Community Research and Development Information Service - CORDIS

FP7

PROSPAWN Report Summary

Project ID: 232305
Funded under: FP7-SME
Country: Norway

Final Report Summary - PROSPAWN (IMPLEMENTATION OF NATURAL SPAWNING FOR MARINE FISH SPECIES IN CULTURE - IMPROVING THE QUALITY OF OFF-SPRING AND ANIMAL WELFARE)

Executive Summary:
The maintenance of normal reproductive processes in broodstock is crucial to industrialized aquaculture, ensuring a dependable supply of juveniles. Moreover, the ability to significantly manipulate the seasonal timing of spawning and age at first sexual maturation (puberty) through environmental or hormonal control is already a valuable tool in the farming of marine species. Further expansion of this capability would increase the efficiency and total output of fish farms while creating the possibility for development of new production techniques. The aim of PROSPAWN was to investigate new concepts for commercial marine fish broodstock management by specifically addressing major obstacles and bottlenecks facing existing juvenile production; the lack of knowledge surrounding correct environmental and hormonal cues necessary for optimal broodstock performance, the need for protocols for the selection of high value breeders, the lack of quick and accurate determination of egg quality, and the relatively high operation costs associated with current practices. The specific overall objectives of PROSPAWN were;
1. To obtain high-quality eggs from naturally spawning flatfish through the use of breeding nests.
2. To obtain high-quality eggs from naturally spawning roundfish in a shallow raceway system.
3. To devise protocols for optimal environmental conditions for the induction of natural spawning in broodstock of selected species.
4. To devise protocols to minimise environmental disturbances which may disrupt the induction of natural spawning in broodstock of selected species.
5. To quantify the benefits obtained, as reflected by egg quality, by utilizing new broodstock management protocols.
6. To develop and test an egg quality detection package (hardware and software) - prototype (EQuaP)
7. To outline future breeding plans regarding selection of broodstock on the basis of behaviour.

With the completion of the experimental work, the final results and impacts of PROSPAWN may be examined. While several avenues of the research undertaken during the project have been fruitful, other anticipated results have not been achieved, in short;
1. At the current time, the use of spawning beds and shallow raceways in the management of flatfish and roundfish broodstock appears unfeasible. Neither of the two technologies has resulted in naturally spawning fish during several years of trials.
2. A software package and the use of automation for the quick determination of egg quality proved to be a very promising technique and merits further development.
3. Valuable information regarding environmental conditions were obtained. This includes a new understanding of the effect of auditory disturbances on broodstock performance as well as unexpected results on the effect of temperature on natural spawning in Dover sole.
4. Work with hormonal manipulation on the two sole species has yielded valuable information to aid in the identification of the source of maturational inhibition in these species.
5. The use of marker analysis and parental assignment has produced valuable information on the importance of mate choice in the breeding of the two sole species. This information is an important element of the breeding plans presented in the full report.

Project Context and Objectives:
The maintenance of normal reproductive processes in broodstock is crucial to industrialized aquaculture, ensuring a dependable supply of juveniles. Moreover, the ability to significantly manipulate the seasonal timing of spawning and age at first sexual maturation (puberty) through environmental or hormonal control is already a valuable tool in the farming of marine species. Further expansion of this capability would increase the efficiency and total output of fish farms while creating the possibility for development of new production techniques. The challenges might be addressed in several ways. In the current proposal, the focus is placed on improving the broodstock environment in order to induce natural spawning. Although natural spawning is currently the normal procedure for both sole species in culture, industry-wide fertilization rates remain below 50% and the potential fecundity of the females is still far from being fully exploited. Flatfish species are generally characterised as exhibiting high fecundity. If rates of fertilisation and embryo/larvae survival are significantly improved, an intended achievement of the current proposal, one significant bottleneck for flatfish aquaculture could be removed and the overall production cost of flatfish could be reduced.

In general, marine fish species have a very short period (a “quality window” of a few hours) when ovulated eggs are of outstanding quality and when fertilization results in high quality embryos and juveniles. One possible method of capitalizing on this fact is to ensure that the environmental conditions and broodstock tank construction is conducive to the occurrence of natural spawning, ensuring the collection of embryos of the highest possible quality. Under the correct environmental conditions, the most important of these being temperature and photoperiod regimes, combined with the proper physical surroundings, courtship and mating may take place in populations of captive broodstock. In addition to increasing the quality of the collected embryos, animal welfare will be significantly improved by replacing stripping with natural spawning. Furthermore, it should be noted that some fish species considered to be good candidates for aquaculture are so susceptible to handling that the only feasible method of obtaining embryos will be through natural spawning.

The PROSPAWN project was based on the idea that the main obstacle preventing large-scale juvenile production in these species, and other commercially important species in aquaculture, is caused by inadequate holding facilities and environmental conditions. For that reason the most important elements of the proposed project was to 1) develop and test two new types of features for the holding systems of broodstock populations (breeding nest and shallow raceway systems), 2) determine species-specific optimal environmental conditions (photo-thermal regimes) mimicking natural habitats and investigate the potential distressing effects of physical perturbations (vibrations, noise) created by surrounding equipments and staff 3) develop an automated egg quality assessment tool for marine fish based on blastomere scoring 4) Provide recommendations and strategies in the planning of breeding schemes to optimize broodstock performance.

Two new holding facilities were tested during the project. The first feature was designed for flatfish species and is called a “Breeding Nest”. It is well known that flatfish congregate in groups in one part of the tank, thus making courtship difficult and chaotic. The nest was designed to allow females to maintain identified isolated territories and be approached by males in a more natural manner, akin to natural courtship taking place at spawning grounds in the wild. This concept has been proven in halibut where the production of high quality fertilised eggs has been achieved through natural spontaneous spawning after providing the broodstock with appropriate environmental cues in conjunction with socially acceptable rearing conditions. In that study, each of the 12 females (size range 30-80 kg) was provided with a nest and 25 m2 of bottom surface in a 3 m deep tank. Each female maintained its own nest for the duration of the spawning season. The proposed project intended to confirm these preliminary findings in large scale Atlantic halibut breeding tanks. This technology was also tested for turbot. The second feature was designed for demersal round-fish species and is called the “Shallow Raceway System” (SRS) (Øiestad, 1999). This is also a behaviour-adjusting solution for the holding of free-swimming fish species. In contrast to round tanks, the fish held in the SRS are exposed to a rigid one-directional homogeneous current pattern which facilitates schooling or shoaling behaviour. The chaotic swimming pattern observed in round tanks is less likely and the system will discipline the fish and encourage courtship as males can move easily up to females and examine those ready to mate. Some of the fish species in question will spend part of their time resting at the bottom and even those will organise themselves along the axis of the current direction. Many fish species that have great potential as aquaculture species are extremely sensitive to handling and therefore unsuited for stripping or even injection of maturation inducing hormones. Egg collection in SRS would be rapid and thorough, even in raceways of 10-15 m length. Moreover, the placement of the water outlet in the SRS is such that unfertilized eggs which have settled to the bottom may be retrieved in addition to the unfertilised pelagic eggs. This would allow for much more precise estimates of fecundity and fertilization rates, and in turn provides breeders with a more accurate overview of the status and performance of the broodstock. The size and depth of the raceways are easily adjustable and may be designed according the species and fish size.

The project also had a goal to investigate and determine species-specific environmental conditions for broodstock spawning. Photoperiod and temperature regimes are known to be key parameters for the entrainment and regulation of broodstock reproduction as well as mating behaviour, timing of spawning and egg quality. However, although temperature regimes mimicking ambient profiles experienced by fish in their natural environment have been developed, little effort has been made in recreating natural lighting conditions or determining optimal photic conditions for broodstock from a range of species. This might be one key to successful spawning in many species. Light is more complex than it looks as it is characterized by its quantity (intensity), quality (spectral content) and duration (photoperiod). In natural conditions, species are exposed to very diverse photic conditions depending on their spawning grounds. As an example, Atlantic halibut spawns in deep sea waters (>100m) in which only very dim blue light can be perceived as a result of water absorbance whereas sole species spawns in shallow waters where high intensities and broader wavelengths can be perceived. Furthermore, in other species, fish undergo migration during reproduction from deep to shallow waters. However, these natural conditions are not well recreated in culture and have not been studied appropriately to date. Light levels are usually set in hatcheries for convenience of the staff working instead of the well-being and physiology of the fish. This probably also explain the high variability in results (timing of spawning, fecundity, egg quality) reported within the industry as no standardised regimes are available. This project set out to recreate optimal (natural) lighting regimes for each species under investigation. To do so, new lighting technologies (LED and cathode lighting) were used to adjust the intensity, the spectrum and daily variations (dawn and dusk) for a range of species. Both the rearing systems and environmental conditions contribute to create “alien” environments which might explain the lack of normal breeding behaviour (mating, pheromones) and natural spawning resulting in poor egg quality. Other perturbations such as physical/auditory disturbances (vibrations, noises resulting from rearing environments e.g. pumps, compressors etc) might also act as stressors. Through the recording of “sounds” in the breeding tank, both external disturbances and broodstock communication (known to exist in a number of species including gadoids such as Atlantic cod) were monitored during several trials.

