• Essam Almadaly
  • Fady Tawfik
  • Ismail El-Kon
  • Bassiouni Heleil
  • El-Sayed Fattouh




This study aimed to investigate the effect of different cryoprotectants, glycerol (GLY) or ethylene glycol (EG) or dimethyl sulfoxide (DMSO) on sperm characteristics, and in vivo fertility of frozen-thawed buffalo-bull semen. A total of 85 ejaculates collected by artificial vagina from buffalo-bulls of proven fertility were used in this study. The collected ejaculates were examined for volume, motility, viability, morphology, and sperm cell concentration. The qualifying ejaculates (≥ 3 mass motion, > 70% progressive motility and viability, < 15% abnormal morphology and > 1 x 109 sperm cells/mL) were pooled and diluted with Tris-based diluent containing either 7% GLY or 5% EG or 5% DMSO. After 4 h equilibration time, the diluted semen was loaded in 0.5 mL straws, labeled, sealed and frozen stored until analysis. Frozen straws were thawed and evaluated for progressive motility, viability, hypo-osmotic swelling test (HOST), acrosomal membrane integrity, and acrosome reaction (AR) in response to calcium ionophore A23187. Moreover, in vivo fertility was calculated after artificial insemination (AI) of 75 buffalo-cows (25 female/cryoprotectant) treated with double doses of prostaglandin F (PGF) 11 days interval. The proportions of progressive motility, viability and intact-acrosome were higher (p < 0.05) in extender containing 7% GLY compared to 5% EG and 5% DMSO. The proportion of intact-plasma membrane was comparable (p ≥ 0.05) between GLY and EG but higher than that of DMSO. A time-dependent increase in the % AR and % relative AR was recorded in the three cryoprotectants with clear significant (p < 0.01) difference among them at 30 and 60 min incubation, respectively. Moreover, GLY yielded higher pregnancy rate (52%) than EG (32%) and DMSO (16%). In conclusion, GLY is recommended for preservation of buffalo-bull semen in order to maintain sperm plasma membrane integrity and improve in vivo fertility of frozen-thawed buffalo-bull semen.

Key words: buffalo-bull; frozen-thawed semen; cryoprotectant; estrus synchronization; In vivo fertility

Author Biographies

Essam Almadaly

Ismail El-Kon


Food and Agricultural Organization (FAO). FAOSTAT, Global livestock production and health atlas. Animal Production and Health Division, Roma, Italy, 2018. Available from http://www.fao.org/faostat/en/#data.

Andrabi SMH. Factors affecting the quali-ty of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reprod Domest Anim 2009; 44: 552–69.

Swelum AA, Mansour HA, Elsayed AA, Amer HA. Comparing ethylene glycol with glycerol for cryopreservation of buffalo bull semen in egg-yolk containing extenders. Theri-ogenology 2011; 76: 833–42.

Singh P, Kumar D, Kumar P, Singh I, Yadav PS. Cryopreservation and quality as-sessment of buffalo bull semen collected from farmer’s doorstep. Agri Res 2013; 2: 148–52.

Lei SU, Xilong LI, Jiexia Quan SY, Yahui LI, Xiechao HEX, Ianghui T. A comparison of the protective action of added egg yolks from five avian species to the cryopreservation of bull sperm. Anim Reprod Sci 2007; 104: 212–9.

Watson PF. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci 2000; 60–61: 481–92.

Mazur P. Freezing of living cells: mecha-nisms and implications. Am J Physiol 1984; 247: 125–42.

Ansari MS, Rakha BA, Andrabi SMH, Ul-lah N, Iqbal R, Holt WV, Akhter S. Glutathione supplemented tris-citric acid extender improves the post-thaw quality and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa. Reprod Biol 2012; 12: 271–6.

Chohan KR, Iqbal J, Asghar AA, Chaudhry MA. Fertility of liquid and frozen semen in Nili Ravi buffaloes. Pak Vet J 1992; 12: 4–5.

