The Precision and Accuracy of Six Different Methods to Determine Sperm Concentration

Abstract

ABSTRACT:The development of new technologies and software that are routinely used in laboratories has now allowed for a more diverse novel range of methods to determine sperm concentrations more rapidly. The aim of this study was to compare 3 such novel methods developed in our laboratory, including a new flow cytometry approach, image analysis, and a fluorescent plate reader, with more conventional methods (hemocytometry, spectrophotometry, and Microcell analysis). Fifteen ejaculates were collected from 13 bulls at an artificial insemination center. The semen samples were analyzed for sperm concentration using a spectrophotometer, hemocytometry, and a novel flow cytometry technique based on counting a fixed volume of fluid. The raw ejaculate was also diluted fivefold in a longterm 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 coefficient of variation (20.0%). There was no significant difference between any of the methods used, and none of them consistently over-or underestimated 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 based on a range of factors, including cost, convenience, sample size, and number of ejaculates to be assessed.

Automation is the key to standardized semen analysis using the automated SQA-V sperm quality analyzer

“…In this study, we showed that the precision of the SQA-V is considerably higher compared with the manual method; CVs of sperm concentration assessed by the SQA-V and manually by first and by the second operator were 1.4%, 6.0%, and 5.1% respectively (Table 3). It was also found that the CV of the SQA-V in assessing sperm concentration (1.4%) was lower than the CVs of six other methods reported in the literature (27). Prathalingam et al (27) compared 3 novel methods for assessing sperm concentration (flow cytometry, image analysis, and a fluorescent plate reader) with conventional methods (hemacytometer, spectrophotometer, and Microcell counting chamber, Conception Technologies, San Diego, CA).…”

Cytometric solutions in veterinary andrology: Developments, advantages, and limitations

“…Later, spectrophotometric methods were introduced, potentially more accurate but suffering from the fact that nonsperm debris in the sample could interfere and bias results. Subsequently, cytometric methods were developed, which give better precision and accuracy [for comparison of methods see (53)]. …”

Determination of Sperm Concentration for Small-Bodied Biomedical Model Fishes by Use of Microspectrophotometry

“…6 In addition, due to the small sample volume (1-2 mL) required for the microspectrophotometer, this method is beneficial for aquarium fish in particular because of the limited volumes of sperm available. Further, this technique of establishing a correlation curve and equation for the estimated sperm concentration has been accepted as a quick and accurate method 13 and would greatly increase the efficiency of cryopreservation and fertilization procedures.…”

“…In addition, variation among counting chambers and hemocytometers made by different manufacturers and the people who use them can create problems. 6 Therefore, development of a fast and accurate method for use with small sample volumes is necessary for sperm concentration determination of model fishes and can be achieved by utilizing microspectrophotometers that have microliter sample size capabilities. The principle of spectrophotometry necessitates calibration of a given instrument for its use in determining the concentration of a given sample.…”