Participating organizations (WP leader in bold): P1. HCMR, P3. IRTA, P4. IOLR, P13. UNIBA, P14. IFREMER, P15. ULL, P24. ITTICAL AND P25. DOR
Task 7.1 Evaluation of the effectiveness of hormone-based treatments on synchronizing gonadal development (led by IOLR). Wild-caught fingerlings reared to maturity under captive conditions will be separated into ten broodstock groups and acclimated to ambient photo-thermal conditions. Towards the onset of the reproductive cycle (mid-July in the Mediterranean Sea) females will be injected intramuscularly with either (i) GnRHa slow-release implants, (ii) the dopamine antagonist domperidone or (iii) a combination of the two. Males will be injected with either (i) recombinant FSH, (ii) 17α -methyltestosterone (MT) loaded slow-release implants, or (iii) a combination of the two. The control fish will be injected with saline only. Recombinant bioactive gonadotropins (i.e., FSH and LH) will be produced in a yeast expression system. Fish will be biopsied during the progression of gametogenesis (July-October), and their reproductive status will be evaluated using (a) wet mounts of ovarian biopsies, (b) histological evaluations of biopsies, and (c) analyses of reproductive hormones including sex steroids and LH. In addition, characterization of grey mullet sperm will be done using CASA, looking at % mobile spermatozoa, velocity, linearity of tracks, sperm membrane integrity by eosin/nigrosin, sperm viability test and sperm energetic status using ATP measurement.
Task 7.2 Development of hormone-based treatments for inducing spawning (led by IOLR). Reproductively mature fish (as determined by gonadal biopsy; Task 7.1) will be selected and treated with either (i) GnRHa alone or in combination with a dopamine antagonist, metaclopramide, and/or recombinant LH. At each spawn, eggs will be collected and evaluated for fecundity and fertilization. The average diameter of the spawned eggs, total egg volume and the volume of fertilized viable eggs (=floating eggs) will be recorded for each spawn, and correlated with larval quality (hatching, larval survival to yolk absorption).
Task 7.3 Optimization and scale-up of a breeding protocol for grey mullet in captivity (led by IOLR). The best performing hormonal therapy for accelerating gonadal development (based on results of Task 7.1) and spawning induction (based on Task 7.2) will be employed to test our ability to further extend the reproductive season (i.e., multiple spawns per individual, manipulated photo-thermal regimes), as well as improve reproductive productivity by mass-spawning. For that purpose captive-reared sexually mature broodstocks will be acclimated to either ambient or shifted photo-thermal conditions. Spawning induction trials will be carried out using broodstocks differing in their size (2 to 20 individuals) and sex ratio (i.e., equal, skewed in favour of males, skewed in favour of females). Spawned females will be subjected again to the same hormonal treatment (as above), in order to obtain further spawns. At the same time, the selected hormonal therapy will be applied to induce spawning in wild-caught mature fish vs. wild fish that will be acclimated to and matured in captivity. This step is expected to confirm the consistency and reliability of the spawning induction protocol. Furthermore, the biochemical composition of wild fish and the spawned eggs from both groups will be analyzed to provide an important reference for identifying specific nutritional requirements for improved egg quality that will be correlated with larval quality (hatching, larval survival to yolk absorption).
Task 7.4 Assessment of the effects of captivity on first sexual maturity of wild-caught and hatchery-produced fish (led by IOLR). Grey mullet fingerlings will be maintained under different stocking densities until sexual maturity. Fish (n=20 of each group) of two age categories (2 and 3 year-old) will be sacrificed at two critical periods during the reproductive season (August at early vitellogenesis and October at late vitellogenesis). Morphometric parameters including the weight of the body and internal organs (i.e., gonads, liver and fat-body) will be recorded and the respective indices (i.e., GSI, HIS and FSI) will be calculated. In addition gonad samples will be subjected to histological analyses and oocyte yolk accumulation will be monitored using image analysis software. Advanced and spontaneous sexual maturity under captive conditions can facilitate grey mullet roe production (bottarga) as a high valued product from grey mullet.
Task 7.5 Establish a shipping protocol for grey mullet eggs (led by DOR). Using surplus eggs from the spawning experiments, DOR will carry out in-house simulation. These trials will be based on protocols developed in a previous FP7 project (SELFDOTT) to transport Atlantic bluefin tuna eggs to different locations in the Mediterranean. In this protocol special attention was paid to the plastic transport container (20 l cubitainers used in the wine industry), type of disinfection agent and concentration, egg density (10-15,000 eggs/l), volume of seawater (10-15 l), size and placement of ice packs, insulating material surrounding the cubitainer and the use of pure oxygen in the cubitainer. In these trials temperature will be constantly monitored by the use of a data logger.
