Andrologia. 2017;e12830. wileyonlinelibrary.com/journal/and | 1 of 6 https://doi.org/10.1111/and.12830 © 2017 Blackwell Verlag GmbH Accepted: 11 February 2017 DOI: 10.1111/and.12830 ORIGINAL ARTICLE Comparison of three different extenders on Murrah buffaloes (Bubalus bubalis) semen freezability M. F. Zorzetto 1 | I. Martin 2 | Y. F. R. Sancler-Silva 1 | S. Zoca 1 | C. P. Freitas-Dell’Aqua 1 | F. O. Papa 1 | A. A. Ramos 3 | J. F. Nunes 4 | C. C. M. Salgueiro 5 | E. Oba 1 1 Department of Animal Reproduction and Veterinary Radiology, College of Veterinary Medicine and Animal Science, Sao Paulo State University, Botucatu, Sao Paulo, Brazil 2 University of Uberaba, Uberaba, Minas Gerais, Brazil 3 Department of Animal Production, College of Veterinary Medicine and Animal Science, Sao Paulo State University, Botucatu, São Paulo, Brazil 4 Department of Veterinary Medicine, State University of Ceará, Integrated Nucleus of Biotechnology, Fortaleza, Ceará, Brazil 5 Health School, Potiguar University, Natal, Rio Grande do Norte, Brazil Correspondence Eunice Oba, Department of Animal Science and Veterinary Radiology, College of Veterinary Medicine and Animal Science, Sao Paulo State University, Botucatu, São Paulo, Brasil. Email: euniceoba@fmvz.unesp.br Funding information CNPq (PQ), Brazil Summary The use of frozen semen for artificial insemination is the main approach utilised for the genetic improvement of most domesticated species. The advantages include lower transportation costs, continuous availability of semen, fewer occurrences of sexually transmitted diseases and the incorporation of desirable genes in a relatively short amount of time. Nevertheless, the use of frozen semen in buffalo herds remains lim- ited due to the loss of sperm quality when buffalo semen is frozen. So, the goal of this study was to evaluate the pre- and post-cryopreservation quality of buffalo semen diluted in three distinct freezing media: Tris-egg yolk, Botu-bov® (BB) and ACP-111®. Thirty-two ejaculates from four bulls were analysed in terms of kinetics, morphology and sperm viability by epifluorescence microscope. Thawed samples were also evalu- ated for capacitation-like damage, DNA fragmentation and plasma and acrosomal membrane integrity using flow cytometry. The Tris-egg yolk and BB® extenders yielded better results than the ACP-111® extender for kinetics parameter (total motil- ity, progressive motility and percentage of rapid cells). However, semen samples were similar for parameters evaluated by flow cytometry. Taken together, the data indicate that in comparison with Tris-egg yolk and BB extender, ACP-111® can also be used as an extender for buffalo semen cryopreservation. KEYWORDS computer-assisted sperm analysis, cryopreservation, epifluorescence microscopy, flow cytometry, sperm viability 1 | INTRODUCTION The prominence of buffalo in the global economy reflects their tri- ple ability, production of meat and milk as well as pulling force. These advantages have attracted investment in technology programmes for buffalo herds similar to those established for cattle. The goals of these programmes are to genetically improve existing buffalo herds using reproductive biotechnologies, which are of particular importance be- cause they permit the dissemination of superior genes within the spe- cies to improve animal performance. One of these reproductive biotechnologies is the semen cryopres- ervation that allows the distribution of the genetic material from a superior bull. So, the quality of frozen-thawed semen is one of several factors affecting conception rate and its use for artificial insemination remains limited in buffalo due to poor semen freezability when com- pared to bull semen (Kumaresan, Ansari, & Abhishek, 2005; Waheed et al., 2012). The process of semen cryopreservation has many risks related to sperm viability, such as changes in temperature, osmotic and toxic stress caused by exposure to the cryoprotectant and the forma- tion and dissolution of ice crystals (Watson, 2000). The major cellular damage related to cold shock occurs between 5 and 15°C (Stornelli, Tittarelli, Savignone, & Stornelli, 2005; Watson, 2000) and significantly reduces sperm motility and metabolic activity (Sansone, Nastri, & Fabbrocini, 2000). Another critical change that may occur during