Deres ref.
Siri Hanson
Hordaland fylkeskommune
Arbeidslag for Verdiskaping i kystsona
Pb 7900, N-5020 Bergen
200601782-2/146.310/SHAN
Vår ref.:
Forsker:
Atle Foss
Telefon:
+47 9829 4071
E-mail:
af@akvaplan.niva.no
Dato:
Bergen, 10.01.2008
Årsrapport PROCOD (Tilsagn BM08-06)
Viser til innvilgede midler fra Hordaland fylkeskommune og tidligere innsendt prosjektplan.
Årsrapport for 2007 med utfyllende beskrivelse av aktiviteter og opparbeidede resultater er
vedlagt.
Med vennlig hilsen
Atle Foss
Senior rådgiver akvakultur
Akvaplan-niva AS
Head Office
Polar Environmental Centre
N-9296 Tromsø, NORWAY
Telephone: +47 77 75 03 00
Telefax:
+47 77 75 03 01
Email: tromso@akvaplan.niva.no
Oslo Office
Ovre Damvei 4
N-2050 Jessheim, NORWAY
Telephone: +47 63 97 82 95
Telefax:
+47 63 97 29 51
Email: oslo@akvaplan.niva.no
Bergen Office
Nordnesboder 5
5005 Bergen
Norway
Telephone/Telefax: +47 55302250
E-mail: af@akvaplan.niva.no
Akvaplan-niva AS
Rådgivning/FoU miljø og akvakultur
Consultancy/R&D environment and aquaculture
Foretaksnr./ Enterprise No.:
NO 937 375 158 MVA
Web: www.akvaplan.niva.no
P79
PROCOD - Statusrapport 2007
Prosjektets overordnede mål er å modernisere og optimalisere produksjonen av juvenil torsk ved å
innta en multidisiplinær tilnærming mot å løse sentrale flaskehalser assosiert med juvenil kvalitet,
vannkvalitet og fiskevelferd i en intensiv produksjon. Prosjektet fokuserer på utfordringer knyttet til
kommersiell skala produksjon av larver og yngel av torsk, og vil integrere biologiske, vannkjemiske,
logistiske og teknologiske faktorer, hvor det overordnete mål er å foreslå anvendbare løsninger for de
adresserte problemer.
Prosjektet er delt inn i 7 hoved ”tasks”
Task 1. Optimalisering av rotatoriekvalitet
Task 2. Optimale startfôringsregimer for torsk – zooplankton vs. rotatorier
Task 3. Interaktive effekter av salinitet, temperatur og genetisk bakgrunn på juvenil kvalitet
Task 4. Transport av torsk
Task 5. Forståelse og forbedring av kritiske vannkvalitetsparametre ved intensiv drift
Task 6. Vannkvalitet og mikrofauna i åpne vs. resirkuleringssystemer
Task 7. Neurogenese i relasjon til fotoperiode hos larver og juvenil torsk
Aktiviteter og sammendrag av resultater i prosjektperioden:
Task 1. Optimizing rotifer quality
Different methods for high density production of rotifers have been developed during the last years,
but all methods have problems with accumulated ammonia and high number of bacteria in the
production water. To optimize the water quality in the tanks different range of water flow-through has
been tested in this project. Rotifers were cultivated in tanks (270 l) with, 0, 100, 200 and 400% water
flow-through per day. The rotifer growth was highest in tanks with 100% water flow-through per day
(Fig. 1). In all tanks with water flow-through bacterial level and ammonia level was lower than in
stagnant cultures.
Fig. 1. Rotifer density in relation to different water exchange rates.
Rotifer can be nutritional enriched by “short term enrichment” and “long-term enrichment”.
Experiment with Brachionus plicatilis fed yeast, Marol E and algal paste, resulted in higher amount of
unsaturated fatty acids in the phospholipids, by using “long-term enrichment. The data indicate a much
higher proportion of DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) omega-3 fatty
acids in the phospholipids in the long time enriched rotifers (Fig. 2).
Fig. 2. Fatty acids proportion in the phospholipids of short time (KT) and long time (LT) enriched rotifers.
Rotifer cultures have a high bacterial load, and one method to clean the culture before use in
firstfeeding tanks can be “algal-cleaning”. In this experiment rotifers were cultivated, washed and
transferred to a cooling tank with algae, algal paste or clear water. Lowest total CFU and lowest
Vibrio CFU was found in tanks added live algae.
