The purpose of this study was to determine whether the analysis of dismount semen samples from Thoroughbred stallions, could be used to predict whether a given mating would result in a pregnancy. The analysis was based on 143 matings of 141 mares by a cohort of 7 Thoroughbred stallions over a 4-week period at an Australian Stud. The criteria of semen quality utilized in this analysis involved a preliminary assessment of the raw dismount sample in terms of semen volume, sperm number and sperm movement characteristics using an iSperm® Equine portable device. Following this initial assessment, a subpopulation of progressively motile spermatozoa was isolated by virtue of the cells’ ability to migrate across a 5 µm polycarbonate filter in a Samson™ isolation chamber over a 15 minute period. These isolated cells were again evaluated for their number and quality of movement using the iSperm® system and, in addition, assessed for their ability to reduce WST-1, a membrane impermeant tetrazolium salt. These data were then combined with additional information describing the ages of the animals used in this study, their historical breeding records and mating frequency during the breeding season. The total data set was then used to predict the occurrence of pregnancy, as confirmed by ultrasound at ~14 days post mating. The criteria used to predict fertility in the ensuing multivariate discriminant analysis were optimized for each individual stallion. Using this strategy, we were able to successfully predict the outcome of a cover with an overall accuracy of 94.6%.

Stallion sperm membranes comprise of a high proportion of poly-unsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane Lipid Replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo. The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stallion spermatozoa, the subsequent addition of GPL reduced the percentage of 4-hydroxynonenal (4-HNE; a key end product of lipid peroxidation) positive cells (32.9±2.7 vs 20.9±2.3%; P≤0.05) and increased the concentration of 4-HNE within the spent media (0.026±0.003 vs 0.039±0.004 μg/mL; P≤0.001), suggesting that oxidized lipids had been replaced by exogenous GPL. Lipid replacement improved several motility parameters (total motility, 2.0±1.0 vs 68.8±2.9%; progressive motility, 0±0 vs 19.3±2.6%; straight line velocity, 9.5±2.1 vs 50.9±4.1 μm/s; curvilinear velocity, 40.8±10 vs 160.7±7.8 μm/s; average path velocity 13.4±2.9 vs 81.9±5.9 μm/s; P≤0.001), sperm viability (13.5±2.9 vs 80.2±1.6%; P≤0.001) and reduced mitochondrial ROS generation (98.2±0.6 vs 74.8±6.1%; P≤0.001). Supplementation with GPL during 17 oC in vitro sperm storage over 72 h improved sperm viability (66.4±2.6 vs 78.1±2.9%; P≤0.01) and total motility (53±5.6 vs 66.3±3.5%; P≤0.05). It is concluded that incubation of stallion spermatozoa with sub-mm-sized GPL micelles results in the incorporation of exogenous GPL into sperm membranes, diminishing lipid peroxidation and improving sperm quality in vitro.

MTT is a commonly used cell vitality probe, due to its ability to form insoluble formazan deposits at cellular locations of intense oxidoreductase activity. Although this response is considered a reflection of mitochondrial redox activity, extra-mitochondrial sites of MTT reduction have been recognized within the spermatozoa of several mammalian species. Therefore, the aim of this study was to determine the major sites and causative mechanisms of MTT reduction in stallion spermatozoa. Our results show that stallion spermatozoa displayed substantial mitochondrial formazan deposition, as well as a single extra-mitochondrial formazan deposit on various locations on the sperm head in approximately 20% of cells. The quality, and capacitation status of stallion spermatozoa was positively correlated with the presence of an extra-mitochondrial formazan granule. Additionally, extra-mitochondrial formazan deposition was suppressed by the presence of an NADPH oxidase (NOX) inhibitor (VAS2870; active against NOX2, NOX4 and NOX5), MnTMPyP (SOD mimetic), and zinc (NOX5 inhibitor) suggesting that extra-mitochondrial MTT reduction may be facilitated by NOX-mediated ROS generating activity, conceivably NOX5 or NOX2. When comparing MTT to resazurin, another well-known probe used to detect metabolically active cells, MTT reduction had a higher correlation with sperm concentration and motility parameters (R2 = 0.91), than resazurin reduction (R2 = 0.76). We conclude that MTT reduction in stallion spermatozoa follows a species-specific pattern due to a high dependence on oxidative phosphorylation and a degree of NOX activity. As such, MTT reduction is a useful diagnostic tool to assess extra-mitochondrial redox activity, and therefore, the functional qualities of stallion spermatozoa.

No abstract

This study aimed to determine whether an analysis of stallion ejaculate could accurately predict the likelihood of pregnancy resulting from artificial insemination in mares. This study involved 46 inseminations of 41 mares, using 7 standardbred stallions over a 5-week period at an Australian pacing stud. Semen quality was assessed immediately after collection and again after chilling at ~5 °C for 24 h. The assessment involved evaluating ejaculate volume, sperm concentration, and motility parameters using an iSperm® Equine portable device. After the initial evaluation, a subpopulation of cells was subjected to a migration assay through a 5 µm polycarbonate filter within a Samson™ isolation chamber over a 15 min period. The cells were assessed for their concentration, motility parameters, and ability to reduce the membrane impermeant tetrazolium salt WST-1. The data, combined with the stallion and mare’s ages, were used to predict the likelihood of pregnancy, as confirmed by rectal ultrasound sonography performed 14 days post ovulation. The criteria used to predict pregnancy were optimized for each individual stallion, resulting in an overall accuracy of 87.9% if analyzed pre-chilling and 95% if analyzed post-chilling. This study suggests that an analysis of stallion ejaculate can be used to predict the likelihood of pregnancy resulting from artificial insemination in mares with a high level of accuracy.