Sperm Characteristics in Cryopreserved Buffalo Bull Semen and Field Fertility

Document Type : Research Article


1 Department of Animal Reproduction and Artificial Insemination, National Research Center, Dokki, Giza, Egypt

2 General Organization for Veterinary Services, Dokki,Giza, Egypt

3 Department of Theriogenology, Faculty of Veterinary Medicine, Benha University, Kaliobia, Egypt


Semen quality and its relationship to fertility are major concern in animal production. The aim of this study was to assess the relationship between frozen thawed sperm characteristics and fertility following in vivo fertility through Artificial Insemination(AI). Semen samples were collected from four buffalo bulls. Semen volume and sperm concentration appeared to be significantly different (P<0.05) among bulls, while sperm motility and live sperm percentage did not vary in fresh semen. Frozen thawed semen was evaluated for motility, viability, sperm abnormalities, membrane integrity and in vivo fertility. A significant variation was found in semen parameters among bulls after thawing. Highly significant (P>0.001) differences in membrane integrity between fresh vs. frozen semen samples were noticed. Pregnancy rate was significantly (P<0.05) different among bulls. Significant correlations were found between motility and sperm abnormalities (r=-0.64; P<0.05) and membrane integrity (r=0.64; P<0.05). A significant negative correlation (r=-0.73; P<0.01) has been reported between sperm abnormalities and membrane integrity. In addition, a correlation between pregnancy rate and live sperm percentages (r=0.65; P<0.05), has also been reported. In conclusion, in this study motility was correlated with sperm abnormalities and membrane integrity. Live sperm percentage was the only parameter correlated with fertility. Motility and live sperm percentage can be used as a predicative measure in semen evaluation.


