Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa Alexei Santiani a, * , Shirley Evangelista a , Néstor Sepúlveda b , Jennie Risopatrón b , Juana Villegas b , Raúl Sánchez b a Laboratory of Animal Reproduction, Faculty of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Perú b Center of Biotechnology in Reproduction (BIOREN-CEBIOR), Faculty of Medicine, Universidad de La Frontera, Temuco, Chile article info Article history: Received 13 November 2013 Received in revised form 30 June 2014 Accepted 1 July 2014 Keywords: Superoxide dismutase Tempo Tempol Oxidative stress Ram sperm Cryopreservation abstract High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5  C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10  C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35  C or at 35  C/5  C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capac- itated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5  C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10  C) protects ram sper- matozoa from oxidative stress. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Oxidative stress, related to altered sperm function [1], is caused by high levels of reactive oxygen species (ROS) produced by ram [2] and human spermatozoa [3] or by activated leukocytes present in the seminal plasma [4]. Ram spermatozoa produce high levels of hydrogen peroxide [5]. High ROS levels result in reduced sperm motility [6,7], peroxidation of unsaturated fatty acids in sperm membranes [8], and sperm DNA damage [7]. Increased ROS levels have been observed during cryo- preservation of human spermatozoa, specifically during the cooling process, with maximum levels observed at 4  C [9,10]. Similarly, gradual reduction of temperature stimu- lated the generation of superoxide anion in bovine sper- matozoa [11]. At temperatures below 0, sperm ROS levels * Corresponding author. Tel.: þ51 1 7229917; fax: þ51 1 4361027. E-mail address: asantiani@hotmail.com (A. Santiani). Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.07.002 Theriogenology 82 (2014) 884–889