Cryobiology of cephalopod (Illex coindetii) spermatophores

7 pages, 4 figures

Cephalopod culture is expected to increase in the near future and sperm cryopreservation would be a valuable tool to guarantee sperm availability throughout the year and to improve artificial insemination programs. We have studied the tolerance of spermatophores from the oceanic squid Illex coindetii to several cryoprotectants, in two toxicity experiments and a cryopreservation test. Five permeating cryoprotectants were tested: Dimethyl sulfoxide (Me2SO), methanol, glycerol, propylene glycol and ethylene glycol. In the first experiment, spermatophores were exposed to the five cryoprotectants at 5% (v/v) and 15% (v/v) at 4 °C for 5 min. In the second experiment, spermatophores were exposed to the cryoprotectants at 15% using different exposure times: 5, 15 and 30 min. In a third experiment, we tested two cryopreservation protocols: LN2 vapor or −80 °C freezer, using a 15% cryoprotectant and 15 or 30 min of exposure. Viability and mitochondrial activity were assessed using Mitotracker deep red, YOPRO1 and Hoechst 33342, by flow cytometry. Spermatozoa in this species remain viable after cryoprotectant exposure but their quality decreased considerably after cryopreservation, only 5–10% of spermatozoa being motile. Flow cytometry demonstrated that Me2SO may be the most appropriate cryoprotectant for I. coindetii spermatozoa, and shows a first approach on cephalopod sperm cryopreservation, opening new possibilities for the research and culture of this group of molluscs

Vanesa Robles and Felipe Martínez-Pastor were supported by the Ramón y Cajal program (RYC-2008-02339 and RYC-2008-02560), and Marta F. Riesco was supported by a Junta de Castilla y León PhD grant (European Social Fund). We thank the Spanish Ministry of Science and Innovation (MICINN) (research projects AGL2009-11546 and AGL2009-06994) and the Fundación Ramón Areces, and Cintia Miranda for her technical support. We gratefully acknowledge Mr. J.M. Fortuño (Institut de Ciències del Mar) for assistance and advice in obtaining SEM images

Peer reviewed