The effect of melatonin on the quality of extended boar semen after long-term storage at 17 °C D. Martín-Hidalgo a , F.J. Barón b , M.J. Bragado a , P. Carmona a , A. Robina c , L.J. García-Marín a , M.C. Gil a, * a Research Team of Intracellular Signaling and Technology of Reproduction (SINTREP), Veterinary School, University of Extremadura, Cáceres, Spain b Biostatistics, Department of Preventive Medicine and Public Health, Medicine School, University of Málaga, Málaga, Spain c Department of Anatomy, Cellular Biology and Zoology, Veterinary School, University of Extremadura, Cáceres, Spain Received 19 August 2010; received in revised form 14 December 2010; accepted 19 December 2010 Abstract Melatonin (MLT) is an efficient antioxidant that protects cells and tissues and initiates a host of receptor-mediated effects. In order to enhance the life span of refrigerated boar semen, our aim was to evaluate the effects of addition of 1 M MLT to commercially produced pig semen (33 seminal doses from 14 boars) that had been preserved at 17 °C for 7 days. Samples without MLT served as controls. On Days 1, 4 and 7, we evaluated motility parameters and the percentage of total motile and progressively motile spermatozoa by a computer-aided sperm analysis system. Viability (SYBR-14/PI), acrosomal status (FITC-PNA/PI), membrane fluidity (M-540/YoPro-1) and mitochondrial membrane potential status (JC-1) were evaluated by flow cytometry. MLT treatment significantly enhanced the percentage of static spermatozoa after 7 days of storage and significantly reduced the percentage of progressively motile spermatozoa on Day 7. The velocity characteristics (VCL, VSL and VAP) were significantly higher for MLT-treated samples on Day 1 and were their lowest on Day 7. With regard to flow cytometry results, the percentage of viable spermatozoa with an intact acrosome was higher in MLT samples throughout the entire storage period. In addition, there was a significantly higher proportion of live spermatozoa on Day 7 in the samples that had not been treated with MLT. The proportion of spermatozoa showing a high mitochondrial membrane potential remained at similar levels (P  0.05) throughout the trial. Although the findings of the present study revealed that 1 M MLT increased the proportion of live sperm with an intact acrosome, this treatment did not enhance the spermatic quality of refrigerated boar semen. © 2011 Elsevier Inc. All rights reserved. 1. Introduction Artificial insemination (AI) during pig production is widely applied throughout the developed world by the use of semen that is preserved in the liquid state, which is stored at 15–20 °C for several days until it is used for AI [1]. To preserve spermatozoa for prolonged periods, their metabolic activity needs to be reduced by dilution into an appropriate medium and by lowering the tem- perature. Several commercial boar semen extenders, which have been proposed for long-term use (i.e., for use during 5 days of storage), are currently available [2]. However, the fertility outcome of liquid semen is gradually lost during extended storage periods and is significantly affected when the number of fertile sperm is below a certain threshold level [3]. Both functional and structural sperm attributes are susceptible to dam- age during storage. The most common of these attri- * Corresponding author. Tel.: +34-927257100 Ext: 1303; Fax: +34-927257110. E-mail address: crgil@unex.es (M.C. Gil). Available online at www.sciencedirect.com Theriogenology xx (2011) xxx www.theriojournal.com 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.12.021