Assessing staining methods for amphibian sperm viability and morphological assessment: several different methods are...

Assisted reproduction methodologies are essential to protect species from extinction. But to improve reproductive outcomes, we need to understand gamete quality. Gamete quality assays are powerful functional endpoints for comparative endocrinology, especially when they are integrated with hormone measurements and experimental manipulations. However, the methodologies for assessing amphibian gamete quality do not have published validation or comparisons within the literature. In this study we analysed sperm samples from two bell frog species, looking at morphology (measuring sperm head and sperm tail length) using both wet mounts and dry mounts of three different stains (unstained mounts, Coomassie blue and eosin nigrosin). We also assessed viability in the same sperm samples using three different preparations (eosin nigrosin dry mount, eosin nigrosin wet mount, and fluorescent SYBR-14 propidium iodide). We found that the methods varied in mean morphological measurements. For example, sperm tail lengths were measured longer when the slides were dry mounts, and when the eosin nigrosin stain was used. There were differences among species as well, where, for example, stain type influenced the sperm head length in one species, but not in the other species. Importantly, all methods had similar variance, indicating that all staining techniques are suitable given that the methods used are consistent within a study. For viability assessment, the dried eosin nigrosin staining method showed lower viability, while the wet mount of eosin nigrosin and the fluorescent SYBR-14 propidium iodide stain were statistically equivalent. These results indicate that there are a wide range of methodologies that can be used for sperm quality assessment of amphibian sperm, and researchers must choose the methodology best suited for the study.