not superior to rapid freezing of normozoospermic spermatozoa: effects on sperm parameters, DNA fragmentation and hyaluronan binding. Reproductive biomedicine online, 28(3), 352-358.
  • Amirat-Briand, L., Bencharif, D., Vera-Munoz, O., Pineau, S., Thorin, C., Destrumelle, S.,... & Tainturier, D. (2010). In vivo fertility of bull semen following cryopreservation with an LDL (low density lipoprotein) extender: Preliminary results of artificial inseminations. Animal reproduction science, 122(3-4), 282-287.
  • Arando, A., Gonzalez, A., Delgado, J. V., Arrebola, F. A., & Perez-Marín, C. C. (2017). Storage temperature and sucrose concentrations affect ram sperm quality after vitrification. Animal reproduction science, 181, 175-185.
  • Baiee, F. H., Wahid, H., Rosnina, Y., Ariff, O. M., Yimer, N., Salman, H.,... & Khumran, A. M. (2017). Hypo-Osmotic Swelling Test Modification to Enhance Cell Membrane Integrity Evaluation in Cryopreserved Bull Semen. Pertanika Journal of Tropical Agricultural Science, 40(2).
  • Baiee, F. H., Wahid, H., Rosnina, Y., Ariff, O., Yimer, N., Jeber, Z.,... & Harighi, F. (2018). Impact of Eurycoma longifolia extract on DNA integrity, lipid peroxidation, and functional parameters in chilled and cryopreserved bull sperm. Cryobiology, 80, 43-50.
  • Baiee, F., Haron, W., Yusoff, R., Ariff, O., Yimer, N., Hammadi, S.,... & Kaka, A. (2018). Modification of Electro-Ejaculation Technique to Minimise Discomfort during Semen Collection in Bulls. Pakistan Journal of Zoology, 50(1).
  • Crespilho, A. M., Sá Filho, M. F., Dell’Aqua Jr, J. A., Nichi, M., Monteiro, G. A., Avanzi, B. R.,... & Papa, F. O. (2012). Comparison of in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or new lecithin based extenders. Livestock Science, 149(1-2), 1-6.
  • Fleisch, A., Malama, E., Witschi, U., Leiding, C., Siuda, M., Janett, F., & Bollwein, H. (2017). Effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen. Theriogenology, 89, 255-262.
  • Hadi, H., Abd, W. H., Rosnina, Y., Daliri, M., Dashtizad, M., Holmes, R., & Abas, M. O. (2011). Nuclear maturation of immature bovine oocytes after vitrification using open pulled straw and cryotop methods. African Journal of Biotechnology, 10(12), 2334-2339.
  • Isachenko, E., Isachenko, V., Katkov, I. I., Dessole, S., & Nawroth, F. (2003). Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success. Reproductive biomedicine online, 6(2), 191-200.
  • Isachenko, E., Isachenko, V., Weiss, J. M., Kreienberg, R., Katkov, I. I., Schulz, M.,... & Sanchez, R. (2008). Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction, 136(2), 167-174.
  • Isachenko, V., Isachenko, E., Katkov, I. I., Montag, M., Dessole, S., Nawroth, F., & van der Ven, H. (2004). Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biology of reproduction, 71(4), 1167-1173.
  • Kaka, A., Wahid, H., Rosnina, Y., Yimer, N., Khumran, A. M., Sarsaifi, K.,... & Ebrahimi, M. (2015). α-Linolenic acid supplementation in BioXcell® extender can improve the quality of post-cooling and frozen-thawed bovine sperm. Animal reproduction science, 153, 1-7.
  • Khoshvaght, A., Towhidi, A., Zare-shahneh, A., Noruozi, M., Zhandi, M., Davachi, N. D., & Karimi, R. (2015). Dietary n-3 PUFAs improve fresh and post-thaw semen quality in Holstein bulls via alteration of sperm fatty acid composition. Theriogenology, 85(5), 807-812.
  • Khumran, A. M., Yimer, N., Rosnina, Y., Ariff, M. O., Wahid, H., Kaka, A.,... & Sarsaifi, K. (2015). Butylated hydroxytoluene can reduce oxidative stress and improve quality of frozen–thawed bull semen processed in lecithin and egg yolk based extenders. Animal reproduction science, 163, 128-134.
  • Kumar, A., Prasad, J. K., Srivastava, N., & Ghosh, S. K. (2019). Strategies to minimize various stress-related freeze–thaw damages during conventional cryopreservation of mammalian spermatozoa. Biopreservation and Biobanking, 17(6), 603-612.
  • Lawson, A., Ahmad, H., & Sambanis, A. (2011). Cytotoxicity effects of cryoprotectants as single-component and cocktail vitrification solutions. Cryobiology, 62(2), 115-122.
  • Magnotti, C., Cerqueira, V., Lee‐Estevez, M., Farias, J. G., Valdebenito, I., & Figueroa, E. (2018). Cryopreservation and vitrification of fish semen: a review with special emphasis on marine species. Reviews in Aquaculture, 10(1), 15-25.
  • Martins, R., et al.(2005)., Effects of vitrification of immature bovine oocytes on in vitro maturation. Animal Reproduction, 2005. 2(2): p. 128-134.
  • Mazur, P., Leibo, S. P., & Seidel Jr, G. E. (2008). Cryopreservation of the germplasm of animals used in biological and medical research: importance, impact, status, and future directions. Biology of reproduction, 78(1), 2-12.
  • Naz, S., Umair, M., & Iqbal, S. (2019). Ostrich egg yolk improves post thaw quality and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Theriogenology, 126, 140-144.
  • Parthipan, S., Selvaraju, S., Somashekar, L., Arangasamy, A., Sivaram, M., & Ravindra, J. P. (2017). Spermatozoal transcripts expression levels are predictive of semen quality and conception rate in bulls (Bos taurus). Theriogenology, 98, 41-49.
  • Schuster, T. G., Keller, L. M., Dunn, R. L., Ohl, D. A., & Smith, G. D. (2003). Ultra‐rapid freezing of very low numbers of sperm using cryoloops. Human Reproduction, 18(4), 788-795.
  • Shahzad, Q., Mehmood, M. U., Khan, H., ul Husna, A., Qadeer, S., Azam, A.,... & Ahmad, M. (2016). Royal jelly supplementation in semen extender enhances post-thaw quality and fertility of Nili-Ravi buffalo bull sperm. Animal reproduction science, 167, 83-88.
  • Sieme, H., Oldenhof, H., & Wolkers, W. F. (2016). Mode of action of cryoprotectants for sperm preservation. Animal reproduction science, 169, 2-5.
  • Taher-Mofrad, S. M. J., Topraggaleh, T. R., Ziarati, N., Bucak, M. N., Nouri, M., Seifi, S.,... & Shahverdi, A. (2020). Knockout serum replacement is an efficient serum substitute for cryopreservation of human spermatozoa. Cryobiology.
  • Tarig, A. A., Wahid, H., Rosnina, Y., Yimer, N., Goh, Y. M., Baiee, F. H.,... & Ebrahimi, M. (2017). Effect of different concentrations of soybean lecithin and virgin coconut oil in Tris-based extender on the quality of chilled and frozen-thawed bull semen. Veterinary World, 10(6), 672.


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