OOA Progress Report for the period 1/01/01 through 12/31/01

Principal Investigator: Dr. David Berlinsky and Dr. David Fredrikkson

I. Accomplishents

A. Scheduled Tasks

• Determine optimal time of feeding and optimal feeding frequency.
• Document the presence of food anticipatory activity in juvenile Atlantic halibut.
• Utilize acoustic and light stimuli to condition Atlantic halibut to the presence of food.

B. Progress on Tasks
Experiment 1- Optimal Time and Feeding Frequency
Five hundred juvenile halibut (5-10 g) were purchased from R and R Finfish Development Company (Nova Scotia, Canada) and held at the Coastal Marine Laboratory (Newcastle NH) prior to experimentation. During the feeding experiment the fish were held in 100 l, circular tanks incorporated in a recirculating sea water system, housed in the animal care facility in Rudman Hall (UNH). Water temperature was held at 13C + 1C and 24 hour artificial lighting (200 Lx). Water quality parameters (pH, temperature, salinity, ammonia, nitrite, alkalinity, and dissolved oxygen) were monitored daily.

Fish were fed to satiation once (0900), three times (0900, 1430 and 2000 hours), and five times (0900, 1200, 1500, 1700, 2000 hours) with three replicates of each treatment (20- 20 g fish/replicate daily). A pre-weighed quantity of feed was dispersed by hand, to each tank, and the amount consumed was determined by subtracting the quantity of uneaten feed. At 21-day intervals the fish were netted, weighed (0.1g) and measured (0.1mm). Average weight, total feed consumption and feed consumption at each feeding period, were calculated. Feed conversion ratios (FCRs) were also calculated as: total dry diet fed (g)/total wet weight gain (g). Differences between means (p<0.05) were determined by a one-way ANOVA using Systat 10.

Experiment 2-Food Anticipatory Activity (FAA)
Fish were fed once daily by automatic feeders and the activity level of juvenile halibut was monitored under 1) constant lighting (24L:0D) and 2) a 12 hour light:12 hour dark (12L:12D) photoperiod. The light phase was initiated at 0600 hours. Ten juvenile halibut (100-120g) were housed in a cylindrical tank (208 L), maintained at 10C, and were continuously monitored for 72h using a video monitoring system. Fish movement was quantified using surveillance software (BTVpro) and data was stored in an Excel file. The data were analyzed by one-way ANOVAs using Chart 4.01b and Systat 10. FAA was characterized by elevated pre-feeding activity relative to baseline.

Experiment 3- Operant Conditioning
The acoustic stimulus apparatus was designed and constructed (see Appendix 1 below). Testing on halibut will begin in January, 2003.

C. Important Results or Findings
Experiment 1- Optimal Time and Feeding Frequency
Mean fish weights for each feeding treatment over the 84-day period are shown in Figure 1. There was a significant difference in mean weight for fish fed once and five times daily. Total food consumption during the experiment differed significantly between fish fed once and five times daily (Table 1). There was no significant difference in FCR's among treatment groups (Table 1).

Experiment 2-Food Anticipatory Activity
A representative recording of swimming activity for fish in the 24L:0D and 12L:12D photoperiods are shown in Figures 2a and 2b, respectively. A significant increase in activity in the 24L:0D occurred only during feeding. Fish maintained on the 12:12 photoperiod regime displayed greater activity prior to feeding, but this was not significantly different from baseline levels as determined by ANOVA. FAA was not detected under either of the photoperiod regimes.

D. Difficulties Encountered
A second feeding experiment was initiated in August, 2002. This experiment examined the effect of feeding frequency of fish fed once (2100 hours), thrice (0900, 1700 and 0100 hours) and five (0412, 0900, 1348, 1836, and 2324 hours) times daily over a 24-hour period. The experiment proceeded for 3 weeks but was terminated because of an equipment malfunction (chiller).

While data analysis of the FAA experiments by ANOVA did not reveal any feed anticipatory activity, additional, relevant statistical analyses are currently being performed.

E. Anticipated Success in Meeting Project Objectives in the Scheduled Project Period
It is anticipated that the project objectives will be met by the end of the scheduled project period.

F. Reports, Manuscripts, and presentations resulting from the project
A graduate student working on this project (Gwynne Schnaittacher) presented a poster at the Flatfish Biology Conference, December 10-11, 2002 in Westbrook,CT. The title of the poster was "The Feeding Behavior of Atlantic Halibut, Hippoglossus hippoglossus." Data on the feeding frequency and feed anticipatory experiments were presented.

II. Tasks and activities for next reporting period

A. Tasks for the next upcoming reporting period
The operant conditioning experiments will begin in January 2003, and are expected to run for approximately two months. The second feeding experiment will be repeated, beginning January, 2003 and will proceed for 12 weeks.