Pandey AK, Dhaliwal GS, Ghuman SPS, Agarwal SK. Impact of pre-ovulatory follicle diameter on plasma estradiol, subsequent luteal profiles and conception rate in buffalo (Bubalus bubalis). Anim Reprod Sci 2011; 123: 169–74.

Watson PF. The roles of lipid and protein in the protection of ram spermatozoa at 5 oC by egg-yolk lipoprotein. J Reprod Fertil 1981; 62: 483–92.

Hammerstedt RH, Graham JK, Nolan john P. Cryopreservation of mammalian sperm: what we ask them to survive. J Androl 1990; 11: 73–88.

Holt WV. Basic aspects of frozen storage of semen. Anim Reprod Sci 2000; 62: 3–22.

Gao DY, Mazur P, Critser JK. Funda-mental cryobiology of mammalian spermato-zoa. In: Karow AM, Critser JK eds. Reproduc-tive Tissue Banking. Scientific Principles. San Diego: Academic Press, 1997, 263–328.

Wündrich K, Paasch U, Leicht M, Glan-der HJ. Activation of caspases in human sper-matozoa during cryopreservation - an im-munoblot study. Cell Tissue Bank 2006; 7: 81–90.

Guthrie HD, Liu J, Critser JK. Osmotic tolerance limits and effects of cryoprotectants on motility of bovine spermatozoa. Biol Reprod 2002; 67: 1811–6.

Ball BA, Vo A. Osmotic tolerance of eq-uine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, via-bility, and mitochondrial membrane potential. J Androl 2001; 22: 1061–9.

Almadaly E, Hoshino Y, Ueta T, Mukoujima K, Shukry M, Farrag F, El-Kon I, Kita K, Murase T. Desalted and lyophilized bovine seminal plasma delays induction of the acrosome reaction in frozen-thawed bovine spermatozoa in response to calcium ionophore. Theriogenology 2015; 83: 175–85.

Rasul Z, Ahmed N, Anzar M. Antagonist effect of DMSO on the cryoprotection ability of glycerol during cryopreservation of buffalo sperm. Theriogenology 2007; 68: 813–9.

Bjoerndahl L, Soederlund I, Kvist U. Evaluation of the one-step eosin-nigrosin stain-ing technique for human sperm vitality assess-ment. Hum Reprod 2003; 18: 813–6.

Graham, JK. Analysis of stallion semen and its relation to fertility. Vet Clin North Am Equine Pract 1996; 12: 119–29.

Gil J, Soderquist L, Rodriguez-Martinez H. Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen. Theriogenology 2000; 54: 93–108.

Masuda H. Preservation of semen in liq-uid and frozen state, In: Artificial Insemination Association of Japan (Eds.), Textbook for the Course of Artificial Insemination in Domestic Animals., Fuji Planning. 2006, 343–352.

Abbas A, Andrabi MSH. Effect of dif-ferent glycerol concentrations on motility be-fore and after freezing, recovery rate, longevity and plasma membrane integrity of Nili Ravi buffalo bull spermatozoa. Pak Vet J 2002; 22: 1–4.

Rohilla RK, Tuli RK, Goyal RL. Com-parative study of the effects of cryoprotective agents in freezing Murrah buffalo bull semen. Ind J Vet Res 2005; 14: 37–43.

Chutia T, Biswas RK, Tamuli MK, Deka BC, Sinha S, Goswami J, Banik S, Kayastha RB. Effect of holding of semen and washing of seminal plasma on quality and fertility of Hampshire boar semen preserved at liquid state. Anim Reprod Sci 2014; 45: 141–9.

Waheed S, Ahmad N, Rahman NU, Jamil-ur-Rahman H, Younis M, Iqbal S. Evalua-tion of duck egg yolk for the cryopreservation of Nili-Ravi buffalo bull semen. Anim Reprod Sci 2012; 131: 95–9.

