CINEMar/Open Ocean Aquaculture Annual Progress Report for the period 1/01/04 through 12/31/04
Principal Investigator: David L. Berlinsky
I. Accomplishments
A. Scheduled Tasks
1) Identify the time course for the stress response in juvenile Atlantic cod 2) Evaluate the growth and survival of juvenile cod reared in an offshore nursery cage 3) Investigate the potential benefits of feeding diets containing immunostimulants to juvenile cod prior to transfer to inshore net pens
B. Progress on Tasks
1) Identify the time course of the stress response in juvenile Atlantic cod:
During the first year, two assays were developed and validated (cortisol and glucose) for quantifying the stress response in cod. Several experiments were conducted to identify the time course of the stress response to and recovery from a single standardized stressor. Additionally, routine hatchery procedures such as handling, confinement and transport were evaluated for their ability to elicit a stress response. Results of these studies are important for the proper design of future experiments aimed at developing mitigation strategies to effectively precondition cod to net pens. A third technique to extract cortisol from homogenates of whole fish is being validated for use on cod less than 30 g. This methodology will allow us to conduct experiments on larvae and small juveniles since the fish cannot provide the quantity of blood needed.
2) Evaluate the growth and survival of juvenile cod reared in an offshore nursery cage:
On July 7, 2004 12,500 fish were stocked into an 600 m3 offshore cage. The fish were harvested on September 13, 2004.
3) Investigate the potential benefits of feeding diets containing immunostimulants to juvenile cod prior to transfer to inshore net pens:
Approximately 12,500 fish (5.0 + 0.3 g) were placed into one of 4 nursery cages under the pier at the Coastal Marine lab on June 5, 2004. Two cages were fed a control diet and 2 cages were fed an INVE diet with an immunostimulant package that they had been eating in the hatchery. Initial stocking water temperature was 8° C but rose to 15° C by the end of June. Ranges in diurnal water temperature readings of 4° C were common. The fish were fed daily to satiation. The study was terminated on September 22, 2004. Body weight was 16.9 + 2.5 g at termination, nearly 40% larger than the offshore cohort. Only 16% of the initial number of fish stocked were present at the end of the study. There was no evidence of mass mortality (carcasses in the net pen). Poaching or bird predation is suspected. Growth data was collected and a hypersalinity stress test was performed twice during the study period. Whole cod were also collected at these times for analysis of cortisol.
C. Important Results or Findings
1) Identify the time course of plasma cortisol and glucose following an acute stressor:
Approximately 75 juvenile cod (41 + 6.2 g = body weight; 175 + 7.4 mm = total length) were transported in a tank containing oxygenated seawater (10° C) from the Great Bay Aquaculture Facility at a density of ~95 g/L. Just prior to transport, a separate group of undisturbed fish (n = 8) were rapidly captured (< 5 seconds) and placed into a metomidate bath (6 mg/L). Metomidate has been shown to inhibit the cortisol response to stress in other species. Once sedated, these fish (pre-stress) were bled using a 26 gauge needle and a heparinized tuberculin syringe. Their cortisol levels were considered as the baseline or “resting cortisol” level for this population of juvenile cod. Upon arrival at UNH, the fish were removed from the tank in groups of 8, held in a net for 30 seconds and then placed into one of five - 75 L aquaria covered with black plastic sheeting and containing 40 L of filtered, aerated seawater in a constant-temperature room (9° C). At 0.5, 1, 3, 7 and 24 hr after the net stressor, all of the individuals in one tank (n = 8) were rapidly captured and bled as described above. All plasma was collected and stored at -20° C. Plasma was analyzed for cortisol using an ELISA validated for use with cod plasma. Plasma glucose was analyzed using the hexokinase method. Results indicate that plasma levels of cortisol and glucose follow a response and recovery profile similar what has been described for other fish species exposed to similar stressors (Figure 1).
Effect of transport from the Wentworth net pens to GBA on plasma cortisol levels:
All of the juvenile cod (26 + 5.3 g; 151 + 7.0 mm) in the Wentworth Marina net pens were transferred to Great Bay Aquaculture, Portsmouth, NH (GBA). Just prior to transfer and before any disruption of the fish occurred, a net was passed through one of the pens and 8 fish were captured and immediately anesthetized and bled as described above. The remaining fish were netted from the cages and placed into one of five Xactics on the boat. Each Xactic contained fresh seawater, supplemental oxygen and cod at ~200 g/L. After a 20 minute transport by boat to the Coastal marine lab, a crane was used to offload the Xactics from the boat directly onto trucks. Oxygen lines were disconnected for ~5 min at this time. Once loaded, the fish were transported to GBA where they were placed into nursery tanks. Analysis of cortisol levels showed that there was a significant elevation in cortisol that occurred during the capture and transport procedures (post-trans; Figure 2), but levels recovered within 72 hours after the transport. Because of the small volumes of blood that can be recovered from < 30 g cod, there was insufficient plasma available for glucose analysis.
