Comparison of two processing techniques on the sperm quality of semen contaminated with urine.

Urospermia in stallions can occur intermittently, consistently, or as an isolated event, and may result in reduced sperm quality which is often assumed to reduce fertility. Although sperm quality declines in urospermic ejaculates, fertility has not been assessed in mares bred with urine contaminated semen. The aims of this study were to compare sperm quality after simple dilution (SD), cushioned centrifugation (CC) alone, or cushioned centrifugation combined with a 40 % silane-coated silica solution (SC) in semen contaminated with 0, 20, or 40 % (v/v) urine. Sperm quality values tended to decrease as the percent urine increased within all treatments (SD, CC, SC) after 24 h of cooled storage. However, SC treated groups had higher sperm quality compared to SD and CC when exposed to 20 or 40 % (v/v) urine. Differences in pregnancy rates among treatment groups (SD with 0 or 40 % (v/v) urine, or 40 % (v/v) urine followed by SC) were unable to be detected.

A matter of agreement: The effect of the technique and evaluator on the analysis of morphologic defects in stallion sperm.

Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High: >57% normal sperm, Moderate: 23-56% normal sperm, or Low: <23% normal sperm (four stallions per category). The data were analyzed using three different statistical methods: Analysis of Variance (ANOVA), correlative analysis, and Bland-Altman method (agreement). A higher level of agreement among techniques was observed between DIC and PH for morphologically normal sperm. The agreement between the alternative methods (EN, DQ, or PH) and the standard method (DIC) varied, depending on the sperm quality level (High, Moderate, or Low). Some morphological defects (e.g., AH, AMP) were constantly underestimated with the staining methods (DQ, EN) compared to DIC and PH, particularly in ejaculates with low sperm morphology. Underestimation of some abnormalities, due to the technique or the evaluator, has the potential to alter the clinical interpretation of stallion fertility.

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.