title: Sperm cryopreservation of green swordtail Xiphophorus helleri, a fish with internal fertilization.
abstract: Sperm cryopreservation for fishes with internal fertilization is essentially unexplored although many species of these fishes are valuable biomedical research models. To explore methods for sperm cryopreservation within the live-bearing genus Xiphophorus, this study used X. helleri to evaluate the effects of cryoprotectant, osmotic pressure, cooling rate, equilibration time, and sperm-to-extender ratio. Sperm motility and survival duration after thawing showed significant differences among different cryoprotectants with the highest motility at 10 min after thawing obtained with 14% glycerol. With subsequent use of 14% glycerol as the cryoprotectant, the highest motility after thawing was observed with Hanks' balanced salt solution (HBSS) at 300 mOsmol/kg. Samples cooled from 5 to -80 degrees C at 20 degrees C/min yielded the highest post-thaw motility although no significant difference was found in the first 4h after thawing for cooling rates across the range of 20-35 degrees C/min. Evaluation of equilibration time revealed no significant difference between 20 min and 2h, but the highest motility at 10 min after thawing was found with a 20-min equilibration. Dilution ratios of sperm-to-extender at 1:20, 1:60, and 1:120 showed no significant differences in motility and survival duration after thawing, but the dilution of sperm solutions with HBSS (320 mOsmol/kg) immediately after thawing reduced the decline of sperm motility, and significantly prolonged the survival duration. Based on these findings, the highest average sperm motility (77%) at 10 min after thawing was obtained when sperm were suspended in HBSS at 300 mOsmol/kg with 14% glycerol as cryoprotectant, diluted at a ratio of sperm to HBSS-glycerol of 1:20, equilibrated for 10 min, cooled at 20 degrees C/min from 5 to -80 degrees C before being plunged in liquid nitrogen, and thawed in a 40 degrees C water bath for 7s. If diluted immediately after thawing, sperm frozen by the protocol above retained continuous motility after thawing for more than 8 days when stored at 4 degrees C.
authors.affiliation: Aquaculture Research Station, Louisiana State University Agricultural Center, Louisiana Agricultural Experiment Station, Baton Rouge, LA 70820, USA.
authors.forename: Changjiang
authors.initials: C
authors.lastname: Huang
authors.forename: Qiaoxiang
authors.initials: Q
authors.lastname: Dong
authors.forename: Ronald B
authors.initials: RB
authors.lastname: Walter
authors.forename: Terrence R
authors.initials: TR
authors.lastname: Tiersch
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000662:veterinary*; D003532:Cyprinodontiformes / Q000502:physiology; D005306:Fertilization; D005615:Freezing; D006358:Hot Temperature; D008297:Male; D008855:Microscopy, Electron, Scanning; D019188:Organ Preservation Solutions / Q000494:pharmacology; D009994:Osmolar Concentration; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013081:Sperm Motility / Q000187:drug effects; D013997:Time Factors
chemical_list: D019188:Organ Preservation Solutions