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 sperm atozoa before and after the processes of 'sex-sorting' by flow cytomet ry. Three semen diluents are examined, hepes-buffered SOF (HSOF), Belt sville thawing solution (BTS) and phosphate-buffered saline in the abs ence or presence of Ca2+ and Mg2+ salts (PBS or dPBS), with and withou t homologous or heterologous seminal plasma. Motility and acrosome int egrity of spermatozoa were observed by light microscopy and the viabil ity was assessed by flow cytometry after dual-staining with Hoechst 33 342 and propidium iodide. The inclusion of 10% ram seminal plasma in t he 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 motilit y in BTS. Heterologous seminal plasma had a negative effect on the via bility 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 viab ility of ram spermatozoa but not to the same extent as ram seminal pla sma. The proportion of viable and motile spermatozoa did not always co rrelate. To study this further, sorted populations of 'viable' and 'de ad' spermatozoa were assessed for motility and acrosome integrity. Mot ility, but not acrosome integrity of ram and boar spermatozoa was redu ced following sorting by flow cytometry. In the 'viable' sorted popula tion, 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 cyt ometer could improve the quality of sex-sorted spermatozoa.