J Vet Sci.  2012 Sep;13(3):315-322. 10.4142/jvs.2012.13.3.315.

Improved assessment of frozen/thawed mouse spermatozoa using fluorescence microscopy

• Diercks AK
• Burgers HF
• Schwab A
• Schenkel J

• KMID: 1389772
• DOI: http://doi.org/10.4142/jvs.2012.13.3.315

Abstract

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).

Keyword

cryopreservation; fluorescence microscopy; genetically modified (GM) mice; membrane integrity; 3Rs

MeSH Terms

Animals
Benzimidazoles/chemistry
Cryopreservation/veterinary
Embryo Transfer/veterinary
Female
Fertilization in Vitro/veterinary
Fluorescent Dyes/chemistry
Male
Mice
Mice, Transgenic
Microscopy, Fluorescence/*methods/veterinary
Propidium/chemistry
Semen Analysis/*methods/veterinary
Semen Preservation/veterinary
Spermatozoa/*physiology