Development of a sperm cryopreservation protocol for the purple-striped jellyfish (Chrysaora colorata)

Cryopreservation of gametes is a valuable strategy for the long-term preservation of genetic material in marine invertebrates, especially for species facing ecological pressures or requiring controlled breeding strategies. The purple-striped jellyfish ( Chrysaora colorata ) holds ecological and scientific significance due to its role in coastal marine ecosystems. Despite increasing interest in cnidarian biology and aquaculture, standardized methods for the cryopreservation of jellyfish sperm remain underdeveloped. Establishing a reliable cryopreservation protocol for C. colorata sperm is crucial for supporting ex situ conservation efforts, facilitating reproductive research, and enabling assisted gene flow between fragmented populations. C. colorata sperm was opportunistically collected from live specimens and shipped in filtered seawater overnight at 4°C from the Monterey Bay Aquarium to the San Diego Zoo Wildlife Alliance. Initial motility and speed of progression (SOP, on a scale of 1-5) were evaluated upon arrival. Sperm was extended in three different cryoprotectants, 10% DMSO, 1.5 M ethylene glycol or 1.5 M propanediol, with or without the presence of 0.5 M trehalose in either filtered seawater or artificial saltwater. Sperm in 200 µl of extender was frozen in cryovials at 50 °C/min before liquid nitrogen storage. Duplicate vials were thawed at 30°C, and samples were diluted 1:1 with filtered seawater before assessing motility and SOP. Sperm frozen in 1.5 M propanediol without trehalose, regardless of water type, yielded the highest post-thaw motility score of all treatments. This sperm cryopreservation protocol serves as a foundation for future advancements in cnidarian germplasm preservation. • This is the first report demonstrating the effective cryopreservation of jellyfish sperm • Multiple cryoprotectants were tested to evaluate post-thaw jellyfish sperm recovery • Water type did not affect jellyfish sperm cryo-survival post-thaw