VIABILITY AND MEMBRANE INTEGRITY OF SPERMATOZOA AFTER DILUTION AND FLOW CYTOMETRIC SORTING IN THE PRESENCE OR ABSENCE OF SEMINAL PLASMA

Boar, bull and ram spermatozoa were examined after staining with the D NA-permeant Hoechst 33342 fluorochrome and flow cytometric sorting in the presence or absence of seminal plasma. Spermatozoa were assessed f or viability with flow cytometry using the live cell nucleic acid stai n SYBR-14 and propidium iodide (PI), and for membrane integrity Using fluorescein isothiocyanate-conjugated Pisum sativum (FITC-PSA) and PI; motility and acrosome integrity were estimated by microscopy. Flow cy tometric sorting was compared with pipette dilution of boar and bull s permatozoa into: (1) medium [boar: Test buffer containing 2% yolk (TY) or Beltsville thawing solution (BTS); bull: TY or HEPES buffer contai ning 0.1% bovine serum albumin (HEPES-BSA)] with or without 10% (v/v) seminal plasma; or (2) an empty tube containing no medium. Sorted sper matozoa were either not centrifuged or centrifuged before assessment d uring a 4-h holding period. The viability, motility and membrane integ rity of boar, bull and ram spermatozoa centrifuged after sorting were also examined when seminal plasma was present or absent from the stain ing extender and/or the TY collection medium. The results indicate tha t the viability and membrane integrity of spermatozoa in vitro would b e improved if: (1) seminal plasma (10%) was routinely included in the BTS and HEPES-BSA staining extenders for boar spermatozoa and ram sper matozoa, respectively, when used in preparation for flow cytometric so rting; and (2) 10% and 50% seminal plasma were included in the TY coll ection medium for boar or bull spermatozoa and ram spermatozoa respect ively.