In an ongoing study (start up in October 2007) a detailed comparison (culture conditions, nutritional
value, particle size, viability and swimming speed) between two different rotifer species is being
performed (a master study). Detailed results will be given in the next progress report.
Task 2. Optimal start feeding scheme for cod – zooplankton vs. rotifers
In a previously described experiment (see last progress report) the effect of first feeding diet on growth
performance and developmental success of cod larvae was investigated. Larvae grew faster when fed
copepods as a first feed compared to larvae fed on enriched rotifers. This indicated that the maximum
growth potential in cod larvae under intensive conditions is not fully utilized and first feeding diet
could potentially improve growth speed.
Larvae fed on copepods and enriched-rotifers were analyzed for their fatty acid content in whole
larvae or in larvae without gut. Figure 3 shows the n3/n6 fatty acid ratio of larvae fed copepods or
rotifers as a first feed. Copepod-fed larvae have a much higher n3/n6 ratio compared to rotifer-fed
larvae. There is hardly a difference found between larvae that were gutted or not, indicating a very
rapid turnover of fatty acids in larval tissues. This indication is strengthened by the observation that a
change in diet from rotifers to copepods or vice versa results in an immediate change in fatty acid
content in the larvae.
Larvae were weaned onto formulated feed from 36 dph onwards. During weaning the n3/n6 fatty acid
ratio decreases in copepod-fed larvae and slightly increased in rotifer-fed larvae.
Fig. 3. Fatty acid ratio (n3/n6) for whole or gutted larvae: starved (sult), yolk larvae (yolk), larvae fed on
copepods (zoo), rotifers (rot), or larvae that were changed in diet from rotifers to copepods (RZ) or vise versa
(ZR).
Task 3. Interactive effect of salinity, temperature and strains on juvenile quality
Juvenile cod were exposed to two temperatures (10°C or 14°C) and two salinity levels (15‰ or 32‰)
for three months and the effect of temperature and salinity on growth performance and physiological
response were measured. Over the total experimental period the fish exposed to reduced salinity
(15‰) showed a significantly higher specific growth rate at 14°C indicating an energetic advantage of
reduced salinity in cod juveniles, but no effect of reduced salinity was observed at 10°C (Fig. 4).
Fig. 4. Mean SGR (±SE) for the 4 treatments during 4 consecutive periods and the total. Different letters denote
significant differences within the period (SNK post hoc test)
The energetic efficiency between the different treatments was determined by measuring enzyme
activities in both muscle and liver tissue. The enzymes measured were Lactate Dehydrogenase (LDH)
as indicator of the anaerobic metabolism, Malate Dehydrogenase (MDH) as an indicator for the
aerobic metabolism and Aspartate Aminotransferase (AAT) as an indicator for protein turnover.
Both the anaerobic capacity (Fig. 5) as well as the aerobic capacity (Fig. 6) of muscle cells increases
with increasing fish size. A stagnant or decreasing trend was observed in liver cells.
Fig. 5. Concentrations of Lactate Dehydrogenase (units / gram tissue), at three consecutive sampling days in four
treatments and two tissues (muscle and liver tissue). Different letters denote significant differences between
treatments within sampling date (SNK post-hoc).
The concentration of both LDH and MDH is at a higher level in liver cells at the onset of the
experiment and no large changes occur as the experiment progresses. MDH shows a slight reduction in
liver cells over time. However, the concentration of these two enzymes increases rapidly in muscle
cells.
No major differences or trends between treatments were found and the effect of reduced salinity seems
to have no major impact on these two specific enzyme concentrations, while the increased growth rate
indicates higher energy efficiency in the 15‰, 14°C treatment.
a bc c ab
a
a ab ab b
b b b
Fig. 6. Concentrations of Malate Dehydrogenase (units / gram tissue), at three consecutive sampling days in four
treatments and two tissues (muscle and liver tissue). Different letters denote significant differences between
treatments within sampling date (SNK post-hoc).
