127 ISSN 0032-9452, Journal of Ichthyology, 2019, Vol. 59, No. 1, pp. 127–130. © Pleiades Publishing, Ltd., 2019. Russian Text © Iu.S. Baiandina, A.N. Khanaychenko, 2019, published in Voprosy Ikhtiologii, 2019, Vol. 59, No. 1, pp. 117–120. Optimization of the Method for Determining the Motility Characteristics of Fish spermatozoa Using ImageJ Software and Excel Macros Iu. S. Baiandina a, * and A. N. Khanaychenko a a Kovalevsky Institute of Marine Biological Research, Russian Academy of Sciences (IMBR RAS), Sevastopol, 299011 Russia *e-mail: sepulturka@mail.ru Received November 20, 2017; revised January 24, 2018; accepted March 1, 2018 Abstract—A method for computer-based determination of the motility characteristics of fish spermatozoa using ImageJ software and a number of original Excel macros that help optimizing the processing of the data obtained using the wrMTrck_Batch plugin are described. The described method has been tested on sperm of the Black Sea turbot Scophthalmus maeoticus. Keywords: Black Sea turbot Scophthalmus maeoticus, sperm, sperm motility, ImageJ, CASA, wrMTrck_Batch DOI: 10.1134/S0032945219010016 The spawning performance of fish in natural pop- ulations, as well as their reproduction under artificial conditions in aquaculture, are largely determined by the reproductive characteristics of males. Fertilization success is the most reliable characteristic of the quality of spermatozoa (upon condition of the appropriate quality of the female gametes). The fertilization suc- cess is influenced by many factors, both during the period before the penetration of the sperm through the micropyle to the egg cell and during the process of the pronuclei fusion of spermatozoid and egg cell (Holt et al., 2004). Sperm quality is assessed by its motility characteristics in accordance with its morphological, physiological, and biochemical parameters. Positive correlation is often observed between fertilization and sperm motility. Therefore, the most informative meth- ods for determining the quality and viability of semen are the functional tests that determine motility charac- teristics of spermatozoa (Fauvel et al., 2010). These include several parameters, such as the motility speed of spermatozoa in straight and curved distances and the proportion of motile spermatozoa. Qualitative and rapid preliminary assessment of sperm quality is necessary for the successful fertiliza- tion of fish eggs under artificial conditions (Pavlov, 2006). Recently, Computer Assisted Sperm Analysis (CASA) methods have become widespread, greatly simplifying the acquisition and processing of quantita- tive data (Rurangwa et al., 2004). Many researchers use ImageJ software with CASA plugins (Sanches et al., 2013; Suquet et al., 2016) or MTrack2 plugins (Pavlov, 2006; Emelyanova et al., 2015). Both plugins imply a graphical modification of each video and manual transfer of data received in ImageJ into statis- tical programs. When the number of samples is high, such processing and analysis of data require a huge time investment from the researcher. The work aims to develop and optimize an algo- rithm for determining the characteristics of fish sper- matozoa motility using the ImageJ program on a model object, the Black Sea turbot Scophthalmus mae- oticus. In order to determine/assess the characteristics of sperm, we applied an original modification of the computer analysis method (Baiandina, 2013) using three computer programs: VirtualDubMod for image capture and image processing, ImageJ for image anal- ysis, and Microsoft Excel for analysis of the data obtained. Video filming was performed using a NikonEclipse inverted microscope with a mounted analog video camera. The curvilinear velocity and straight line velocity (μm/s) and the proportion of motile spermatozoa for each sample (%) were calculated using the lengths of the path of each spermatozoon obtained in ImageJ. In accordance with the adopted system for assessing the fish sperm motility (Burness et al., 2005; Pavlov, 2006) and our own preliminary experiments, spermatozoa with a speed of <20 μm/s were considered to be inac- tive and were not taken into account when calculating average motility speed. The individual characteristics of sperm were deter- mined in 15 males of the Black Sea turbot caught in the Black Sea in 2012 (eight ind.) and 2013 (seven ind.). Sampling was carried out by sterile disposable syringes onboard a research vessel immediately after the live SHORT COMMUNICATIONS