CINEMar/Open Ocean Aquaculture Annual Progress Report for the period 1/01/03 through 12/31/03

Principal Investigator: Larry Buckley, Dave Berlinsky, George Nardi

I. Accomplishments

A. Scheduled Tasks

1. Provide healthy embryos to OOA investigators and others involved in culture and research of Atlantic cod and haddock.
2. Develop methods for induction of ovulation and for strip spawning of wild and domesticated cod broodstock
3. Develop an effective method for cryopreservation of cod sperm
4. Efficiently produce juveniles for stocking in the OOA pens.

B. Progress on Tasks
1) Provide healthy embryos to OOA investigators and others involved in culture and research of Atlantic cod and haddock.
Cod and haddock broodstocks were maintained at the Narragansett Laboratory through the reporting period. The established brood stocks were supplemented with collections of cod and haddock from the setline fishery out of Chatham, MA. Broodstock numbers were maintained at about 40 fish of both species. Mortality through the period was low. Photoperiod and temperature were maintained on a schedule previously found to be effective for production of high-quality eggs over an extended season in these species (Buckley et al 2000). A number of haddock brood fish were used in studies of hormone induction of spawning (below). Natural spawning of cod commenced in the beginning of December and extended through early April of 2003. Spawning of haddock was delayed apparently due to the cold winter. Presently, the Narragansett Aquarium has no provision for heating the large broodstock tanks. Incoming water was as low as -0.4°C. Water temperature in both brood tanks was below 4°C for much of the time between mid January to mid March (56d). Natural spawning of haddock commenced in mid March and only extended through the first week of April. Eggs were collected daily with egg collectors placed in the tanks and effluent streams.

Egg production and egg quality were monitored over the production period. Egg quality for cod was excellent with some batches exceeding 90% viable hatch. Egg quality in haddock was poor through much of the season with low % buoyant eggs and low fertilization rates. Embryos of both species were provided to OOA investigators and others working on a variety of projects related to aquaculture and sustainable fisheries. Small numbers (hundreds) of cod and haddock juveniles were produced at the Narragansett Laboratory as part of a variety of studies related to larval and juvenile feeding, growth and metabolism. Juveniles produced at the Narragansett Laboratory tested negative for nodavirus.

Replicated experiments were completed on the response of cod larvae to photoperiod and feeding level. Optimum photoperiod depended on developmental stage. Young larvae did best on intermediate photoperiod (14.5h), while older larvae did best on continuous light.

2) Develop methods for induction of ovulation and for strip spawning of wild and domesticated cod broodstock
Ovarian tissue was sampled from wild cod and captive haddock. Fish with fully developed follicles, determined by biopsy, were held in 2-m diameter tanks and implanted with a 95% cholesterol and 5% cellulose pellet containing 150 ug Gonadotropin releasing hormone analogues (des-Gly10 ,[D-Ala6] LH-RH Ethylamide) or a salmon gonadotropin releasing hormone analogue (D-Arg6-Trp7-Leu8-des-Gly10 LHRH ethylamide; Syndel International. Inc, Vancouver, B.C. Canada). The dose was based on earlier experiments with cod. Other fish were not implanted to serve as controls. Ovarian development was monitored by catheterization until ovulation occurred. The approximate time of ovulation was verified as the time when the eggs flow freely from females given slight manual pressure on their abdomens. The eggs were fertilized with the sperm from one male using the “dry” method as previously described (Berlinsky et al. 1996). Large volumes of viable cod and haddock eggs were obtained using this procedure.

3) Develop an effective method for cryopreservation of cod sperm
During the natural spawning season (December, 2002), 19 male cod were collected by otter trawl off the coast of New Hampshire and brought to Great Bay Aquaculture (GBA). Semen from spermiating males was obtained by abdominal massage, collected in 3-ml syringes, and kept on ice prior to cryopreservation experiments. Sperm cell density, osmolality seminal plasma, and spermatozoa viability and motility were determined.

