Please cite this article in press as: Mui ˜ no, R., et al., Effect of different thawing rates on post-thaw sperm viability, kinematic parameters and motile sperm subpopulations structure of bull semen, Anim. Reprod. Sci. (2008), doi:10.1016/j.anireprosci.2007.11.028 ARTICLE IN PRESS +Model ANIREP-3497; No. of Pages 15 Animal Reproduction Science xxx (2008) xxx–xxx Effect of different thawing rates on post-thaw sperm viability, kinematic parameters and motile sperm subpopulations structure of bull semen R. Mui ˜ no a , M.M. Rivera b , T. Rigau b , J.E. Rodriguez-Gil b , A.I. Pe ˜ na a,∗ a Unit of Reproduction and Obstetrics, Department of Animal Pathology, Faculty of Veterinary Medicine of Lugo, University of Santiago de Compostela, 27002 Lugo, Spain b Unit of Animal Reproduction, Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra, Spain Received 13 June 2007; received in revised form 21 November 2007; accepted 23 November 2007 Abstract The aim of the present study was to evaluate three thawing rates for bull semen frozen in 0.25-ml straws: placing the straws in a water bath at 37 ◦ C for 40 s, at 50 ◦ C for 15 s or at 70 ◦ C for 5 s. In a first experiment, the three thawing rates were compared in relation to post-thaw sperm motility, determined subjectively, and sperm plasma and acrosomal membrane integrity, examined by flow cytometry, after 0 and 5h of incubation at 37 ◦ C. In a second experiment, the three thawing rates were evaluated based on post-thaw sperm motility, determined using a CASA system, after 0 and 2 h of incubation at 37 ◦ C. In addition, for the motile spermatozoa, the individual motility descriptors were analysed using a multivariate clustering procedure to test the presence of separate sperm subpopulations with specific motility characteristics in the thawed bull semen samples. Finally, it was investigated if the thawing rate had any influence on the relative frequency distribution of spermatozoa within the different subpopulations. In terms of overall post- thaw motility or plasma and acrosomal sperm membrane integrity there were no significant differences between the three thawing methods evaluated. The statistical analysis clustered all the motile spermatozoa into four separate subpopulations with defined patterns of movement: (1) moderately slow and progressive sperm (27%); (2) “hyperactivated-like” sperm (15.4%); (3) poorly motile non-progressive sperm (34.3%); (4) fast and progressive sperm (23.3%). The thawing rate had no significant influence on the frequency distribution of spermatozoa within the four subpopulations, but there was a significant effect (P < 0.05) of the interaction between thawing rate and incubation time. Higher proportions of spermatozoa with fast and progressive movement were observed after 2h of post-thaw incubation when the thawing was at the faster ∗ Corresponding author. Tel.: +34 982 25 23 50; fax: +34 982 28 59 40. E-mail address: anaipena@lugo.usc.es (A.I. Pe˜ na). 0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.11.028