Cryopreservation of amphioxus (Branchiostoma belcheri) spermatozoa

Amphioxus, Branchiostoma belcheri, a closest relative of vertebrates, is at a high risk of extinction due to a combination of low effective population size, altered native habitats and environmental pollution, yet little is known about cryopreservation of its gametes. This study deals with the cryopreservation of amphioxus semen. The main findings are that (1) the extender of Yao et al. is the best one among the four extenders examined; (2) the appropriate ratio of semen to extender of Yao et al. plus cryoprotectant is from 1:5 to 1:7; (3) dimethyl sulfoxide (DMSO) and methanol are the better cryoprotectants than glycerol, with DMSO giving the best results; (4) the eggs fertilized with post-thaw spermatozoa are capable of developing to at least hatching stage, and the highest hatching rate is (12.4±3.0)%. This is the first report on freezing and thawing of amphioxus spermatozoa, providing a simple and practical protocol for cryopreservation of amphioxus spermatozoa and laying a foundation for safeguarding this endangered species.