Task 7.1 Evaluation of the effectiveness of hormone-based treatments on synchronizing gonadal development (led by IOLR). Wild-caught fingerlings reared to maturity under captive conditions will be separated into ten broodstock groups and acclimated to ambient photo-thermal conditions. Towards the onset of the reproductive cycle (mid-July in the Mediterranean Sea) females will be injected intramuscularly with either (i) GnRHa slow-release implants, (ii) the dopamine antagonist domperidone or (iii) a combination of the two. Males will be injected with either (i) recombinant FSH, (ii) 17α -methyltestosterone (MT) loaded slow-release implants, or (iii) a combination of the two. The control fish will be injected with saline only. Recombinant bioactive gonadotropins (i.e., FSH and LH) will be produced in a yeast expression system. Fish will be biopsied during the progression of gametogenesis (July-October), and their reproductive status will be evaluated using (a) wet mounts of ovarian biopsies, (b) histological evaluations of biopsies, and (c) analyses of reproductive hormones including sex steroids and LH. In addition, characterization of grey mullet sperm will be done using CASA, looking at % mobile spermatozoa, velocity, linearity of tracks, sperm membrane integrity by eosin/nigrosin, sperm viability test and sperm energetic status using ATP measurement.
Task 7.2 Development of hormone-based treatments for inducing spawning (led by IOLR). Reproductively mature fish (as determined by gonadal biopsy; Task 7.1) will be selected and treated with either (i) GnRHa alone or in combination with a dopamine antagonist, metaclopramide, and/or recombinant LH. At each spawn, eggs will be collected and evaluated for fecundity and fertilization. The average diameter of the spawned eggs, total egg volume and the volume of fertilized viable eggs (=floating eggs) will be recorded for each spawn, and correlated with larval quality (hatching, larval survival to yolk absorption).
Task 7.3 Optimization and scale-up of a breeding protocol for grey mullet in captivity (led by IOLR). The best performing hormonal therapy for accelerating gonadal development (based on results of Task 7.1) and spawning induction (based on Task 7.2) will be employed to test our ability to further extend the reproductive season (i.e., multiple spawns per individual, manipulated photo-thermal regimes), as well as improve reproductive productivity by mass-spawning. For that purpose captive-reared sexually mature broodstocks will be acclimated to either ambient or shifted photo-thermal conditions. Spawning induction trials will be carried out using broodstocks differing in their size (2 to 20 individuals) and sex ratio (i.e., equal, skewed in favour of males, skewed in favour of females). Spawned females will be subjected again to the same hormonal treatment (as above), in order to obtain further spawns. At the same time, the selected hormonal therapy will be applied to induce spawning in wild-caught mature fish vs. wild fish that will be acclimated to and matured in captivity. This step is expected to confirm the consistency and reliability of the spawning induction protocol. Furthermore, the biochemical composition of wild fish and the spawned eggs from both groups will be analyzed to provide an important reference for identifying specific nutritional requirements for improved egg quality that will be correlated with larval quality (hatching, larval survival to yolk absorption).
Task 7.4 Assessment of the effects of captivity on first sexual maturity of wild-caught and hatchery-produced fish (led by IOLR). Grey mullet fingerlings will be maintained under different stocking densities until sexual maturity. Fish (n=20 of each group) of two age categories (2 and 3 year-old) will be sacrificed at two critical periods during the reproductive season (August at early vitellogenesis and October at late vitellogenesis). Morphometric parameters including the weight of the body and internal organs (i.e., gonads, liver and fat-body) will be recorded and the respective indices (i.e., GSI, HIS and FSI) will be calculated. In addition gonad samples will be subjected to histological analyses and oocyte yolk accumulation will be monitored using image analysis software. Advanced and spontaneous sexual maturity under captive conditions can facilitate grey mullet roe production (bottarga) as a high valued product from grey mullet.
Task 7.5 Establish a shipping protocol for grey mullet eggs (led by DOR). Using surplus eggs from the spawning experiments, DOR will carry out in-house simulation. These trials will be based on protocols developed in a previous FP7 project (SELFDOTT) to transport Atlantic bluefin tuna eggs to different locations in the Mediterranean. In this protocol special attention was paid to the plastic transport container (20 l cubitainers used in the wine industry), type of disinfection agent and concentration, egg density (10-15,000 eggs/l), volume of seawater (10-15 l), size and placement of ice packs, insulating material surrounding the cubitainer and the use of pure oxygen in the cubitainer. In these trials temperature will be constantly monitored by the use of a data logger.