A practical novel method for ensuring stable capacitation of spermatozoa from cryopreserved C57BL/6J sperm suspension.
A large number of genetically modified mouse strains have been produced in recent years. Sperm cryopreservation is the most effective means of preserving these valuable strains, most of which have a C57BL/6 genetic background. However, the fertilization efficiency of sperm from several cryopreserved strains, including C57BL/6, is quite low. While new and improved methods of cryopreservation have been developed, the majority of sperm stocks have already been cryopreserved using traditional methods, such as storage in 18% raffinose and 3% skim milk (R18S3). Therefore, new thawing methods for these frozen stocks are needed. We have developed a new thawing method that involves selective collection of motile sperm and a preincubation medium that enhances capacitation. Motile sperm are selected simply by collecting a sample from the center of a dish, and capacitation is induced by the addition of methyl-beta-cyclodextrin, D-penicillamine, sodium citrate, and hypotaurine to modified Tyrode's solution. The fertilization rate of sperm prepared using this method was increased significantly compared to that of sperm thawed using the traditional method (63.9 vs 16.5%, P<0.01). These results demonstrate that this new in vitro fertilization method is an effective means of reviving C57BL/6 sperm cryopreserved in R18S3.
Taguma
Kyuichi
K
Technology and Development Team for Mouse Phenotype Analysis, RIKEN BioResource Center, Koyadai, Tsukuba, Ibaraki, Japan.
Nakamura
Chika
C
Ozaki
Ai
A
Suzuki
Chigusa
C
Hachisu
Akiko
A
Kobayashi
Kimio
K
Mochida
Keiji
K
Ogura
Atsuo
A
Kaneda
Hideki
H
Wakana
Shigeharu
S
D000818:Animals; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D005260:Female; D005298:Fertility / Q000187:drug effects / Q000502:physiology; D005307:Fertilization in Vitro; D050498:Live Birth; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D012662:Semen Preservation / Q000379:methods*; D013075:Sperm Capacitation / Q000187:drug effects* / Q000502:physiology; D013081:Sperm Motility / Q000187:drug effects / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
D003451:Cryoprotective Agents