Characterizing the effects of trypsin on fresh and previously frozen butterfly sperm cell motility

Despite numerous decades-long conservation efforts, monarch butterfly (Danaus plexippus) populations in North America have declined, leading to both eastern and western monarch populations nearing suspected quasi-extinction risk. Cryobanking is an alternative conservation practice that uses ultra-low temperatures and specialized cryoprotective agents to preserve the structure and function of biological materials, such as germplasm, that could be frozen and later thawed to assist population revival. A common indicator of sperm cell quality is sperm motility. Trypsin-Like Serine Proteases has been identified in the accessory gland of males in several moth species and has been hypothesized to be an important activator of sperm. Subsequently animal-derived trypsin has been used to induce sperm motility in several moth species. It is unknown how trypsin affects butterfly sperm cell motility. We will isolate sperm cells from the seminal vesicle, the primary sperm storage organ, of mature adult monarch butterflies and then assess the effects of different trypsin concentrations on fresh and thawed sperm cells that were previously cryopreserved. We suspect that trypsin will have a dose-dependent effect on butterfly sperm motility as has been seen in several moth species. This study will show whether trypsin can be used to induce butterfly sperm cell motility as well as indicate the quality of thawed sperm cells through trypsin-induced motility assays.