Functional and Flow Cytometric Analysis of Buffalo Cryopreserved Spermatozoa: Comparison of Different Breeds and Incubation Times

Document Type : Original Article


1 Department of Anatomical Sciences, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.

2 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Selcuk University, Konya, Turkey

3 Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran

4 Department of Anatomical Sciences, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran

5 Department of Urology, Selcuk University School of Medicine, Konya, Turkey

6 Department of Medical Services and Techniques, Denizli Vocational School of Health Services, Pamukkale University, Denizli, Turkey

7 Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran.

8 Buffalo Breeding and Training Extension Center, Jabal, Urmia, Iran

9 Department of Animal Science, Faculty of Veterinary Medicine, Selcuk University, Konya, Turkey

10 Reproductive Epidemiology Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran


Background: The purpose of this research was to compare the functional parameters of frozen-thawed Iranian Azari
buffalo spermatozoa with imported semen samples of Italian Mediterranean buffalo (IMB) after the thawing process
and 4 hours of incubation.
Materials and Methods: In this experimental study, a total of twenty-four ejaculates from four Iranian Azari buffalo
bulls were collected. Semen samples were diluted in AndroMed extender at a concentration of 50×106 spermatozoa/
ml. The diluted samples were filled in 0.5 ml straws and were frozen in a programmable freezer. For imported semen
samples, twenty-four samples of four IMB were used, which were diluted in AndroMed extender and frozen by the same
procedure. Frozen-thawed sperm motion patterns, mitochondrial activity, membrane integrity, DNA integrity, reactive
oxygen species (ROS), and apoptosis status were evaluated immediately after thawing and 4 hours of incubation.
Results: Post-thawed sperm motility, progressive motility (PM), mitochondrial activity, membrane integrity were
significantly higher in imported semen samples in compare with Iranian Azari buffalo. After 4 hours of incubation,
sperm velocity patterns were superior in Iranian Azari semen samples. Moreover, the percentage of sperm cells with
un-damaged DNA was higher in Iranian semen samples compared to imported samples at the time 0 of incubation.
Following 4 hours of incubation, a significant increase in intracellular ROS level leads to reduced membrane integrity,
mitochondrial activity, and DNA integrity in both buffalo breeds. At time 4, Iranian samples showed significantly
lower apoptosis and higher dead spermatozoa compared to imported semen samples.
Conclusion: Our study showed that the post-thawed quality of Iranian Azari buffalo semen was comparable with imported samples after 4 hours of incubation. Further investigations are recommended to assess the in vitro and in vivo
fertility rate of both buffalo breeds.


