title: Towards a vitrification-based cryopreservation protocol for the coral Pocillopora damicornis L.: Tolerance of tissue balls to 4.5 M cryoprotectant solutions.
abstract: In this study, we tested the tolerance of tissue balls (TBs, 100-400 μm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions. TBs were treated for 20 min at room temperature with individual, binary, ternary or quaternary CPA solutions with a total molarity from 2.0 to 5.0M. Four CPAs were used: ethylene glycol (EG), dimethylsulfoxide (Me2SO), methanol (Met) and glycerol (Gly). In some experiments, the molarity of the CPA solutions was increased and decreased in a stepwise manner. The tolerance of TBs following CPA treatment was evaluated using two parameters. The Tissue Ball Regression (expressed in μm/h) measured the diameter regression of TBs over time. The % Undamaged TBs quantified the proportion of TBs, which remained intact over time after the CPA treatment. TBs tolerated exposure to binary solutions with a total molarity of 4.0 M containing 2.0 M EG+2.0 M Met and 2.0 MEG+2.0 M Gly. TBs displayed tolerance to ternary solutions with a total molarity up to 3.0 M, containing each CPA at 1.0 M. Quaternary solutions with a total molarity of 4.0M containing each CPA at 1.0 M were not tolerated by TBs. When the molarity of the CPA solutions was increased and decreased in a stepwise manner, TBs withstood exposure to a CPA solution with a total molarity of 4.5 M, containing 1.5 M EG+1.5 M Gly+1.5 M Me(2)SO. This study confirmed the interest of using TBs to test CPA solutions, with the objective of developing a vitrification-based cryopreservation protocol.
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, UMS 2348, Centre de Ressources Biologiques Marines, Observatoire Océanologique, F-66650 Banyuls/Mer, France; IRD, UMR DIADE, 911 Avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Feuillassier
authors.affiliation: Aquarium de Canet-en-Roussillon, 2 boulevard de la Jetée, 66140 Canet-en-Roussillon, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Masanet
authors.affiliation: Sorbonne Universités, UPMC Univ Paris 06, UMS 2348, Centre de Ressources Biologiques Marines, Observatoire Océanologique, F-66650 Banyuls/Mer, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Romans
authors.affiliation: Océanopolis, Port de plaisance du Moulin Blanc, BP91039, 29210 Brest Cedex 1, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Barthélémy
authors.affiliation: IRD, UMR DIADE, 911 Avenue Agropolis, BP 64501, 34394 Montpellier Cedex 5, France. Electronic address: florent.engelmann@ird.fr.
authors.forename: Florent
authors.initials: F
authors.lastname: Engelmann
mesh_terms: D000818:Animals; D037421:Anthozoa / Q000187:drug effects*; D057894:Coral Reefs; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D000432:Methanol / Q000494:pharmacology; D058989:Vitrification / Q000187:drug effects*
chemical_list: D003451:Cryoprotective Agents; D019855:Ethylene Glycol; D005990:Glycerol; D000432:Methanol; D004121:Dimethyl Sulfoxide
keywords: Coral; Cryoprotectant solutions; Pocillopora damicornis; Tissue ball