By

John E. Halver

HALVER CORPORATION

Seattle, Washington, USA

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TABLE OF CONTENTS

Terms of Reference
Formulation and Initiation of Research Programme
In Service Training
Nutrition Laboratory Equipment
Recommendations
Personnel Consulted

Proforma for Projects Annex A
Work Unit for Projects Annex B
Supplies for 100 Sample Assays Annex C
Fish Feed Formulae Annex D
Erythrocyte Fragility Test Annex E
Prothrombin Time Annex F

TERMS OF REFERENCE:

1. To formulate and initiate a research programme designed to develop appropriate diets for Clarias and Pangasius catfish including:

   1. demonstration of vitamin and enzyme analysis techniques.

   2. designing of fish nutrition experiments relating particularly to vitamins and enzymes.

   3. advise on formulating appropriate diets for catfish.

2. To give in-service training to counterpart officers.

3. To advise on upgrading of the equipment of the nutrition laboratory with emphasis on vitamin nutrition research.

4. To submit a technical report at the end of assignment.

Upon arrival the TOR were refocused upon planning and design for research capability in areas of vitamin and coenzyme analysis, amino acid analysis and fatty acid analysis of fish tissues, feedstuffs and finished feeds. Equipment dilivered during 5–9 June was cleared for meeting specifications and was installed. A Shimadzu Gas - Liquid Chromatograph and a Waters system High Performance Liquid Chromatograph was initially operated with reference standards for methyl esters of fatty acids and for 9 standard of 18 amino acids.

1. FORMULATION AND INITIATION OF RESEARCH PROGRAMME (DIETS FOR CLARIAS AND PANGASIUS).

Research planning was initiated by developing research project proforma for the major projects to be implemented under THA/87/004. A type proforma can be found in Annex A.

Research work unit proforma were developed for subprojects with the three major projects identified under the THA/87/004 Programme.

1. Research Capability in Amino Acid Analysis.
2. Research Capability in Fatty Acid Analysis.
3. Research Capability in Vitamin Analysis.

Example of a typical work unit can be found in Annex B.

1.1 Vitamin and Enzyme Analysis Capability was initiated by delivery and installation of a Waters HPLC analytical system. The instrument was delivered on 12 June 89 and was test run for unit operations. It was decided to focus on amino acid and fatty acid analysis capability during the 1989 consultancy and then emphasize vitamin and clinical enzyme chemistry during the 1990 consultancy period. The same Waters instrument will be used for the vitamin analyses but different columms will be used than those on hand for amino acid analysis. Two Bondapak C₁₈ 30 cm columms were not purchased with the Waters instrument and will need to be obtained before vitamin resolution and quantification can be done. Roche Thailand is very interested in this assay capability, visited the facility and has offered to try to provide the two C₁₈ columns needed.

1.2 Fish nutrition experiments was designed to estimate Protein to Energy ratios for feeds for Clarias and Shrimp. Dr. Mali Boonyaratpalin has obtained feedstuffs for diets for shrimp and is currently assaying these for gross nutrients to estimate crude protein and metabolizable energy ratios for each dietary ingredient. Formulations for a matrix of Crude Protein at 35, 39, 43 % with ME values of 6, 8, 10 kcal per gram of protein were designed. Triplicate lots of shrimp will be reared on these diets and biological response measured. Growth Diet Conversion, Protein Efficiency Ratios and Net Protein Utilization will be calculated to yield the most effective Protein to Energy ratios for farm diets.

The same techniques will be used to measure Protein to Energy ratios in diets for Clarias reared at the experimental facilities of NIFI. These experiments are scheduled to begin in late 1989 or early 1990.

1.3 Experiments were designed to measure L-ascorby1-2-polyphosphate (C₃) as a vitamin C source for Clarias. A matrix of C₃ at 0, 100, 500, 1000 mg C₁ equivalent will be compared with diet treatments of 0, 100 and 500 L-ascorbic acid (C₁) dietary intake using a standard Clarias diet base at the NIFI Center. Supplies of C₃ were obtained by a gift from Vitamin Technologies, Inc. of Buhl, Idaho, USA. Vitamin C₁ will be obtained from Roche, Thailand.

