ADDRESSING QUALITY CONTROL FOR DEVELOPMENT OF SPERM REPOSITORIES OF PROBLEMATIC FISH SPECIES

Quality control (QC) strategies are essential for the development of sperm repositories. However, some species, such as biomedical research models, most imperiled species, and aquarium fishes, present QC challenges due to their small body sizes (< 5 cm) and miniscule sperm volumes (< 10 μL), and some present viviparity and unusual sperm morphology. Zebrafish (Danio rerio), Redtail Splitfin (Xenotoca eiseni), and Green Swordtail (Xiphophorus helleri) were chosen to model these QC challenges such as the estimation of sperm concentration, motility, membrane integrity, and fertilization in these problematic groups. In Zebrafish, sperm collected by stripping (≤ 5 μL per male) or dissection (≤ 6 mg of testis), were suspended in Hanks' balanced salt solution at an osmolality of 300 mOsmol/kg (HBSS300) with a ratio of 1:25 for sperm volume (μL) or testis weight (mg): μL HBSS. Computer-Assisted Sperm Analysis (CASA) and a Makler® counting chamber were used to assess sperm concentration and motility, with 1-5 μL of diluted sperm mixed with water at a ratio of 1:4. Sperm concentration was adjusted to 2 ×10⁸ sperm/mL, and 5 μL of sperm suspension were used to estimate membrane integrity by flow cytometry using the fluorescent dyes propidium iodide and SYBR-14. In Redtail Splitfin (a viviparous fish) sperm are packed into specialized 'bundles' (spermatozeugmata). To estimate and adjust their concentration bundles were released from anterior sections of testes with forceps on a glass slide and collected with a pipette. The concentration of suspended bundles was measured by use of the counting chamber (Fig.1), and motility was estimated by mixing with activation medium (160 mM CaCl2, pH 9.0) at a ratio of 1:4. Ten representative bundles in the center of the chamber were categorized by evaluating their motion and morphology into four phases. The frequency of each phase was used to evaluate sperm quality. In Green Swordtail the sperm volume needed for insemination of females was limited to a few µL and the cryoprotectant can be toxic to thawed sperm and the female. Washing of the cryoprotectant by centrifugation led to prolonged motility of thawed sperm for as long as 72 h. The harvest of live young demonstrated that use of 4-6 μL of concentrated sperm at a density of at least 10⁹ sperm/mL after thawing was necessary for artificial insemination. In summary, maximizing the amount of information collectable from minimal volumes of sperm is necessary to make quality control for these problematic fishes possible.