Influence and Total Antioxidant Capacity of Non-enzymatic Antioxidants on the Quality and Integrity of Extended and Cryopreserved Semen of Murrah Buffalo (Bubalus bubalis)

Kimberly I B. Turaja1, Renato SA. Vega1, Thelma A. Saludes2, Abraham G. Tandang2,
Jose Arceo N. Bautista2, Agapita J. Salces2, and Carmelita M. Rebancos3

1Animal Physiology Division, Institute of Animal Science,
College of Agriculture and Food Sciences
2Philippine Carabao Center
3School of Environmental Science and Management
University of the Philippines Los Baños, College, Laguna 4031 Philippines

*Corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.




The study compared the effect of non-enzymatic antioxidants on the % motility, livability, and plasma membrane integrity of the cryopreserved sperm from four purebred Murrah bulls aged 10–17 years old. The level of antioxidants was also determined.  Experiment 1 was analyzed using 4 x 4 factorial design in a randomized complete block design (RCBD) with bulls blocked in four; in Experiment 2, it analyzed the levels of MegaAntiox using PROC ANOVA of SAS software. Results revealed no significant (P > 0.05) difference in post-thaw sperm motility (%) between antioxidants but a significant decrease (P < 0.05) in the post-thaw sperm motility was observed at Week 3. The addition of non-enzymatic antioxidants did not affect (P > 0.05) the live sperm though difference was observed on the livability of sperm cells in different collection weeks. In extended semen, the addition of non-enzymatic antioxidants did not show any significant effect (P > 0.05) on post-thaw sperm motility (%) and livability. After six months of cryopreservation, the addition of vitamin E (one of the non-enzymatic antioxidants) had a negative effect on the post-thaw sperm motility. Livability and PMI were not significantly affected (P > 0.05) by the different treatments. In conclusion, the addition of 0.1 mg/ml non-enzymatic antioxidants did not improve sperm motility (%) and the livability of the sperm.



Reactive oxygen species (ROS) production is a normal physiological process; however, an imbalance between ROS generation, scavenging activity, and uncontrolled production of ROS that exceeds the antioxidant capacity of the seminal plasma will lead to oxidative stress and can damage the sperm membrane integrity, DNA, acrosome, metabolic activity, and fertilizing potential of the sperm (Bansal and Bilaspuri 2010, Tariq et al. 2015). In the stages of freezing and thawing, the endogenous defense system of the buffalo semen is not enough to counter oxidative stress due to low concentration of naturally occurring antioxidants in buffalo semen (Ansari et al. 2012). Sperm viability is also decreased by 50% whereas fertilizing capacity is affected after cryopreservation (Lessard et al. 2000). However, the spermatozoa are provided with protection from oxidative damages with the presence of various antioxidants and antioxidant enzymes in the seminal plasma (Tariq et al. 2015) and buffalo semen is equipped with anti-oxidative stress system consisting of enzymatic and non-enzymatic antioxidants (Lone et al. 2016). With this, supplementation of antioxidants in extenders was observed to provide a cryoprotective effect on bull, ram, goat, boar, canine, and human sperm quality and minimize the detrimental effect of ROS as well as improve post-thaw spermatozoa (Amidi 2016). Balance measurement of both ROS and total antioxidant capacity (TAC) is essential in the assessment of oxidative stress in sperm and semen (Kashou 2013). . . . . read more