1.4 Clinical chemistry techniques to measure nutritional status of catfish for thiamin and pyridoxine were outlined to staff officers. Erythrocyte transketolase preparation from red blood cells and subsequent assay for activity to reflect thiamin status was outlined. A reprint of latest details developed by Dr. Ron Hardy of National Marine Fisheries Service in Seattle, Washington was left with Nanthiya Unprasert for NIFI laboratory assay files.

A similar outline of sample preparation of liver tissue for amino transferase to measure pyridoxine status was discussed in detail with Nanthiya Unprasert. A late reprint of details for assay of aspartate amino transferase was left for NIFI lab assay files. Techniques for assaying for pyridoxine by HPLC and for pyridoxal and amino propanal were included in the detailed procedures. It is anticipated that Nanthiya Unprasert, the trained scientist for HPLC analytical techniques on the Waters system instrument will be able to prepare appropriate samples from tissues and then to assay for both the chemical vitamin compounds on the HPLC and for the clinical enzyme activities for these two metabolic systems which do reflect thiamin and pyridoxine status of the animal.

1.5 Estimates of costs for running 100 samples of fish tissues or of feedstuffs or feeds for (1) fatty acids, (2) amino acids, (3) Pyridoxine, (4) Pantothenic acid, (5) Thiamin, (6) Thiamin pyrophosphate, (7) Vitamers C₁, C₂ & C₃ (8) Serum amino transferase, (9) Erythrocyte transketolase, (10) Prothrombin time, (11) Erythrocyte fragility, (12) Riboflavin, (13) Biotin and (14) Tocopherols was assembled and left with the project manager for future research effort planning. Annex C.

1.6 A review of standard methods used for proximate analysis disclosed unacceptable variance in assay results for crude fat, ash and fiber. A new fiber extraction apparatus has been purchased and was delivered. A standard reference protein (vitamin-free casein) was adopted and samples reserved for at least 5 years of reference standard analysis. More training in finite analytical techniques was emphasized because proximate analysis forms the basis for gross nutrient calculations in diet formulations.

1.7 A standard reference diet (H440) and the same standard reference protein (V.F. Casein) was adopted for formulation and amino acid assay techniques. One lot of material was identified and properly subdivided, sealed and stored for subsequent use for the next several years as reference material for various laboratory analysis. In addition a standard reference fish, Clarias of about 100 g weight and fed on NIFI# 12 diet for at least 6 weeks, was adopted for blood and other tissue reference material.

1.8 Practical diet formulae which had been used successfully to grow catfish in Thailand were obtained from Prasert Sitasit the National Programme Manager THA/87/004. From this list gross nutrient analysis of components used in these diets could be used to factor out some common denominators to form a general basis for estimating nutrient requirements and a first approximation of acceptable formulae limits. Conversion values indicated considerable variation in metabolic value of components in the feedstuffs used. For example, fish meal used in the formulae could be either: good value at 60% crude protein, fair value at 55% CP, or low value @ 50 % CP, and these values would be reflected in the diet conversion and growth responses. These formulae did however provide a foundation for nutrient requirement trial design. See Annex D.

1.9 A National Survey for Feedstuffs was discussed and urged to be included in the future plans for NIFI. The INFIC system of nomenclature and AOAC methods of analysis need to be adopted in order for gross and fine nutrient data to be acceptable for the International Network of Feed Information Centers to incorporate into their Feed Data Bank. Type entry forms are available from INFIC sites or from the Agricultural Research Service, National Agriculture Library in Beltsville, Maryland, USA.

2. IN SERVICE TRAINING

In service training was concentrated with Nanthiya Unprasert who - is the person with background and analytical technique experience capable of operating the advanced analytical equipment newly purchased and delivered to NIFI.

