Presentation: 2025 ND EPSCoR Annual conference 

October 21, 2025, NDSU Memorial Union, Fargo, North Dakota

Comparative Effects of Cryopreservation on Lepidopteran Sperm Quality: Monarch and Painted Lady Butterflies

Some reports have suggested that the western monarch butterfly (Danaus plexipus) has a >99% chance of being extinct by 2080. Biotechnological tools such as cryopreservation may serve as a safeguard against species extinction, because it allows storage of germplasm, like sperm cells from genetically diverse populations, in liquid nitrogen until they can be used in downstream applications such as artificial insemination. In this study, we assessed the effects of cryopreservation by vitrification on sperm cells from the painted lady butterfly (Vanessa cardui) and the monarch butterfly. The seminal duplex is the primary storage organ for mature sperm cells and contains two types of sperm, parasperm and eusperm. In the seminal duplex, eusperm are stored in bundles of 256 sperm cells that dissociate and become motile after being transferred to the female during mating. This study focuses on the quality of eusperm cells because parasperm do not fertilize eggs. The seminal duplex was removed from the butterfly, cryopreserved through vitrification, then stored in liquid nitrogen for at least 24 hours until thawed. After thawing, sperm cell viability was assessed using fluorescent labels (Helixyte™ and propidium iodide). We measured the width of eusperm bundles and compared those to unfrozen sperm to assess for changes over time. Despite the need for further studies to assess the functionality of thawed cells, our findings suggest that cryopreservation by vitrification may be a useful tool for preserving genetic diversity in important lepidopteran species (moths and butterflies).