Note: Schedule has been revised. See First Addendum to the Eighth Work Plan

Objectives
Significance
Anticipated Benefits
Identification of Beneficiaries
Collaborative Arrangements
Experimental Design
Schedule/Time Line
Methods

Objectives

1) Evaluate substitution of preserved samples for standard freeze-dried samples for use in isotope analysis;

2) Study partitioning of food sources utilized by stocked tilapia in fertilized ponds or fed and fertilized ponds by isotope analysis.

Significance

Research on efficient utilization of nutrients and feeds requires knowledge both of nutritional requirements and of feeding strategies. Efficient use of pond inputs is expected to result in reduced effluent loads and thus reduce environmental degradation. Isotope analysis may be a useful tool to measure the relative nutritional contribution of supplemental feeds and natural foods to tilapia production at different rates of supplemental feeding. Verification of the acceptability of a modified sample preparation method using preserved samples rather than freeze-drying samples could expand the use of the isotope method to areas where freeze-drying is not available or easily accessible.

Anticipated Benefits

Quantitative indications of the relative dependence of tilapia on pond versus supplemental feeds would allow modifications in feed rates and result in savings in feed costs. Isotope analysis could be simplified and expanded by the modified techniques proposed.

Identification of Beneficiaries

Fish farmers, extension agents, researchers.

Collaborative Arrangements

UAPB will collaborate with AU, OSU and host country personnel at the African site.
This study will be performed in conjunction with the study entitled "Relative contribution of supplemental feed and inorganic fertilizers in semi-intensive tilapia production" (Kenya Research 3). Samples for isotope analysis will be obtained from that site and results will be evaluated in terms of the conditions of that study.

Experimental Design

Study site: Sagana Fish Culture Farm, Kenya

Isotope Sampling Plan:

1) A pilot study will be performed at UAPB to compare the effects of preservation methods (formalin-preserved versus freeze-dried) on stable isotope content of samples. Tilapia, stomach contents, plankton and mud will be collected from ponds at UAPB. (See detailed methods below). Half of each sample will be preserved using each method. Samples will be analyzed for stable isotopes. Results of this pilot study will be used to determine the preservation method(s) used on samples obtained in Kenya. A trip to the study site before of near the beginning of the study would greatly facilitate a personal assessment of the research and analytical potential of the Sagana site and neighboring facilities (i.e., Nairobi lab). It is particularly critical to interact with personnel who will be directly involved in collecting samples and data pertinent to stable isotope analysis.

2) Samples of stocked fish tissue and stomach contents, rice bran, plankton, and pond mud will be obtained initially, at monthly intervals during the study, and at harvest. The samples will be treated according to predetermined methods and shipped to UAPB. Isotope analysis will be performed by Coastal Science Laboratories, Inc., Austin, Texas.

Schedule/Time Line

UAPB pilot - Start November 1996, end January 1997; Kenya portion - Start March 1997, end December 1997

Methods

We will perform a pilot study to determine the relative effect of different sample preservation methods on stable isotope content. Results will be applied to the Kenya study. In addition, we will act as a clearinghouse for samples gathered and processed in Africa. We will process some of the samples further if necessary and send them to a commercial laboratory for stable carbon isotope analysis. We will select the appropriate samples to be analyzed based on the actual conditions of the experiment. We will assimilate and interpret the isotope data and try to tie it in with standard production data obtained in Africa. The objectives are: 1) to establish the effects of two different methods of sample preparation (preservation in formalin, then freeze-dried vs. freeze-drying only) on isotope analysis; 2) to establish the relative contributions of plankton and rice ban to tilapia production under the conditions of the Kenya Research 3.

To conduct isotopic analysis of the relative contributions of live foods and rice bran the following samples will be needed:

Initial samples:

1) initial fish (10 individuals) - take representative samples from the fish to be stocked into the study, weigh and measure length of each fish individually (means can be computed later), then freeze fish in labeled plastic bags (always put date, pond #, and I.D. the item). Further processing will be necessary - detail provided later. Accurate, thorough labeling of all samples is critical!