2.1 A convenient rapid method for a Folch type extraction and purification of liquids from fish tissue was demonstrated. Livers from Clarias catfish were excised on necropsy, were homogenized in 5 parts per volume of 2/1 chloroform methanol in a high speed homogenizer. More solvent (15 parts) was added and thoroughly mixed. The precipitate was filtered through Whatman 41 paper in a long stems funnel and dropwise washed as it dripped through distilled water into a beaker on the bottom of the water container. After standing for 30 minutes the chloroform layer containing the extracted lipid was separated and chloroform evaporated off at 50 with an air flow in the hood. The final 5 mL of liquid was removed under vacuum. This dried oil was dissolved in n-hexane and sealed for use as the reference lipid for fatty acid analysis.

2.2 Methylations of fats and oils was performed by the standard technique detailed in Watanabe's manual for preparation of samples for GLC analysis. Nanthiya Unprasert reviewed the technique she had learned from Watanabe and produced good samples of methyl esters for assay.

2.3 The Shimadzu Gas Liquid Chromatograph model 14A was installed on 14 June 89 in a clean room set up for that purpose. Upon operation it was found that electricity leaked back through the metal couplings and therefore a ground circuit was installed in the room to protect workers. The machine was test run by Shimadzu personnel and found fairly adequate for analysis. A special 2 meter glass coil column was used on the basis of Shimadzu guarantee of better methylated fatty acid resolution. Nanthiya Unprasert was trained in normal operation of the instrument for fatty acid quantitation. An n-2 hydroxyl octadecanoic acid standard was substituted for the n-octadecanoic standard ordered and the replacement methylester fatty acid had indiscrete resolution. At time of this report the instrument, while operational, has not been standardized for parameters guaranteed by Shimadzu for quantitative fatty acid analysis. Shimadzu promises to calibrate the gas flow gauges and demonstrate temperature and carrier gas flow parameters for the instrument which give good quantitative resolution of C₆ - C₂₄ fatty acids before acceptance of instrument and payment of purchase voucher. This should be a minor problem easily resolved by providing the operation parameters for the new column or by providing the column originally ordered. NIFI should have the capability for routine GLC analysis of fatty acids present in fish tissues and feedstuffs in fats and oils, and in finished feeds.

2.4 The Waters High Performance Liquid Chromatograph with preparative station for protein hydrolysis and for derivatization of amino acids was delivered on 15 June 89. A clean room area was provided for the pumps, the automatic sample injector, the high pressure columns, the graph plotter and the integrator. The phenylisothiocyanate derivative station and major electric power supplies were also grounded to prevent accident. One tubing connection for exterior exhaust of the vacuum pump will be necessary for SAFETY of personnel during the PITC derivative preparation. Nanthiya Unprasert was trained in all steps necessary to prepare samples by acid hydrolysis of protein, by derivatization of the resultant amino acids and to prepare samples for automatic injection into the apparatus. She has demonstrated proficiency in programming the apparatus for amino acid analysis. The air line for the automatic sample injector needs to be connected to the large air pump and air tank rather than use the inefficient small pump provided by Waters Associates. NIFI now has the capability to perform amino acid analysis of fish tissues, feedstuffs and finished feeds. The clean room is crowded with the GLC and HPLC with prep stations in the one room. The sample preparation areas are scattered in various laboratory rooms of NIFI. A central fatty acid, amino acid, and vitamin analysis prep area would be very desirable to keep sample preparation very clean and uncontaminated for the nanogram and picogram quantities of chemical compounds analyzed by these very sensitive and very accurate instruments.

2.5 Personnel were trained in developing a project performa for fish nutrition research. Items included summary of budget with class accounting estimates, background, objectives, literature review, methods of approach, needs, relation with other projects, work phasing, references, personnel, benefits, supplies, space, equipment and monitoring. The development of a sound research proposal with full accountability for personnel, time, facilities, equipment and research scheduling was emphasized as the foundation for research programme planning in perspective. See Annex A.

2.6 Personnel were trained in developing work units for the projects within THA/87/004. A short descriptive title with objectives, methods, needs, personnel, research schedule review periods, and budget for the work unit was emphasized as logical interim steps for annual work plans. See Annex B.

2.7 Methods for estimating vitamer E status by measuring erythrocyte frogility were outlined with Dr. Mali Boonyaratpalin and Nanthiya Unprasert. The detailed procedure can be found in Annex E.

