Understanding Computer Assisted Sperm Analysis (CASA) Options: IDENT Fluorescence


Written by Jennifer Ringwelski

One of the primary applications of a computer assisted sperm analysis (CASA) system in production animal species is in the post-thaw quality assessment of frozen-thawed sperm doses.   In these instances, laboratories are particularly interested in acquiring an objective, standardized assessment for post-thaw concentration, motility as well as progressive motility to ensure doses released for sale adhere to minimum quality control standards. However, many cryopreservation medias currently used today contain a protein base such as egg yolk or milk that can distort the CASA systems ability to properly identify and differentiate sperm from detritus in the background.  

When accurate and repeatable concentration and motility are required and the clarity of the media is an obstacle, the technology of the CASA system must advance to meet the evolving needs of the customer. The IVOS II with IDENT system offers a unique solution to ensure an accurate and precise analysis.  Through the use of a specialized, DNA-specific fluorescent dye (Hoechst 33342), a strobed fluorescent light source, as well as the IVOS II integrated optics;  the system is able to identify and differentiate sperm cells from debris based on the presence of DNA as opposed to typical sperm identification traits such as size, shape and intensity from background.  

Using a simple staining procedure consisting of Hoechst 33342 and a clear buffer, the stain is incubated with sperm cells during which time the dye is readily absorbed into the highly condensed DNA of the sperm head. This dye is then excited using a strobed, fluorescent (xenon or LED) light source, and emits an image that is detected by a camera when viewed through a fluorescent filter. In contrast, most debris present in raw ejaculates or cryopreservation media does not contain enough DNA to absorb the DNA-specific dye and to emit fluorescence at the same level of excitation. 

The utilization of a strobed xenon or LED fluorescent light source limits the exposure of the sample to UV light which dramatically reduces any phototoxic effects on the spermatozoa.   This makes it possible to utilize the CASA for motility analysis in addition to absolute counts under fluorescence. 

To learn more about the benefits of the IVOS II with IDENT, visit with one of our global CASA experts or visit our website

The views expressed in IMV Technologies' blog do not necessarily represent the views of the IMV Technologies Group but solely those of the blog post's author.