Original Research Influence of Seminal Plasma Antioxidants and Osteopontin on Fertility of the Arabian Horse M.M. Waheed DVM, MVSc, PhD a , S.M. El-Bahr DVM, PhD b , A.K. Al-haider DVM, MVSc, PhD a a Department of Clinical Studies, College of Veterinary Medicine and Animal Resources, King Faisal University, Al Ahsa, Kingdom of Saudi Arabia b Department of Biochemistry, Faculty of Veterinary Medicine, Alexandria University, Egypt article info Article history: Received 13 July 2012 Received in revised form 24 August 2012 Accepted 9 November 2012 Available online 17 March 2013 Keywords: Stallion Antioxidants Uric acid Glutathione peroxidase Osteopontin abstract This study was designed to investigate enzymatic antioxidants’ activity and nonenzymatic antioxidants’ levels in seminal plasma of stallions and to relate them with season, age, and fertility of stallions. Fifty ejaculates were collected from six healthy Arabian stallions, 4-22 years old. Ejaculates were evaluated by conventional methods. Five milliliters of each semen sample was centrifuged, and the supernatant seminal plasma was stored at 20  C. Five antioxidants, in addition to osteopontin (OPN) and testosterone, were determined in stallion seminal plasma by using commercial enzyme-linked immunosorbent assay kits. Results revealed that uric acid, ascorbic acid, OPN, and testosterone concentrations and glutathione peroxidase (GPx) activity in stallions’ seminal plasma were high (P < .05) during spring. GPx activity was higher (P < .05) in age group B (11-18 years old) than in age group A (4-10 years old). The effect of stallions’ age on GPx activity in the fertility groups was highly significant (P <.01). OPN concentration was highest (P <.05) in age group A. Uric acid and OPN concentrations and GPx activity in stallions’ seminal plasma and percent of sperm motility were higher (P < .05) in fertility group III (>70%) than in fertility group I (<50%). However, ascorbic acid concentration, catalase activity and percentage of sperm abnor- malities were lower (P < .05) in fertility group III than in fertility group I. It was concluded that season and stallion age may affect antioxidant defense systems in stallions’ seminal plasma. The impairment of seminal antioxidants and OPN could lead to low fertility. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Reactive oxygen species (ROS) are known to be involved in lipid peroxidation, as well as DNA and protein damage that may lead to cell death [1,2]. The balance between ROS production and their detoxification may be an important factor in sperm survival and function [3]. Oxidative stress has been associated with perturbation of normal sperm function, including damage to chromatin, proteins, and membrane lipids resulted in a decrease in sperm motility, a decreased capacity for acrosome reaction, and loss of fertility [4-6]. ROS are found in all tissues in the body and also in semen, where they are produced by damaged and dead spermatozoa [7], from cytoplasmic remnants in immature spermatozoa [7], and also by leukocytes [8] and cellular debris [9]. High levels of ROS are associated with infertility in humans [10] but were previously thought not to cause sperm damage in equine semen, on the grounds that the antioxidative systems present in stallion seminal plasma (glutathione peroxidase [GPx], superoxide dis- mutase [SOD], and catalase [CAT]) would neutralize ROS [9,11]. Seminal plasma is a rich source of enzyme scaven- gers of ROS, such as SOD, CAT, and GPx. These act to degrade superoxide anion, hydrogen peroxide or lipid peroxides [6], and nonenzymatic low-molecular-weight factors such as lactate, urate, taurine, hypotaurine, pyruvate, ascorbic acid, tocopherol, ergothioneine, and albumin are capable of removing certain ROS [12,13], whereas the sperm cell has limited antioxidant capacity, related to its small Corresponding author at: Professor M.M. Waheed, DVM, MVSc, PhD, College of Veterinary Medicine and animal resources, King Faisal University, Clinical Studies, Qatar road, Al-Ahsa, Saudi Arabia. E-mail address: mmwaheed@kfu.edu.sa (M.M. Waheed). Journal of Equine Veterinary Science journal homepage: www.j-evs.com 0737-0806/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2012.11.006 Journal of Equine Veterinary Science 33 (2013) 705-709