Animal Reproduction Science 104 (2008) 119–131 Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin Eneiva Carla Carvalho Celeghini a , Rubens Paes de Arruda a,* , Andr´ e Furugen Cesar de Andrade a , Juliana Nascimento a , Cl´ audia Fernandes Raphael a , Paulo Henrique Mazza Rodrigues b a Laboratory of Semen Biotechnology and Andrology, Department of Animal Reproduction, Veterinary Medicine and Animal Science School, University of S˜ ao Paulo (USP), Brazil b Department of Animal Nutrition and Production, School of Veterinary Medicine and Animal Science, University of S˜ ao Paulo (USP), Brazil Received 17 April 2006; accepted 5 February 2007 Available online 9 February 2007 Abstract The process of cryopreservation impairs sperm cell function, potentially leading to a reduction in fertility. The objectives of the present study were to evaluate the effects that cryopreservation using two different extenders has on sperm motility and mitochondrial function, as well as on the integrity of plasma mem- branes, acrosomal membranes and chromatin, using practical and objective techniques. The focus of the present study was to identify correlations between alterations in sperm membranes and sperm motility in cryopreserved bovine spermatozoa. Seven ejaculates were collected from eight Simmental bulls (n = 56). After collection, semen volume and concentration were assessed for purposes of dilution. Sperm motility was evaluated subjectively and by computer-assisted semen analysis, morphological characteristics were evaluated by differential interference microscopy, the integrity of plasma and acrosomal membranes, as well as mitochondrial function, were determined using a combination of fluorescent probes containing fluorescein isothiocyanate–Pisum sativum agglutinin, propidium iodide or 5,5 ′ ,6,6 ′ -tetrachloro-1,1 ′ ,3,3 ′ - tetraethylbenzimidazolcarbocyanine iodide. Chromatin integrity was evaluated using the acridine orange technique. The semen was subsequently divided into two aliquots and diluted with one of two extenders (Bioxcell ® or Botu-Bov ® ), after which both were packaged in 0.5 mL straws and frozen using an automated * Corresponding author at: Av. Duque de Caxias Norte, 225, Post Box 23, 13-635-900 Pirassununga, SP, Brazil. Tel.: +55 19 3565 4262/21; fax: +55 19 3565 4060. E-mail address: arrudarp@usp.br (R.P. de Arruda). 0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2007.02.001