Development of assays for the characterization of sperm motility parameters, viability, and membrane integrity in the epididymis and vas deferens of the greater rhea (Rhea americana)

The objectives of the study were to (1) describe the kinematic parameters of spermatozoa (2) compare methods of evaluating sperm viability (3) validate assays of functionality and integrity of the sperm membrane and (4) evaluate possible changes between spermatozoa from the epididymis and the vas deferens of the greater rhea. Semen samples were recovered from 7 adult individuals. Sperm motility was characterized by adjusting the set-up for Computer-assisted semen analysis (CASA) to that new species. For sperm viability evaluation, smears of bromophenol blue and eosin-nigrosine dyes were used. Five solutions of different osmolarities were then tested for the hypoosmotic swelling test (HOST). The combination of fluorescent probes (propidium iodide - IP and Hoechst 33342) was also used to assess plasma membrane integrity. Data were presented as mean +/- SEM. Rhea spermatozoa from the vas deferens had an overall motility of 14.6 +/- 2.5%. The bromophenol blue staining technique revealed that 64.6 +/- 5.2% sperm were viable, while that proportion was 72.1 +/- 2.5% using eosin-nigrosine. An average of 77.6 +/- 4.8% of spermatozoa reacted to the HOST with distilled water at 0 mOsm/l. Fluorescent probes indicated that 65.3 +/- 2.6% of spermatozoa had intact membranes. Interestingly, no statistical differences were observed between the parameters analyzed in the epididymal spermatozoa and the vas deferens. These new assays set reference values that can now be used to further exploration of sperm handling conditions and freezing protocols in rheas.