AHMAD S, ZHANG T, LEE F, LIU Y, LI X, GUO M. 2013. Seasonal variations in chemical composition of buffalo milk. Buffalo Bulletin 2013, Vol. 32.
ANSARI MS, RAKHA BA, ANDRABI SM, ULLAH N, IQBAL R, HOLT WV, AKHTER S. 2012. Glutathione-supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa. Reproductive Biology I 12 (2012) 271-276.
AMIDI F, PAZHOHAN A, SHABANI NM, KHODARAHMIAN M, NEKOONAM S. 2016. The role of antioxidants in sperm freezing: A review. Dordrecht: Springer Science+Business Media.
ANDRABI MH, ANSARI MS, ULLAH N, AFZAL M. 2008. Effect of non-enzymatic antioxidants in extender on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa. Pakistan Vet J 28(4): 159–162.
BANSAL AK, BILASPURI GS. 2010. Impacts of oxidative stress and antioxidants on semen functions. Veterinary Medicine International, Vol. 2011.
BHAKAT M, MOHANTY TK, GUPTA AK, PRASAD S, CHAKRAVARTY HK, KHAN HM. 2015. Effect of season on semen quality parameters in Murrah Buffalo bulls. Buffalo Bulletin 34(1): 100–112.
BILODEAU JF, CHATTERJEE S, SIRARD MA, GAGNON C. 2000. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Molecular Reproduction and Development 55: 282–288.
BINIOVÁ Z, DUCHÁČEK J, STÁDNÍK L, DOLEŽALOVÁ M, HÁJEK R. 2017. Effects of climatic conditions on bovine semen characteristics. Acta Universitatis Agriculturae Et Silviculturae Mendelianae Brunensis 65(2): 17–23.
EGHBALI M, SHOUSHTARI SMA, REZAEI SA, HASSAN M, ANSARI K. 2010. Calcium, magnesium and total antioxidant capacity (TAC) in seminal plasma of water buffalo (Bubalus bubalis) bulls and their relationships with semen characteristics. Veterinary Research Forum 1(1).
HU JH, TIAN WQ, ZHAO XL, ZAN LS, WANG H, LI QW, XIN YP. 2010. The cryoprotective effects of ascorbic acid supplementation on bovine semen quality. Animal Reproduction Science 121(2010): 72–72.
JEEVA JS, SUNITHA J, ANANTHALAKSHMI R, RAJKUMARI S, RAMESH M, KRISHNAN R. 2015. Enzymatic antioxidants and its role in oral diseases. Journal of Pharmacy & Bioallied Sciences 7(Suppl. 2): S331–S333.
KADIRVE G, KUMAR S, GHOSH SK, PERUMAL P. 2014. Activity of antioxidative enzymes in fresh and frozen thawed buffalo (Bubalus bubalis) spermatozoa in relation to lipid peroxidation and semen quality. Asian Pacific Journal of Reproduction 3(3): 210–217.
KASHOU AH, SHARMA R, AGARWAL A. 2013. Assessment of oxidative stress in sperm and semen. In: Carrell DT, Aston KI eds. Spermatogenesis: Methods and protocols. Methods in Molecular Biology. New York: Springer. p. 351–361.
LESSARD C, PARENT S, LECLERC P, BAILEY JL, SULLIVAN R. 2000. Cryopreservation alters the levels of the bull sperm surface protein P25b. Journal of Andrology 21(5): 700–707.
LONE SA, PRASAD JK, GHOSH JK, DAS SK, KUMAR GK, BALAMURUGAN N, KATIYAR B, VERMA R. 2016. Effect of cholesterol loaded cyclodextrin (CLC) on lipid peroxidation and reactive oxygen species levels during cryopreservation on buffalo (Bubalus bubalis) spermatozoa. Asian Pacific Journal of Reproduction 5(6): 476–480.
MAMUAD FV, VENTURINA HV, VENTURINA EV, MORCOSO RT, ATABAY EC, KAZUHIRO K. 2005. Artificial insemination manual for water buffaloes. Water Buffaloes and Beef Cattle Improvement Project (WBBCIP). p. 51–66.
MARAI IF, HABEEB AAM. 2010. Buffaloes reproductive and productive traits as affected by heat stress. Tropical and Subtropical Agroecosystems 12(2010): 193–217.
PATEL HA, SIDDIQUEE GM, CHAUDHARI DV, SUTHAR VS. 2016. Effects of different antioxidant additives in semen diluent on cryopreservability (–196 °C) of buffalo semen. Veterinary World 9(3): 299–303.
RATHER HA, ISLAM R, MALIK AA, LONE FA. 2016. Addition of antioxidants improves quality of ram spermatozoa during preservation at 4° C. Small Ruminant Research 141(2016): 24–28.
SHARMA M, BHAT Y, SHARMA N, SINGH A. 2018. Comparative study of seasonal variation in semen characteristics of buffalo bull. Journal of Entomology and Zoology Studies 6(1): 947–951.
SHIMADA K, FUJIKAWA K, YAHARA K, NAKAMURA T. 1992. Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. J Agric Food Chem 40: 945–948.
TARIQ M, KHAN M, SHAH MG, NISHA AR, UMER M, HASAN SM, RAHMAN A, RABANNI I. 2015. Exogenous antioxidants inclusion during semen cryopreservation of farm animals. Journal and Chemical Pharmaceutical Research 7(3): 2273–2280.
WATSON PF. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fertil Dev 7: 871–891.