The activities of the AQUAGAMETE COST Action will be organized under four Working groups:

WG1. Techniques for evaluation of gametes quality

Leader: Dr Julien Bobe
Vice-Leader: Dr Christian Fauvel

The WG1 main goals will be to standardize current techniques for gamete quality evaluation and to develop new protocols for field, lab and industry applications.

Objectives of WG1:
1. To organize and implement a survey of updated gamete quality assessment methods, both through cross information within the partners and through contacts with other groups of animal realm such as human and mammals.
2. To be incentive (or stimulating) for the test and use of updated methods in the research programs of the partners.
3. To try and collect original data in order to standardize and propose adapted or dedicated analytical as well as field techniques of gamete assessment in aquaculture and reproduction biology academic studies.

Expected deliverables from WG1
– Results and conclusions to be shown in the IWBFGs organized by WG5.
– Suggestion of subjects for training courses organized by WG4.
– Description of WG1 activities and conclusions will be included in progress (annual + final) reports to COST.
– Specific reports published as user-friendly instructions manuals for the industry.
– On-line delivered information (COST webs, AQUAGAMETE web and blog, Partners webs).
– Collaboration in the periodic edition of COST Action newsletters and press releases.
– Collaboration in academic activities (on-line resources, links with Aquaculture Masters).

Milestones for WG1 includes at least 1 coordination meetings/year (month 9; coinciding with IWBFGs or European Aquaculture Society Annual Meeting), at least one training course per year (month 3) and 1 reporting period/year (month 12). Other milestones include:
M1 Survey organization set up (month 6).
M2 Survey results acquired and proposition for new tools in running programs (month 12).
M3 Original results collected (month 24).
M4 Proposition of standardized methods (end of the project).

WG2. Gametes storage and preservation

Leader: Dr Elsa Cabrita
Vice-Leader: Dr Vanesa Robles

The WG2 goals will be the standardization of protocols for sperm cryopreservation, and manipulation, short-term sperm storage and cryopreservation of spermatogonia; optimization of egg storage at sub-zero temperatures; coordination of tests of new cryoprotectants, and standardization of protocols for cryobanking of endangered species, aquaculture-selected or transgenic lines.

Objectives of WG2
1. To standardize cryopreservation protocols, proceedings during freezing/thawing, equipment and identify key bottlenecks in cryopreservation of different types of reproductive cells/tissues (oocytes, embryos, gametes, germ cells, reproductive tissues).
2. To provide techniques for cell and tissue preparation for cryopreservation.
3. To simplify procedures and adapt protocols for specific situations: a) germplasm cryobanking of endangered and threatened species, b) selection of gametes from specific breeders for aquaculture production, c) broodstock reproductive management d) model species or specific transgenic lines (e.g zebrafish).
4. To articulate knowledge within workgroups (especially WG1 and WG3) to identify possible causes for cryodamage (oxidative stress, DNA fragmentation, etc.).
5. To integrate industry interests (fishfarms, cryobanks and cryogenic companies) with research knowledge and identify gaps in collaboration and needs.
6. Networking.

Expected deliverables from WG2
– Results and conclusions to be shown in the IWBFGs organized by WG4.
– Suggestion of subjects for training courses organized by WG4.
– Description of WG2 activities and conclusions will be included in progress (annual + final) reports to COST.
– Specific reports (i.e.: standardization of cell collection, isolation and freezing techniques) published as friendly user’s manuals for the industry.
– On-line delivered information (COST webs, AQUAGAMETE web and blog, Partners webs).
– Collaboration in the periodic edition of COST Action newsletters and press releases.
– Collaboration in academic activities (on-line resources, links with Aquaculture Masters).