2.8 A method to estimate vitamer K status by blood clotting time measurement was described to Dr. Mali Boonyaratpalin and Nanthiya Unprasert. The detailed procedure can be found in Annex F.

2.9 A Ph.D. research programme proposal was developed to measure development of the proteolytic digestive system of Clarias catfish. Histology and histochemistry techniques will be used to record sequential development of the gut system of larvae to fry for protein digestion capability as a basis for larval to fry feed formulation. Time permitting, lipolytic enzymes will also be measured histochemically. This thesis research plan for Ms. Amornrat Sermwatanakul was accepted by Dr. Beryl March, her major professor, at the Department of Animal Sciences, University of British Columbia, Vancouver, B.C., Canada.

3. NUTRITION LABORATORY EQUIPMENT

The nutrition laboratory equipment of NIFI was reviewed resulting in several suggestions for improvement.

3.1 The clean room housing the nearly purchased Shimadzu GLC and Waters HPLC systems needs to be provided with better electrical service with multiple grounded outlets to supply the various electrical apparatus present in the room.

3.2 The aircompressor and airtank need to be connected with shut off valves to both the GLC and HPLC equipment.

3.3 A copper tube exhaust should be drilled through the outer wall immediately above the vacuum station pump, and then connected to the exhaust port of the pump to prevent inadvertant contamination of the work area when toxic cyanide gas is generated during the amino acid derivative preparations.

3.4 The hood designed for preparation of samples located adjacent to the clean room should be provided with a good exhaust fan vented through the outer building wall.

3.5 Remodeling of the outer room adjacent to the clean room into a general vitamin fatty acid and amino acid sample preparation area would be most desirable. The present sink area should be relocated and walled off from the drain line drip from the ceiling which is currently contaminating the work area. A good laboratory work bench equipped with sink, multiple grounded electric supply, good lights, hot and cold water supply, and a good sink drain should be a high priority item in developing the vitamin, enzyme, fatty acid and amino acid assay capability for NIFI. It is ludicrous to spend nearly $100,000 on analytical instruments and then have inadequate sample preparation area available for personnel.

3.6 An HPLC quality water supply system is essential if good reproducible assay for nanogram quantities of vitamins and vitamin metabolites are contemplated. The analytical capability of the instrument is compromised when trace quantities of contaminating proteins, fats, vitamins, and intermediates are present in the water used. It will become too expensive to purchase high resistance HPCL quality water from Waters or some other supplier when multiple samples are scheduled for assay in major nutrient requirement and diet development studies.

3.7 A laboratory model Wiley Mill should be purchased to grind feedstuffs and finished feeds into fine particles which can then be representatively sampled for the small quantities of material used in the final sample preparations for the vitamin, amino acid and fatty acid assays.

3.8 A small micro wave oven will be necessary for sample preparation for vitamin assay. Vitamers C₁, C₂, C₃ cannot be assayed quantitatively without microwaving the tissue material to denture the protein and release the water soluble components. This microwave oven is also used for releasing vitamins and intermediates from feedstuffs and finished feeds.

3.9 The air conditioned clean now works only during business hours where as this room containing the GLC and HPCL systems should be air conditioned 24 hours per day for most efficient and most accurate assay conditions Equipment life will also be extended and maintenance costs minimized. Therefore a small room commercial type air conditioner should be installed in this clean room.

3.10 The quality control proximate analysis room located above the wet laboratory has inadequate water supply and electrical supply with grounded outlets. It would be more efficient to move the protein, fat, and fiber assay equipment downstairs, leaving the moisture and ash ovens upstairs.

3.11 The budget developed for feed milling equipment was based on 1986 prices whereas funds are inadequate to purchase the Wenger or Sprout-Walden high grade steam extruder for manufacture of extruded expanded floating feeds. Also the present hammer mill at NIFI does not grind fine enough for good pellet compaction and water stability. Therefore it will be necessary, baring a major increase in funding for the project, to select alternate options for feed milling equipment. In consultation with the NPM it may be necessary to purchase a small hammer mill with extra hammers and small screens; a Thai manufactured ribbon mixer of 200 Kg batch capacity; a Thai manufactured dryer; a Taiwan manufactured steam process extruder; and a steam generator. The current cost estimates for these items totals about $75,000 which will leave about $25,000 balance in the equipment budget which can be used for minor support equipment and for repair and remodeling of the existing experimental feed laboratory area to accommodate the revised feed manufacturing lines.