Fukui Y, Togawa M, Abe N, Takano Y, Asada M, Okada A, Iida K, Ishikawa H, Ohsu-mi S. Validation of the sperm quality analyzer and the hypo-osmotic swelling test for frozen-thawed ram and minke whale (Balanenoptera bonarensis) spermatozoa. J Reprod Dev 2004; 50: 147–54.

Bamba K. Evaluation of acrosomal integ-rity of boar spermatozoa by bright field micros-copy using an eosine-nigrosine stain. Theri-ogenology 1988; 29: 1245–51.

Murase T, Mukohjima K, Sakaguchi S, Ohtani T, Tsubota T, Kita I. Characterization of frozen-thawed Japanese black bull spermatozoa by standard semen analysis, mucus penetration test and the ability to undergo the acrosome reaction in response to calcium and the calcium ionophore A23187. J Reprod Dev 2001; 47: 237–43.

Barile VL. Technologies related with the artificial insemination in Buffalo. J Buffalo Sci 2012; 1: 139–46.

Snedecor, GW, Cochran, WG. Statistical Methods, 9th edition. Iowa State University Press Ames, Iowa, United States 1994.

Galli A, Bornaghi V, Balduzzi D, Buttaz-zoni L, Aleandri R. Sexual behavior and semen quality relating to italian buffalo. Proc 3rd World Buffalo Congr, Varna, Bulgaria 1993; 1: 562–70.

Tasdemir U, Buyukleblebici S, Tuncer PB, Coskun E, Ozgurtas T, Aydın FN, Buyukle-blebici O, Gurcan IS. Effects of various cryo-protectants on bull sperm quality, DNA integrity and oxidative stress parameters. Cryobiology 2013; 66: 38–42.

Harrison RAP, Ashworth PJC, Miller NGA. Assessment of sperm function under fertilizing conditions. Reprod Dom Anim 1996; 31: 25–30.

Guerrero CA. Cryopreservation and in-tracytoplasmic sperm injection with bovine epi-didymal spermatozoa (PhD Thesis). Fac Agric and Mechanical College, Louisiana State Uni-versity; Mexico, 2006.

Gabr AA. Assessment of in vitro and in vivo fertilizing capacity of buffalo sperm treat-ed with various cryoprotectants (PhD Thesis). Fac Agric, Mansoura University, Egypt, 2009.

El-Harairy MA, Eid LN, Zeidan AEB, Abd El-Salaam AM, El-Kishk MAM. Quality and fertility of the frozen-thawed bull semen as affected by the different cryoprotectants and glutathione levels. J Am Sci 2011; 7: 791–801.

Sarmah BC, Kaker ML, Razdan MN. Ef-fect of cold shock and freezing on loss of total lipids and phospholipids of buffalo spermato-zoa (Bubalus bubalis). Theriogenology 1984; 22: 621–4.

Alvarez JG, Storey BT. Taurine, hy-potaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa. Biol Reprod 1983; 29: 548–55.

Rota A, Milani C, Cabianca G, Martini M.

Comparison between glycerol and ethylene gly-col for dog semen cryopreservation. Theri-ogenology 2006; 65: 1848–58.

Ansari MS, Rakha BA, Akhter S. Cryo-preservation of Nili-Ravi buffalo (Bubalus bu-balis) semen in AndroMed® extender; in vitro and in vivo evaluation. Reprod Dom Anim 2017; 52: 992–7.

Layek SS, Mohanty TK, Kumaresan A, Parks JE. Cryopreservation of bull semen: Evo-lution from egg yolk based to soybean based extenders. Anim Reprod Sci 2016; 172:1–9.




How to Cite

Almadaly, E., Tawfik, F., El-Kon, I., Heleil, B., & Fattouh, E.-S. (2019). EFFECT OF DIFFERENT CRYOPROTECTANTS ON THE POST-THAW SPERM CHARACTERISTICS AND IN VIVO FERTILITY OF BUFFALO (Bubalus bubalus) BULL SEMEN. SLOVENIAN VETERINARY RESEARCH, 56(22-Suppl). https://doi.org/10.26873/SVR-792-2019



Veterinary medicine: Research and application