High pressure flow cytometric sorting damages sperm

Sexing sperm by high-speed flow cytometry subjects them to high pressure. The routine operating pressure of the MoFlo® SX flow cytometer for sperm sorting for commercial production has been 50 pounds/square inch (psi), with a standard 70 μm standard nozzle tip. It was hypothesized that lowering the sorting pressure could reduce sperm damage. Therefore, a series of experiments using semen from six bulls, sorted with three MoFlo® SX sorters, was conducted to determine optimal pressure. An additional experiment was done with stallion spermatozoa. In Experiment 1, sorting at 30 psi compared to 50 psi with the 70 μm nozzle tip increased sperm motility post-thaw at 30 min and 2 h from 40.5 to 48.0% and 30.0 to 40.2%, respectively (P < 0.05). In Experiment 2, 49, 43, 37, 31, and 25 psi resulted in 24.2, 32.8, 35.6, 37.5, and 39.8% progressively motile spermatozoa post-thaw (P < 0.05). In Experiment 3, 3 pressures (50, 40, 30 psi) × 2 sorting methods were further evaluated. At 50, 40, and 30 psi, respective mean sperm motilities at 30 min were 44.8, 48.6, and 49.6% (P < 0.05), and percentage of live spermatozoa were 51.7, 55.7, and 57.8% (P < 0.05). The improvement of post-sort sperm quality with lowered pressure was also evident in stallion spermatozoa. After sorting at 30, 40 and 50 psi were 40.6, 34.5 and 30.1% motile spermatozoa (P < 0.1), and were 76.7, 72.5 and 67.8% (P < 0.05) live spermatozoa (determined by SYBR-14/propidium iodide staining). In Experiment 4 sorter performance was evaluated with two pressures (40 and 50 psi) × 2 staining concentrations of bovine spermatozoa (75 × 106 and 100 × 106 mL−1). Lowering pressure to 40 psi did not lower sort rate and purity when compared to 50 psi (P > 0.05), and higher sperm concentration during staining increased sort rate (P < 0.05). In conclusion, lowering pressure of the MoFlo® SX flow cytometer for sperm sorting from 50 psi (standard pressure) to 40 psi clearly improved sperm quality without a significant decrease in sorter performance.