Milestones for WG2 includes at least 1 coordination meetings/year (month 9; coinciding with IWBFGs or EAS Annual Meeting), at least one training course per year (month 6) and 1 reporting period/year (month 12). Other milestones include:
M1 Standardization of cryopreservation protocols for sperm and germ cells (month 10).
M2 Identification of bottlenecks in cryopreservation of different types of reproductive cells/tissues (oocytes, embryos, gametes, germ cells, reproductive tissues) (month 18).
M3 Establishment of techniques to collect, isolate and visualize cells for cryopreservation.
M4 Evaluation of cryodamage in different reproductive cell/tissue types as new tools in reproductive biotechnology (month 28).

WG3. Basic and applied research on gametes biochemistry and physiology, including omics

Leader: Dr Andrzej Ciereszko
Vice-Leader: Dr Catherine Labbé

The WG3 effort will be centered on providing a critical overview of the knowledge on the mechanisms responsible for the function of gametes (sperm motility mechanisms, gamete proteins, water & ion control in gametes, gametes oxidative stress and antioxidative protection, molecular mechanisms controlling oocyte competence) at physiological and pathological conditions. Special attention will be paid to integration of existing knowledge and development of emerging methods such as transcriptomics, proteomics and analysis of epigenetic modification of gametes. The acquired knowledge will be used for identification of critical factors of gamete quality.

Objectives of WG3
1. To provide critical overview of current knowledge related to following key areas: 1.1. Primordial germ cells (PGCs).
1.2 Epigenetic modification of gametes.
1.3. Genome quality in gametes.
1.4. Mechanisms of sperm motility.
1.5. Identification and characterization of gamete proteins and maternal mRNAs. 1.6. Water and ion control in gametes.
1.7. Oxidative stress to gametes and antioxidative protection.
1.8. Role of maternally-inherited factors in embryonic developmental success/potential.
2. To integrate research related to above key areas through:
2.1. Networking such as STSMs, organizing specialized sessions in conferences, technical workshops and training schools (through WG4).
2.2. Addressing identified gaps in the scientific knowledge.
3. To prepare joint proposals addressing identified gaps and to use basic knowledge to develop objectives related to applied research, such as gamete quality and biotechniques related to reproduction in aquatic animals.

Expected deliverables from WG3
– Results and conclusions to be shown in the IWBFGs organized by WG4.
– Suggestion of subjects for training courses organized by WG4.
– Description of WG3 activities and conclusions will be included in progress (annual + final) reports to COST.
– Specific reports (Scientific reviews on fundamental knowledge and its potential applied significance related to areas 1.1-1.8).
– Important gaps in the scientific knowledge will be identified and a report on this issue will be prepared.
– On-line delivered information (COST webs, AQUAGAMETE web and blog, Partners webs).
– Collaboration in the periodic edition of COST Action newsletters and press releases.
– Collaboration in academic activities (on-line resources, links with Aquaculture Masters).
– Direction of future studies will be defined and joint proposals will be prepared.

Milestones for WG3 includes 1 coordination meeting/year (month 9), at least one training course per year (month 10) and 1 reporting period/year (month 12).

-Overview of current knowledge
First, specific areas will be defined and leaders for particular topics will be named. Then, thematic sessions, covering key areas 1.1-1.8 will be planned for IWBFG as described in WG4. Each session will include a review lecture and several reports concerning specific issues. At the second meeting of IWBFG specific gaps will be named and addressed.

-Proposed training courses
Name of the Course: Micromanipulation techniques for Zebrafish Primordial Germ Cells (PGCs).
Program: Techniques for zebrafish genital ridge extraction, PGC, visualization and selection.
Techniques for sterilization of recipient animals and transplantation of PGCs from donor embryos.

Name of the Course: Identification and characterization of gamete proteins
Program: Extraction of proteins, electrophoresis and chromatography, identification and characterization of proteins, introduction to proteomics.

Name of the Course: Gene expression during gamete formation and early embryonic development in relation with developmental potential.
Program: Gene expression using quantitative PCR and in situ / in toto maternal mRNA localization during gametogenesis and embryonic development. Microarray analysis including design and data analysis. Use of next-generation sequencing (NGS).

During preparation and execution of training courses, opportunities for STSMs will be identified and offered through WG4 activities.