Abdel Dayem A.M.H., Mahmoud K.Gh.M., Nawito  M.F.,  Ayoub  M.M. and Scholkamy T.H. (2009). Fertility evaluation in Egyptian buffalo bulls using zona pellucida binding and in vitro fertilization assays. Livest. Sci. 122, 193-198.
Aitken R.J. (2006). Sperm function tests and fertility. Int. J. Androl. 29, 69-75.
Aitken R.J., Clarkson J.S. and Fishel S. (1989). Generation of reactive oxygen species, lipid peroxidation and human sperm function. Biol. Reprod. 41, 183-197.
Akhter S, Ansari M.S., Andrabi S.M.H., Ullah N. and Qayyum M. (2007). Effect of antibiotics in extender on fertility of liquid buffalo bull semen. Pakistan Vet. J. 27, 13-16.
Al Naib A., Hanarahan J.P., Lonergan P. and Fair S. (2011). In vitro assessment of sperm from bulls of high and low fertility. Theriogenology. 76, 161-167. 
Alvarez J.G. and Storey B.T. (1995). Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. Mol. Reprod. Dev. 42, 334-346.
Anzar M., Farooq U., Mirza M.A., Shahab M. and Ahmad N. (2003). Factors affecting the efficiency of artificial insemination in cattle and buffalo in Punjab, Pakistan. Pakistan Vet. J. 23, 106-113.
Barile V., Galasso A., Pacelli C., Francello M., Cigliano A., Penna L., Panfili M., Fiorinr M. and Borghese A. (1999). Conception rate in synchronized and artificially inseminated buffalo cow in different season under field conditions. Pp. 262-264 in Proc. 13th A.S.P.A. Cong. Piacenza. 1, 262-264.        
Barros P.M.H., Nichi M., Cortada C.N.M., Carvalho N.A.T., Baruselli P.S., Barnabe R.C. and  Barnabe V.H. (2007). Semen evaluation of Murrah buffalo bulls using sperm functional tests. Italian J. Anim. Sci. 6, 772-774.
Bavister B.D. (1990). Test of sperm fertilizing ability. Pp. 77-105 in Gamete Physiology. R.H. Asch, J.P. Balmaceda and I. Johnston Eds. Serono Symposia, USA.
Blom E. (1983). The spermiogram of the bull, Nordisk Vet. Med. 35, 105-111.
Brackett B.G. and Oliphant G. (1975). Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. 12, 260-274.
Campbell R.C., Dott H.M.  and Glover T.D.  (1956). Nigrosin-eosin as stain for differentiating  live and dead  spermatozoa. J. Agric. Sci. 44, 1-8.
Chacon J. (2001). Assessment of sperm morphology in zebu bulls, under field conditions in the tropics. Reprod. Domest. Anim. 36, 91-99.
Chohan K.R., Iqbal J., Asghar A.A. and Chaudhry M.A. (1992). Fertility of liquid and frozen semen in Nili Ravi buffaloes. Pakistan Vet. J. 12, 4-5.
Correa J.R. and Zavos P.M. (1994). The hypoosmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane. Theriogenology. 42, 351-360.
Correa J.R., Pace M.M. and Zavos P.M. (1997). Relationships among frozen- thawed sperm characteristics assessed via the routine semen analysis, sperm functional tests and fertility of bulls in an artificial insemination program. Theriogenology. 48, 721-731.
Courtens J.L., Ekwall H., Paquignon M. and Ploen L. (1989). Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing. J. Reprod. Fertil. 87, 613-626.
Den Daas N. (1992). Laboratory assessment of semen characteristics. Anim. Reprod. Sci. 28, 87-94.
Drevius L.O. and Eriksson H. (1966). Osmotic swelling of mammalian spermatozoa. Experimen. Cell. Res. 42, 136-156.
El-Sheshtawy R.I., El-Natat W.S. and El-Sisy G.A. (2009). Improvement of buffalo bulls semen cryopreservation by adding low density lipoprotein to diluents. Egypt J. Basic. Appl. Physiol. 8, 33-40.
El-Sheshtawy R.I., El-Sisy G.A., Mohamed A.A. and El-Natat W.S. (2010). Effect of egg yolk from different avian species on cryopreservability of buffalo semen. Global J. Biotechnol. Biochem. 5, 211-215.
El-Sisy G.A., El-Sheshtawy R.I., Mohamed A.A. and  El-Nattat W.S. (2010). Correlations between semen parameters and conception rate in buffaloes. Global. Vet. 5, 15-21.
Fukui Y., Glew A.M., Gandolfi F. and Moor R.M. (1988). Ram-specific effects on in vitro fertilization and cleavage of sheep oocytes matured in vitro. J. Reprod. Fertil. 82, 337-340.
García Álvarez O., Maroto-Morales A., Ramón M., Del Olmo E., Montoro V., Dominguez-Rebolledo A.E., Bisbal A., Jiménez-Rabadán P., Pérez-Guzmán M.D. and Soler A.J. (2010). Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa, Theriogenology. 74, 979-988.
Gordon I., (1996). Controlled Reproduction in Farm Animals  Series: controlled Reproduction in Cattle and Buffaloes. CAB International, UK.
Haugan T., Gröhn Y.T., Kommisrud E., Ropstad E. and Reksen O. (2006). Effects of sperm concentration at semen collection and storage period of frozen semen on dairy cow conception. Anim. Reprod. Sci. 97, 1-11.
Janny L. and Ménézo Y.J. (1994). Evidence for a strong paternal effect on human preimplantation embryo development and blastocyst formation. Mol. Reprod. Dev. 38, 36-42.
Januskauskas A.  and Žilinskas H. (2002). Bull semen evaluation post-thaw and relation of semen characteristics to bull’s fertility. Vet. Zootec. 17, 29-36.
Januskauskas A., Soderquist L., Haard M.G. and Rodriguez-Martinez H. (1996). Influence of sperm number per straw on the post-thaw sperm viability and fertility of Swedish red and white A.I. bulls. Acta. Vet. Scand. 37, 461-470.
