Name: fish sperm assessment using software and cooling devices
Uploaded: 2018-07-28T12:30:00
Description: Watch this Scientific Journal Video about Fish Sperm Assessment Using Software and Cooling Devices at JoVE.com

This project has received funding from the COST Association (Food and Agriculture COST Action FA1205: AQUAGAMETE, and the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie project IMPRESS (GA No 642893). We would like to thank the scientific team of PROiSER, specifically to the student Alberto Vendrell Bernab&#233;u, for his active participation in the video recording of this project.

Procedures involving animal subjects have been approved (2015/VSC/PEA/00064) by the General Direction of Agricultural Production and Livestock at the Universitat Polit&#232;cnica de Val&#232;ncia.
1. Collect Sperm from Mature European Eels in Captivity
NOTE: Use European eel males maintained in tanks with seawater and a recirculation system at constant temperature (20 &#176;C). Treat with hormones through weekly intraperitoneal injection (human chorionic gonadotropin ...

The sperm analysis software used in this protocol has been used by researchers worldwide for different species, including fish. However, fish have some specific features that can affect the sperm assessment. Fish spermatozoa showed high speed in the moment of activation which declines quickly and leads to a short time of motility after activation. Besides, the temperature of reproduction is species-dependent and, in some cases, could be around 4 &#176;C2,4,<...

The efficacy of reproduction depends on the quality of both gametes (eggs and sperm)1,2. This is the major factor that contributes to successful fertilization, allowing the development of viable offspring3,4. The convenient evaluation of gamete quality is the best tool for defining the fertility potential of a specimen. 
Mixing sperm from multiple males is a common practice in the production of many aquatic commercial species4. However, the sperm variability between males can lead to sperm competition and, consequently, not all the males are equally contributing to the gene pool5. In this sense, the correct evaluation of individual ejaculate/spermatozoa features, such as motility, is fundamental to obtain discriminatory information regarding individual male fertility potential. Direct observation of sperm motility can produce inaccurate and subjective data as it requires time and experience, which leads to a lack of consistency and incompatibility of results6,7. However, there are many innovative, rapid and quantitative techniques that can provide a reliable sperm quality analysis2,4. 
Computer-assisted sperm analysis was developed to offer accurate data about sperm quality8. This technology includes the development of software associated with a phase contrast microscope that allows the assessment of sperm motility. However, a limiting factor of motility parameter is the frame rate of the video camera. Individual spermatozoa trajectories are based on spermatozoa head centroid position in consecutive frames of video recordings, which is correlated with the flagellar movement patterns3,9,10,11. The main kinetic parameters measured are the straight-line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP). VSL is the distance between the start and end-point taken by the spermatozoa divided by time. VCL is the real velocity along the precise trajectory taken by the spermatozoa. VAP is the velocity along a derived smoothed path of trajectory. These parameters allow additional kinetic information, including linearity (LIN), straightness (STR), wobble (WOB) and beating measurements like amplitude of lateral head movement (ALH) and beat-cross frequency (BCF)4,10.
The sperm analysis system was originally used for mammalian species, and one of the requirements for the system is to operate at the body temperature of the donor (about 37 &#176;C). This software could also be used for fish species; although, it is necessary to make some adaptations to reduce the error of sperm analysis results. In some fish species, such as salmonids and eel8,12, fertilization occurs at low temperature (around 4 &#176;C)2,4. Thus, cooling devices should be developed to avoid uncomfortable working conditions. In addition, fish spermatozoa are immotile in seminal fluid and require an osmotic shock to activate motility. For freshwater species, the activator medium should have hypotonic osmolality, while for marine species the medium should be hypertonic. However, for some species, as salmonids, the ion concentration could also be important3,4,9. After activation, fish sperm is characterized by a rapid decrease of motility (less than 2 min)13,14 and high velocity, being vital to determine the optimal frame rate to obtain reliable data15.
The objectives of this study are to design and apply refrigeration systems for fish sperm samples. In addition, this protocol defines how to determine the optimal frame rates for the establishment of standard protocols depending on the species. The use of this protocol opens new doors in the context of fish seminal evaluation, using the European eel as a model.

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fish sperm assessment using software and cooling devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

Analysis of the time effect on sperm motility
In the case of the European eel, the percentage of static spermatozoa increased from 15 s to 120 s after activation (from 24.4% to 40.7%), and the percentage of mobile progressive spermatozoa decreased (from 36.9% to 20.9%) (Figure&#160;1A and 1B). Based on speed, spermatozoa cells showed a decrease in velocity over time...

Watch this Scientific Journal Video about Fish Sperm Assessment Using Software and Cooling Devices at JoVE.com

Fish Sperm Assessment Using Software and Cooling Devices

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

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