WG4. Organization of training courses, coordination meetings, and two next International Workshops on Biology of Fish Gametes

Leaders: Dr Sonia Martínez, Dr Oliana Carnevali

The WG4 will be dedicated to the organization of training courses, coordination meetings, IWBFGs and will help with the organization of STSMs. The Executive Group will receive the groups’ proposals for the use of funding and select year activities.

Objectives of WG4
– Student training courses. Working Groups 1-3 will define the necessities and topics of the most relevant training courses, and WG4 will coordinate with the hosting groups the organization of each of the courses.
An initial list of possible training courses including several subjects has been proposed by participant institutions: use of CASA/ASMA (sperm quality testing), sperm cryopreservation, egg and larvae microinjection, PGCs and spermatogonia handling, in vitro fertilization procedures, assessment of egg quality using embryonic survival and larval malformations, unfertilized egg holding at low temperature, analysis of egg transcriptome using microarray, gene expression in the oocyte using qPCR and in situ hybridization, protein methods (identification, proteomics), flow cytometry, FTI spectroscopy.
However, this is an open list. In fact, some of these courses could be repeated or offered in upgraded levels, and new topics can be incorporated depending on final participants (bringing new disciplinary perspectives), funding, network interest, etc.
– Coordination meetings. Focused on the standardization of techniques, creation of new tools, and coordination of research. Initially, proposed subjects are: CASA software development, gamete cryopreservation and quality evaluation (standardization), short-term storage of gametes (standardization), cryobanking (standardization, up-scaling know-how), and links with know-how in mammals and industry needs.
– STSMs. They will constitute an integral part of training within this action. STSM-s will be awarded to postgraduate or PhD students or other early stage research scientists based on proposals submitted to calls that will be announced preferably three times as year (depending on the budget). Their development will be under decision of the Executive Group.
– International Workshops on Biology of Fish Gametes (IWBFG). As it has already been mentioned, most of the applicant groups have a common background consisting in the series of IWBFGs.
The WG4 will organize the 4th and 5th IWBFG, as a forum for exchange of state-of-the-art scientific knowledge. But, more specifically its detailed objectives will include hosting AQUAGAMETE coordination meetings, exploratory sessions for EU projects application and plenary sessions for the presentation of conclusions made by AQUAGAMETE WGs, and including practical sessions presenting new techniques and standardized protocols using existing techniques.
The Action funding will allow WG4 leaders to increase the impact of coming IWBFGs, trying to increase the number of attendees (they were 82 from 17 countries during the last meeting) and presented works (79 works at the last meeting).

Expected deliverables from WG4.
– Results and conclusions to be shown in the IWBFGs organized by WG5.
– AQUAGAMETE has the preliminary intention of organizing every year 1 coordination meeting/WG1-4, 1 training courses/WG1-3 (see timetable), and a budget-dependent number of STSMs (making 3 calls annually).
– Description of WG4 activities and conclusions will be included in progress (annual + final) reports to COST.
– On-line delivered information (COST webs, AQUAGAMETE web and blog, Partners webs).
– Participation in periodic edition of COST Action newsletters and press releases.
– Academic activities (on-line resources, links with Aquaculture Masters).
– Grants for STSMs for exchange of students and researchers (seminars, tutorials, practical exercises).
Proceedings of coming IWBFGs that will be published as special numbers in the Journal of Applied Ichthyology. In this case summaries and recommendations will be especially important because they will include the main goals of AQUAGAMETE Action, both in terms of research coordination and standardization on the application of lab techniques.

Milestones for WG4.
At least initially, the time table includes every year one training course suggested per each one of the WGs 1-3 (months 3, 6 and 10 respectively).
The 4th IWBFG will take place in the Universidade do Algarve in Faro (Portugal) in September- October 2013, while the 5th IWBFG will be host by the Università Politecnica delle Marche in Ancona (Italy) in September-October of 2015. During the last edition (Budapest, September 2011) researchers from both institutions committed themselves as coming Workshops organizers. Funding from this Action will help guarantying the successful organization of these meetings.