Standardization of sperm motility analysis by using CASA-Mot for Atlantic salmon (Salmo salar), European eel (Anguilla anguilla) and Siberian sturgeon (Acipenser baerii)

Highlights

• In captivity, the fertility potential of each male is defined by the accurate evaluation of the sperm quality.

• The assessment of sperm quality with rapid and quantitative techniques can be considered a useful tool for aquaculture.

• The kinetic parameters of fish sperm can be affected by the frequency of images used on the motility analysis.

• A standard methodology for sperm analysis enhances the reliability and comparability of data between research groups.

Abstract

It is essential to define an optimized standard method to assess the fish sperm quality to minimize the differences between the results obtained by different laboratories. Only this optimization and standardization can make them useful from academia to industry. This study presents the validation of sperm motility assessment using a CASA-Mot system for three endangered diadromous fish species: European eel (Anguilla anguilla), Atlantic salmon (Salmo salar) and Siberian sturgeon (Acipenser baerii). To attain this goal, different technical and data processing methods were tested: 1) magnification lens (×10 and ×20), 2) Spermtrack® reusable chambers (10 and 20 μm depth) and 3) different frame rates (50 ≥ FR ≤ 250). The results suggested that the sperm motility assessment for eel, salmon and sturgeon should be performed at 200, 250 and 225 frames s⁻¹, respectively. Moreover, to obtain a high number of analysed spermatozoa in less time and a natural movement of the sperm cells, it is recommended to use ×10 objective and 20 μm depth. In conclusion, different technical settings influence sperm kinetic parameters and should be validated for each fish species to allow the comparison of results between laboratories.

Introduction

The marked decline of wild stocks of some diadromous fish species such as European eel (Jacoby and Gollock, 2014), Atlantic salmon (NASCO, 2016) and Sturgeon sp. (Ruban and Bin Zhu, 2010) due to construction of dams, pollution, poaching and overfishing, together with their economic importance and high commercial demand, aroused a great interest in their production in captivity. The efficacy of aquatic fertilization in captivity depends on the accurate evaluation of the sperm quality, which nowadays is the best way to define the fertility potential of each male (Kime et al., 2001; Rurangwa et al., 2004). For the assessment of sperm quality, it is needed to have available rapid and quantitative techniques as a useful tool for aquaculture purposes (Kime et al., 2001; Gallego and Asturiano, 2018a). Sperm motility is one of the most important parameters of sperm quality and is sensitive to biological and technical conditions during analysis (Rurangwa et al., 2004; Castellini et al., 2011).

Computer-assisted sperm analysis (CASA) is an accurate, reliable and objective technology which offer several spermatozoa quantitative parameters (Rurangwa et al., 2004; Caldeira et al., 2018). A complete CASA-Mot system, which is a CASA devoted to motility analysis (Soler et al., 2016; Holt et al., 2018), includes a software associated to a phase contrast microscope equipped with a video camera. However, in the market, there are a range of products or even different versions of the same product (Holt et al., 1994; Castellini et al. 2011). Besides the different CASA-Mot systems can follow the same general principle, each one has specific algorithms which can result in the incompatibility of results (Holt et al., 1994). This common principle consists in the individual measurement of spermatozoa motility based on the detection of spermatozoa head in consecutive images in order to obtain spermatozoa tracks (Mortimer et al., 1997; Bobe and Labbé, 2010; Fauvel et al., 2010). In addition, the sperm quality assessment is also sensitive to the hardware systems, such as the optical microscope, video camera and counting chambers (Castellini et al., 2011; Soler et al., 2012; Gallego et al., 2013; Del Gallego et al., 2017; Bompart et al., 2018).

The frequency of images used on the motility analysis can be a limiting factor (Acosta and Kruger, 1996) in the reconstruction of the trajectories and, consequently, some kinetic parameters are frame rate (FR) dependent for both mammals and fish (Morris et al., 1996; Castellini et al., 2011Boryshpolets et al., 2013; Valverde et al., 2018). Therefore, it is necessary to know the optimal frame rate that provides enough detail about spermatozoa trajectory avoiding redundant information (Castellini et al., 2011). Sperm trajectory and velocity can also be affected by counting chamber depth due to the natural movement of spermatozoa (Kraemer et al., 1998; Bompart et al., 2018). This issue depends on the different motility patterns, head shape and flagellum size and could be species-specific. In this respect, a reliable and standardized method to analyse the sperm quality is needed for each species. Thereby, it is important to enhance the reliability and comparability of data provided by different research groups through the application of a standard methodology for sperm analysis (Wilson-Leedy and Ingermann, 2007; Gallego et al., 2013).

The aim of this study was to evaluate different technical settings such as frame rate, counting chamber models and lens magnification to define a standard method for the analysis of sperm motility of these three endangered fish species (Anguilla anguilla, Salmo salar, Acipenser baerii) using a CASA system.