Finally, the project aimed to develop an egg quality tool for marine fish species that can be used by farmers. Egg quality is one of the main bottlenecks in marine finfish aquaculture with very low survival (5 to 15% at best at the weaning stage in species such as halibut or cod). One of the best indicators to assess egg quality in marine fish species is the scoring of blastomere morphology. However, this assessment is labour intensive when performed manually. This project aimed at developing an automated blastomere scoring tool through the use of modern image analysis software. Such a tool would allow the marine finfish industry for a relatively small investment to significantly improve accuracy, reproducibility and importantly speed of egg quality assessment ensuring that production investment is not wasted on sub-optimal egg batches.

In short, the overall objective of the project was to strengthen the aquaculture industry and European research area for marine finfish culture by;
1. Improving the foundation of sustainable broodstock management of emerging and developing new commercially important species within the EU (WP1),
2. Validating new rearing systems and protocols based on species specific behavioural adaptations (WP2),
3. Developing a real time diagnostic tool for egg quality assessment in marine finfish (WP2),
4. Improving broodstock performance by recreating species specific natural rearing environments in culture (WP3),
5. Identifying selection criteria for breeders based on parentage assignment and reproductive performance (WP4),
6. Specifically quantifying the benefits that may be obtained, with a special emphasis on fecundity and egg quality (i.e., embryo survival), from utilizing new broodstock management protocols
7. Overall, the results from the project will deliver new knowledge and technologies to improve the sustainability and welfare standards of broodstock in culture from a range of new and emerging marine fish species across Europe. The improved broodstock management and subsequent seed quality will be achieved through the refinement of the physical, environmental and social rearing conditions. Ultimately, this will allow fish farmers across Europe to expand and diversify their production.

The main project results as specified in Annex I to the Grant Agreement are as follows:
*Obtaining high-quality eggs from naturally spawning flatfish by use of breeding nest.
*Obtaining high-quality eggs from naturally spawning roundfish in shallow raceway system.
*Protocol for optimal environmental conditions for the induction of natural spawning in broodstock of selected flatfish species.
*Protocol to minimise disturbances for the induction of natural spawning in broodstock of selected flatfish species.
*Protocol for optimal environmental conditions for the induction of natural spawning in broodstock of selected demersal species.
*Protocol to minimise disturbances for the induction of natural spawning in broodstock of selected demersal species.
*Quantification of benefits obtained, from improved egg quality by utilizing new broodstock management protocols.
*Egg quality detection package (hardware and software) - prototype (EQuaP)
*Outline future breeding plans regarding selection of broodstock on the basis of behaviour.
*A list of published micro-satellites for the selected species containing primers, allele sizes and numbers of alleles.

Project Results:
WP 1
Period 1; At the start of the project a broodstock workshop was organised for the consortium members to review the current status of knowledge concerning broodfish and hatchery production strategies – both in a commercial and scientific setting – for the selected group of marine species. The main goal of the workshop was to identify gaps in the current knowledge and to identify more exactly the priorities the project should aim to fulfil, especially taking into account the changes in the consortium. The workshop was held in Wageningen, The Netherlands, on March 18, 2010, and was both well attended and productive. After the first session of the working group, during which all participants presented their respective institutes and production facilities, short presentations were given covering various aspects of the status quo regarding broodstock management. These presentations were followed by more detailed group discussions where areas of special interest were fleshed out and strategies for further investigations were discussed. The following proceedings are composed mainly of the reports given by the presenters and the following discussion of the chief areas of concern (written up after the workshop).

Objectives of the Workshop:
*Review and discuss the current production situation of the participants and the fish species which exhibit the most promise for the European fish farming industry.
*Review current knowledge for these species regarding their reproduction biology.
*Review current broodstock management and egg production techniques, including environmental and hormonal manipulation.
*Review the existing knowledge on environmental tolerances for the key species.
*Review the existing knowledge regarding the reproductive physiology of the species under discussion.
*Discuss the possible effects and consequences of natural spawning on genetic composition of offspring and utilization in breeding programmes.
*Identify strengths and weaknesses with current broodstock management practices as well as knowledge gaps requiring further research.

The topics and speakers were as follows;
1. Innovative tank design with regards to natural spawning
Led by Prof. Victor Øiestad, APN
2. Egg quality and its measurement
Led by Dr. Andrew Davie, UoS
3. Environmental conditions and natural spawning
Led by Dr. Herve Migaud
4. Broodstock quality and parental effects
Led by Dr. Robbert Blonk (WU) and Prof. Maria Teresa Dinis (CCMAR)

Period 2; At the internal broodstock workshop it was suggested that the most sensible outcome of this task would be one or several technical applied articles in wide audience journals ( Fish Farming International or similar), rather than a review article covering all the topics mentioned in the original DoW. To this end, a review template has been drafted to which the RTD partners will contribute state-of-the-art summaries most related to their role in the project. Additionally, a review on the impact of light and sound on fish spawning performance focusing on round fish (cod) has been written and will be submitted for publication soon. Results from the work on minimising genotyping in breeding programs for natural spawning species have also been submitted. Another review paper is in press in Reviews in Aquaculture on Egg quality and broodstock management in fish as part of the Cost Action Larvanet and the Prospawn project. An overview of the project has also been presented in the Spanish trade magazine " Ruta Pesquera". Finally, an experimental paper “Anthropogenic noise in enclosed aquaculture facilities can act as a stressor in Atlantic cod gadus morhua.” has been written and will be submitted for publication soon. At this time, the list of reviews and popular-science publications stands as such;

http://prospawn.akvaplan.com/
http://www.acuinuga.com/actividad.php?id=27
http://www.acuinuga.com/actividad.php?id=33
http://www.ardtoemarine.co.uk/2011/03/07/did-you-know-that-cod-are-chatty/
http://www.eatip.eu/default.asp?SHORTCUT=121
http://www.netalgae.eu/profile-viking-fish-farm.php
http://www.icman.csic.es/proyectos.php?idd=1
http://www.akvaplan.niva.no/en/aquaculture/new_rearing_methods
http://www.aqua.stir.ac.uk/about/people/herve-migaud#/sect/projects
http://www.efishent.eu/pages/participants.php
http://es.scribd.com/doc/100595051/Noise-a-source-of-stress-for-farmed-fish
http://www.ist-world.org/ProjectDetails.aspx?ProjectId=deb4e6363ce241028236d159c09e1d07
http://www.bioemprende.eu/files/InformesVigilancia/3_5_InformeVigilanciaTecnologica_BiologiaReproductiva.pdf
http://opidi.org/files/presentacion/ACTIVIDADES%20FINANCIADAS%20FP7-GALICIA%202007-2011-p.pdf
http://ec.europa.eu/research/bioeconomy/pdf/marine_sme_2011_v4.pdf
http://www.campusdelmar.es/eng/news/1/2011/2/el-proyecto-europeo-prospawn-eu-fp7-research-for-smes-232305-se-reunira-los-proximos-4-y-5-de-abril-en-faro--133
http://aquafeed.co/peces_de_cultiv/
http://www.kg.eurocean.org/proj.jsp?load=30131

WP 2
The description of work as specified in Annex I to the Grant Agreement is as follows::
Task 2.1 Installation of breeding nests in flatfish broodstock tanks
In the current task, spawning nests will be installed in conventional broodstock tanks for two species which generally do not spawn in captivity (halibut and turbot), necessitating the stripping of eggs and sperm. The number of nests used will be dependent upon the total area of the tank and vary from species to species, but in all cases there will be one nest per female. The ratio of males to females will also act as an additional parameter to be studied in relation to behavioural studies in WP4. Water quality measurements (e.g., temperature, O2) will be monitored as specified by each of the SME partners involved in the trials. Eggs will be collected and incubated using commercially standardized procedures, and quantified to produce data describing the success of the breeding (e.g., fecundity, per cent fertilization, embryo survival). This task will also be incorporated to tasks 3.1 and 3.2 to study the combined effects of the physical environment and photoperiod on spawning halibut and turbot.
Task 2.2. Installation of shallow raceways in demersal round-fish broodstock tanks
The focus of this task will be on commercially important species for which this technology may be suitable and will be installed and tested in commercially or lab-based conditions in species in which commercial scale is either not feasible due to the small size of the industry or too risky (sea bream and mullet). SRS systems will be designed (size, current requirements, temperature and lighting controlled systems) according to available knowledge in the species (literature review) and set up during the first year of the project. This task will be in synergy with WP 3 regarding environmental control of spawning and WP4 for the behavioural monitoring. If possible, two spawning seasons will then be monitored during the project.
Task 2.3 Egg quality obtained from natural spawning
Two to three spawning seasons will be monitored during the project in a range of species in both systems developed in tasks 2.1 and 2.2. R&D partners will design a standardised protocol for each species to monitor egg quality (month 2 of the project). This protocol will include a number of parameters to be analysed by SMEs (or R&D in the case of task 2.2) during each spawning season including spawning window (start and end date), daily egg production (if possible time of the day), drop out percentage, fertilisation rate, developmental stages (blastomere), fecundity, egg survival, hatching rate, larvae survival and first feeding success if possible as well as environmental data (water quality, flow rate, oxygen) and broodstock background data. Data on all these parameters will be compiled by R&D partners.
Task 2.4 Development of an automated egg quality monitoring system
A computer-based scoring macro will be designed to allow the industry for a relatively small investment to significantly improve accuracy, reproducibility and importantly speed of egg quality assessment ensuring that production investment is not wasted on sub-optimal egg batches. Blastomere scoring will be used according to five characteristics; symmetry, uniformity, margins, inclusion bodies and cell adhesion. An image analysis software will be used to automate the scoring usually done manually. The tool will first be developed and validated for cod and then transferred to other species of interest in this project. The use of this tool outside of WP2 will be chiefly in WP 4 as a way to monitor egg quality in the sole species.