2) initial fish stomach contents - remove material from the anterior portion of the stomach where food is least likely to have been altered chemically or physically. Treat as for initial fish. Important note: for this data to be useful it is important to label the stomach contents so that they can be matched up with the fish they came from (to enable later comparison of gut contents with fish tissues).

3) rice bran - several samples will be needed (1 g each) due to the variable nature of rice bran that will be used at the study site. Ideally, collect and date a homogeneous sample of rice bran from the batch currently in use along with each monthly pond sampling. Keep frozen, but otherwise no special treatment is necessary if it is already in a powdered form.

4) initial mud - take approximately 5 g (total) of mud from beneath the water surface from each pond. Samples will be taken as described by K. Veverica. These can be put in individual bags (one per pond), labeled, and frozen.

5) plankton - initial samples - one per pond. For each sample, use a phytoplankton net (80 micron mesh) & combine the material collected in 10 tows of the net. You might need to see how much dry material you get in 10 tows and increase the number of tows if necessary. Alternatively, plankton may be concentrated from bulk water samples by filtration through glass filters as discussed with K. Veverica. Collection methodology should be consistent throughout the study. We need at least a gram of freeze-dried material for analysis. Take the initial sample after fertilization and before stocking fish if possible. Some of each sample (about a quarter) should be preserved in 10% buffered formalin, and the rest should be freeze-dried. When these samples are sent to UAPB, we will filter the formalin-preserved samples onto glass filter papers, freeze-dry those and send them on for isotope analysis with the samples that were freeze-dried in Africa without being preserved first. (Preservation methods for all samples may be modified according to results of the pilot study).

During each sampling period try to collect all samples as close in time as possible - no samples should be collected more then a few days apart. Isotope values can change quickly!

Intermediate Samples:

Plankton from each pond should be collected, labeled and freeze-dried once a month throughout the study. Carefully date specimens.

The intermediate samples do not have to be preserved for isotope analysis.

Mud samples should be taken from each pond once a month and frozen.

About 5 (minimum of 2) fish of similar size should be taken from each pond once a month, weighed, and frozen (the 5 fish from each pond can be frozen together in the same bag).

Stomach contents of the fish should be collected, labeled, and frozen as for initial samples.

Final Samples:

Shortly before harvest, take final plankton samples from each pond and freeze-dry them.

Take final mud samples and freeze.

Final fish: take about 5 representative fish from each pond, weigh individually, and freeze them.

Obtain stomach contents of fish and treat as described previously.

Note: All initial and final samples will be analyzed for stable carbon isotope (d¹³C) ratios at Coastal Science Laboratories, Inc., in Austin, Texas. Only some of the intermediate samples will be analyzed. We will have to make our selections after the study ends, based on growth rate of the fish and whether or not the rice bran becomes incorporated into the plankton (rendering the rice bran and plankton isotopically indistinguishable), and the time frame in which this occurs. The weight/length data of the fish will still be useful, however. If the rice bran label (d¹³C) is similar to that of the plankton at the beginning of the study or becomes so during the study, the isotope technique will not be useful in distinguishing relative contributions of the rice bran and plankton to tilapia growth. However, it is necessary to establish whether or not this happens and on what time frame before the isotope technique can be applied to other studies, so useful data will still be generated.

It would be useful to have backup information on the plankton to help interpret the isotope ratio results. Will any chlorophyll measurements be made during the study? Chlorophyll measurements taken at each plankton sampling from each pond would be very helpful. (Karen Veverica has confirmed that chlorophyll measurements will be taken).

Will quantitative estimates of tilapia reproduction be made? (Yes). The amount of reproduction that occurs during the study will affect the nutrient partitioning between adult and juvenile fish. We do not need any juvenile fish samples but we would like to know the relative yields of juvenile and adult fish at harvest.