  1. Mokhber M, Moradi-Shahrbabak M, Sadeghi M, Moradi-Shahrb­abak H, Stella A, Nicolzzi E, et al. A genome-wide scan for sig­natures of selection in Azeri and Khuzestani buffalo breeds. BMC Genomics. 2018; 19(1): 449.
  2. Ghoreishifar SM, Moradi-Shahrbabak H, Fallahi MH, MoradiShahrbabak M, Abdollahi-Arpanahi R, Khansefid M. Genomic measures of inbreeding coefficients and genome-wide scan for runs of homozygosity islands in Iranian river buffalo, Bubalus buba­lis. BMC Genet. 2020; 21(1): 1-12.
  3. Safari A, Shadparvar AA, Hossein-Zadeh NG, Abdollahi-Arpanahi R. Economic values and selection indices for production and re­production traits of Iranian buffaloes (Bubalus bubalis). Trop Anim Health Prod. 2019; 51(5): 1209-1214.
  4. Neglia G, de Nicola D, Esposito L, Salzano A, D’Occhio MJ, Fatone G. Reproductive management in buffalo by artificial insemination. Theriogenology. 2020; 150: 166-172.
  5. Nasr MA. The impact of crossbreeding Egyptian and Italian buf­falo on milk yield and composition under subtropical environmental conditions. J Dairy Res. 2016; 83(2): 196-201.
  6. Costa A, Neglia G, Campanile G, De Marchi M. Milk somatic cell count and its relationship with milk yield and quality traits in Italian water buffaloes. J Dairy Sci. 2020; 103(6): 5485-5494.
  7. Serafini R, Love CC, Coletta A, Mari G, Mislei B, Caso C, et al. Sperm DNA integrity in frozen-thawed semen from Italian Medi­terranean Buffalo bulls and its relationship to in vivo fertility. Anim Reprod Sci. 2016; 172: 26-31.
  8. Baldassarre H, Karatzas CN. Advanced assisted reproduction technologies (ART) in goats. Anim Reprod Sci. 2004; 82-83: 255-266.
  9. Ezz MA, Montasser AE, Hussein M, Eldesouky A, Badr M, Hegab AE, et al. The effect of cholesterol loaded cyclodextrins on post-thawing quality of buffalo semen in relation to sperm DNA damage and ultrastructure. Reprod Biol. 2017; 17(1): 42-50.
  10. Qadeer S, Khan MA, Ansari MS, Rakha BA, Ejaz R, Husna AU, et al. Evaluation of antifreeze protein III for cryopreservation of Nili-Ravi (Bubalus bubalis) buffalo bull sperm. Anim Reprod Sci. 2014; 148(1-2): 26-31.
  11. Minervini F, Guastamacchia R, Pizzi F, Dell'Aquila M, Barile VL. As­sessment of different functional parameters of frozen–thawed buf­falo spermatozoa by using cytofluorimetric determinations. Reprod Domest Anim. 2013; 48(2): 317-324.
  12. Topraggaleh TR, Shahverdi A, Rastegarnia A, Ebrahimi B, Shafie­pour V, Sharbatoghli M, et al. Effect of cysteine and glutamine add­ed to extender on post-thaw sperm functional parameters of buffalo bull. Andrologia. 2014; 46(7): 777-783.
  13. Shahverdi A, Rastegarnia A, Topraggaleh Rezaei T. Effect of ex­tender and equilibration time on post thaw motility and chroma­tin structure of buffalo bull (Bubalus bubalis) spermatozoa. Cell J. 2014; 16(3): 279-288.
  14. Öztürk AE, Bodu M, Bucak MN, Ağir V, Özcan A, Keskin N, et al. The synergistic effect of trehalose and low concentrations of cryo­protectants can improve post-thaw ram sperm parameters. Cryobi­ology. 2020; 95: 157-163.
  15. Mahmoud KGM, El-Sokary AAE, Abou El-Roos MEA, Abdel Ghaf­far AD, Nawito M. Sperm characteristics in cryopreserved buffalo bull semen and field fertility. Iran J Appl Anim Sci. 2013; 3(4): 777-783.
  16. Ansari MS, Rakha BA, Akhter S. Cryopreservation of Nili-Ravi buf­falo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation. Reprod Domest Anim. 2017; 52(6): 992-997.
  17. Akhter S, Rakha BA, Ansari MS, Iqbal S, Khalid M. Evaluation of pigeon egg yolk for post thaw quality, enzyme leakage and fertil­ity of buffalo (Bubalus bubalis) bull spermatozoa. Theriogenology. 2018; 119: 137-142.
  18. Kadirvel G, Kumar S, Kumaresan A. Lipid peroxidation, mitochon­drial membrane potential and DNA integrity of spermatozoa in rela­tion to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen. Anim Reprod Sci. 2009; 114(1-3): 125-134.
  19. Mahfouz RZ, du Plessis SS, Aziz N, Sharma R, Sabanegh E, Agar­wal A. Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress. Fertil Steril. 2010; 93(3): 814-821.
  20. Motlagh MK, Sharafi M, Zhandi M, Mohammadi-Sangcheshmeh A, Shakeri M, Soleimani M, et al. Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze–thawing process of ram sperm. Cryobiol­ogy. 2014; 69(2): 217-222.
  21. Andrabi SMH. Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reprod Domest Anim. 2009; 44(3): 552-569.
  22. Sansone G, Nastri MJ, Fabbrocini A. Storage of buffalo (Bubalus bubalis) semen. Anim Reprod Sci. 2000; 62(1-3): 55-76.
  23. Rastegarnia A, Shahverdi A, Rezaei Topraggaleh T, Ebrahimi B, Shafipour V. Effect of different thawing rates on post-thaw viability, kinematic parameters and chromatin structure of buffalo (Bubalus bubalis) spermatozoa. Cell J. 2013; 14(4): 306-313.
  24. Rastegarnia A, Shahverdi A, Rezaei Topraggaleh T, Shafiepour V. In vitro comparison of soybean lecithin-based extenders for cryo­preservation of buffalo (Bubalus bubalis) semen. Comp Clin Path. 2014; 23(4): 893-900.
  25. Sharma M, Bhat Y, Sharma N, Singh A. Comparative study of sea­sonal variation in semen characteristics of buffalo bull. J Entomol Zool Stud. 2018; 6(1): 52-109.
  26. Bhakat M, Mohanty TK, Gupta AK, Prasad Sh, Chakravarty AK, Khan HM. Effect of season on semen quality parameters in Murrah buffalo bulls. Buffalo Bull. 2015; 34(1): 100-112.
  27. Rahman MB, Vandaele L, Rijsselaere T, El-Deen MS, Maes D, Shamsuddin M, et al. Bovine spermatozoa react to in vitro heat stress by activating the mitogen-activated protein kinase 14 signal­ling pathway. Reprod Fertil Dev. 2014; 26(2): 245-257.
  28. Nelli G, Pamanji SR. Di-n-butyl phthalate prompts interruption of spermatogenesis, steroidogenesis, and fertility associated with in­creased testicular oxidative stress in adult male rats. Environ Sci Pollut Res Int. 2017; 24(22): 18563-18574.
  29. Kim SH, Yu DH, Kim YJ. Apoptosis-like change, ROS, and DNA status in cryopreserved canine sperm recovered by glass wool filtration and Percoll gradient centrifugation techniques. Anim Re­prod Sci. 2010; 119(1-2): 106-114.