Task 4. Transport of Atlantic cod
Experimental start was planned in March 2007 at Fosen Aqua Senter AS. Fosen Aqua Senter AS went
bankrupt in March 07, and planned experiments had to be postponed and relocated to Marine Harvest,
Tustna. Experiments will be performed during October/November 07 Juvenile cod (75 g) will be used
in a simulated open transport study at 3 densities. In November 2007 juvenile cod will be used in a
simulated closed transport at the same location. A full scale study of water quality and stress during
commercial transport(s) of juvenile cod in a truck and a well boat will be carried out during second
half of 2007 and first half of 2008. During spring 2007, a technical investigation of two well boats has
been carried out. This to understand the dynamics of water quality changes during open and closed
systems. A risk for supersaturation with nitrogen was discovered during loading of water into the well,
and the start of circulation pumps. This is due to air inside the pipes (circulation systems) that is
pressurized and has limited possibility for escape through the hatches in the deck. A solution with
degassers in the hatches was found to be an effective solution to get rid of the supersaturation before
fish is loaded onboard the vessel.
This information will be useful during the experiments that will be carried out in October/November
2007 and is completely new knowledge for the transporters. The results are therefore already
published in by Rosten et al (2007) in the book “Vannkvalitet og smoltproduksjon” edited by Vilhelm
Bjerknes. The figure below shows the results from one wellboat.
Fig 7. The development in total gas pressure, nitrogen super saturation and oxygen saturation at filling, start and
degassing of well water. A degasser (type: “Snippen”) was used in the hatch to degas the excessive nitrogen
during the loading process. It took approximately 2 hours to degas a well of 330 m3 with this type of degasser.
Task 5. Improving and understanding critical water quality aspects in rearing of juvenile cod
The first attempts to perform aorta cannulation of cod were done in June 2006 (see previous progress
report). The technique will be used to measure gasses, ions and stress parameters in the blood of freeswimming cod at rest exposed to varying concentrations of oxygen and different water treatments
common in cod aquaculture. At present, we use a protocol developed successfully for Atlantic salmon,
but instead of performing a “head-aorta” cannulation we have so far only succeeded by a ventral
lateral aorta cannulation. We therefore have to adjust the procedures to be able to perform head-aorta
cannulation as planned in our experiments. A project employed PhD student, Anders Karlsson, started
in medio September 2006. In the reporting period, he has taken a courses in statistics related to
experimental work and a course being licensed to work with animal experiments (Forsøksdyrlære).
The experimental laboratory at NIVA Marin Research Station Solbergstrand (NIVA-MFS) has been
partly upgraded and new equipment for anaesthesia and monitoring of blood gas pressure have been
purchased. The PhD candidate has started to build procedures for cod cannulation based on
experiences from the Atlantic salmon cannulation. Contact with groups in Iceland (Dr. Helgi
Thorarensen) and France (Dr. Jean-Charles Massabuau) have been established. The PhD candidate has
been working on the procedures for cod cannulation. So far, a “blind” cannulation method seems
promising, as the “Atlantic salmon cannulation method” is difficult to perform due to anatomical
differences between the two species.
International field work
Anders Karlsson had in the period September 17 – October 17 2007 a one month stay at Holar
College, Island, to work with the group of Dr. Helgi Thoraresen (Head of Department) on cannulation
of marine fish species. In the period September 28 to October 5, 2007, Dr. Jean-Charles Massabuau
(France) and Bjørn Olav Rosseland joined in the field work, and comparative methods for cannulation
of cod was be performed. Water quality parameters were analysed according to protocols.
Plan for next period



Further develop the manual for cod cannulation, and continue experiments with oxygen.
Start experiments with UV/ozon and metal speciation in seawater.
Participate in transport experiments with NIVA.
Task 6. Water quality, survival, growth and cannibalism: comparison of open and recirculation
systems
The experiment was started in early November 2006 and terminated 7 February 2007. In this
experiment two different water qualities were tested and compared. WQ1 was recirculated water
treated in a TMC Marine 5000 filter system. The WQ2 was water sampled from 150 m depth in
Bjørnefjorden and pumped to the tanks via filter (approximately 25 µM) and heat exchanger to
stabilise water at 12 °C. Preliminary conclusions are that the recirculation fish did not tolerate the
transfer from a recirculation system to a system receiving water from an open source, probably due to
non-resistance to open water bacteria.
