Presentation: 2025 ND EPSCoR Annual conference 

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

Comparing Slow-Freezing and Vitrification for Cryopreservation of Female Lepidopteran Ovarian Primordial Cells

Cryopreservation is a conservation method to safeguard species’ genetic material by storing cells at cryogenic temperatures for future restoration. Studies on several lepidopteran (moths and butterflies) species worldwide have shown evidence of declining populations. To better understand the most viable cryopreservation techniques for lepidopteran germplasm, we assessed the effects of two cryopreservation methods, slow freezing and vitrification, on the ovarian primordial cells isolated from ovariole tips of the adult painted lady butterfly (Vanessa cardui). The butterflies were anesthetized, and the abdominal contents were removed and placed in a petri plate with phosphate-buffered saline. The primordial cells were isolated from the tips of four ovarioles and immediately frozen (frozen-thawed samples) using either the vitrification or slow freeze methods. They were later thawed and assessed for cell viability, and the control samples were immediately assessed for cell viability (never-frozen). To assess cell viability, samples were stained using Helixcyte™ (497/521) and counterstained with propidium iodide (537/618). Under a fluorescent microscope, the nuclei of the cells with intact membranes fluoresce green, while cells with compromised membranes appear red. Our initial findings suggest that both slow-freeze and vitrification cryopreservation methods show potential for upholding cell viability in frozen-thawed samples. Optimization of both protocols is ongoing. This work represents some of the first applications of vitrification techniques to lepidopteran germplasm. Comparison of the two cryopreservation methods contributes to the field of cryobiology specifically for lepidopterans, while also establishing a foundation for future development of insect cryobanks.