The effects of metabolic substrates glucose, pyruvate, and lactate added to a skim milk-based semen extender for cooled storage of stallion sperm.

Under in vitro conditions, stallion sperm might preferentially use energy substrates that primarily undergo mitochondrial metabolism. The present study sought to determine the effects of glucose, pyruvate, lactate, or their combinations on the quality of stallion sperm subjected to cooled storage at different temperatures, when using a skim milk-based semen extender. In Experiment 1, no substrate (Control), glucose (40 mM; Glu-40), pyruvate (2 mM, 19.8 mM; Pyr-2, Pyr-19), lactate (2 mM, 19.8 mM; Lac-2, Lac-19, respectively), or their combinations (G/P/L-2 or G/P/L-19, respectively) were added to a milk-based extender and their effects were determined on motion characteristics, viability/acrosomal intactness (VAI), lipid peroxidation status (VLPP), and DNA integrity (COMPα-t) of sperm incubated for 1 h at 37 °C, or sperm stored for 24 h at either 10 or 20 °C. At any period and temperature tested, Glu-40, G/P/L-2, and G/P-L-19 resulted in similar motion characteristics (P > 0.05) but were higher than that of other treatment groups (P < 0.05). Mean VAI was highest in Glu-40 (P < 0.05). Mean VLPP was highest in G/P/L-2 and G/P/L-19 groups (P < 0.05), and mean COMPα-t was lowest in Control, Glu-40, G/P/L-2 and G/P/L-19 groups (P < 0.05). All measures of sperm quality were higher in semen stored at 10 °C than 20 °C (P < 0.05). In Experiment 2, increasing concentrations of either pyruvate or lactate (Pyr-40, Lac-40 or Pyr-80, Lac-80) were added to the extender as energy substrates and compared to glucose (40 mM), following storage for 72 h at either 10 or 20 °C. Groups Glu-40 and Pyr-40 yielded similar sperm motion characteristics and VAI, while VLPP and COMPα-t were reduced in these treatment groups, as compared to Pyr-80, Lac-40, and Lac-80 (P < 0.05). All measures of sperm quality were higher in semen stored at 10 °C vs 20 °C (P < 0.05). This study demonstrates that at storage temperatures of 10 or 20 °C, stallion sperm quality is optimized by the presence of glucose in a skim milk-based semen extender. The addition of substrates that readily support oxidative phosphorylation (i.e., pyruvate or lactate) did not improve the quality of stallion sperm over that of glucose alone and resulted in deleterious effects on sperm quality over time. These effects appeared to be associated with oxidative stress. Use of pyruvate (40 mM) as an alternative energy substrate to glucose generally yielded similar results to that of glucose when sperm were stored at 10 °C only.

Effects of glucose concentration in semen extender and storage temperature on stallion sperm quality following long-term cooled storage.

In Experiment 1, the effects of glucose concentration in extender (0 mM, 67 mM, 147 mM, 270 mM; G0, G67, G147, and G270, respectively) and storage temperature of extended semen (5, 10, 15 and 20 °C) were evaluated after storage for up to 5 days (T0h to T120h). For all time points tested, mean total (TMOT) and progressive (PMOT) sperm motility were lower in G0 than all other treatment groups (P < 0.05). Mean curvilinear velocity (VCL) was lower in G0 than other treatment groups at all time points tested except T0h (P < 0.05). Mean percentage of plasma membrane/acrosome intact sperm (VAI) was similar among treatments at T0h, T72h, and T120h (P > 0.05). Mean TMOT and PMOT, were lower for semen stored at 20 °C than all lower storage temperatures (P < 0.05) at all time points. In Experiment 2, semen was stored at 10 °C in extender containing no added glucose (G0) or 147 mM glucose (G147). Following storage, semen was centrifuged and resuspended in extender containing no added glucose (G0 - G0 or G147 - G0, respectively) or 147 mM of glucose (G0 - G147 or G147 - G147, respectively). Mean TMOT, PMOT, and VCL were higher in G147 than G0 at all time periods tested (P < 0.05), whereas mean VAI was similar between these treatment groups throughout the experiment (P > 0.05). Mean TMOT and PMOT were higher in G0 - G147 than G0 - G0 at T72h and T120h (P < 0.05) and mean VCL was higher in G0 - G147 than G0 - G0 for all time periods. Mean TMOT, PMOT, and VCL were higher in G147 - G147 than G147 - G0 at all time points tested (P < 0.05), whereas mean VAI was similar between these two treatment groups for each of the time points (P > 0.05). In Experiment 3, the minimum concentration of glucose required to maintain sperm quality following long-term cooled storage (T120 h) was evaluated (G0, G5, G10, G20, G40, G67, G147 mM). At T120 h, mean TMOT was lowest in G0, G5, G10, and G20 (P < 0.05), whereas mean PMOT and VCL were lower in G0, G5, G10, and G20 than in G40, G67, and G147 (P < 0.05). Mean VAI was higher in G10 than G67, but similar among G10 and other treatment groups (P > 0.05). In conclusion, the absence of added glucose in extender reduced the motion characteristics of stallion sperm during long-term storage (5 days), but VAI was not affected. The use of temperatures between 5 and 15 °C for long-term storage (5 days) best maintained sperm motility and VAI. The threshold concentration of added glucose in extender required to optimize sperm motion characteristics was 40 mM.