Validation of a comercially available fluorescence-based instrument to evaluate stallion spermatozoal concentration and comparison to photometric systems
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Accurate measurement of stallion spermatozoal concentration is important to equine breeding operations. The hemacytometer is considered the standard for measuring spermatozoal concentration but is time consuming and may be imprecise. The flow cytometer is considered precise and accurate, but only practical for research purposes due to sample preparation time and high cost. Photometric systems are commonly used but can be inaccurate outside a relatively narrow concentration range and can be rendered inaccurate in the presence of contaminants. A new instrument, the NucleoCounter SP-100 is reported to enumerate spermatozoa at wider concentration ranges and can identify spermatozoa in opaque semen extenders. Epididymal, neat (raw) ejaculates, and ejaculates diluted in various semen extenders were analyzed with the NucleoCounter, the Densimeter, the Spermacue, flow cytometric and hemacytometric methods. Results were compared statistically by: 1) regression analysis, 2) the agreement of two instruments, whereby the difference in values between two instruments was plotted on the y-axis against the mean of those values on the x-axis  and 3) a modified method that measured the percentage deviation, whereby the percentage (of the difference in values between two instruments divided by the mean) of the same two values was plotted on the y-axis against the mean value of the two instruments on the x-axis. The NucleoCounter showed more agreement with both the flow cytometer and hemacytometer for epididymal, neat ejaculated and extended spermatozoa over a range of concentrations than the Densimeter or the Spermacue. The NucleoCounter showed more agreement with the flow cytometer for epididymal and neat ejaculated spermatozoa and more agreement with the hemacytometer for spermatozoa diluted in semen extenders. The Spermacue showed the least agreement with both standards for all spermatozoal comparisons. All coefficients of variation for the flow cytometer, hemacytometer and NucleoCounter were >10% for all spermatozoal comparisons. This study indicates that the NucleoCounter shows more agreement with the flow cytometer and hemacytometer than photometric systems when evaluated with epididymal, neat ejaculated and extended spermatozoa. The instrument is also more repeatable than either photometric system, but may be cost-prohibitive for some operations.
Comerford, Kathryn L. (2009). Validation of a comercially available fluorescence-based instrument to evaluate stallion spermatozoal concentration and comparison to photometric systems. Master's thesis, Texas A&M University. Available electronically from