Please cite this article in press as: Qadeer, S., et al., Evaluation of antifreeze protein III for cryopreservation of Nili-Ravi (Bubalusbubalis) buffalo bull sperm. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.04.013 ARTICLE IN PRESS G Model ANIREP 4965 1–6 Animal Reproduction Science xxx (2014) xxx–xxx Contents lists available at ScienceDirect Animal Reproduction Science jou rn al hom epage : w ww.elsevier.com/locate/anir eprosci Evaluation of antifreeze protein III for cryopreservation of Nili-Ravi (Bubalusbubalis) buffalo bull sperm S. Qadeer a , M.A. Khan a , M.S. Ansari b , B.A. Rakha a , R. Ejaz a , A.U. Husna a , Q1 M. Ashiq c , R. Iqbal b , N. Ullah a , S. Akhter a,∗ a Animal Physiology Laboratory, Department of Zoology, PirMehr Ali Shah Arid Agriculture University, Rawalpindi 46300, Pakistan b Department of Zoology, University of Gujrat, Gujrat 50700, Pakistan c Semen Production Unit Qadirabad, Sahiwal 57000, Pakistan a r t i c l e i n f o Article history: Received 16 November 2013 Received in revised form 25 April 2014 Accepted 27 April 2014 Available online xxx Keywords: Buffalo bull sperm, Antifreeze proteins, Ice crystal a b s t r a c t Lower fertility in buffaloes with frozen-thawed semen is attributed to sperm damage that is believed to be due to formation of ice crystals during freeze/thaw process. It was hypoth- esized that antifreeze proteins in the extender may improve the post thaw quality of buffalo bull sperm. For this purpose, two separate experiments were conducted to eval- uate antifreeze proteins III (AFPIII) at 0 (control), 0.1, 1 and 10 g/mL (Experiment I) and0 (control), 0.01, 0.1 and 1 g/mL (Experiment II) for its effect on post thaw quality of buf- falo bull semen. Semen was collected from three Nili-Ravi buffalo (Bubalusbubalis) bulls with artificial vagina (42 ◦ C) for three weeks (replicate) per experiment. For each experi- ment, qualifying ejaculates (6 ejaculates/bull) were divided into four aliquots and diluted (at 37 ◦ C having 50 × 10 6 sperm/mL) in tris-citric acid extender containing above mentioned concentrations of AFP III. Diluted semen was cooled to 4 ◦ C in 2 h, equilibrated for 4 h, filled in 0.5 mL straws, kept over liquid nitrogen vapors for 10 min and plunged in the liquid nitrogen. After 24 h of storage, semen straws were thawed at 37 ◦ C for 30 s to assess sperm motility (SM), plasma membrane integrity (PMI), viability (live sperm with intact acrosome) and normal epical ridge (NAR). In experiment I, improvement (P < 0.05) in percentage SM and sperm PMI was recorded in extender containing 0.1 g/mL AFP III compared to control, the higher concentrations (1 g/mL and 10 g/mL) being inefficient. While evaluating the lower concentration (experiment II), 0.01 g/mL of AFP III in the extender it was found to be ineffective to improve semen quality parameters, while 0.1 g/mL AFP III in extender was found better in terms of progressive motility and plasma membrane integrity of buffalo bull semen compared to control. Sperm viability and NAR remained similar (P > 0.05) in extend- ers containing different concentrations of AFP III and control in both of experiments. In conclusion addition of AFPIII in the extender at0.1 g/mL improved the progressive motility and plasma membrane integrity of cryopreserved buffalo bull semen. © 2014 Published by Elsevier B.V. ∗ Corresponding author. Tel.: +92 333 5206056; fax: +92 51 9290160. Q2 E-mail addresses: sashraf1993@gmail.com, shamim@uaar.edu.pk (S. Akhter). 1. Introduction Since the first successful pregnancy in buffalo using cryopreserved semen (Basirov, 1964), artificial insem- ination has been adopted; however, it has remained unpopular because of poor fertility with frozen–thawed semen (Kumaresan et al., 2005, 2006; Shukla and Misra, http://dx.doi.org/10.1016/j.anireprosci.2014.04.013 0378-4320/© 2014 Published by Elsevier B.V. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26