ANNEX A
STANDARD MENHADE OIL METHYL ESTERS

NMFS, SEATTLE

10 OCTOBER 91

by Dr. Supis

LIST WIDTH (2) SHIMADEU GLC w/3 m packed SS column
ANALYSIS PARAMETER FILE 2

ANNEX B
Proforma for Lipid Research Programme at NIFI

A. INTRODUCTION

Lipid requirements of fish and shrimp are poorly defined, but lipid needs and functions in growth and physiology are growth and life determinants. General knowledge exists on the need for fats in the diet of warm water fish and shrimp, but quantitative levels for optimum growth and health under various environmental conditions encountered in aquaculture are not known. Since it is known that W-6 and W-3 type fatty acids are needed for proper membrane function, current feed formulations include 1–3% of these fatty acids in the added fat. Fish and shrimp can use neutral lipid sources for energy, thus sparing protein in the diet for growth, but exact levels for various species and growth conditions are only estimated. Protection of labile fatty acids in the diet and in the tissues requires adequate extra cellular and inter cellular antioxidant systems, and these need to be investigated for various fat sources used in aquaculture feeds. Dividends from better knowledge of amount and type of lipid to be used, and for proper protection of these important compounds will promote more economical diets for aquaculture and will assure more positive and reliable aqua-industrial development in Thailand.

B. OBJECTIVES

1. Determine the lipid levels in feeds which will satisfy the essential fatty acid and sterol requirements of priority species of animals for aquaculture in Thailand.
2. Priority species to be examined are :
   1. Penaeus monodon shrimp
   2. Clarias catfish
   3. Puntius catfish
   4. Machrobrachium shrimp
   5. Pangasius catfish
   6. Tilapia
   7. Sea bass
3. Investigate and determine essential fatty acid profiles of 10 high priority oil and fat sources for aquaculture feeds. These are :
   1. 4 marine oil types
   2. 3 vegetable oil types
   3. 3 special fats and oils

C. LITERATURE REVIEW

The current state of knowledge on fat and sterol requirements of fish and shrimp will be reviewed with special emphasis on the lipids present in the 10 selected diet oils and on proper protection for essential fatty acids in diet formulations and on fish/shrimp tissue protection. Physiological functions of both neutral lipids and essential fatty acids reported in fish and other animals will be included together with assay techniques to determine both lipid intermediates present in feeds and in growing animals. From the literature survey, obvious voids in knowledge will furnish specific work units for high priority investigations.

D. METHODS

1. Growth response of various diet treatments will be evaluated by statistical analysis of properly experimentally designed trials following standard techniques for conduct of fish nutrition experiments (see Standard Methods adopted by IUNS, ICFS, EIFAC and NRC)
2. Analytical techniques will be AOAC or improved analytical techniques reported in the literature by refered laboratories.
3. Parameters significantly affecting results will be identified together with interactions which influence or compromise conclusions.

E. NEEDS

1. Materials (stock, feed, chemicals, etc.) should be estimated for each work unit approved.
2. Facilities (ponds, tanks, lab equipment, etc.) will be itemized to develop the schedule and budgets.
3. Staff requirements (man hours of labour and type) will be specified for each work unit.

F. RELATION WITH OTHER PROJECTS

The lipid research programme will be an integral part of the NIFI nutrition and diet development programme. It will complement projects in vitamin and amino acid research and in feed development technology. In addition it will interface with other NIFI and NICA projects in fish/shrimp health for improved aquaculture in Thailand.

G. PHASING OF WORK

A barchart indicating scheduling of phases of each work unit will be maintained to show progress and achievement of goals and objectives of the overall lipid research programme.

H. REFERENCES FOR LITERATURE

A complete list of citations for the literature review will be submitted with titles and complete authorship for articles listed.

I. BUDGET

A complete budget estimate together with allocation segments requested will be submitted according to budget class accounting listed in accompanying, sheets 5, 6, 7 of the NIFI Project Proforma form.

