Assessment of ram and boar spermatozoa during cell-sorting by flow cytometry

The aim of this study was to improve the quality of ram and boar spermatozoa before and after the processes of 'sex-sorting' by flow cytometry. Three semen diluents are examined, hepes-buffered SOF (HSOF), Beltsville thawing solution (BTS) and phosphate-buffered saline in the absence or presence of Ca2+ and Mg2+ salts (PBS or dPBS), with and without homologous or heterologous seminal plasma. Motility and acrosome integrity of spermatozoa were observed by light microscopy and the viability was assessed by flow cytometry after dual-staining with Hoechst 33342 and propidium iodide. The inclusion of 10% ram seminal plasma in the diluent increased motility and viability of ram spermatozoa, except in BTS where seminal plasma had no effect on viability. Boar seminal plasma increased the viability of boar spermatozoa in HSOF and motility in BTS. Heterologous seminal plasma had a negative effect on the viability of ram and boar spermatozoa but increased the motility of boar spermatozoa diluted in BTS and ram spermatozoa diluted in BTS and PBS. The presence of Ca2+ and Mg2+ salts in PBS enhanced motility and viability of ram spermatozoa but not to the same extent as ram seminal plasma. The proportion of viable and motile spermatozoa did not always correlate. To study this further, sorted populations of 'viable' and 'dead' spermatozoa were assessed for motility and acrosome integrity. Motility, but not acrosome integrity of ram and boar spermatozoa was reduced following sorting by flow cytometry. In the 'viable' sorted population, the acrosome integrity was always high (> 72%) but motility was variable (range 10-85%). The 'dead' sorted population, however, had a low percentage of intact acrosomes (< 40%) and no motile spermatozoa. Careful selection of a pre-sort diluent containing homologous seminal plasma and detection of viable spermatozoa while sorting on a flow cytometer could improve the quality of sex-sorted spermatozoa.