title: Effect of cooling mouse spermatozoa to 4 degrees C on fertilization and embryonic development.
abstract: Attempts to freeze mouse spermatozoa in liquid nitrogen (-196 degrees C) have met with limited success. In an attempt to identify the factor(s) that damage mouse spermatozoa during cryopreservation, the effect of slow cooling to 4 degrees C was examined. Epididymal spermatozoa were collected into a variety of media at 37 degrees C, cooled slowly to 4 degrees C over 4 h and warmed in a water bath at 37 degrees C for 5 min. Survival of spermatozoa was assessed by motility, membrane integrity and acrosome status. Labelling with chlortetracycline showed that > 80% of spermatozoa were capacitated and had intact acrosomes immediately after warming compared with < 20% of freshly collected (control) spermatozoa. The rate of fertilization in vitro was similar using spermatozoa cooled in Dulbecco's phosphate-buffered saline and then mixed with oocytes immediately after warming and with control spermatozoa incubated for 2 h before mixing with oocytes (85%). Fewer oocytes were fertilized with spermatozoa cooled in either a modified HEPES-buffered Tyrode's medium or a simple HEPES-buffered medium with a high osmolarity (D3), 63% and 58%, respectively. Two-cell embryos were transferred to the oviducts of pseudopregnant recipients. Implantation was similar in all groups (81-88%) and 54-74% of embryos formed normal late stage fetuses.
authors.affiliation: MRC Experimental Embryology and Teratology Unit, St George's Hospital Medical School, London, UK.
authors.forename: S J
authors.initials: SJ
authors.lastname: Fuller
authors.forename: D G
authors.initials: DG
authors.lastname: Whittingham
mesh_terms: D000177:Acrosome / Q000502:physiology; D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure; D002470:Cell Survival; D015925:Cryopreservation; D005314:Embryonic and Fetal Development; D005307:Fertilization in Vitro; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D013075:Sperm Capacitation; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure