Flow cytometric evaluation of mitochondrial function and membrane integrity of marine invertebrate sperm

A method was developed to evaluate the mitochondrial function and membrane integrity of marine invertebrate sperm using flow cytometry in combination with rhodamine 123 (R123) and propidium iodide (PI) staining. The method was evaluated with sperm of sea urchins (Evechinus chloroticus), mussels (Perna canaliculus) and abalone (Haliotis iris). Using R123 and PI simultaneously, it was possible to distinguish live sperm with functioning mitochondria and intact membranes from dying or dead sperm. The mitochondrial inhibitor rotenone was used to confirm that R123 was only accumulated by sperm with functioning mitochondria. The stain combination was further validated using known ratios of fresh to killed sperm. Among individual males evaluated, the percentage of sperm identified as live varied considerably, irrespective of the species studied. Among samples from the same male, however, the percentage of sperm identified as live was similar. The method developed has application for assessing the quality of sperm used in cryopreservation and bioassays.