4. RECOMMENDATIONS

4.1 Complete the clean room for efficient operation of the GLC and HPLC equipment.

4.2 Install an air condition unit in clean room.

4.3 Complete a sample preparation lab area adjacent to clean room for vitamin, amino acid and fatty acid samples for analysis.

4.4 Recruit and train a person with analytical chemistry background and training to operate the GLC and HPLC systems when Nanthiya Unprasert may be absent.

4.5 Install SAFE electrical and room exhaust systems to clean room and adjacent lab prep areas.

4.6 Purchase a laboratory Wiley mill for sample reduction and representative sampling of feedstuffs and feeds.

4.7 Have NIFI join the INFIC system as a contributing member.

4.8 Purchase a small microwave oven with a good timer for biological sample preparation for vitamin and vitamin intermediate analysis.

4.9 Reassign quality control and proximate analysis equipment in wet laboratory area.

4.10 Readjust the budget for milling equipment and purchase Thai manufactured equipment and a Taiwan extruder.

4.11 Remodel experimental feed mill area and repair existing equipment.

4.12 Adopt a major project proposal proforma system to plan and implement research effort according to documented priorities.

4.13 Develop work units for all subproject elements together with phase monitoring and rescheduling strategy.

4.14 Recruit the feed technology expert as soon as possible to advise and implement the feed formulation; feed manufacturing and feed testing projects.

4.15 Recruit the fatty acid expert as soon as possible and implement the fatty acid analysis - diet design and requirement studies for the n-3 and n-6 fatty acid needs for fish and shrimp.

4.16 Recruit the amino acid expert as soon as possible and implement the amino acid analysis - diet design and requirement studies for indispensible amino acids for fish and shrimp.

4.17 Send at least two young scientists to the International Intensive Fish Feed Technology Course at the University of Washington 15 September - 15 December 1989.

4.18 Request return of vitamin - enzyme consultant in May, June 1990.

4.19 Adopt the ammended schedule and FAO request to supply a Research Planning and Diet Development Project Coordinator to assist implementation of all elements of THA/87/004 during the remaining tenure of the project.

PERSONNEL CONSULTED

ANNEX A
Proforma for Projects

A. INTRODUCTION

1. definition of the problem

2. background/justification

   • perspectives (practicability, scale)

   • impact (production, farmers, nation)

B. OBJECTIVES

1. purpose

2. segmented subunits

C. LITERATURE REVIEW

What is known about subject leading to VOIDS IN KONWLEDGE

D. METHODS

1. design of experiments, treatment procedures, sampling, etc.

2. record keeping

3. evaluation of results

E. NEEDS

1. materials (fish stock, fertilizer, feed, etc.)

2. facilities (ponds, tanks, lab, equipment, etc.)

3. staff requirements (manhours of labour)

F. RELATION WITH OTHER PROJECTS

G. PHASING OF WORK (barchart of activities)

H. REFERENCES FOR LITERATURE REVIEW

I. BUDGET

ANNEX B
Work Unit Plan for Projects

ANNEX B1: WORK UNIT PLAN - NIFI - NDD

Work Unit Code: (Programme; Project; Unit No.)

THA/87/004 : Amino Acid Capability : Unit No. A-001

Title: (Short descriptive title)

Capability for amino acid analysis with HPLC

Objectives: (List clearly 1, 2, 3 etc. succinctly)

1. Install HPLC with the Waters Instrument.
2. Standardize Methodology for the a.a. analysis with the amino acid cocktail standard.
   1. check the hydrolysis
   2. check derivatization
   3. check column for capacity factor
   4. check sensitivity resolution
   5. calculation and error correction factor
3. Sample analysis for amino acid profide

Methods: (Short description of method of approach)

1. Install machine to Waters Specification.
2. Eluents and reagents preparation and standard preparation.
3. Redrying the cocktail standard.
4. Hydrolysis and Derivatization using Pico-tag station.
5. Check the column for capacity factor.
6. Programme the parameter on Gradient Controller.
7. Analysis to measure the resolution.
8. Calculate experimental error on analysis.
9. Try on the specimen sample (H440).