Task 2.1
Period 1
Turbot nest trial 2010
Materials and Methods
A prototype turbot nest box was built in April 2010 constructed out of high density polyethylene, the nest box measured approximately 1.2m x 0.8m with the internal nest area measuring 0.9m(H) x 06.m (W) x 0.08m(D). Due to financial limitations only one box was constructed and placed in a 16 m3 tank with a bottom tank area of 10.75 m2. The tank bottom was clear with a mix of sand and small gravel collected from the local shoreline being place in the nest box only. The broodstock population held at Ardtoe (n=38) was manually inspected and sexed using ultrasonography on 8th April 2010, with 9 turbot (sex ratio 1♀:2♂ (Mean total length ~550mm) being stocked into the tank containing a nest and the remaining fish (14♀:15♂ were stocked into a similar size tank without a nest. The 9 fish in the “nest tank” were photographed to ensure consistent/accurate visual identification of nest occupation. Both tanks were supplied by sand filtered sea water at 1.7 m3 hour-1 with an ambient temperature regime and additional aeration was provided through diffused air blowers. Illumination was provided by an open 1m2 hatch in the tanks lid as well as two 60W tungsten filament bulbs running on a simulated natural photoperiod. Fish were fed twice a week with a commercial pelleted diet.
Occupation of the nest was observed and recorded daily from June to September 2010. In addition egg collection nets were placed on the tank overflows and were inspected daily with any collected eggs being removed, weighted (± 0.1g) and examined under a microscope to record fertilisation rates. Furthermore, every third day, all females were manually inspected and hand stripped if identified to have ovulated eggs within their ovaries. These eggs were then fertilised ex vivo. This is routine practice to produce juveniles and ensure the parent females did not develop complications (e.g. blockages) associated with the retention of decomposing oocytes.

Results
It was observed that fish readily occupied the nest and would bury themselves within the sand layer. Noticeable colour changes were observed when fish occupied the nest. While both sexes were seen to use the nest, in general it was occupied by females most frequently. On occasions two females were seen to co-habit the nest but a mixed sex or all male pair were never seen to share the nest.

Eggs were collected from both treatments from the end of July to the end of August . Freely released eggs were collected from both tanks, on 5 occasions from the “nest” tank and on two occasions from the “no nest” tank. In all cases, oocytes were not fertilised. All fish were hand stripped and produced good quantities of viable eggs. Volumes were as expected and showed no apparent quality difference between populations.

Halibut nest trial 2010
Materials and Methods
During the summer of 2010 an experimental population of halibut broodstock was transferred to an isolated fiberglass tank (7m diameter, depth approximately 1.5 m) at the rearing facilities of Norsk Kveite (Askøy, Norway). The fish were supplied with ambient temperature seawater (200 l min-1). The tank was fully enclosed by a canvas tent, blocking direct sunlight but allowing enough light that the fish are able to detect and maintain physiological rhythms. No additional lighting was supplied, in accordance with the operating protocols of Norsk Kveite. Fish were fed a commercial pellet daily until satiated. The population consisted of 4 females and 8 males. Both the female and male populations were split evenly between wild-caught broodstock and first generation farmed fish. Four prototype spawning nests for halibut (one for each female) were constructed and installed between September and December 2010 and the fish were allowed to become acclimated to the new environment until the start of the spawning season (typically February to April). The dimensions of the nests were 1m x 1.8m x 0.1m and were filled with a smooth 5 mm gravel. An infrared camera was installed at the peak of the tent, allowing for all four spawning nests to be observed constantly. The fish were inspected several times a week to assess their maturational status. An exterior egg collection system was designed and installed so that eggs could be collected without disturbing the fish.
Results
Over the course of the months leading to the normal spawning period, it was observed that the spawning nests were used extensively by both females and males. During the earliest weeks of this trial, when only one bed was completed and installed, the largest fish (a female approximately 60 kg) was most often observed occupying the nest. However, after all four nests were installed no obvious signs of hierarchies or preference for any particular nest by any particular female was noted. The females were most often observed in the nests, although males were also frequently observed to be using the nests. Additionally, 2 to 3 fish were often observed sharing a nest for extended periods of time. No aggressive behaviour was observed with regards to the nests, i.e., there did not appear to be any effort made by any of the fish to defend any particular nest.

No spawning was recorded during the normal spawning season. Three of the females appeared to develop normally as the spawning season approached, with the abdomen swelling as would be expected. The fourth female did not show any signs of sexual development at all. All four females have released eggs naturally during previous spawning seasons, which is in part why they were chosen for this trial, but no eggs were found in the egg collection device or seen in the broodstock tank at any point during the season. By mid to late April the swelling in the abdomen began to regress in all females without a single incidence of egg release.

Period 2
At the site in Norway (Norsk Kveite), several changes were made to the experimental set-up in response to the previous year's results. As no spawning was recorded during 2011 despite the apparently normal physiological development in the broodstock, several possibilities were considered for the inhibition and an effort was made to correct for these. As mentioned above, all the spawning beds were re-filled with gravel rearranged such that there was the maximum distance possible between spawning beds, as well as positioning the beds as far away as possible from the entrance to the tent and water inlet.

The egg collection system was also extensively modified during the off-season in order to alleviate two possible sources of disturbances. The collection tank was increased to 1500 l with a 1000 l collection net, allowing the full flow of the broodstock tank to flow throw the collection system at all times, without damaging any potential eggs that would be collected. This allowed for any eggs to be collected more rapidly and easily inspected, thus reducing the frequency with which the broodstock tank needed to be inspected for eggs and therefor reducing the amount of disturbances to the broodstock. The second modification to the egg collection system allowed for the water depth in the broodstock tank to be increased to over 170 cm, the maximum depth for the given tank. This was done in response to the prospect that the broodstock were not able to complete proper spawning behaviour in the shallower depth during the previous season.

The most serious shortcoming of the previous spawning season was the inability to film the halibut in the complete darkness of the tent covering the tank. During the spawning season of 2011 a single infrared camera hanging over the tank was used, and this proved to be inadequate in penetrating the water column to capture any images of the fish. After several consultations with the Institute of Marine Research in Bergen, several alternative strategies were employed over the course of the 2012 season. Two overhead IR spotlights were installed around the edges of the tank to further illuminated the halibut but proved to not increase the effectiveness of the camera. Two powerful underwater IR lamps were acquired from the Institute of Marine Research and installed in the tanks near one of the spawning nests, but this also proved to be ineffective. Lastly, lamps utilizing far-red light of differing frequencies and intensities were installed in the tanks and used as the sole source of illumination as well as in combination with the different IR lamps. This proved also to be insufficient in providing enough light to capture video or still images of the fish in the tanks. As the only way to capture images of the fish was to leave the tent door open to let in light, the hope of capturing spawning activity on film was abandoned so as not to disrupt the natural spawning rhythms of the broodstock.

Results
Over the course of the 2 years of the trials, it was observed that the spawning nests were used extensively by both females and males. However, there were few indications that the spawning beds were utilized as intended. During the earliest weeks of this trial (2010), when only one bed was completed and installed, the largest fish (a female approximately 60 kg) was most often observed occupying the nest. However, after all four nests were installed no obvious signs of hierarchies or preference for any particular nest by any particular female was noted. The females were most often observed in the nests, although males were also frequently observed to be using the nests. Additionally, 2 to 3 fish were often observed sharing a nest for extended periods of time. No aggressive behaviour was observed with regards to the nests, i.e., there did not appear to be any effort made by any of the fish to defend any particular nest. This was true for both the 2011 and 2012 spawning seasons.

As opposed to the 2011 season, when no spawning or egg release was recorded, there was ample evidence of egg release during the 2012 season. All four of the females appeared to develop normally as the spawning season approached, with the abdomen swelling as would be expected. In addition, the majority of males exhibited running milt during the spawning season. When viewed closely to assess maturation, several of the males at any given time could be seen to be running, without any external influences. Similarly, eggs were collected in the collection system daily and eggs were seen to be floating in the broodstock tank often. However, no spawning activity was observed at any time and no fertilized eggs were collected during the entire course of the spawning season.

As opposed to the 2011 season, where there was no evidence of final maturation among the broodstock fish and the reasons for the absence of fertilized eggs were quite obvious, the source of inhibition of successful spawning in 2012 is not entirely evident. Female broodstock were producing and releasing eggs and males exhibited free-running milt, yet no fertilized eggs were procured during the entirety of the spawning season. Given the apparently random use of the spawning beds, it would seem evident that their presence alone is not enough to induce correct spawning behaviours in halibut. As compared to successful trials with natural spawning in the 1980s, the one parameter that stands out is tank size and depth. The previous successful induction of natural spawning incorporated a broodstock tank that was over 3 meters deep. It is possible that the relatively shallow broodstock tank utilized in the current project provide insufficient room for natural spawning.

In February 2011 a population of Atlantic halibut at Ardtoe marine laboratory were inspected, and their sex/stage of maturity was assessed using ultrasonography. These fish are a long established proven breeding stock and are held in a 40 m3 volume tank with a bottom surface area of 38.5m2 and to date these fish have been manually stripped. In April 2011 three commercially available trays (Metre square tray, IGE, UK) (1 tray per female) measuring 100x100x12cm which were filled with a mixture of sand and small gravel as in the turbot setup and were placed within the tank. Nest occupation and egg release was monitored through the 2011 spawning season (June to September 2011). Each fish was photographed to help identification.

