Viability assessment of turkey sperm using fluorescent staining and flow cytometry

Turkey sperm viability was evaluated using several fluorescent stains both singularly and in combination. Dilution curves and several extenders were used to determine optimal stain concentrations. Semen was collected from eight toms, pooled, diluted, stained, and evaluated microscopically within 2 h of collection. Replicates were assessed for both viable and nonviable sperm (green and red fluorescence, respectively) using flow cytometry. SYBR-14, which likely requires membrane potential for optimal fluorescence, or Calcein AM (CAL), which assesses the membrane integrity of cells (both green fluorescence), in combination with propidium iodide (PI) to stain the dead or degenerating cells (red fluorescence) provided optimal results. Sperm were killed by unprotected freeze-thawing to provide mixed aliquots containing known amounts of fresh:killed sperm. The percentage of viable sperm, as determined by SYBR-14 with PI or CAL with PI staining, were 76.6, 58.8, 39.3, 20.1, 8, and 73.5, 55.8, 36.0, 17.1, .4, respectively, for ratios of 100:0, 75: 25, 50:50, 25:75, and 0:100 of fresh:killed mixtures. Semen from 30 individual toms was collected on 2 d and examined in replicate using these staining combinations. The proportion of viable sperm, as indicated by uptake of SYBR-14 or CAL stain, ranged from 55.8 to 86.7 and 38.0 to 86.1, respectively. Staining combinations were effective in estimating the viability of turkey sperm and could be useful for monitoring sperm viability before and after storage.