使用软件和冷却装置进行鱼类精子评估

The main goal of this procedure is to analyze fish sperm quality based on spermatozoa motility in order to contribute towards successful artificial fertilization. I'm Dr.Carles Soler, professor at Valencia University, and scientific director of PROiSER R+D. I'm Carina Caldeira, a fellowship from Impress Project.

CASA technology was developed some decades ago for the semen analysis of mammalian species by using a computer-assisted semen analysis. In the case of fish, it is needed to take into account two things. The first one, is the fact that these animals are normally fertilizing in low temperatures, around four degrees, and for this reason, it is not possible to analyze the semen in home temperature, but is needed to develop specific tools for refrigerating the samples.

The other problem, is that the motility decreases very fast after activation, and it is needed to analyze this fact, to define what is a good moment for the analysis of the quality, or of semen quality. So, we are going to show the analysis of fish sperm, and this protocol will be a modification of mammal protocol, and will include motility analysis using So, let's get started. Fish are anesthetized for hormone injection, and sperm samples are collected 24 hours later.

The genital area is cleaned to avoid contamination. Sperm is collected by applying gentle pressure. Sperm samples are kept at four degrees, until motility analyses are carried out.

The diluter and activator are prepared beforehand. Set the cooler block at four degrees to maintain samples at a constant temperature. Wait until it stabilizes.

Dilute the sperm samples using the diluter specific for each species. For European eel, sperm is diluted in the P1 medium, with a ratio of 1:50. Prepare 500 microliters of diluted sperm, adding 450 microliters of P1, and 50 microliters of fresh sperm sample.

Mix well. Take an aliquarter of activator solution, and place in the cooler block. Set the cooler stage at four degrees.

Wait until it stabilizes. Select motility module, and if necessary, use a name and password. Choose properties.

Click on species, and select fish. Select frames per second and number of images you wish to use. It is mandatory to use negative contrast.

Select corresponding counting chamber and scale. For each species, adapt particle area and connectivity. For European eel, the minimum particle area should be two, and connectivity seven.

Save the configuration. Open capture. Take four microliters of activator solution, and place in the chamber.

Homogenize well the diluted sperm. Take 5 microliters of sperm, and mix with activator. Place the cover.

Focus sample, and find the best visual area. Select capture video to obtain spermatazoa tracks, which are separated according to speed. More fields can be captured.

Select capture and proceed as before. Click on exit to obtain a general view of all fields. Sperm captures were taken at specific time intervals post-activation.

However, sperm analysis was carried out for each time interval replicate. The partial report for each time interval can be obtained by selecting fields and clicking on partial. The Excel sheet provides mean values of partial data, charts, and images of spermatozoa tracks.

Repeat the same procedure for other fields. The other option is general data. These data provide kinetic values for individual spermatozoa of all fields.

With the partial report, the percentage motility and progressivity for the different times can be compared immediately, using pie charts. General data give the option to obtain several statistical analyses using the kinetic parameters. Spermatozoa tracks give three parameters.

VCL, curvilineral velocity, VSL, progressive velocity, and VAP, path velocity. VCL is the most important parameter, and changes with the number of frames per second. By increasing the number of frames, the VCL also increases, allowing a near perfect track analysis.

The best frame rate can vary according to species. The objective is to find the highest value of VCL, after which any potential differences with other tools, such as counting chambers, may be observed. Subpopulations is currently an important statistical analysis which gives more information regarding the sperm sample.

This is an example of Atlantic salmon sperm samples. Different spermatozoa subpopulations can be observed in different generations produced in captivity. We have shown how to use a computer system for fish sperm analysis, and also how to optimize technical and methodological aspects like counting chamber, temperature, and frame rates.

So, with this data, we can do a lot of analysis in different fields, like fish breeding, not only for aquaculture, but also for conservation programs, phylogenetic studies, or to increase knowledgement about spermatozoa population. So, I hope this protocol will help you in your studies.