Results showed relatively poor nest occupancy as shown in figures 6 and 7. Overall, fish occupancy was only 11%, with 6 and 5% in the morning and afternoon respectively. This percentage is based on a maximum occupancy of 173 days x 3 nests subdivided in am and pm (total of 1038). No natural egg release was observed.

Conclusions
*There is no evidence of positive effects of nests on egg production in turbot and natural spawning in halibut broodstock. The increase in volume and quality of turbot eggs in the nest setup is most likely due to reduced stock density (9 vs. 29 in nest vs. control populations)
*Nest occupancy was overall low in both species and from a limited number of broodstocks.
*The incorporation of nests in a turbot/halibut broodstock tanks would not be recommended as it showed no evidence for the promotion of natural spawning.

While the exact nature of the inhibition remains elusive, after two full spawning seasons with halibut and turbot, the conclusion has been reached that any new production techniques involving the use of spawning beds or similar structures to induce the occurrence of natural spawning and egg collection are commercially unfeasible at this time. While the potential outcome of a successful implementation of this form of production could be great, it is far too costly to develop in a commercial setting. Valuable tank space and broodstock were essentially idle during this period, and any eggs produced by the fish that may have been used in normal production were lost. The further development of this production technique must occur in a purely research driven project.

Task 2.2
In April 2011 an adequate facility for the experimental work was secured at the Aquarium de O Grove in NW Spain (Pontevedra, Galicia, Spain)[Figure 1]. This is a private facility with both saltwater and freshwater exhibitions, and with a strong policy for the promotion of divulgation, training and research activities within the field of aquatic animal biology. The main building facilities, with a covered area of 3,000 square meters, consists of three well differentiated areas running in partially recirculated regimes: the aquaria exhibition area, the tank exhibition area and the quarantine area.
Water inlets were specifically designed in the PVC pipes in order to ensure a smooth mixture of new and reused water without generating turbulences; the water depth reached 17 cm within the raceways and the pumping regime was designed in order to deliver an optimal current speed of 0,3 body lengths per second during the offseason. The plan was to increase water speed up to 0,1 body lengths per second during the spawning season, after full acclimation to the containment system had taken place. Careful selection of sufficiently sized (10 cm in diameter) PVC drainage pipes in each tray allowed for quick water passage, an essential requisite for the maintenance of the required water speeds within the raceways. An egg collector made of suitable size (500 micron) nylon mesh was attached to the water outlet of the raceway system and a protocol for daily examination of the egg collector implemented. The goal was to incubate any collected eggs and to assess the spawning quality on the basis of egg symmetry and viability, while any surplus of viable eggs could be distributed to interested parties.
The raceways were enclosed by plastic sheeting curtains in order to reduce the amount of exposure to environmental light and sound disturbances. The installation of these raceways was completed at the experimental facilities and broodstock were introduced to the system during the month of June 2011 [Figure 3]. The shallow raceway was stocked with blackspot seabream (besugo, Pagellus bogaraveo) and white seabream (sargo, Diplodus sargus) at a rate of fifteen adult individuals per tray in order to ensure low fish densities (30-50 g/L) and an approximate sex ratio of 1F:3M, based on previously published data for these species. The body length range for the black spot seabream was 25-35 cm and the body weight 400-700 g. For the white seabream, the body length range was 20-25 cm and the body weight range was 240 to 400 g. Behaviour and growth was observed as the fish adjusted to their new rearing system, and water quality parameters were monitored weekly.

In general both species adapted well to their new containment system. Both species showed good appetite as they were fed ad libitum with moist feed (mussels, squid, whitefish etc.). Mortalities were high (i.e. up to 50% of the introducedbroodstock) within the first days of stocking, but remained low (i.e. under 5%) after the remaining individuals adapted to their new containment system. Generally Pagellus bogaraveo was observed to be a much more sensitive and stressful species than Diplouds sargus, with mortalities always recorded higher for the blackspot seabream broodstock than for the white seabream broodstock. Water quality parameters analysed (temperature, pH, conductivity, dissolved oxygen, ammonia, nitrites, nitrates) remained within normal ranges throughout the experimental period. The water height in the raceways appeared to be not deep enough for both species tested. The dorsal fins of the largest individuals protruded out of the water, and a diving or downwards swimming behaviour was observed for these fish. No courtship behaviour or spawning was detected throughout the trial period, which was extended until December 2011. No eggs were collected in the egg collector and it was decided that the fish were not progressing towards maturation because of the inadequacy of the containment system, i.e. higher water heights were required.

In view of the lack of spawning detected during the first experimental trials, a second shallow raceway system was designed for installation in the tank exhibition area of the Aquarium de O Grove, NW Spain, during early 2012. This design called for the installation of a larger containment system within a large (20 x 5 m) concrete seawater basin, 1 meter deep, in this area of the Aquarium. Approximately 20 maturing broodstock of white spot seabream from the Aquarium collection were transferred to the new raceway in late June 2012. Their length range was 20-25 cm and their weight range was 240-400 g. White seabream adapted well to the new raceway, readily accepting moist feed and showing no skin lesions with negligible mortalities. These fish were repeteadly sampled for the follow-up of morphometric parameters and the assessment of gonadal maturation. They were kept in the new raceway with water speeds ranging from 0,5 to 0,3 body lengths per second, while their swimming behaviour was monitored and recorded during both day time and night time.

With regards to spawning events during the drials carried out at the larger shallow raceway within the tank exhibition area of the Aquarium, no eggs were observed in the egg collector, which was examined and cleaned daily.

Conclusions
The achievement of the goals considered for this task, mainly the observation of spawning behaviour, the collection of eggs and the assessment of spawning quality in shallow raceway systems for demersal round-fish species was very much conditioned by different technical problems. Some of them were difficult to overcome, i.e. the need for longer acclimation periods to the tested containment system for the target species, the availability of maturing broodstock in larger numbers, and the high susceptibility to handling stress shown by the initial target species (Mullus surmuletus and Pagellus bogaraveo). Nevertheless and in spite of the shortcomings, we consider the information gathered in these trials very valuable. The main implication of the swimming behaviour registered at the SRSs is that both species are well suited for stocking in shallow raceways, particularly the white seabream, which registered very low mortalities throughout the experimental period. This is in contrast to some fish species which are swimming continuously and do not maintain a stationary swimming position in the flow, swimming up and down the raceway in repetitive cycles. This behaviour has been observed for gilthead seabream and is expected to be the behaviour of species like yellowtail kingfish (Seriola lalandi).
Finally the general results obtained for this task may be evaluated within the context of our perception of this novel technology (SRS) and its potential for the industrial scaling up of fish production. The production potential of this containment systems has been demonstrated for some species, mainly flatfish (sole Solea solea, Senegal sole, Solea senegalensis; turbot Scophthalmus maximus, Atlantic halibut Hippoglossus hippoglossus) and appears to be particularly well suited for some phases of the production cycle. A number of advantages make it a very appropriate choice for the nursery phase, when fish handling is frequent and must be performed on large numbers of individuals i.e. during grading, sorting, vaccination, marking, weaning etc. Amongst these advantages, the high stocking densities accepted, the high biomass to water volume ratios –facilitating intensive recirculation and water filtration and sterilisation-, the easy access to and observation of the fish derived from low water heights, which could be further improved by using transparent materials for the trays, the self-cleaning derived from high water speeds etc.
Questions on the suitability of this system remain, though, for other phases of the production cycle, mainly hatchery and grow-out, and for demersal round-fish species. Some of these species may well be particularly adaptable to the SRS conditions, especially well domesticated, docile or gregarious species (i.e. salmonids, spotted wolfish Anarhichas minor, some breams), although others may not accept it, such as the Pagellus bogaraveo tested in these trials. Key areas for the implementation of this technology on an industrial scale are the water recirculation system -which shall require a specific focus for this particular application, given its very unique working conditions-, and the development of specific solutions for routine activities (i.e. grading, handling, harvesting, etc.). For grow-out, the recirculation of large volumes of water under superintensive conditions raises both technical questions and also on the economic feasibility of the technology -i.e. operational costs-. A large effort of applied research must be carried out yet in order to assess the realistic potential for these containment systems under commercial conditions. The potentiality of this technology for the industrial production of seafood within the European context of animal production standards makes this effort worthwhile.

Task 2.4
Period 1
Any methodology which can rapidly define the quality of a batch of eggs and thus allow a hatchery to determine whether to continue to invest labour and resources will greatly help the hatcheries productivity. One such method that was first described in Atlantic halibut but has latterly been proven in a range of other species is the assessment of the evenness of cell divisions at the 8 cell blastomere stage. This method is however very labour intensive and susceptible to wide operator influences. This task aims to translate the methodology of blastomere scoring to a “simple” analytical process using digital image analysis to increase its value to the marine hatchery community.

Materials and methods
In March and April 2011 subsamples from 10 individual crosses of halibut embryos were collected from Otterferry Seafish Ltd and transported to UoS. Embryos were transported in a portable light proofed constant temperature incubator that maintained embryos at 7°C. On arrival at UoS they were transferred to a constant temperature (7°C) tissue culture incubator where they were maintained until photographed.
When embryos reached the 8-cell blastomere stage at 96°C hrs they were stocked individually into a 24 well microplate which was pre-filled with 3ml of 0.2µm filtered, chilled sea water. These were then photographed using a 3Mp digital camera attached to a stereo zoom microscope and illuminated using a reflected incident light to ensure embryos were not subjected to heat shock. Once all embryos were photographed and stocked, the plate was sealed and placed in a 6°C darkened incubator and checked later at regular intervals to record hatch rate of the individual embryos (approximately 14 days).

