Computer assisted sperm analysis (CASA) systems can reduce errors occurring in manual analysis. However, commercial CASA systems are frequently not applicable at the forefront of challenging research endeavors. The development of open source software may offer important solutions for researchers working in related areas. Here, we present an example of this, with the development of three new modules for the OpenCASA software (hosted at Github). The first is the Chemotactic Sperm Accumulation Module, a powerful tool for studying sperm chemotactic behavior, analyzing the sperm accumulation in the direct vicinity of the stimuli. This module was validated by comparing fish sperm accumulation, with or without the influence of an attractant. The analysis clearly indicated cell accumulation in the treatment group, while the distribution of sperm was random in the control group. The second is the Sperm Functionality Module, based on the ability to recognize five sperm subpopulations according to their fluorescence patterns associated with the plasma membrane and acrosomal status. The last module is the Sperm Concentration Module, which expands the utilities of OpenCASA. These last two modules were validated, using bull sperm, by comparing them with visual counting by an observer. A high level of correlation was achieved in almost all the data, and a good agreement between both methods was obtained. With these newly developed modules, OpenCASA is consolidated as a powerful free and open-source tool that allows different aspects of sperm quality to be evaluated, with many potential applications for researchers.

BIOFITER Research Group Department of Biochemistry and Molecular and Cell Biology Faculty of Veterinary Sciences Institute of Environmental Sciences of Aragón University of Zaragoza Miguel Servet 177 50013 Zaragoza Spain

BIOFITER Research Group Higher Polytechnic School of Huesca Institute of Environmental Sciences of Aragón University of Zaragoza Ctra Cuarte s n 22071 Huesca Spain

Department of Computer Science and Systems Engineering Universidad de Zaragoza María de Luna 3 50018 Zaragoza Spain

Department of Engineering Mathematics University of Bristol 75 Woodland Rd Bristol BS8 1UB UK

Department of Mathematics Institute of Mathematics and Applications University of Zaragoza Pedro Cerbuna 12 50009 Zaragoza Spain

Laboratory of Reproductive Physiology Faculty of Fisheries and Protection of Waters South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Research Institute of Fish Culture and Hydrobiology University of South Bohemia in České Budějovice Zátiší 728 2 389 25 Vodňany Czech Republic

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