The use of cryomicroscopy in guppy sperm freezing.
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.
Wang
Xiaohua
X
Institute of Watershed Science and Environmental Ecology, Wenzhou Medical College, PR China.
Wang
Fang
F
Wu
Xiaoxing
X
Zhao
Xingen
X
Liu
Jing
J
Huang
Changjiang
C
Dong
Qiaoxiang
Q
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
D003451:Cryoprotective Agents; D004126:Dimethylformamide; D005990:Glycerol; D000432:Methanol; D004121:Dimethyl Sulfoxide