title: Cryoprotectant-free vitrification of fish (Oncorhynchus mykiss) spermatozoa: first report.
abstract: The aims of this investigation were to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryo-injuries. Spermatozoa were isolated and vitrified using three different media: Group 1: standard buffer for fish spermatozoa, Cortland(®) medium (CM, control); Group 2: CM + 1% BSA + 40% seminal plasma; and Group 3: CM + 1% BSA + 40% seminal plasma + 0.125 m sucrose. For cooling, 20-μl suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM + 1% BSA at 37 °C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility and cytoplasmic membrane integrity with SYBR-14/propidium iodide staining technique. Motility (86%, 81% and 82% for groups 1, 2 and 3, respectively) (P > 0.1) was not decreased significantly. At the same time, cytoplasmic membrane integrity of spermatozoa of Groups 1, 2 and 3 was changed significantly (30%, 87% and 76% respectively) (P < 0.05). All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility. However, cytoplasmic membrane integrity was maximal in Group 2 (CM + 1% BSA + 40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA + 40% seminal plasma.
authors.affiliation: Center of Biotechnology in Reproduction, La Frontera University, Temuco, Chile.
authors.forename: O
authors.initials: O
authors.lastname: Merino
authors.forename: R
authors.initials: R
authors.lastname: Sánchez
authors.forename: J
authors.initials: J
authors.lastname: Risopatrón
authors.forename: E
authors.initials: E
authors.lastname: Isachenko
authors.forename: I I
authors.initials: II
authors.lastname: Katkov
authors.forename: E
authors.initials: E
authors.lastname: Figueroa
authors.forename: I
authors.initials: I
authors.lastname: Valdebenito
authors.forename: P
authors.initials: P
authors.lastname: Mallmann
authors.forename: V
authors.initials: V
authors.lastname: Isachenko
mesh_terms: D000818:Animals; D003451:Cryoprotective Agents; D008297:Male; D017686:Oncorhynchus mykiss; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology*
chemical_list: D003451:Cryoprotective Agents