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

Abstract

The goal of this study was to establish an efficient method for determination of sperm concentration requiring only 1-2 mL 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 ¼ 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.

Determination of sperm concentration using flow cytometry with simultaneous analysis of sperm plasma membrane integrity in zebrafish Danio rerio

“…For microspectrophotometry, the protocol established by a previous study (13) was used following the standard equation of: Y = (3 × 10 8 ) X − 3 × 10 7 with “X” being defined as the absorbance measured at 400 nm. Briefly, a 2 µl sample of sperm suspension from each male was loaded onto the lower pedestal of the NanoDrop, and absorbance was measured at 400 nm.…”

“…Meanwhile, spectrophotometry has been used for determination of cell concentration by identifying the linear relationship between particle concentration and light absorbance (9–12), which provides a more rapid estimation of concentration than actual counting methods. In addition, considering the sperm volume required, microspectrophotometry with 1–2 µl sample sizes has been developed specifically for concentration determination of sperm from small-sized biomedical research model fishes (13). …”

Cryobanking of aquatic species

“…However, small sample sizes from aquarium fishes limit standardization because determination methods such as a hemocytometer require a relatively large amount (~10 μl). Micro-spectrophotometric methods to determine sperm concentration have been developed (Tan et al, 2010), which require less time (seconds per sample), and minimal sample volume (1–2 μl of diluted sperm). Equations relating concentration and absorbance have been established for samples collected by stripping, and by crushing of dissected testis of zebrafish, medaka, and Xiphophorus , and the accuracy of these relationships were verified (Tan et al, 2010).…”

A Procedure-Spanning Analysis of Plasma Membrane Integrity for Assessment of Cell Viability in Sperm Cryopreservation of ZebrafishDanio rerio

“…The absorbance of a 2-lL aliquot of sperm suspension was measured at a wavelength of 400 nm in triplicate, and sperm concentration was calculated from the average absorbance using an equation previously determined in our laboratory. 26 Cell concentration was adjusted to 2 · 10 8 cells/mL by dilution with extenders and samples were filtered through 30-lm nylon mesh to remove any remaining tissues.…”