Results
The eggs batches sampled were from across the spawning season which permitted the assessment of a wide range in quality. Mean fertilisation rate was 39.1 ± 8.0% but ranged from 10.4-77.9%, while mean hatch rate was 21.4 ± 9.5% but ranged from 0 to 66.7%. While casual observation may suggest an association between fertilisation and hatch rate, such a correlation was not significant in this limited sample size (P=0.17).
The aim of this work package is to take the manual blastomere quality scoring criteria (Shields et al., 1997) and translate these into simple empirical criteria that can be applied using digital image analysis software which could ultimately allow for the automation of the quality scoring methodology. To develop and validate an automated egg quality tool prototype there are a number of steps being followed as outlined below:
The original scoring methodology was a subjective 4 point scale assessment of 5 parameters. Methods to digitally measure these (or similar criteria) are being assessed using two different image analysis softwares. The first, Image-Pro plus, is a typical comprehensive “off the shelf” image analysis package, while the second, ImageJ, is a public domain, open source JAVA based image analysis software with some reduced functionality. While the ultimate aim would be to design the analysis on the free open source platform, both are being examined in this prototyping phase as the commercial package has a more comprehensive range of tools. If a specific function was identified as being crucial for successful analysis then it could be developed and added to the ImageJ toolkit. The criteria being tested focus on parameters that are easy and rapid to measure with a view to subsequently stream line the automation of the procedure where possible. Thus most criteria focus on aspects of the symmetry/evenness of the cell division process, however more complicated analysis methodologies that allow single cell identification and measurement are also being tested.
*Symmetry: Bilateral symmetry about the axes of the 8 blastomeres. ‘Normal’ appearance is symmetrical.
*Cell size: Uniformity of cell size between all 8 blastomeres. ‘Normal’ appearance means all cells are of the same size.
*Adhesion: Proximity of adjacent cell membranes. In ‘normal’ blastomeres, adjacent cells are contacting.
*Margins: Discreteness of cell margins. In ‘normal’ blastomeres, margins are well resolved.
*Inclusions: Inclusions are vacuoles (composition unknown) between adjacent blastomere membranes or on the periphery of the blastodisc. ‘Normal’ blastomeres have no inclusions

Quantification of operator variability in quality scoring
In order to demonstrate the value of an image analysis based quality tool, a blastomere scoring test was designed to compare the new system against the old manual scoring methodology. A sample of typical blastomere images was compiled and will be measured using the refined digital method, while the same example images were compiled with a brief guidance note on the manual scoring methodology and circulated to a number of volunteer subjects. The volunteers consisted of a range of people including experienced culturists within the project as well as researchers and students at UoS, with some of the volunteers having experience of the methodology while others were naive. All participants were asked to read the guidance notes before scoring the example images, the volunteers were also asked to record the time taken to score the images. All test results will be completed before the end of May 2011 to provide a measure of the operator variability in the existing methodology which can then be compared to the digital scoring output.

Period 2
In March and April 2011 subsamples from 9 individual crosses of halibut embryos were collected from Otterferry Seafish Ltd and transported to UoS. Embryos were transported in a portable light proofed constant temperature incubator that maintained embryos at 7°C. On arrival at UoS they were transferred to a constant temperature (7°C) tissue culture incubator where they were maintained until photographed.

When embryos reached the 8-cell blastomere stage at 96°C hrs they were stocked individually into a 24 well microplate which was pre-filled with 3ml of 0.2µm filtered, chilled sea water. This was performed in 8 of the 9 collected batches as one batch was unfertilised and thus not used in subsequent analysis. Embryos were then photographed (n = 192) using a 3Mp digital camera attached to a stereo zoom microscope and illuminated using a reflected incident light to ensure embryos were not subjected to heat shock. Once all embryos were photographed and stocked, the plate was sealed and placed in a 6°C darkened incubator and checked later at regular intervals to record hatch rate of the individual embryos (approximately 14 days).

Follow a consideration of image analysis platforms available on the market ImageJ was selected as it is an open source JAVA software freely accessible to all users. The proof of concept digital image analysis was performed using the complete 192 embryo image library. For each of the embryos 15 landmark points were selected to represent the point of contact for each cell as well as the corners of the balstomere group so that as a whole it created an eight cell grid representation of the blastomere itself. Of the 192, 49 blastomeres were removed from the analysis as they had an abnormal cell count (≠8) or margins were sufficiently undefined to allow the placement of landmark points (n.b. None of these blastomeres proceeded to hatch).

Conclusions
At its conclusion this task has demonstrated a ‘simple’ and accurate scoring methodology using digital image analysis to assess blastomere morphology and ultimately predict embryo hatch fate. The method removes the subjective criteria of the past proven methodology and has replaced it with a methodology that relies on precise objective criteria which will not be open to operator variability. In terms of the future application of this tool, any interested party can photograph embryos using any standard dissection microscope and digital camera. These images can then be accessed using the open source package ImageJ. Landmark points can be calculated using the above description and the resulting X,Y, coordinates then imported into the provided excel template. The excel template is set up for a 24 well plate setup however the sheet can be expanded or contracted to the users needs. This template will calculate the 74 individual measurements for each blastomere, gather the 9 summative values and then subject them to a logratio transformation. The resulting dataset can them be imported into a statistical analysis software like Minitab to perform the discriminant analysis. This whole process from imaging to analysis takes in the region of 1 hour once an operator is confident with the process, thus this method can provide reliable hatch predictions within 24 hours of embryos being fertilised. The method and associated data generated have been performed using Atlantic halibut as proof of principle. Literature suggests that the relationship between blastomere formation and quality exists in most species studied to date thus it is highly likely that the above described methodology could be applied to other cultured fish species e.g. Turbot, Sole sp. etc. However it would be advised that a reference dataset is generated for the target species in question following the methodology described, prior to use in a commercial setting.

WP 3
Overview period 1;
Task 3.1 (Species specific lighting environments and new technology lighting systems) Prototype lighting systems that allow spectral and intensity control were designed by INTRA after agreement on the characteristics required and sent to UoS (July 2010). Once the system was delivered to UoS it was apparent that while the system demonstrated the potential functionality that an LED based system could provide the prototype itself was not suitable for field trials. Additionally, a study of the endogenous reproductive rhythms in Senegalese sole has been initiated to study the persistence of the reproductive cycle without a fluctuating photoperiod signal and its potential 12-month periodicity. For this purpose, a group of F1 Senegalese sole breeders were maintained under constant photoperiod (CP, 12L:12D daylength), in parallel with control fish maintained under a natural photoregime. Blood sampling started on February 2011 and will continue on a monthly base until July 2012, covering two reproductive cycles (natural spawning time of the F1 Senegalese sole stock around April-June). Approximately every six months, ELISA analysis (VTG and sex steroids) will be performed for the collected set of samples.
Task 3.2 (Effects of daily light variations (dawn/dusk)) Trials were conducted on Atlantic cod during the winter of 2011. The broodstock were divided into two populations; one was reared under a lighting regime which provided a natural dawn and dusk transition while the other was reared under a regime where the lighting system was switched on or off at sunrise/sunset with no dimming during the crepuscular period. Eggs were collected from both populations to investigate the effect of the lighting regimes. While there was some variation in the egg production between treatments it appears to be nothing other than natural variability between isolated populations.
Task 3.3 (Identification and monitoring of artificial disturbances in culture) To characterise the “sound-scape” in a typical broodstock facility underwater sound (frequency and sound pressure level) was recorded using a Reason TC4013 hydrophone connected via a charge amplifier to a laptop PC. In general, background sound levels within the tank environment were low and generally were around the reported auditory thresholds reported for cod. However, it was clear that routine husbandry practices could create clear sound disturbances within the typical tank environment. Secondly, during the spawning season of spring 2011, cod vocalisations were recorded within the broodstock facilities of Ardtoe to benchmark the frequency range and volume which are used by the stock, providing a useful benchmark of the natural communication bandwidths that the species use. Finally, short term exposure trials demonstrated that sound in the frequency and volume ranges that can be experienced in the tank environment can incite a short but significant stress response.