Needs: (Equipment, Chemicals, Supplies, Fish, etc.)

• HPLC (complete set) / Vortex mixer / Clinical Centrifuge / Dewar flask
• ethanol / Triethylamin / Phenylisothiocyanate / Acenaphthane / Acetonitrile / Helium / Sodium Acetate trihydrate / D1 Sodium acetate trihydrate / disodium hydrogen phosphate, phosphoric acid / ION HCl / Dry ice / disposable gloves
• cocktail standard
• sample of feeds / feed stuff / fishes / shrimp

Who: (Personnel with m/m of effort for each)

Nanthiya
Viratada

Schedule: (Dates for each phase if more than one)

Last phase is always publication if appropriate

Review: (Dates for phase evaluation and redirection)

Budget (Summary of class costs 01 – 09)

(add separate sheet) 50,250 Baht.

Submitted by:

Nanthiya Unprasert

Approval:

Consultant THA/87/004	:
National Project Manager:	:
		Mr. Prasert Sitasit
Director NIFI:	:
		Dr. Kitjar Jaiyen

ANNEX B2: WORK UNIT PLAN - NIFI - NDD

Work Unit Code: (Programme; Project; Unit No.)

THA/87/004 - Fish Nutrition and Aquaculture Diets

Title: (Short descriptive title)

Capability for fatty acid analysis with GLC

Objectives: (List clearly 1, 2, 3 etc. succinctly)

1. Install GLC for fatty acids analysis.
2. Check machine with the standard fatty acid ester.
3. Check machine for the appropriate temperature programme for column, injector and detector for the best resolution and correct analytical results.

Methods: (Short description of method of approach)

1. Install GLC to Shimadzu specification.
2. Preparation of total lipid, fatty acids sponification and methylation.
3. Preparation of column and gas flow rate with the appropriate temperature programme.
4. Test run with the standard and samples.

Needs: (Equipment, Chemicals, Supplies, Fish, etc.)

1. G.L.C. of Shimadzu 14A model.
2. Lipid extraction apparatus with Fohn method.
3. Sponification apparatus and reagent to clearified the fatty acids.
4. Methylation with BF₃ methanol and purified of fatty acid methyl ester.
5. Injection dilution of hexane.
6. Injector of 5 ul sample.

Who: (Personnel with m/m of effort for each)

Nanthiya Unprasert

Schedule: (Dates for each phase if more than one)

Last phase is always publication if appropriate

Review: (Dates for phase evaluation and redirection)

Budget: (Summary of class costs 01 – 09)

10,000 Baht.

ANNEX C
Supplies needed for 100 samples assays

ANNEX D
Recommendation for fish feed formula

Prasert Sitasit

ANNEX E
Erythrocyte Fragility Test

1. Draw 2–5 ml blood from heart or candal vein in heparinized syringe.
2. Expell carefully into cold centrifuge tube.
3. Spin in clinical centrifuge for 3 mins.
4. Decant off serum.
5. Suspend RBC cells in 1% NaCl.

6. Dilute 2 ml blood sample cells to 5 ml.
   5 ml blood sample cells to 10 ml.

7. Prepare 10 sets of tubes containing 5 ml of: 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1% NaCl.
8. Pipette serological pipette 0.1 ml RBC suspension into each tube.
9. Record time and tube number when cells rupture and release hemoglobin into solution.
10. EFT = tube NaCl concentration x time of rupture.

ANNEX F
Prothrombin Time Test

1. Server tail or draw blood in cold syringe.
2. By capillary action fill non-heparinized hematocrit tubes.
3. Wait 10 secs then break portion of blood filled tube and pull apart slowly.
4. Continue breaking portions every 10 secs until 3 breaks occur.
5. Continue break and pull every 5 secs.
6. Record time first fibril of fibrin clot occurs.
7. Prothrombin Time is seconds after filling that clot forms.
   PRT = (30) to (35) seconds.