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Updated: Oct 10, 2025
Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit
Published on: December 12, 2011
A triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in
Daniel de Souza Ramos Angrimani1, Luana de Cássia Bicudo1, Nuria Llamas Luceno2
1Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Orlando Marques de Paiva, 87 -05508-270, São Paulo, Brazil.
Abstract:
In order to accurately analyze the possible side effects of sperm cryopreservation, an in-depth screening of post-thaw sperm status is necessary. Thus, this study aimed to identify thorough effects of sperm cryopreservation, by evaluating the integrity of all specific structures of the canine spermatozoa. Thirteen (n = 13) mature dogs of different breeds were selected. Six dogs (n = 6) were subjected to sperm cryopreservation, whereas seven dogs (n = 7) were used as semen donors to validate a simultaneous assessment of sperm plasmatic, acrosomal, and mitochondrial membranes (triple stain) by fluorescent probes. Fresh and post-thaw semen samples were evaluated through a computer-assisted analysis of sperm motility, sperm morpho-functional evaluation, triple stain and sperm DNA integrity. Post-thaw semen samples had lower total and progressive motility, as well as higher percentage of minor and major defects. Moreover, post-thaw samples had higher percentage of sperm with plasma membrane and mitochondrial damage but intact acrosome, and also sperm with simultaneous damaged plasma, acrosomal and mitochondrial membranes. Furthermore, post-thaw sperm had higher protamination deficiency and DNA fragmentation. In conclusion, cryopreservation has a broad impact in sperm morphology and function, altering motility patterns, plasma, acrosome and mitochondrial membranes integrity, as well as sperm DNA.