I. BUDGET

NATIONAL INLAND FISHERIES INSTITUTE - RESEACH PROJECT PROFORMA

ANNEX C
PROTOCAL FOR FEED C ASSAYS

(C range 5–50 ppm)

A. Cold Extraction (for C1, C2, C3 MP)

1. Take 1 gm sample

2. Add to 10 total with 5% Metaphosphoric Acid

3. Homogenize in Polytron 1 minute

4. Centrifuge hispeed 5–10 min (until clear)

5. Filter thru 0.45 Um disc

6. Inject 10 mL for assay in HPLC

B. Hot Extraction for (C1, Coated C, C2, C3 MP)

1. Take 1 gm sample

2. Add 5% Metaphosphoric acid to 10 gm

3. Homogenize in Polytron 1 minute

4. Pour into 100 mL beaker, weigh, (cover with cover glass)

5. Heat in microwave 30 seconds medium heat

6. Stand 5 minutes, make back to weight with water

7. Centrifuge until clear

8. Filter thru 0.45 Um disc

9. Inject 10 mL for assay in HPLC

C. TCA Extraction (for C1, C2, C3 MP)

1. Take 1 gm sample

2. Add 5% TCA to 10 gm

3. Homogenize in Polytron 1 minute

4. Centrifuge until clear

5. Filter thru 0.45 Um disc

6. Inject 10 mL for assay in HPLC

ANNEX D
Vitamin C Assays with NOVA PAK Columns

Demonstration of use of the Waters HPLC using tandem NOVAPAK 150 mm C18 columns and a mobile phase of 0.1 M sodium acetate plus 200 mg Na₂EDTA and 0.17 mL of n–octylamine per liter was completed with Dr. Wimol. Standards of C₁ and C₂ were prepared and run for time storage stability tests. C₁ in water decreased in activity from 90 to less than 30% in one day at ambient temperature. C₂ in water solution was stabile at 95–100% activity for at least 5 days. C₁ in mobile phase was stabile for one working day (6 hours) but lost over half activity within 24 hours. C₁ in 5% metaphosphoric acid solution was stabile for at least 3 days retaining greater than 90% activity. Standards of 10 ppm C₁ were used and injections of 10 uL (100 ng C₁) were measured on the HPLC yielding 6000 to 7000 area units per ng of C₁ injected. Feed mash containing 1000 ppm coated C₁ were assayed with 3 extraction techniques to determine best method for feed samples. Raw extruded feed and final dried feed pellets were also extracted and assayed.

Assay results were as follows :

1. 1 gm sample + 9 gm of 5% cold metaphosphoric acid, then homogenized in Polytron, centrifuged 10 minutes, filtered thru 0.45 um disc,
   
   injected 5 uL = 960 mg C₁/Kg mash
   = 666 mg C₁/Kg raw diet
   = 300 mg C₁/Kg dry diet
2. 1 gm sample + 9 gm 5% HPO₃, homogenized 1 minute, poured into beaker and microwaved at medium heat 1 minute, cooled, made back to weight with distilled water, centrifuged 10 minutes, filtered thru 0.45 um disc, injected
   
   5 uL = 400 mg C₁/Kg mash
3. 1 gm sample + 9 gm 5% TCA, homogenized 1 minute, centrifuged 10 minutes, filtered, and injected
   
   5 uL = 760 mg C₁/Kg mash
   = 250 mg C₁/Kg dry diet
4. 1 gm sample + 9 gm mobile phase, homogenize 1 min centrifuge, filter, inject
   
   5 uL = 250 mg C₁/Kg mash
5. 1 gm sample + 9 mL mobile phase, homogenize 1 minute, defat with diethyl ether, centrifuge 10 min, filter, inject
   
   5 uL = 200–250 mg C₁/Kg mash
6. 1 gm sample dry pellet defat with diethyl, ether, add 9 gm 5% HPO₃, homogenize 1 min., centrifuge 10 min, filter inject
   
   5 uL = 90–100 mg C₁/Kg dry pellet

Routine assays of mash feed, raw extruded feed, and dried finished feed could be run on tandem NOVAPAK columns when cold 5% metaphosphoric acid was used to extract and stabilize C₁. Coated C₁ was fairly stabile in mash mixtures but when water and extrusion plus drying final pellet was done, this C source rapidly degraded, and only 10% of original C₁ material was present in the dried pellet. Pellet residue life was not measured as HPLC was replumbed for amino acid analysis until the BONDAPAK tandem columns were delivered.