Refrigerated Storage - Triplicate samples of cod sperm were stored neat or diluted 1:1, 1:2, 1:3, 1:5, 1:10 in the modified Mounib’s extender containing 50 IU/ml penicillin/streptomycin (Mediatech Cellgro, Herndon, Virginia). The sperm samples were transferred to labeled, 1.5-ml polypropylene microcentrifuge tubes (Fisher Scientific, Pittsburgh, PA), and refrigerated at 3°C. At 2-day intervals, 5 ml of each sample was removed by pipet, activated, and examined for motility as described above.

Cryopreservation Experiment 1 - Cod sperm were frozen using one of three extenders (Table 1), two DMSO concentrations (5 and 10 %), two dilution ratios (1:1 and 1:3), and two freezing rates (-5 and -10°C min-1) and frozen in liquid nitrogen. At 30 days post freezing (DPF) and again at 90 DPF, spermatozoa samples were immediately activated and motility was determined.

Cryopreservation Experiment 2 - The results from experiment 1 indicated that the highest post-thaw motility for cod sperm was achieved using MME. This extender was used to dilute cod sperm (1:3) to evaluate two cryoprotectants (10% DMSO and 10% glycerol) and two freezing rates (-1 and -5° C min-1). Motility and viability were assessed 5 and 30 DPF.

Fertilization - Freely flowing, ovulated eggs were examined with a dissecting microscope (25X magnification) prior to fertilization trials for confirmation of normal cytological appearance. Frozen and fresh cod sperm from the same individuals were compared. After 4 h of incubation, fertilization success was determined by examining a minimum of 100 eggs and enumerating those undergoing cleavage.

4) Efficiently produce juveniles for stocking in the OOA pens.
While GBA’s entire production of cod, as last reported (2002 report), was lost due to an outbreak of nodavirus at the hatchery, GBA researched the virus and management strategies being used by other commercial marine hatcheries to prevent its further introduction within the facility to develop a plan for continued production. Our findings indicated that the following changes would be necessary:

• Increase seawater disinfection UV dose
• Use ozone to disinfect fertilized eggs
• Rear fish at colder than optimum temperatures
• Develop nodavirus screening protocol

In order to minimize the introduction of nodavirus into the hatchery seawater for the production of juvenile cod, GBA undertook significant upgrades to its disinfection of incoming and recirculating seawater supplies to the culture systems. New UV units capable of dosing the water at 200,000 mws were installed. Fertilized eggs were disinfected with ozone at levels shown to kill the virus if present on the surface at 1.5 mg/l for 90 seconds. In addition, samples from egg to 5 g juvenile were periodically screened.

Rearing temperatures were also held at 10°C with chillers and an area reefer system to prevent any spikes in temperature, which seems to be one of the triggers to activate the virus. While 14°C has been shown to be the optimum for cod growth it is very close to temperatures known to have been present during a nodavirus outbreak (~15°C and higher). Therefore, our target was set at 10°C, with an upper limit of 12°C.

All contributing parents were sacrificed and their CNS tissue, eggs and sperm were tested for the virus. As the embryos developed in the incubators, samples were preserved in RNA later for further screening by PCR along with hatched yolk sack fry. If these proved to be negative, they were stocked into the larval culture tanks and testing continued by screening selected larvae at day 25 and again after the first grade at about day 65-70. Finally, prior to leaving the hatchery a full health inspection was performed along with viral screening by both PCR and cell culture.

These measures appeared to pay off as our first production in January 2003 resulted in over 250,000 5g juveniles and again in the May production the hatchery produced 130,000 5g juveniles. A total of 40,000+ juveniles were provided to the OOA project from the January production for eventual stocking into the OOA project’s 3000m³ Ocean Spar submersible Sea Station cage. Thirty three thousand were initially placed into near shore cages in the spring of 2003. During August the juveniles weathered an outbreak of Vibrio, but lost a few thousand fish. GBA provided the project additional fish to bring the numbers back up above 30,000 at that time. The fish were stocked from the nursery cages into the Sea Station offshore during the fall of 2003.

