Determination of sperm concentration for small-bodied biomedical model fishes by use of microspectrophotometry

The goal of this study was to establish an efficient method for determination of sperm concentration requiring only 1-2 microL of sample by use of microspectrophotometry. The objectives were (1) determination of wavelengths with absorbance profiles appropriate for analysis of sperm suspensions from zebrafish Danio rerio, green swordtail Xiphophorus helleri, and medaka Oryzias latipes collected by crushing of dissected testis or by stripping of live males; (2) generation of standard curves and equations between sperm sample absorbance and sperm concentration estimated by hemocytometer counts; (3) accuracy verification of equations for estimating concentration by microspectrophotometry; and (4) analysis of the precision in generating equations and estimation of sperm concentration. Within the visible wavelengths (380-750 nm) there was no single maximal absorbance peak. For zebrafish, a linear correlation was established with an effective absorbance range of 0.034-0.936 for crushed samples, and 0.028-0.961 for stripped samples at 400 nm. For Xiphophorus, the effective absorbance range was 0.014-1.154 for crushed samples, and the effective range was 0.038-1.082 for stripped samples. For medaka, the effective range was 0.041-0.896 for crushed samples. The accuracy of these equations was verified by comparison of sample concentrations counted with hemocytometer and calculated with equations, and no significant differences (p = 0.447) were observed. Measurement of serially diluted aliquots from pooled samples verified the precision of techniques used. Overall, this confirmed that microspectrophotometric estimation of sperm concentration is accurate, efficient, and sample-saving for use with small-bodied fishes.