Period 2

Task 3.1; To study the influence of photoperiod (light regime) on Senegalese sole reproduction, a long-term experiment was designed to determine if reproduction occurs in the absence of the photoperiodic signal, which would indicate the existence of an endogenous clock timing the process of reproduction. For this purpose, a group of G1 Senegalese sole breeders were maintained under constant photoperiod (CP, 12L:12D daylength), in parallel with control fish maintained under a natural photoregime. The experiment lasted for two years, to study two consecutive reproductive cycles (springs of 2011 and 2012). The reproductive process was monitored by analyzing spawning (timing, quantity and quality) and blood levels of reproductive hormones.
In August 2010, fish were selected and distributed in four circular 3,000 L fiberglass tanks. In September 2010, two experimental groups were established (duplicate tanks), controls (natural photoperiod) and the constant photoperiod group (CP, 12L:12D daylength). The constant photoperiod was started on the autumn equinox (21 September 2010), coinciding with the natural 12L:12D daylength and then maintained onwards. Spawning (quantity and quality) was checked daily in all four tanks and the following parameters determined: 1) egg quantity, 2) % of buoyancy, 3) egg quality (observation of egg morphology under the binocular) and, 4) % of hatching (after incubation of floating egg for 48 h).
Results of spawning showed that both groups spawned spontaneously on each reproductive period (springs 20111-2012), indicating that the absence of the photoperiodic signal did not inhibit the occurrence of natural spawning. The timing of spawning was similar in both groups; 19 apr to 31 may for controls versus 13 apr to 23 may for the constant group on year 2011 and 25 apr to 29 may for controls versus 2 to 22 may for the constant group, on year 2012. Thus, reproduction under constant photoperiod occurred on yearly bases, similar to controls, suggesting the existence of an endogenous reproductive rhythm in this species. This result may highlight the importance of temperature on driving spawning time. The quantity of yearly released eggs was similar in both groups, indicating that photoperiod did not affect female fecundity. As usually observed in G1 Senegalese sole, all spawns were unfertilized and exhibited similar values of egg buoyancy.
In conclusion, spawning in Senegalese sole occurs on yearly bases under constant photoperiod, similar to controls maintained under seasonally fluctuating photoperiod, suggesting the existence of an endogenous rhythm of reproduction and highlighting the influence of temperature on driving spawning in this species. Constant photoperiod did not have negative effects on spawning parameters (fecundity, egg quality) or the endocrine reproductive system (hormone levels in the blood).

Task 3.3; Experiments were carried on at the facilities of Viking Fish Farms Ltd., Ardtoe Marine Laboratory, Acharacle, in Scotland (N 56°46’ W 05°53’). The marine hatchery facilities include cod broodstock holding tanks. A representative sound mapping was done through broodstock tanks. The analysis of perturbations consisted in first determining the background sound levels, followed by monitoring of common disturbances. Those disturbances were later used to determine the sound level to be tested.
Atlantic cod broodstock (total length 59.9 ± 5.6 cm, body weight 3.4 ± 1 Kg) were maintained in a circular holding tank (5.3 m diameter) under ambient photoperiod. Fish population was split into two identical tanks two weeks prior to the initiation of the experiment. No replication could be done due to facility and fish limitations.10 females and 6 males were used for the experiment in each tank. Fish were fed three times a week using commercial marine fish diets (Biomar). Cod eggs were collected daily from an overflow egg collector at 3:00 pm every day during the spawning season and egg quality parameters were monitored.
Both broodstock tanks were supplied with a suspended omnidirectional underwater loudspeaker (EV UW30, Frequency response 100-10,000 Hz; Impedance 8 ohms). One of the tanks was exposed daily to six hours of a randomized linear sweep exposure starting 2 weeks prior the first egg collection. The sound pressure level (SPL) was 34 dB re 1µPa above background noise levels, the equivalent of the same energy input from the previous experiment.
No significant differences were found between treatments in terms of the quantity of egg production, egg size and fertilisation rate. However the fertilization rates and thus total viable egg yield was significantly reduced (≈12.4Kg viable eggs, no sound vs. ≈6.3Kg viable eggs, sound exposure). The reduced yield (6.1Kg) equates to a loss in the region of 3 million viable embryos which would translate to a loss of ≈150,000 weaned fry if a pessimistic survival rate of 5% to weaning was applied.
During the second period of the project Prospawn, the effects of sound exposure on spawning performances of Atlantic cod broodstock were studied. A similar sound level than the one used in juveniles (i.e. 10 minute exposure, randomly dring the day, of a repetitive 10 second linear sweep from 100 to 1000Hz) was used. Our results showed negative effects of noise on Atlantic cod broodstock performance. There were no significant differences in egg quality parameters (total egg volume, egg diameter, eggs per gram and floating fraction percentage) but fertilization rates were significantly reduced, almost by half (control: 58.7 %, noise exposure: 34.7 %.). The negative effect of noise on fertilization success and larvae development is probably the consequence of several factors caused by the noise exposure. Regardless of the exact pathway of action these study shows that noise might be affecting fish performance and explain variations between tanks in commercial and research facilities.
WP 4
In 2010 and 2011, several experiments were performed to test the effects of GnRH, HCG and DHP hormonal treatments, supply of natural pheromones, and of adding wild reproductive males that exhibit natural reproduction behaviour, on increase of egg release and fertilisation in G1 common sole broodstocks. None of the experiment al treatments showed positive effect on production of (fertilised) eggs.
However, in spring 2011, successful reproduction of G1 common sole was detected and validated with genetic markers and pedigree reconstruction in outside aquaculture ponds that experience (semi)natural temperature fluctuations (daily and seasonal) and natural light. It was hypothesised that the strong winter of 2010-2011 and in particular the low temperatures were a reason for the successful spawning event. This outcome gave an indication of the circumstances that are required for spawning if G1 sole. In the previous periodic reports, in May 2011 and Feb 2012, the first experiments performed by CCMAR team during 2010 and 2011 were described. Briefly, during autumn experiments (2010), in which spring conditions were simulated for the experimental group (tanks A3 and A4), no spawning events were observed in any group, although some swelling of the abdomen was noticed in the larger females from tank A4. When analyzing the daily rhythms of locomotor activity, for both groups it was clear that fish were preferently active during night time, with lower levels of activity during day time. It was observed an anticipation of the night in both groups: in the latest hours on the day, the activity started to increase, right before sunset. As to differences between groups, in the experimental group, which was exposed to a longer daylength and the sunset occurred 1 hour later, the increase in activity also occurred latter. Different melatonin profiles were observed among groups under different photoperiodic conditions, demonstrating the synchronization of this species to photoperiod.
In what refers to spring experiments (2011), as described in the previous report, fish from the experimental group were exposed to a faster lengthen of the photoperiod from winter solstice onwards (reaching “spring equinox” sooner than the control group) to test if an advance of the spawning season was produced. During this period just one spawning event was registered in tank A1. When analysing this day’s activity profile, there was an important increase of activity right after sunset, when spawning begins (Oliveira et al., 2009), probably corresponding to the moment of courtship. As to locomotor activity rhythms, a very clear influence of the lengthening of the day in the patterns of activity was observed in the actograms of both control and experimental groups. Locomotor activity was highly synchronized with photoperiodic information, and the length of the activity period mirrored that of the photoperiod. Furthermore, a strong influence of the daily phase shift from light to dark or vice versa was also registered in all cases, with a very intense activity being observed in “dawn” and “dusk” for both groups. During the autumn experiments we added fish to our broodstock at CCMAR facilities and individuals had to go through an adaptation period to captivity and new environmental conditions. Consequently the lack of a spawning event during the autumn season was mainly due to this adaptation period. During spring spawning season Senegalese sole was already established and adapted, and all experiments were set up, however there was record of a single spawning event. The reasons that lead to these results are still under discussion.

Period 2
Task 4.1; (Behaviour Of Naturally Spawning Broodstock) During this last period of the project we have performed two different experiments focusing on the behavior of the broodstock under different rearing conditions and according to each individual’s origin:

*Light intensity influence on basal cortisol and locomotor activity of Senegalese sole broodstock.
*Daily rhythms of locomotor activity and stress response in wild and G1 individuals of Senegalese sole
For the first experiment, we utilized just two of the four tanks assembled, in order to use always the same individuals for each phase. These fish were exposed to natural conditions of temperature, and simulated natural photoperiod. This experiment intended to describe the best light intensity to rear Senegalese sole broodstock, focusing on the influence of light intensity in parameters such as basal cortisol levels in plasma and locomotor activity rhythms.
The second experiment, was intend to study the daily rhythms of stress response in this species, based on the commencement hypothesis that if fish present daily rhythms of basal cortisol levels, it is very likely that stress response will not be the same when fish are handled during the day or during the night.
In the first experiment, intended to describe the best light intensity to rear Senegalese sole broodstock, a strong influence of light intensity has been observed both in basal cortisol levels and in locomotor activity rhythms. As to cortisol levels, significant differences were observed between the levels obtained during the period fish were exposed to 100 lx when compared to the period they were under 200 lx. However, when fish were exposed to an intensity of 50 lx, values were medium and didn’t resemble any statistical difference with the values of the other intensities. These results suggested that the most suitable light intensity would be 100 lx, as under 200 lx cortisol basal levels are significantly elevated, probably jeopardizing fish welfare. In what refers to locomotor activity rhythms, the results also revealed an influence of light intensity in fish behavior.
During the first period, under 50 lx, both tanks showed a very marked daily activity rhythm, with activity coinciding with daytime. When these animals were switched to an intensity of 100 lx, they remain strictly diurnal, and in one of the tanks the levels of activity increase. Finally, when soles were reared under 200 lx their behavior changed greatly. Both tanks seemed to become arrhythmic: soles in tank A3 showed irregular patterns of activity, and in tank A4, a total loss of rhythmicity was detected towards the end of these phase. These results support cortisol levels data described previously, in terms of suggesting that 200 lx is an inadequate intensity to rear Senegalese sole broodstock. Between 50 and 100 lx, behavior did not show major differences, with animals presenting marked rhythmicity under both intensities. Thus, both seem to be adequate.
The fact that during the present experiment, soles presented mainly diurnal activity, although Senegalese sole is known as a nocturnal species is probably related with the fact that these group fish is fed during the day, and this way they are optimizing food availability.
In the second experiment, we pretended to determine whether Senegalese sole responds differently to stress when manipulated during the day or during the night, and also according to their origin. In what refers to cortisol levels results, samples are still to be analyzed in our facilities, and we hope to have it done soon. As to locomotor activity rhythms, marked differences in patterns of behavior have been recorded depending on fish origin. Soles which were already born in captivity showed no daily activity rhythm at all, and very low levels of activity, while both wild groups presented a very marked rhythmicity, with the active phase coinciding with day time. Among these two groups of wild individuals, the levels of activity varied greatly. While soles which were brought from nature just 6 months prior to the experiments presented very high levels of activity during the day, the long adapted group showed much lower levels of movement. These results suggest that G1 soles were probably exposed to extreme conditions in captivity during their life cycle, jeopardizing their circadian system, and not allowing them to synchronize rhythms with environmental cycles. On the other hand, wild fish all exhibited a very marked rhythmicity. The higher levels of activity observed in the new wild group when compared to the older group, gives the impression that these fish are still adapting to captivity conditions, and thus still present a very agitated behaviour.

