Cryopreservation of the euryhaline rotifer Brachionus plicatilis embryos

Abstract

A method for the separation and cryopreservation of Brachionus plicatilis embryos is described. Juveniles with uniform development were collected from a cultured stock by passing them through a series of nets. Collected juveniles were cultured and the embryos separated by vigorous vortex mixing as soon as the majority had laid their first eggs. Separated embryos at stage I (cleavage stage), stage II (invagination stage), stage III (symmetrical embryo stage), or stage IV (“eyed” stage) were frozen to −196°C using various concentrations of DMSO and a two-step freezing procedure. No stage I embryos survived freezing and the highest post-thaw survival was obtained with stage III embryos. A DMSO concentration of 10% of the freezing medium resulted in high post-thaw survival while concentrations higher than 10% appeared to be harmful to embryos. Prolonged incubation in 10% DMSO for up to 30 min before freezing increased post-thaw survival.

Incorporating the above results, stage III embryos from a single batch culture were incubated in 10% DMSO for 30 min and frozen to −196°C. Post-thaw survival rates of 63%, 62%, 53%, and 55% were obtained after 3, 7, 15, and 30 days of storage in liquid nitrogen, respectively. Survivors fed actively on marine chlorella and started to lay eggs 2–3 days after thawing.