C. Important Results or Findings
Viable cod and haddock eggs were produced by both natural spawning and hormone induction of wild and captive fish. Replicated experiments with cod larvae using different photoperiods, indicated that the optimal photoperiod varied with development. Contrary to common aquaculture practice, a brief dark period favored growth and survival of young larvae.

In each sperm preservation experiment, the highest post-thaw motility was achieved using the modified Mounib’s extender. Higher post-thaw motility was also achieved using a 1:3 dilution ratio and 10% DMSO. When a -5°C min^-1 freezing rate was used, higher post thaw motility was attained using DMSO rather than glycerol. There was no significant difference between eggs fertilized with fresh or post-thaw frozen sperm frozen in the modified Mounib’s extender (p < 0.05). The percent spermatozoa observed motile was highly correlated with those incorporating SYBR 14® stain (Adjusted R² = 0.94; R = 0.97; p < 0.05).

GBA made considerable progress in optimization of procedures fro production of juvenile cod. Over 250,000 juvenile cod were produced at GBA.

D. Difficulties Encountered
Unlike last year, nodavirus did not compromise production of cod juveniles. The cold winter temperature apperared to compromise haddock egg production at the Narragansett Laboratory. The facility will be modified in the coming year to facilitate heating of the broodstock tanks.

E. Anticipated Success in Meeting Project Objectives on Schedule
All objectives under the original proposal were met. We anticipated that we will be able to meet the new project objectives.

F. Reports, manuscripts, and presentations resulting from the project
DeGraaf, J.D. and D.L. Berlinsky. Cryogenic and refrigerated storage of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) spermatozoa. Aquaculture (in review).

Peck, M.A., Buckley, L.J., and Bengtson, D.A. (2003) Energy losses due to routine and feeding metabolism in young-of -year juvenile Atlantic cod (Gadus morhua). Can. J. Fish. Aquat. Sci. 60:1-9.

Peck, M.A., Buckley, L.J., O’Bryan and A.E. Lapolla. Efficacy of egg surface disinfectants in captive spawning Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus). J. World. Aquacultuew Soc. (in review)

II. Tasks and Activities for Next Reporting period

A. Tasks for the next reporting period
Although all three groups will continue to receive funding from OOA for related activities, GBA and URI will be funded separately with GBA working on cod and URI working on haddock.

1. The group in Rhode Island will concentrate on haddock broodstock management and production of haddock embryos, larvae and juveniles.
2. The group at UNH will continue to work on hormone induction of spawning in both cod and haddock.
3. GBA will concentrate it efforts on cod broodstock management and juvenile cod production.
4. All will continue to work on disease prevention and management in marine aquaculture.

B. Brief work plan to accomplish tasks

1. Haddock broodstocks will be maintained at the Narragansett Laboratory through the next spawning season and egg production and quality monitored. Since nodavirus is present in the environment and there is presently no reliable non-lethal test for its presence in cod and haddock, we will implement several measures to minimizing its impact on larval and juvenile production. These will include UV treatment of incoming and recirculated water.
2. Additional haddock will be collected to establish to broodstocks of 40 to 60 fish each. The aquarium systems will be upgraded to allow heating of the broodstock tanks.
3. Additional haddock will be collected for further optimization of methods for hormone induction of spawning.
4. Experiments will be completed to optimize methods for juvenile haddock production.

C. Anticipated concerns or difficulties
Haddock is a more difficult species to work with than cod. The adults are more sensitive to the stress of capture and confinement. We are very concerned with the possibility of an outbreak of nodavirus and will be taking a number of measures to minimize the chances of another outbreak.

III. Expenditures
Expenditures were within the expected range for the work accomplished to date.

References
Berlinsky, D.L., King V, W., Smith, T.I.J, Hamilton, R.D., Holloway, J. and C.V. Sullivan. (1996) Induced Ovulation of Southern Flounder (Paralichthys lethostigma) Using Gonadotropin Releasing Hormone Implants. Journal of the World Aquaculture Society 27 (2) 143-152.

Buckley, L.J., T.M. Bradley, and J Allen (2000) Production, quality and low temperature incubation of eggs in Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus). J. World Aq. Soc. 31:22-29.