ANNEX E
Calculations for C₁ Assays on HPLC

1. Weigh sample X
2. Add 9 × of Extractant = 1/10 dilution
3. Use std. 10 ppm C₁ in 5% metaphosphoric acid
4. Measure area of 10 uL injection by HPLC
5. Divide area (100 ppm) by 100 = area/ng
6. Measure area of Extracted sample
7. Divide area by area/ng = ng in injected sample
8. Divide ng by uL injected
9. Multiply by dilution factor = ng/uL of sample

10. Calculate sample size (divide by sample wt.)
    = ppm C₁ in sample

Example

1. 10 ppm C₁ @ 10 uL injection = 654300 area units/100 ng
2. 654300/100 = 6543 area units/ng C₁
3. Sample wt. 0.7 g + 6.3 metaphosphoric acid = 7.0 g (1/10 dilution)
4. HPLC of 8 uL injection = 3814322
5. 3814322/6543 = 583 ng in 8 uL
6. 583/8 = 73 ng/uL sample
7. 73 × 10 = 730 ppm. C₁ in sample or 730 mg C₁/Kg sample

ANNEX F

BONDAPAK C18 Column C Assays

Mobile phase : 0.1 M NaAc + 200 mg EDTA + .17 mL n-octylamine per Liter

HPLC conditions : Isocratic flow elution rate 1.5 mL/min

Detector 254 nm. Temperature : ambient

Tandem columns : 4 × 300 mm u BONDAPAK C18

Retention times : C₁ @ 4–5 min. C₂ @ 8–9 min. C₃ @ 20–22 min.

Note: In addition to C₁, C₂, peaks, trout liver shows another peak midway between C₂ and C₃MP retention times which, upon treatment with acid, rapidly diminishes with commensurate rise in C₁ peak.

(1) Feed with 1000 mg C₁ added per kilogram mash before pelleting.

(2) Feed with 100 mg C₂ added per kilogram mash before pelleting.

(3) Feed with 100 mg C₃ added per kilogram feed.

(4) Feed with 200 mg C₃TP added per kilogram mash before pelleting.

ANNEX G
Vitamin Requirements Research Proforma

A. Water soluble vitamin requirements for warm water fish and shrimp have not been established for quantitative levels to be added to feeds. Deficiency signs have been described for C₁ thiamin, pyridoxine, pantothenic acid and niacin which are similar to those reported for salmonds and channel catfish. Vitamin supplements for feeds generally follow NRC recommendations with added amounts for safety. New analytical techniques are now available at NIFI to determine levels in feeds and in fish tissues to give a better assessment of vitamin status in animals on different aquaculture feeds. Therefore it should be possible to determine more exact recommended levels for these vitamins for these expensive feed supplements. Dividends from examination of critical health status vitamin needs in the feed related to health status in the fish and shrimp should enhance aquaculture production in Thailand both in lowered feed costs and in improved fish/shrimp production. Priorities for vitamin investigation should be C, thiamin, pyridoxine pantothenic acid, E, niacin and K. This national program should be an integral part of the Research & Development efforts of NIFI for the next 5 years with ammendments in direction and emphasis made on annual review of progress.

B. OBJECTIVES OF THE VITAMIN RESEARCH PROGRAMME include :

1. Determination of critical health status water soluble and fat soluble vitamins needed in aquaculture feeds.
2. Priorities for vitamins to be examined will be (a) C levels and stability; (b) Thiamin; (c) Pyridoxine; (d) pantothenic acid; (e) E levels and stability forms; (f) niacin; and (g) K.
3. Species to be examined in priority are :
   1. Penaeus monodon
   2. Clarias catfish
   3. Puntius catfish
   4. Machrobrachium rosenbergii
   5. Pengasius catfish
   6. Tilapia
   7. Sea bass

C. LITERATURE REVIEW :

The current literature should be reviewed as to vitamin requirements and functions reported for fish and shrimp species.

D. METHODS

Individual work units will be developed to determine what specific vitamin requirements and feed supplement levels are necessary to guarantee good production and sound health. Growth experiments should be coupled with tissue and clinical chemistry to determine response to different diet treatments. Experimental design will allow statistical analysis of date to determine reliability of results.

E. NEEDS

Specific needs for each work unit will be developed specifying fish/shrimp stock, facilities, (ponds, tanks, lab equipment, etc.), materials (feed, chemicals, supplies) and staff requirements (manhours of labour).

F. RELATION WITH OTHER PROJECTS

The vitamin research efforts are an integral part of the nutrition Research & Development programme and will complement both field and laboratory efforts in lipid and amino acid research to provide sound data on feeds formulation and use in aquaculture in Thailand.

G. PHASING OF WORK

A barchart of approved work units within the vitamin research program will be posted at headquarters and in offices of principal investigatives.

H. REFERENCES

A complete listing of references used in developing the project and work units will be maintained.

I. BUDGET

The budget will be developed according to line items listed in the budget proforma attached.

I. BUDGET

NATIONAL INLAND FISHERIES INSTITUTE - RESEARCH PROJECT PROFORMA