Development and implementation of new technologies and protocols in the study of fish reproductive ecology
The project aims to use new technologies to solve one of the bottlenecks that have raised the analysis of the dynamics of fish populations: to have accurate estimates of population, reproductive potential. For this calculation are required, among other variables, the proportion of mature individuals in the population and fecundity at each age class, two parameters that are the main subject of this project. It aims to develop methods and tools that facilitate and automate the evaluation of reproductive parameters for ultimately, determine the population reproductive potential.
To achieve this overall objective we will address the problem from very different methodological approaches:
i. Resolving key issues affecting reproductive ecology methodologies applied: the duration of regression of postovulatory follicles and the onset of sapwning.
ii. Designing an automatic system based on computer vision that operates on digital images preparations ovaries. The system must operate quickly and accurately enough to be used later in the routine analysis.
iii. Applying new histological and microscopic tools that allow rapid diagnosis of the state of maturation and activity of the individual.
- Duration of POF
Knowledge of the rate of degeneration of this structure and, therefore, the age determination, it is essential to estimate the spawning fraction. This is an essential parameter for calculating fecundity in indeterminate fecundity species such as hake , sardine, or mackerel . This estimate is made by experiments with captive species.
- Early detection of maturation
The presence of cortical alveoli oocytes is generally asume as the best signal for determining fish maturity. However, very preliminary investigations suggest that there are earlier stages that allow early identification of maturation, allowing the estimation of spawning biomass a year in advance.
- Implementing staining techniques
Application of different staining techniques that allow rapid diagnosis of the presence of atretic oocytes and POF, both in samples and in whole tissue sections. The project also try to implement histology and fluorescence techniques to facilitate the automatic detection of oocytes.
- Application of autodiametric method
The autodiametrico method is fast and effective to estimate fertility, but only useful for determinate fecundity speceis with group synchronous ovarian development. Most of the species are asynchronous Galician so this method is not applicable at all. We intend to study variations of this method for species of the Galician shelf (hake, whiting, megrim).
- Improved estimation fecundity protocols
Fecundity estimations require the use of highly toxic chemicals or carcinogenic ( formaldehyde , mercury, … ) at present. The project aims to develop alternative methods that facilitate the counting of eggs without the use of these products.
- Automation of stereological techniques
To develop an automatic method based on computer vision for determining the number of oocytes in an ovary from histological sections using stereological techniques.
- Oocytes count automation
To develop an automatic method based on computer vision to determine fecundity based og oocyte count of digital images of ovarian samples (whole mount). The fundamental problem is to estimate the number and size of the eggs when they form agglomerates.
- Automation Dissector
The dissector method is the most adequate for estimating atresia (which reduces fertility) and fecundity, but requires the use of serial histological sections and identifying each oocyte along the series of slides. Using image analysis to recognize automatically each atretic oocyte in the different images and thus automate counting can be a good alternative.
- Implementing a software tool
Finally, we develop a software tool that automates the laboratory work (reduction in time analysis and independent of the level of expertise of the worker) and improve quality in calculating reproductive parameters of the studied species. The aim is to increase the capacity for the systematic study of a larger number of stocks and species.
The most striking finding of this project is embodied in “GOVOCITOS: Free software for determining reproductive parameters in fish“