A deficiency in NAD+ has previously been linked with increased occurrences of congenital abnormalities and embryonic death in humans and mice. Early embryonic death is a major factor involved in pregnancy loss in mares, and very little is known regarding the NAD+ requirements for optimum reproductive function in horses. The aim of this study was to determine the effect of supplementing the diet of mares with nicotinic acid (NA) on the composition of NAD+ metabolites in the blood and follicular fluid. Vehicle alone or NA (3 g per os) were administered to seven mares over a minimum of 3 consecutive days during the follicular phase of the oestrous cycle. Blood samples were collected immediately prior to supplemental feeding and follicular fluid aspiration. Follicular fluid was collected from the dominant follicle through transvaginal ultrasound-guided aspiration. Blood and follicular fluid samples were processed and analysed by mass spectrometry. The concentration of nicotinamide mononucleotide (NMN) in the follicular fluid of NA-fed mares was 4-fold greater than that in the corresponding plasma and 10-fold greater than that in the follicular fluid of vehicle-fed mares. The concentrations of NA, nicotinamide (NAM) and nicotinuric acid (NUR) tended to be greater in the follicular fluid of NA-supplemented mares than in the corresponding plasma. The results show that NA supplementation increased the bioavailability of NAD+ precursors in the follicular fluid of the dominant follicle, which is proposed to better promote the maturation of good quality oocytes, especially in older mares.

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.

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