B. Brief work plan to accomplish tasks
Operant Conditioning Experiment:
These experiments will consist of three treatments with three replicates per treatment (20-100g fish per replicate). Fish in treatment 1 will be exposed to a 10 second acoustic tone (300 Hz) 10 seconds prior to feeding. Fish in treatment 2 will be exposed to a light pulse 10 seconds prior to feeding and control fish will be fed without receiving any prior sensory stimulation. The fish will be fed by automated feeders (Sweeney) and the time interval between feed release and initiation of feeding will be documented by video recording. Fifteen minutes after feeding, uneaten feed will be removed and weighed. Feed consumption rates and time to feeding initiation will be calculated and compared among groups.
Expected Time of Completion: March, 2003.

Feeding Frequency Experiment
Two hundred juvenile halibut (5-10g) will be purchased from R and R Finfish Development Company in January 2003. The feeding experiment outlined above will be performed at the Aquaculture Research Center (January-April 2003)

C. Anticipated Concerns or difficulties
None

III. Expenditures
Expenditures were in the range anticipated for the work accomplished to date.

In support of the conditioning experiments, a device was needed to be designed and constructed to acoustically transmit a predetermined signal, automatically, at regular intervals. Specifically, the design requirements were:

1. To operate autonomously in three separate grow out tanks in a hatchery laboratory.
2. The system was to transmit a variable length pulse at a frequency of 300 Hz at a power level higher than the background noise (as indicated by the PI).
3. The acoustic signal power must not be too great, however, to cause health damage in the fish. In the design process, it was determined that typical ambient noise sound pressure levels (SPL) in hatchery facilities that included 14 meter circular fiberglass tanks are distributed between 90 -130dB in the 315Hz one-third octave band (Bart et. al, 2001). As a starting point, it was decided to use a SPL 20dB higher than this range to condition the halibut.

B. Progress on Tasks

The design of the system consisted of thee parts: (1) development of the frequency generator and clock, (2) amplification of the generated signal, and finally (3) packaging of the system. A BASIC Stamp 2sx (BS2sx) microcontroller from Parallax was implemented to generate the variable length tone. The microcontroller, shown in Figure 1, features the ability to produce a tone in the range of 0 to 82 kilohertz for a period of twenty-seven seconds in 0.4ms increments.

Once the circuitry was checked for accuracy and repeatability, the next step was to size the transducers and the amplification equipment of the system. Three Model GZ001 underwater transducers (Figure 3) were purchased from Sunpride Co. (www.esunpride.com). These speakers have a maximum input power rating of twenty watts and an impedance of 4/8ohms. The impedance and maximum input power are key factors in the design of the amplifier circuit.

The impedances of the amplifier and speaker must be matched or else damage might occur to the speaker and/or the microprocessor. In addition, the maximum output power of the amplifier must not exceed the rating of the speaker or damage can occur to the transducer. Based on these two design criteria, a National Semiconductor, Model LM1875, 20W audio power amplifier was used in the circuit. The LM1875 amplifier was configured in a typical single supply orientation (for circuit details, refer to Figure 4). Three amplifiers were constructed, one for each of the three underwater speakers. Since each of the amplifiers can have a maximum output of 25W, a 75W power supply at 24V had to be used.

Once all the components were bench tested and determined to be in working order, printed circuit boards were created for each of the amplifiers and the real time clock components and all of the components assembled. The parts were then laid out to determine the appropriate size enclosure. It was decided to go with a economical plastic container enclosure. The container was fitted with a panel which provided an area to mount the user interface (Figure 5) and separate the user from the electronics (Figure 6).

C. Important Results or Findings

As mentioned in the previous section, the system was first tested on an oscilloscope to verify that the microcontroller was producing the correct frequency. The data was digitized, plotted and examined for performance. As shown on Figure 7, it was found that a signal with a frequency of 301.8 Hz was computed. This is within the expected error of the instrument and therefore the microcontroller acceptable to use.

A raw data files from each of the tests was then loaded into Matlab and sectioned into matrices based on a 2n power of 512 points, which yielded eleven bins. In each one of these bins, an acoustic pressure (acpr) was calculated for each voltage point (v) based on the equation:

where the sensitivity and gain of the receiving hydrophone are 186 dB and 30 dB, respectively. Through the use of a Fast Fourier Transform the noise spectrum level (NSL) was computed using the following technique:

where

Once all eleven bins were converted from voltages in the time series to a power spectral density in the frequency domain, they were ensemble averaged to create a single matrix of NSL. The two NSL matrices for the ambient noise were then ensemble averaged to produce a single matrix of NSL for all the ambient noise data sets. This same procedure was repeated for the three data sets where the ACD was active. The processed data was then plotted against each other, with the ambient noise data represented in blue and the output of the acoustic device data in red (Figure 9).

REFERENCES

Abbott, RR (1972). Induced aggregation of pond-reared rainbow trout (Salmo gairdneri) through acoustic conditioning Trans. Am. Fish. Soc. 101(1):35-43

Bart, A.N, J.Clark, J. Young, and Y Zohar, (2001). Underwater ambient noise measurements in aquaculture systems: a survey. Aqua. Eng. 25 (2001), 99-110.

Williams, J. (1997). It's time to get real using the Dallas Semiconductor 1302. Parallax, Inc. November, 1997. pg 365-378.