Effect of weighing and measuring procedures
Approximately 100 cod (22 + 4.3 g; 140 + 6.6 mm) were captured from the net pen and placed into 1 of 2 plastic fish totes containing 30 L of seawater (~80 g/L). Just prior to this, fish from an undisturbed pen were collected immediately, anesthetized in metomidate and bled within 3 minutes as described. Cortisol levels from these fish were considered “resting”or baseline” No supplemental aeration or oxygenation was provided. Two totes were filled with water and fish. After 15 minutes, 8 fish were collected from one of the totes, anesthetized and bled as described. Fish for the 30 minute time point were obtained from a second tote which was undisturbed until sampling began after 30 minute. The results demonstrate that cortisol increases very rapidly upon exposure to this procedure.
Identifying changes in cortisol using whole bodies of cod
This methodology has been used to measure cortisol levels in eggs and larvae of several salmonids, but has not been used in any larger juvenile fish. Fish ( < 30 g) were euthanized in MS-222 and then flash frozen in liquid nitrogen and stored at -70° C. In the laboratory, fish were thawed and a wet weight was obtained. The fish were cut into small (3 mm) pieces and added to a volume of phosphate buffered saline that was twice their wet weight. This mixture was homogenized until the contents were completely liquefied. Various volumes of homogenate containing 3 ml of ether with and without added cortisol were vortexed vigorously for 45 seconds. The homogenate (aqueous portion) was snap frozen in liquid nitrogen and the ether was decanted into a clean tube. This procedure was repeated twice. Ether extracts were dried down under a stream of nitrogen in a 37° C water bath. Once completely evaporated, each tube was rinsed with an additional 0.5 ml of ether. Dried extracts were resuspended in 0.9% NaCl and aliquots were subjected to the cortisol ELISA. The assay is currently being validated.
2) Evaluate the growth and survival of juvenile cod reared in an offshore nursery cage:
Approximately 12,500 cod (5.5 + 0.8 g) were placed into an offshore cage (600 m3) on July 7, 2004. Staff access was limited to 1-4 times per week and there was no provision for automatic feeding. It was estimated that about 5% of the fish were lost due to the initial transfer: however, > 90% of the remaining fish survived. Because the cage was submerged in a cold thermal layer (6-9 C; surface = 15-16 C during the same period), and feed was limited, fish grew to an average of 11.8 + 0.9 g when they were withdrawn on September 13, 2004.
Approximately 20,000 juvenile cod (13.1 + 0.3 g) were stocked into custom nursery cages at the Wentworth Marina in Rye, NH in November 2003. Each of the 2 cages (3.6 x 3 m each) had 2 sections. Two sections were stocked with 3500 fish each and 2 sections were stocked with 7000 fish each. Water temperature was 6° C at stocking and reached -1.5° C during the winter. Fish were seen eating at 1° C. The fish were removed on May 6, 2004 and averaged 25 + 1.3 g. Mortality was < 5%. The final density in the low-density cage was 4.8 kg/m3 while in the high density cage it was 9.7 kg/m3. Fish transported to GBA exhibited compensatory growth.
D. Difficulties Encountered
1) Limited availability of animals. There have been some production problems which lead to shortages in juvenile fish for experiments. This was only a temporary problem as we have been able to obtain fish from other sources.
2) It is extremely difficult to obtain the volume of blood required for these studies with cod < 30 g BW. To address this we have developed a whole-body cortisol extraction technique for use on these fish. The methodology will allow us to perform experiments on juveniles and larvae.
3) It appears that the feeding frequency was too low to attain significant growth of fish in the off shore cage. This situation has been rectified with the addition of an automated feeder
E. Anticipated Success in Meeting Project Objectives on Schedule
We anticipate no problems in meeting project objectives in the upcoming scheduled project period.
F. Reports, manuscripts, and presentations resulting from the project
Senior Thesis- Brian Hooper, Marine and Freshwater Biology Major
II. Tasks and Activities for Next Reporting period
A. Tasks for the next reporting period
Continue with stress studies. Determine the effect of sudden temperature and salinity depression on the stress response. Examine the benefits of using anesthetics or gradual temperature and salinity reduction as a means to alleviate stress. Develop a protocol for the handling and transporting of cod that mitigates the stress response.
B. Brief work plan to accomplish tasks
Conduct the proposed experiments on the effects of changes in salinity and temperature and anesthesia type and dose on the stress response. Based on these results, develop protocols to precondition cod during times when they must be exposed to stress (eg: grading, sorting, netting and transporting) strategies for cod). Analyze whole bodies for cortisol content in fish that were fed immunostimulant diets. The whole body cortisol extraction methodology is being validated.
III. Expenditures
Expenditures on the project to date have been in the range anticipated for the work that has been completed.