title: The use of cryomicroscopy in guppy sperm freezing.
abstract: The present study employed cryomicroscopy to derive an optimal sperm freezing protocol for guppy (Poecilia reticulata) sperm. Evaluation criteria during the freezing-thawing process were assessed for nucleation temperature (Tn), temperature when more than 50% of sperm display bending mid-piece (Tb), temperature when more than 80% of sperm stop moving (Tm), thawing temperature (Tt), and post-thaw motility. We compared four different cryoprotectants: 5% N-dimethyl formamide (DMF), 6% methanol (MEOH), 10% dimethyl sulfoxide (DMSO), and 14% glycerol, as well as glycerol at different concentrations of 7-50%; cooling and rewarming rates ranged from 5 to 100°C/min. The protocol that yielded the highest post-thaw motility was samples suspended in 14% glycerol, cooled at 25°C/min, and thawed at 100°C/min, which was in complete agreement with our previous findings derived from a controlled-rate freezer. In addition, Tb and Tm were found to be negatively correlated with post-thaw motility, suggesting their possible role in predicting freezing success. The present study for the first time demonstrated the usefulness of cryomicroscopy in deriving an optimal sperm freezing protocol for aquatic species.
authors.affiliation: Institute of Watershed Science and Environmental Ecology, Wenzhou Medical College, PR China.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Wang
authors.forename: Fang
authors.initials: F
authors.lastname: Wang
authors.forename: Xiaoxing
authors.initials: X
authors.lastname: Wu
authors.forename: Xingen
authors.initials: X
authors.lastname: Zhao
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Changjiang
authors.initials: C
authors.lastname: Huang
authors.forename: Qiaoxiang
authors.initials: Q
authors.lastname: Dong
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D058639:Cold-Shock Response / Q000187:drug effects*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004126:Dimethylformamide / Q000494:pharmacology; D005399:Fishes; D005615:Freezing; D005990:Glycerol / Q000494:pharmacology; D006358:Hot Temperature; D008297:Male; D000432:Methanol / Q000494:pharmacology; D008853:Microscopy; D011036:Poecilia; D011309:Preservation, Biological / Q000662:veterinary; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects; D013696:Temperature
chemical_list: D003451:Cryoprotective Agents; D004126:Dimethylformamide; D005990:Glycerol; D000432:Methanol; D004121:Dimethyl Sulfoxide