(Development of an artificial fertilization protocol to control reproduction of G1 Senegalese sole). The experiment was performed during the 2011 reproductive season, in the facilities of ACC (Povoa, Portugal), by personnel from ACC, CCMAR (Portugal) and CSIC (Spain). All AF trials were performed with the same sperm pool, to avoid variations caused by the use of different sperm samples. For this purpose, a cryopreserved sperm bank was prepared during the previous months by personnel from CCMAR and CSIC, conserved under liquid nitrogen and carried to ACC for its use in each AF trial. Sperm quality parameters were always analyzed before and after freezing, by using the CASA software. Females were treated with a single GnRHa injection (25 ug/kg), given at two different times of the day: 1) 08:00 and, 2) 20:00. Occurrence of ovulation was determined by egg stripping at different time post-treatment (24-36-40-44-48-72 h p.t.), by applying gentle abdominal pressure. Simultaneously, biopsies were obtained to analyze, 1) the stage of oocyte development (morphology and diameter), by image analysis and, 2) pH and osmolarity of the ovarian fluid. The quantity and quality (floatability and morphology) of stripped eggs was analyzed. Fertilization was performed for each batch of stripped eggs and fertilization and hatching rates analyzed (see first report for methodological details).
In general and similarly to what was described for the previous parameters, the time of hormonal induction did not affect the output of the AF trials. By contrast, clear effects were observed by the ovulation time. Highest fertilization rates (45-54 %) were observed on trials performed over 36 h stripped eggs, in both groups. Fertilization rates drastically decreased on AF trials performed over eggs stripped at later times (40 to 48 h p.t.), which indicated that ovulation in Senegalese sole should occur at around 36 h p.t. and most important, that overriping is a quick process occurring in approximately 4 h after ovulation.
In conclusion, results showed that GnRHa treatment of females is effective to induce ovulation of good quality eggs, if stripped on time. Time to ovulation is 36-40 h after GnRHa injection, independently of the time of the day when the hormone-induction treatment is applied. Overriping is a quick process in S. senegalensis (aprox 4 h). AF can achieve high fertilization and hatching rates (up to 50 %), enough for an industrial transferable protocol and thus, it can be an alternative for the S. senegalensis aquaculture industry to reproduce G1 broodstock.

Task 4.2; In 2012 an experiment was set up to test the hypothesis of the positive effect of low temperature on reproduction of G1 sole. The two G1 reproductive groups of the previous experiments were subjected to one treatment each:
• Standard temperature: this first group was subjected to temperature conditions as used in routine reproduction of common sole broodstocks, with the lowest temperature being approximately 7 °C.
• Natural temperature: the second group was subjected to natural temperature conditions reaching as low as 5 °C, and comparable to what was achieved in the winter of 2010-2011.
In both treatments, temperatures were controlled with heating and cooling systems. Al groups were housed indoors with artificial lights (40W) and in a recirculating aquaculture system with water filtration. Both broodstocks were fed twice per week with ragworms (Nireis virens) and moist pellets. During the reproductive season, production of eggs was checked daily for fertilisation. Fertilised batches were incubated and hatched to check ture reproductive success.
During the reproductive season of 2012 the “Standard temperature” group did not produce fertilised eggs as expected. However, in the “Natural temperature group”, numerous batches with fertilised eggs were produced. Several batches of fertilised eggs were incubated, hatched and used for ongrowing successfully.
Although there were no replicates in this experiment, the results give a strong indication that relative low temperatures, compared to conventional temperatures, can lead to successful reproduction of G1 soles. The mechanism of temperature effects on reproduction and fertilisation success in sole remains to be investigated because it is not clear yet why the “natural” and more extreme temperature regime was more successful than conventional regimes at which captive wild common sole reproduces routinely.
Part 2; From 2008 to 2010, two broodstocks of G1 soles were conditioned for reproduction in indoor circumstances. Every night that a spawn was fertilised successfully, a batch of eggs was incubated and hatched separately. Of each batch, 24 G2 larvae were sampled and stored in lysis buffer (Nucleospin DNA extraction kit, Machery nagel) at 4 °C. In 2011 DNA from all sampled batches (n = 17) was extracted and all samples were genotyped with 10 microsatellites: AF173855, AF173854, AF173852, AF173849, AY950593, AY950592, AY950591, AY950589, AY950588, AY950587.
Results and Conclusions
As expected, results showed that over several spawning nights, only few parents or parental pairs produced major part of the offspring (skewed distribution of parental contributions to offspring). This is typical for groups of natural mating soles. From one parental pair (animal 1_44 and animal 2.78), 240 offspring were detected. This implies that in reality, this pair had a major contribution to the total G2 offspring. These results indicate the presence of mate choice based on genetic relatedness between partners. In addition, further analysis of the data, e.g. on the possible phenotypic clues may point to other factors influencing mate choice. However, it should be noted that these results are derived from only one G1 broodstock. In order to get stronger evidence of the relationship between genetic relatedness and mate choice in sole, more data is required.

Potential Impact:
In response to the general perception that the communication among partners and the distribution of project results within and beyond the PROSPAWN consortium has been insufficient to date, an agreement was reached between the consortium and Project Officer Michaela Bitsakis during the consortium meeting in Brussels (29.10.2012). It was decided that an SME member of the consortium would be assigned the task of assisting project manager Erik Vikingstad in the efficient distribution and dissemination of project results to the other consortium members in a timely fashion. After several weeks of deliberation, it was decided that Dr. Luis A. Perez Carrasco from ACUINUGA would assume the role of facilitator for the duration of the project. The roles of the project manager PM and "facilitator" will be closely related and require cooperation between the two positions. The following description of the role of facilitator was presented to Project Officer Michaela Bitsakis, who approved of the new role.
• The PM will continue with the task of collecting and editing the progress reports from the Work package leaders, compiling the relevant and related results from each, and submitting the final deliverable reports to the EU.
• In addition to submitting deliverable reports to the EU, the PM will also provide the reports to the facilitator for distribution among the consortium. They will be written in a manner such that the most important and valuable results are prominent and easily internalized. The facilitator will be free to further edit the documents so that items most relevant to the SME partners are communicated in the best possible way, if they deem in necessary.
• The PM will also remain responsible for the compilation and delivery of the periodic and final reports. These will also be made available to the facilitator for distribution to the consortium as a whole.
• With regards to the further dissemination of the results; the deliverables and periodic reports made available to the facilitator will contain results that represent both economic and scientific value. The facilitator will maintain a dialogue with the WP leaders and invested SME partners regarding the extent to which the results will be made available to the scientific community, industrial interests, and the general public. As the SMEs own the rights to the use of the results, it is appropriate that the facilitator represents the SMEs during these discussions. In situations where the WP leader wishes to publish results from PROSPAWN, the facilitator will mediate the discussion between the RTDs and the SMEs regarding the wider publication of the results. As owners of the results, the SMEs will decide, under the guidance of the facilitator, whether a wider publication of said results would be in their own best interest, and participate in choosing the venue of publication.
• The facilitator will also aid the PM in leading the discussion of dissemination matters at the final project meeting (TBA). This will give the facilitator detailed and immediate feedback regarding the potential the most promising results possess, from both an economic and scientific standpoint, from the partners involved .