During the first phase of the experiment, no differences in growth or mortality were registered. An
average survival rates for all 24 experimental units was approximately 35% from onset of first feeding
to day 55 after first feeding. During this period water content of total ammonia was low in both
systems. At day 50 post first feeding, the fish were weaned onto a commercial dry food (Aglonorse 2,
300-500 um) for one week, and then redistributed between the two water qualities. High mortalities
were observed in all groups except for the group that remained in the open water system. The
mortality may probably be due to bacterial infection that had developed in the recirculation system, or
by a non-biotic toxicant that have built up. The results from water quality assessments are not yet
ready, and will probably give firmer indications. The second phase of the experiment was terminated 2
weeks after redistribution due to few survivors in a number of the tanks. In this period increased
aggression/cannibalism was not registered as expected. Final analysis of the main findings will be
given in the nest progress report.
Task 7. Neurogenesis in relation to photoperiod in larval and juvenile cod.
Development of a protocol for visualizing instantaneous cell proliferation (neurogenesis) in the brain
of larval marine fish by immunocytochemical markers has been carried out autumn and winter 2006.
The work is done at Wellesley College, Massachusetts, USA under supervision of professor B. Beltz.
Fish larvae (summer flounder, cobia, and cod) have been provided by Great Bay Aquaculture Inc
(GBA). The established protocol showed good immunofluorescent staining of BrdU-marked cells at
surfaces of whole-mount specimens or 100µm Agar-embedded tissue sections. Cell proliferation
occurred mostly adjacent to the ventricles between the two brain hemispheres and between the
different brain compartments (Fig. 1). However, penetration of the antibodies into the neural tissues
was weak, and a reliable method of quantification in 3D of newborn cells through the whole tissue
specimen could therefore not be established. This problem may be solved by exploring dehydration
and subsequent rehydration techniques for better penetration, or by applying different embedding
media that enables thinner sectioning. It should be noted that the antibodies for other neurological
signal compounds (e.g. serotonin) did not show a similar penetration problem. It was therefore not a
problem to mark serotonergic active cells, even in whole mount larvae (Fig. 2).
Further, in accordance with the project plan, a photoperiod experiment was carried out during spring
2007 for cod larvae at GBA. Due to the limitations set by quantification of neurogenesis, it was
decided to use the 2D area of the eye disc for quantification of cell proliferation (Fig 3). The BrdU will
mark all newborn cells in all tissues during the BrdU incubation, including the eye disc. Two 2.5m3
tanks were used, one with photoperiod of 8h dark and 16h light, and the other with 24h light. About
30250 Atlantic cod larvae of same origin were released in each of the tanks at day 1 posthatch, and
treated according to standard rearing protocols at GBA (including use of green water and enriched
rotifers fed in three batches per day). Feeding time was chosen so that the larvae of the photoperiod
tank would be able to feed at least one hour before the dark period started. Diurnal BrdU incubation
was carried out at day 11, 21 and 32 posthatch. Seven samples were incubated during the 24h
sampling period, and an incubation 3.5h was used for the days 11 and 21, while an 1h incubation was
used for day 32. To maintain light and temperature conditions during incubation, the larvae were
incubated in 400 ml opaque plastic containers floating in the respective rearing tank. Only
proliferation rate and eye disk area of the day 11 samples have so far been determined, together with
eye size, standard length (SL), and wet weight (WW) of 32-day-old larvae. The cell proliferation rates
of the day 21 and 32 samples are in progress.
The results in 11-day-old larvae show that eye disc cell proliferation rate in the photoperiod group
seems independent of the photoperiod itself, but is significantly lower than in the continuous light
group. Eye size at day 11 was not different between the treatments. However, at day 32 the
photoperiod-exposed larvae have developed significantly smaller eyes at similar larval size when
compared to 24h-exposed larvae (Fig. 4). Larval size was also less in the photoperiod treatment, but
only significant for SL. The data suggest that instantaneous cell growth is an excellent tool to assess
immediate effects of extrinsic factors on growth, effects that normally would need to accumulate over
a longer period (and hence would need chronic exposure) to be detected by e.g. standard body
morphometrics.
Welfare studies
During winter/spring 2007, the incidences of spinal deformities and welfare of two groups of cod has
been evaluated in different experiments by combining X-ray, behavioural and physiological
measurements. The two groups were transferred from Bergen in December 2006 and had previous
been fed on either rotifers or zooplankton during first feeding stages.
