13 REACTIVE OXYGEN SPECIES IN STORED STALLION SEMEN
Authors: Morrell J. M. , Lundgren A. , Humblot P. , Johannisson A. 
Published: December 2012

The quality of cooled semen doses for AI varies considerably between stallions. One of the factors affecting sperm quality may be the content of reactive oxygen species (ROS), which are known to affect fertility in some species. The objective of this study was to measure the ROS content of cooled stored stallion semen doses for AI as part of an evaluation of sperm quality and to correlate it with pregnancy rates. Ejaculates (3 per stallion) were collected from 14 stallions at a commercial stud farm and were extended in INRA 96 (IMV Technologies) as standard cooled semen doses for AI. After transporting the semen doses overnight to the laboratory at the Swedish University of Agricultural Sciences (Uppsala, Sweden) in an insulated container with a cold pack, aliquots were evaluated for sperm quality and ROS content by staining with hydroethidine and dihydrodichlorofluorescein diacetate and measuring fluorescence by flow cytometry (Guthrie and Welch 2006 J. Anim. Sci. 84, 2089–2100). Seven sperm sub-populations were quantified: superoxide positive or negative, living (S+L and S–L, respectively); superoxide positive, dead (S+D); and hydrogen peroxide positive or negative, living or dead (P+L, P–L, P+D, and P–D, respectively). Sperm motility was measured by computer-assisted sperm motility analysis (Sperm Vision, Minitube), membrane integrity by flow cytometry, and chromatin structure using the sperm chromatin structure assay (all assays described in Morrell et al. 2011 Theriogenology 76, 1424–1432). Pregnancy rates following AI with cooled semen doses from the same stallions were available later in the year. The effect of stallion was tested by ANOVA. Correlations were calculated between the proportions of sperm stained for S or P and other sperm quality parameters and also with pregnancy rates (SAS version 9.1, SAS Institute Inc., Cary, NC, USA). The effect of stallion was significant on all variables measured (P < 0.01). There were no significant correlations between percentages of S+L or P+L spermatozoa and progressive motility, membrane integrity, or DNA fragmentation index (%DFI). There was a trend for the proportion of P-L spermatozoa to be correlated with progressive motility (r = 0.51; P < 0.07) whereas the proportions of S+D and P–D were negatively correlated with progressive motility (r = –0.66 and –0.61; P < 0.02). The proportion of S+D and chromatin damage were negatively correlated (r = –0.54; P < 0.05). There was a trend for the proportions of S+D and P+D to be negatively related to the overall pregnancy rate (r = –0.52; P < 0.07, and r = –0.58; P < 0.05, respectively). The proportions of superoxide and hydrogen peroxide-containing live spermatozoa were not correlated to stallion fertility or other parameters of semen quality in stored semen samples. In contrast, a significant negative relationship was found between the proportion of dead spermatozoa producing superoxide radicals and progressive motility, and between dead spermatozoa producing superoxide radicals and chromatin damage. However, many factors contribute to sperm quality and fertility, with many interactions between them. More work is needed to unravel these different effects and determine which factors can be used to predict stallion fertility.