Exploitable knowledge and its Use
At the final project meeting in Barcelona (10 – 11.12.2012) the possibilities for dissemination and use of the various project results were discussed at length and are presented here. The original list of project results from the latest DoW is presented first, followed by the consensus of the project consortium regarding the merits of each point. While specific journals/targets for publication were not specified at the time, several results were identified as having potential for publication or wider dissemination. The SMEs will ultimately determine to what degree the scientific results obtained through the proposed project may be presented through publications, international conferences and open workshops. Results available to all EU aquaculture industry will generally be made available through the following channels;

Publications (peer review journals, PRJ)
In cooperation with the SMEs involved, data from both small scale laboratory trials and large scale industrial trials will be published, when feasible and agreed between all partners, in acknowledged scientific journals. Candidate journals are Aquaculture, Aquaculture Research, Fish Physiology and Biochemistry, Aquaculture International, Canadian Journal of Fisheries and Aquatic Sciences and Journal of the World Aquaculture Society. At the internal broodstock workshop it was suggested that the most sensible outcome of this task would be a technical applied article in a wide audience journal. It was agreed that a template should be formed by the task leader (CSIC) and inputs from all other partners should be incorporated into this. The title and theme of the article will fall in the area “Best practice....”. In addition, a review on the impact of light and sound on fish spawning performance focusing on round fish (cod) has been written and will be submitted for publication soon. Another review paper is in press in Reviews in Aquaculture on Egg quality and broodstock management in fish as part of the Cost Action Larvanet and the Prospawn project.
In addition, the following websites feature work from the Prospawn project;

http://prospawn.akvaplan.com/
http://www.acuinuga.com/actividad.php?id=27
http://www.acuinuga.com/actividad.php?id=33
http://www.ardtoemarine.co.uk/2011/03/07/did-you-know-that-cod-are-chatty/
http://www.eatip.eu/default.asp?SHORTCUT=121
http://www.netalgae.eu/profile-viking-fish-farm.php
http://www.icman.csic.es/proyectos.php?idd=1
http://www.akvaplan.niva.no/en/aquaculture/new_rearing_methods
http://www.aqua.stir.ac.uk/about/people/herve-migaud#/sect/projects
http://www.efishent.eu/pages/participants.php
http://es.scribd.com/doc/100595051/Noise-a-source-of-stress-for-farmed-fish
http://www.ist-world.org/ProjectDetails.aspx?ProjectId=deb4e6363ce241028236d159c09e1d07
http://www.bioemprende.eu/files/InformesVigilancia/3_5_InformeVigilanciaTecnologica_BiologiaReproductiva.pdf
http://opidi.org/files/presentacion/ACTIVIDADES%20FINANCIADAS%20FP7-GALICIA%202007-2011-p.pdf
http://ec.europa.eu/research/bioeconomy/pdf/marine_sme_2011_v4.pdf
http://www.campusdelmar.es/eng/news/1/2011/2/el-proyecto-europeo-prospawn-eu-fp7-research-for-smes-232305-se-reunira-los-proximos-4-y-5-de-abril-en-faro--133
http://aquafeed.co/peces_de_cultiv/
http://www.kg.eurocean.org/proj.jsp?load=30131

Conferences and exhibitions
Data obtained in the project that are not subject to limitations, i.e. industrial protection, may be presented orally or as posters in national (within each country in question) and international conferences such as the annual meetings of the European Aquaculture Society.


1. Obtaining high-quality egg from naturally spawning by use of breeding nest.
After several trials with both halibut and turbot, the consortium has reached the conclusion that any new production techniques involving the use of spawning beds or similar structures to induce the occurrence of natural spawning and egg collection are commercially unfeasible at this time. While the potential outcome of a successful implementation of this form of production could be great, it is far too costly to develop in a commercial setting. During the course of 3 years, no successful natural spawning occurred at any of the facilities employing spawning beds. Valuable tank space and broodstock were essentially idle during this period, and any eggs produced by the fish that may have been used in normal production were lost. The further development of this production technique must occur in a purely research driven project. No publications describing the projects activities in this area are currently planned.

2. Obtaining high-quality egg from naturally spawning in shallow raceway system.
The achievement of the goals considered for this task, mainly the observation of spawning behaviour, the collection of eggs and the assessment of spawning quality in shallow raceway systems for demersal round-fish species was very much conditioned by different technical problems. Some of them were difficult to overcome, i.e. the need for longer acclimation periods to the tested containment system for the target species, the availability of maturing broodstock in larger numbers, and the high susceptibility to handling stress shown by the initial target species (Mullus surmuletus and Pagellus bogaraveo). Nevertheless and in spite of the shortcomings, we consider the information gathered in these trials very valuable. The main implication of the swimming behaviour registered at the SRSs is that both species are well suited for stocking in shallow raceways, particularly the white seabream, which registered very low mortalities throughout the experimental period. A large effort of applied research must be carried out yet in order to assess the realistic potential for these containment systems under commercial conditions. The potentiality of this technology for the industrial production of seafood within the European context of animal production standards makes this effort worthwhile. The findings may be suitable for publication, which would be approved by the consortium.

3. Protocol for optimal environmental conditions for the induction of natural spawning in broodstock of selected species.
A lamp was specifically designed for the R&D purposes in this project, according to the following criterea:
• LED RGB (red, green, blue) high intensity light
• Computer controllable and dimmable either by DMX system or by a 0-10V system
• Possibility for manual dimming control
• Adapted for use in indoor aquaculture with both seawater and freshwater.
• Adapted for use in large tank systems.
• Energy efficient
The lamp were designed by Intravision AQUA and engineered in cooperation with our Chinese production partner Hangzhou Opto Electronics Ltd. Each lamp (Fig 12) has 4 x 30W integrated LED chips. The R&D lamps are arranged with 2 x RED chips, 1 x BLUE and 1 x Green Chip. In order to cool the LED’s the lamps have a sunken liquid filled copper cooling cycle which are connected with a plate type heat converter and an air-fan on the top side. The lamp housing is made in seawater resistant aluminum 5083. In this first setup the lamps come with the 240V electronics and the lamp controls arranged in a separate aluminum casing with a 12V connecting cable for the light housing itself. In this way the lamps can be arranged hanging over large tank systems with accessibility for the control at the side of the tank. The computer controls are based on a DMX 512 interface, and Intravision AQUA have arranged for an adaptation of a DMX control system for the PROSPAWN setup. In this case light measurements of the lamps will be programmed into the computer control system in order for the users to have a calibrated system. Then the dusk and dawn light cycles will be designed and programmed as continuous running sequences. Seven lighting units were delivered to UoS in May 2011 where the light units performance will be measured before they are transported to ARDTOE and setup for broodstock spawning trials in spring 2012. In the next phase Intravision plan to develop this control software further, but we will also evaluate the possible use of a 0-10 V or 1-10V control system to make the lamps system more easily used by companies that already have a computer control system running their light regimes. The design of the lamps are the intellectual property of Intravision, although the other SMEs share in the environmental data generated by the trials. Results may be published in trade magazines or peer reviewed publications at the SME partners discretion.

4. Protocol to minimise disturbances for the induction of natural spawning in broodstock of selected flatfish species.
Sound is the least studied physical factor in the culture environment. While it is known that some species like Atlantic cod use sound as part of their mate choice process, almost no research has focused on ensuring that this key part of the spawning ritual is allowed to occur without being masked/interrupted by abiotic disturbances. During the first trials of the project, background sound levels within the tank environment were low and generally were around the reported auditory thresholds reported for cod. However, it was clear that routine husbandry practices could create clear sound disturbances within the typical tank environment. Secondly, during the spawning season of spring 2011, cod vocalisations were recorded within the broodstock facilities of Ardtoe to benchmark the frequency range and volume which are used by the stock, providing a useful benchmark of the natural communication bandwidths that the species use. Finally, short term exposure trials demonstrated that sound in the frequency and volume ranges that can be experienced in the tank environment can incite a short but significant stress response. Together these findings will help to shape modified protocols for minimizing disturbances at not only broodstock facilities, but at all fish farming enterprises. The data will be suitable for scientific publication, with the permission of the SME partners, but may also be suitable as the basis for a workshop or similar activity.

5. Quantification of benefits obtained, from improved egg quality by utilizing new broodstock management protocols.
During the reproductive season of 2012 “Natural temperature group (ambient)”, numerous batches with fertilised eggs were produced. Several batches of fertilised eggs were incubated, hatched and used for ongrowing successfully. Although there were no replicates in this experiment, the results give a strong indication that relative low temperatures, compared to conventional temperatures, can lead to successful reproduction of G1 soles. The mechanism of temperature effects on reproduction and fertilisation success in sole remains to be investigated because it is not clear yet why the more extreme temperature regime was more successful than conventional regimes at which captive wild common sole reproduces routinely. This has the potential to be valuable information for sole producers and may result in a future publication.

6. Egg quality detection package (hardware and software) - prototype (EQuaP)
The aim of this portion of the project is to take manual blastomere quality scoring criteria and translate these into simple empirical criteria that can be applied using digital image analysis software which could ultimately allow for the automation of the quality scoring methodology. A list of chosen digital assessment parameters is finalised and their performance as a quality score in relation to the established methodology and actual measured embryo/batch quality needs to be validated. When completed, the system and the novel aspects of the technology employed will be evaluated as to whether some or all of the system is patentable. Certain aspects may be published in scientific peer review publications, as long as this does not encroach on the intellectual property of the owners.

7. Outline future breeding plans regarding selection of broodstock on the basis of behaviour.
The most promising results from this avenue of research include the improvement of reproductive output and hatchery management protocols by including long(er) periods of conditioning of future parents in order to develop a higher and more stable productivity, resulting in the potential of selection with natural mating of parents. Also, to reduce costs of breeding programs with natural mating species and long term conditioning of selection candidates, effects on accuracy of breeding value estimation for different factors are now analysed for such situations using stochastic simulation. Factors considered are: male-female ratios in the parents, number of tested sibs, number of selected parents and levels of heritability.


Summary
During the final project meeting the consortium identified the following areas as valuable from either a commercial or scientific standpoint and worthy of follow-up work;
• Artificial protocol for the induction of spawning in Senegal sole through hormonal and photoperiod control, publication
• Stress disturbances and their effects on spawning, publication
• General broodstock husbandry of turbot and cod, publication
• Image analysis tool for egg quality, publication as well as a good candidate for follow-up work to further develop a hardware/software package
• Mate choice in solea solea, publication
• Effects of dawn and dusk on Senegal sole, publication
• Further development of the shallow raceway system, publication and further research

List of Websites:
prospawn.akvaplan.com

Related information

Contact

Erik Vikingstad, (Senior advisor)
Tel.: +47 98227798
Fax: +47 55 23 24 95
E-mail
Record Number: 189136 / Last updated on: 2016-09-15