The objective was to examine possible links between rearing conditions during early life stages and
deformities, boldness behaviour and physiological stress coping in juvenile cod.
Growth study
A couple of weeks after arrival in Tromsø, the fish was marked (pit-tags), weighted and length was
measured. The fish from both groups (rotifers or zooplankton) were then kept in the same tank for
about 4 weeks. At the end of the period, weight and length were measured again. The results showed
that the zooplankton group was slightly larger than the rotifer group at the start and that this difference
further increased during the course of the trial. Thus, the zooplankton group seems to have a higher
scope for growth. However, it is possible that the differences in growth rate could be linked to
differences in rearing conditions before arrival to Tromsø and this need to be elucidated.
Evaluation of spinal deformities
All the fish in the two groups (totally 96) were X-rayed soon after the arrival in Tromsø (Fig. 8). The
X-ray study showed low incidences of severe spinal deformities in both groups, but 17 % and 9 % of
rotifer and zooplankton fed fish, respectively, showed some sort of smaller or larger disruption of the
spine or head region. Thus, there were 50 % more deformities in the rotifer group compared to the
zooplankton group. The occurrence of deformities was not related to present size and did not affect
subsequent growth. A majority of spinal deformities were found in the area around vertebrae 10-13,
and 17-23, the former area above and the latter area towards the end of the swim bladder. The mean
vertebrae length did not differ between the two groups.
Fig. 8. X-ray of deformed fish.
There were indications of differences in swim bladder size between the two fed-groups. Possible links
between swim bladder size and spinal deformities will be evaluated. The low incidence of deformities
in this fish material made it impossible to study the welfare consequences of being deformed. Thus,
this remains to be done.
Evaluation of boldness behaviour
During spring 2007, the boldness behaviour was examined in a sub-sample of individual fish (18 and
14 rotifer and zooplankton fed fish, respectively) in a two-chamber tank with a automatic video
tracking system (Ethovision). Preliminary results indicate that there is a difference in behaviour
between the zooplankton and rotifer fed fish (Fig. 9). In this study the boldness of the fish was
investigated by measuring how fast the fish was willing to swim from a dark and secluded to an open,
risky chamber. Fish with high boldness motivation tend to adapt quickly to new challenges. In this
study, the fish in the zooplankton group used shorter time before they entered the open chamber, and
thus, seems to be bolder than the fish in the rotifer group. There were no differences in swimming
speed or duration out in the open field between the two groups of fish.
Fig. 9. Boldness of juvenile cod startfed with zooplankton or rotifers.
Physiological stress coping test
A physiological stress coping test was done by use of a swim tunnel. Three groups of 10 fish from
each of the two groups were used. The groups were subjected to a 22-hour respirometric stress
challenge test where oxygen consumption rates (MO2) was monitored continuously. On day 1, the fish
was bulk-weighed and introduced into the swim-tunnel respirometer (Loligo Systems). On day 2, the
fish was subjected to a standardized acute handling stress, which consists of the fish temporarily being
netted out of the respirometer and hold in air for 30 sec. The fish was sacrificed 4-hours post-stress
and sampled for measurements of plasma cortisol, glucose, lactate, osmolality, blood gas tensions and
pH. Treatment groups was also compared with respect to rate of decline in MO 2 following
introduction to swim tunnel, pre-stress MO2 level, and changes in MO2 post-stress. The physiological
stress coping tests demonstrated a markedly increased oxygen consumption rate in response to acute
handling stress for fish belonging to both feeding groups. Preliminary (non-processed) data for this
parameter suggest no obvious differences between groups, but statistical processing of the data is
needed before conclusions can be made. Similarly, all blood samples have been analysed, with
processing of data being in progress.
Prosjektgruppen har i perioden hatt møter ved Univ. for Miljø- og Biovitenskap på Ås og ved
SINTEFs fasiliteter i Trondheim. Neste møte er planlagt i mai 2008 På Austevoll.
Samarbeidet har så langt fungert utmerket, og ingen problemer med gjennomføring av
prosjektet har så langt hverken oppstått eller er forventet å oppstå, og prosjektet er således i
rute.
Bergen 10/01-2008