Jeyendran R.S., Vander Ven H.H., Perez-Pelaez M., Crabo B.G. and Zaneveld L.J.D. (1984). Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil. 70, 219-228.
Johnson K.R., Dewey C.E., Bobo J.K., Kelling C.L. and Lunstra D.D. (1998). Prevalence of morphologic defects in spermatozoa from beef bulls. J. Am. Vet. Med. Assoc. 213, 1468-1471.
Johnston R.C., Clarke G.N., Liu D.Y. and Baker H.W.G. (1995). Assessment of the sperm quality analyser.  Fertil. Steril. 63, 1071-1076.
Kruger T.F., Menkveld R. and Stander F.S. (1986). Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil. Steril. 46, 1118-1123.
Lewis S.E.M. (2007). Is sperm evaluation useful in predicting human fertility? Reproduction. 134, 31-40.
Mahmoud K.G.M., Ziada M. and Scholkamy T.H. (2004).  Freezability of buffalo semen in relation to its in vitro fertilizing capacity and chromosomal picture. J. Egypt. Vet. Med. Assoc. 64, 239-251.
Maxwell W.M.C. (1984). Current problems and future potential of artificial insemination programmes. Pp. 291-298 in Reproduction in Sheep. D.R. Lindsay and D.T. Pearce. Eds. Canberra:  Australian Academy of Science and Australian Wool.
Mazur P. (1984). Freezing of living cells: mechanisms and implications. Am. J. Physiol. 247,  125-142.
Mohammed K.M.E., Ziada M.S. and Darwish G.M. (1998). Practical trials for freezing semen of buffalo and Friesian bulls. Effect of various  regimens of freezing, different milk extenders and types of straws packages on post thawing semen characters. Assiut. Vet. Med. J. 39, 70-75.
Ollero M., Muiño Blanco T., López Pérez M.J. and Cebrián Pérez J.A. (1996). Viability of ram spermatozoa in relation to the abstinence period and successive ejaculations. Int. J. Androl. 19, 287-292.
Otoi T., Tachikawa S., Kondo S. and Suzuki T. (1993). Effect of different lots of semen from the same bull on in vitro development of bovine oocytes fertilized in vitro. Theriogenology. 39, 713-718.
Petrunkina A.M., Waberski D., Günzel Apel A.R. and Töpfer-Petersen E. (2007). Determinants of sperm quality and fertility in domestic species. Reproduction. 134, 3-17.
Ramos L. and Wetzels A.M. (2001). Low rates of DNA fragmentation in selected motile human spermatozoa assessed by the tunel assay. Hum. Reprod. 16, 1703-1707.     
Rasul Z., Ahmad N. and Anzar M. (2001). Changes in motion characteristics, plasma membrane integrity, and acrosome morphology during cryopreservation of buffalo spermatozoa. J. Androl. 22, 278-283.
Rasul Z., Anzar M., Jalali S. and  Ahmad N. (2000). Effect of buffering systems on post-thaw motion characteristics, plasma membrane integrity and acrosome morphology of buffalo spermatozoa.  Anim. Reprod. Sci. 59, 31-41.
Rowe P.J., Comhaire F.H., Hargreave T.B. and Mellows H.J. (1993). WHO Manual for the Standard Investigation and the Diagnosis of the Infertile Couple. Cambridge University Press.
Saacke R.G., Dalton J.C., Nadir S., Nebel R.L. and Bame J.H. (2000). Relationship of seminal traits and insemination time to fertilization rate and embryo quality.  Anim. Reprod. Sci. 60, 663-677.
Sanchez R., Stale T., Khanago O., Turley H., Gips H. and Schill W.B. (1996). Sperm selection methods for intracytoplasmic sperm injection (ICSI) and andrological patients. J. Assist. Reprod. Gen. 13, 228-233.
Sansone G., Nastri M.J.F. and Fabbrocini A. (2000). Storage of buffalo (Bubalus bubalis) semen. Anim. Reprod. Sci. 62, 55-76.
Scholkamy T.H., Mahmoud K.G.M., El-Zohery F.A. and Ziada M.S. (2009). Evaluation of sephadex filtration for freezability and in vitro fertilizing ability of buffalo semen. Global. Vet. 3, 144-150.
Shoukir Y., Chardonnens D., Campana A. and Sakkas D. (1998). Blastocyst development from supernumery embryos after intracytoplasmic sperm injection: a paternal influence? Hum. Reprod. 13, 1632-1637.
Soderquist L., Janson K., Larsson K. and Einarsson S. (1991). Sperm morphology and fertility in AI bulls. J. Vet. Med. 38, 534-543.
Stanger J.D., Long V., Yovich  J.L. and  Almahbobi  G. (2010). Hypo osmotic swelling test identifies individual spermatozoa with minimal DNA fragmentation, Reprod. Biomed. Online. 21, 474-484.
Sumantri C., Ooe M., Saha S. and Boedino A. (1996). The influence of sperm-oocytes incubation time and breed of bull on in vitro embryo development in cattle. Theriogenology. 45, 264-269.
Tomar N.S. and Singh B. (1970). Grading of semen for better fertility rates. Indian Vet. J. 47, 409-413.
Vale W.G. (1997). Sperm cryopreservation. Bubalus. Bubalis. 1, 129-140.
Vawda A.I., Gunby J. and Younglai E.V. (1996). Semen parameters as predictors of in vitro fertilization; the importance of strict criteria sperm morphology. Hum. Reprod. 11, 1445-1450.
Watson P.F. (1995). Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 7, 871-891.
Zhang B.R., Larson B., Lundeheim N. and Roderiguez Martinez H. (1998). Sperm characteristics and zona pellucida binding in relation to field fertility of frozen-thawed semen from dairy AI bulls. Int. J. Androl. 21, 207-216.