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Published-Ahead-of-Print December 5, 2005, DOI:10.2164/jandrol.05112

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The precision and accuracy of six different methods to determine sperm concentration

Nilendran S. Prathalingam *, William W. Holt , Stuart G. Revell , Sian Jones , and Paul F. Watson

* To whom correspondence should be addressed. E-mail: nprathalingam{at}rvc.ac.uk.

The development of new technologies and software that are routinely used in laboratories have now allowed for a more diverse novel range of methods to determine sperm concentrations more rapidly. The aim of this study was to compare three such novel methods developed in our laboratory including a new flow cytometry approach, image analysis and a fluorescent plate reader with more conventional methods (haemocytometry, spectrophotometry and Microcell analysis). Fifteen ejaculates were collected from thirteen bulls at an artificial insemination centre. The semen samples were analyzed for sperm concentration using a spectrophotometer, haemocytometry and a novel flow cytometry technique based on counting a fixed volume of fluid. The raw ejaculate was also diluted 5 fold in a long-term diluent and sent overnight to another laboratory, where sperm numbers were assessed using Microcells, an image analysis system and a fluorescent plate reader. Each ejaculate was assessed 5 times using each of the methods described in order to determine the coefficient of variation for each method. Comparisons between methods were determined using correlation and limits of agreement. The flow cytometry results showed the lowest coefficient of variation (2.3%), with the plate reader showing the highest (20.0%). There was no significant difference between any of the methods used and none of them consistently over or under estimated numbers when compared against each other. It is concluded that flow cytometry showed the highest repeatability of results. However the method employed by each laboratory should be determined on a range of factors including cost, convenience, sample size and number of ejaculates to be assessed.






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