title: Gamete cryobanks for laboratory research: developing a rapid and easy-to-perform protocol for the cryopreservation of the sea urchin Paracentrotus lividus (Lmk, 1816) spermatozoa.
abstract: Gamete cryopreservation is a biotechnology that can guarantee a continuous supply of gametes, regardless of the seasonal reproductive cycle. In this study we developed a protocol for the cryopreservation of the sea urchin Paracentrotuslividus spermatozoa, with a view to the creation of cryobanks of semen to be used as a model system in laboratory research and ecotoxicological tests. All the key phases of the procedure were separately considered and the effect on sperm motility was evaluated by means of computer assisted analysis. The best results were obtained using 7% dimethylsulfoxide in 1% NaCl plus 0.04 M trehalose as the extender, at a freezing rate of -20 °C/min. On thawing, in semen samples cryopreserved in accordance with this protocol the velocity parameters of the sub-population of rapid sperm (best performing spermatozoa) did not significantly differ from semen on collection; in addition also the fertilization ability was restored, and about 50% of normal developed plutei larvae were obtained by thawed semen. The developed protocol is rapid and easy-to-perform; moreover, the use of gametes from reared urchins makes it unnecessary to continuously collect specimens from natural populations, making this procedure a promising starting point for the creation of alternative and more sustainable methodologies in laboratory research on sea urchin gametes and embryos.
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, sect. Lesina, Lesina (FG), Italy. Electronic address: adele.fabbrocini@fg.ismar.cnr.it.
authors.forename: Adele
authors.initials: A
authors.lastname: Fabbrocini
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, sect. Lesina, Lesina (FG), Italy.
authors.forename: Raffaele
authors.initials: R
authors.lastname: D'Adamo
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, sect. Lesina, Lesina (FG), Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Pelosi
authors.affiliation: Instituto Oceanografico - USP, São Paulo, SP, Brazil; The Capes Foundation, Ministry of Education of Brazil, Brasília, DF, Brazil.
authors.forename: Luis F J
authors.initials: LF
authors.lastname: Oliveira
authors.affiliation: Centro Interdipartimentale di Ricerche per la Gestione delle Risorse Idrobiologiche e per l'Acquacoltura, Università degli Studi di Napoli Federico II, Portici (NA), Italy; The Capes Foundation, Ministry of Education of Brazil, Brasília, DF, Brazil.
authors.forename: Fausto
authors.initials: F
authors.lastname: Silvestri
authors.affiliation: Dip. Biologia, Università degli Studi di Napoli Federico II, Napoli, Italy; Centro Interdipartimentale di Ricerche per la Gestione delle Risorse Idrobiologiche e per l'Acquacoltura, Università degli Studi di Napoli Federico II, Portici (NA), Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Sansone
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005615:Freezing / Q000009:adverse effects; D008297:Male; D047328:Paracentrotus; D012661:Semen / Q000187:drug effects / Q000502:physiology; D055101:Semen Analysis; D012662:Semen Preservation / Q000379:methods*; D012965:Sodium Chloride / Q000494:pharmacology; D013081:Sperm Motility / Q000187:drug effects* / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology; D014199:Trehalose / Q000494:pharmacology
chemical_list: D003451:Cryoprotective Agents; D012965:Sodium Chloride; D014199:Trehalose; D004121:Dimethyl Sulfoxide
keywords: Closed-circuit rearing system; Computer assisted sperm motility analysis; Cryopreservation; Cryoprotectants; Freezing rate; Paracentrotus lividus; Sea urchin; Spermatozoa