Elsevier

Aquaculture

Volume 451, 20 January 2016, Pages 353-356
Aquaculture

Short communication
Usefulness of a portable flow cytometer for sperm concentration and viability measurements of rainbow trout spermatozoa

https://doi.org/10.1016/j.aquaculture.2015.09.027Get rights and content

Highlights

  • Regressions between sperm concentration obtained by flow cytometer, spectrophotometer, microscope, haemocytometer were found

  • Portable flow cytometer offers additional flow cytometric analysis that can be useful for fish sperm characteristics

  • Portable flow cytometer enables flow cytometry analysis to be carried out in field conditions

Abstract

The aim of this study was to test the usefulness of a portable flow cytometer in relation to the measurement of sperm concentration and viability in rainbow trout (n = 12). This method was compared with the currently used methods, including the spectrophotometric method, computer-aided fluorescence microscopy and a haemocytometer. The mean concentration obtained using a spectrophotometer (9.86 ± 3.69 × 109 spermatozoa ml 1) and a fluorescence microscope (10.46 ± 3.60 × 109 spermatozoa ml 1) was significantly lower than that obtained with a flow cytometer (12.35 ± 3.88 × 109 spermatozoa ml 1) and a haemocytometer (11.73 ± 4.78 × 109 spermatozoa ml 1). Significant regressions (P < 0.0001) between sperm concentration obtained by using a flow cytometer, a spectrophotometer (y = 0.93 ×  1.64; r2 = 0.95), a fluorescence microscope (y = 0.90 ×  0.69; r2 = 0.94) and a haemocytometer (y = 1.16 ×  2.57; r2 = 0.87) were found. The sperm viability determinations obtained using a flow cytometer were significantly higher (97.00 ± 0.99%) than the values obtained by using fluorescence microscopy (86.22 ± 1.16%). However, a significant regression was found between these two viability measurements (r2 = 0.26, P < 0.05). To the best of our knowledge, this is the first study related to the usefulness of a portable flow cytometer in vertebrate sperm analysis. The flow cytometer provides fast measurement of sperm concentration and viability. The advantage of a compact flow cytometer is the ability to incorporate the examination of other sperm functions related to apoptosis, mitochondrial potential, oxidative stress and DNA fragmentation in future fish reproductive studies.

Statement of relevance

Study relevant to sperm concentration, viability monitoring.

Introduction

Sperm concentration is one of the major semen parameters that is evaluated as part of the standard fish semen analysis. However, accurate measurement of this parameter is quite challenging. Precise estimation of the number of sperm cells is necessary for fish reproductive studies including the determination of the optimal sperm-to-egg ratio in fertilization trials (Ciereszko et al., 2014, Nynca et al., 2014), the calibration of ultraviolet irradiation to induce gynogenesis, the optimization of staining with fluorescent dyes (Paniagua-Chávez et al., 2006) and nutritional and toxicological studies (Cuevas-Uribe and Tiersch, 2011). Different methods were developed for the measurement of fish sperm concentration; the most popular are based on counting a single sperm cell in a haemocytometer chamber and spectrophotometric measurement of absorbance caused by the turbidity of sperm suspensions (Suquet et al., 1992, Ciereszko and Dabrowski, 1993). Counting cells in a haemocytometer is reliable, but time-consuming and laborious, and thus cumbersome for experiments requiring fast measurement of sperm concentrations of numerous fish males. In the case of the spectrophotometric method, a standard species-specific curve is needed. Other techniques available to determine sperm concentration include fluorescence microscopy and flow cytometry. Nynca and Ciereszko (2009) demonstrated the usefulness of computer-aided fluorescence microscopy with the employment of a NucleoCounter SP-100 for accurate and fast measurement of the sperm concentration and viability of fish semen. Flow cytometry can be potentially used for counting cells, but until now it has been restricted to laboratory conditions (Hossain et al., 2011). However, recently a portable flow cytometer has been developed, which for the first time enables flow cytometry analysis to be carried out in field conditions.

The Muse Cell Analyser is a compact flow cytometer that uses miniaturized fluorescent detection and microcapillary technology to deliver quantitative cell analysis. The Muse Cell Analyser offers a variety of assays, such as determination of cell concentration and viability, DNA fragmentation, detection of cells in various stages of apoptosis, counting cells undergoing oxidative stress, measurement of the changes in cell mitochondrial potential and cell cycle phase measurement. So far, the Muse flow cytometer has been mostly applied in studies related to the multiple aspects of cancer cells, including viability, apoptosis (Neri et al., 2014, Marusiak et al., 2014), cell cycle and cell signalling pathways (Guerriero et al., 2014). To our knowledge, there is no information regarding the usefulness of this instrument for the semen analysis of vertebrates.

The aim of this study was to test the usefulness of a portable Muse Cell Analyser in relation to the measurement of sperm concentration and viability in rainbow trout. This method was compared with the currently used methods, including the spectrophotometric method (Ciereszko and Dabrowski, 1993), computer-aided fluorescence microscopy (Nynca and Ciereszko, 2009) and a haemocytometer.

Section snippets

Source of milt

Mature rainbow trout males (3 years of age, n = 12) were cultivated at the Rutki Salmonid Research Laboratory at the Institute of Inland Fisheries in Olsztyn, Poland. Rainbow trout males with a mean weight of 1188 ± 236 g and length of 44.4 ± 3.5 cm were kept in concrete ponds supplied with water from the Radunia river with oxygen saturation levels maintained at 85–95% and temperatures of 7–10 °C during spawning. Prior to milt collections, the fish were anaesthetized with Propiscin (1 ppm IFI, Żabieniec,

The comparison of sperm concentration measurements

Significant differences between values of sperm concentration evaluated by the applied methods were noticed (Table 1). The mean concentration obtained using the spectrophotometer and NucleoCounter SP-100 was significantly lower (by 15–20%) than that obtained by the flow cytometer and haemocytometer. The flow cytometer produced concentration outcomes similar to the outcomes obtained by the haemocytometer (Table 1). The sperm concentration calculated by the haemocytometer was characterized by the

Discussion

All four methods produced different sperm concentration results. It was not surprising that the highest standard deviation was found for the haemocytometer, since counting the fish sperm heads is difficult because of their small size. The three other methods are based on different principles: the flow cytometer and NucleoCounter SP-100 directly measure the stained cells, while the spectrophotometer estimates the concentration by measuring the suspension turbidity. It is likely that different

Acknowledgements

This work was supported by funds from the National Science Centre granted for research project nr 2011/01/D/NZ9/03738, funds appropriated to the Institute of Animal Reproduction and Food Research, Polish Academy of Sciences.

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