1.1 Nanthiya Unprasert completed 50 assays of amino acids in various feedstuffs and diets used for fish and shrimp diets using the HPLC equipment in the new laboratory.
1.2 The hood was installed and is operational for phenylisocyanate dirivative preparations and also for lipid extractions and methyl ester preparations. Thus samples may now be prepared for both amino acid and fatty acid analysis using the HPLC and GLC equipment.
1.3 Amornrat Sermwatanakul used the GLC equipment to perform fatty acid analysis on a number of brine shrimp strains which she is using in her Ph.D. thesis research programme at University of British Columbia. Results of these artemia analysis will be available to NIFI and DOF staff when needed to plan and implement larval feeding programmes of fresh water or brackish water larval fish species.
1.4 Fellowship training has almost been completed.
1.5 The experimental feed mill has been installed and is operational for manufacture of extruded diets of floating or slowly sinking type.
1.6 The quality control laboratory is equipped for routine crude protein, crude fat, moisture, ash and fiber analysis. Personnel have been trained for these operations for proximate analysis of feedstuffs and finished feeds.
1.7 In-service trining has been given to a number of DOF personnel and field extension courses have been given to farmers.
1.8 The Nutrition unit at NIFI is performing useful. international training to fishery scientists from various countries of S E Asia and is being used as the fish and shrimp nutrition and diet training unit for the Network of Aquaculture Centers of Asia (NACA).
2.1 Dr. Supis Thongrod, on temporary assignment from the
Coastal Aquaculture Division, assayed a number of lipid
samples for fatty acids using the Shimadzu Gas Liquid
Chromatograph (GLC) and the techniques she had learned
at the Tokyo University of Fisheries. Dr. Hardy
brought a standard menhaden oil set of methyl esters
for a comparison run to validate the retention times
and quantitative perk areas when run on the 3 meter
Stainless steel packed column and on the
2.2 Prasert Sitasit NPM of project, furnished a list of 4 marine oils, 3 vegetable oils and 3 special fats which were determined to be high priority potential feedstuffs to supply fat and essential fatty acids for fish and shrimp feed formulations. These materials were assayed for fatty acid profiles and a table of fatty acid quantitative levels in each was assembled for 12C, 14C, 16C, 18C, 20C and 22C acids of W-9, W-6 and W-3 type.
2.3 A priority list of species for lipid requirements studies in the future was developed for likely candidates for aquaculture. These priorities for R & D diet effort in 1992–1995 were:
2.4 A lipid research programme proforma was scheduled to be completed for NIFI with work units within this programme for 1992–1993. Dr. Supis will submit these research planning documents before the end of her temporary assignment. See Annex B.
2.5 Dr. Supis conducted several fatty acid profile analysis of drum dried artemia strains to identify variance in essential fatty acid content of potential larval fish feed material.
2.6 Technicians have been trained to prepare samples and to operate the GLC. This training phase has been completed by Dr. Supis, Dr. Wimol, Dr. Hardy and myself since we all have had some exposure and practice in GLC assay techniques. Therefore the lipid research programme facilities, equipment, and personnel are ready to conduct experiments and assays for lipid requirements and fish/shrimp diet analysis of test and commercial feeds.
2.7 The lipid research capability and research programme planning is substantially completed.
3.1 The Waters High Performance Liquid Chromatograph (HPLC) was delivered with NOVAPAK columns for resolution of the vitamers C. BONDA PAK C18 columns were ordered but not delivered until 28 October 91. In the interum, the NOVAPAK Column were used to demonstrate C1 and C2 analysis.
3.2 A protocol for feed sample preparations was developed and demonstrated to the staff. L-ascorbic acid (C1) assays of feedstuffs and finished feed samples were extracted with cold 5% metaphosphoric acid, with 5% trichloracetic acid, with hot water in the microwave oven, and with the mobile phase used to elute the C compounds from the NOVAPAK Columns. Two columns in tandem were used to extend the column length to separate the C compounds more discretely. Results showed C1 in mash feed preps, in raw feed, cold extruded, and in finished feed was rapidly lost with only a small fraction present in the finished feed. Either 5% MPA or 5% TCA could be used to extract the C1 from the feed and still retain C1 activity in the extract for several hours. Water and heat extractions destroyed the C1 in the extract rapidly. A modification was made in the protocal for feeds with high fat content. The extract was defalted with petroleum ether before filtration and injection into the HPLC. See annex C, D, E.
3.3 Dr. Wimol was trained in all types of sample extraction and clarification techniques, and operated the HPLC for C1 and C2 assays, of C1 added to diet preparations on the NOVAPAK HPLC column adapted procedure.
3.4 After receipt of the BONDAPAK Columns, Dr. Wimol was trained in standard C1 and C2 assays. Tissue preparation technique were demonstrated and a protocol developed for liver and other tissue analysis for the various vitamins C. See annex F.
3.5 A list of vitamins and sources for research priorities was furnished by Prasert Sitasit, NPM. A master plan for developing R & D proforma was discussed and submitted for incorporation into the NIFI plans for 1992–1995. The priorities to develop analytical capability for feed levels and tissue levels was as follows: (1) C; (2) Thiamin; (3) Pyridoxine; (4) Pantothenic acid; and (5) Niacin for the water soluble vitamins and (1) E; (2) Carotinoids and (3) K for the fat soluble vitamins. Since C analytical techniques were the highest priority (this vitamin has several life supporting functions in both fish and shrimp) and since several forms of C are available to the feed industry for diet formulation, this vitamin was emphasized in the research diet development and training functions of the consultant.
3.6 A type proforma for vitamin studies was developed as a model for future work in this area by NIFI and DOF personnel. See annex G, H.
3.7 C1 and C2 stability tests were performed on both cold extruded and hot steam treated expanded pellet diet formulae. Coated C1 was also included in the tests. Pfizer Inc. provided the reference and test lots of C2 and Hoffmann La Roche provided the standard C1 material. Losses of coated C1 and crystalline C1 during processing were dramatic with little C material present in the finished feed. In contrast the C2 was relatively stable and present in the mash, raw feed or processed pellets.
3.8 The HPLC equipment came with a fluorescent detector and a manual injector system plus an extra solvent pump. This equipment was described in detail to Dr. Wimol and it was planned to plumb up and activate this system for thiamin assays. Unfortunately, the long delay in delivery of the BONDAPAK C18 columns precluded any time for laboratory demonstrations or training. Perhaps Dr. Hardy will have some time after completion of the amino acid assays training to undertake demonstration of this nice piece of equipment which will increase the capabilities of the nutrition unit at NIFI, and allow finite measurement of thiamin requirement and sources for fish and shrimp species.
3.9 The sample preparation area next to the clean room holding the HPLC and GLC equipment needs to be remodeled into a regular scientific laboratory containing normal lab work benches, tables, electrical outlets and an animal/vermin proof prep area. This area is now a converted hall with an open center floor trench and improvised work tables, sink, and electrical supply. The hood is adequate and operates well, but the rest of the prep area does not allow adequate space, facilities, utilities to make micro sized samples which are used in the HPLC, GLC equipment. Ultra micro analytical techniques are necessary for reproducible assay when only nonogram or picogram quantities of sample are used. The amino acid sample preps are better controlled in the PICO TAG system provided in the “clean” room, but lab bench portions of sample hydrolysis are performed in contaminated areas. Rats, mice and cockroach crawl over the glassware, tables, sink, equipment and supplies - - most undesirable in an analytical laboratory. Any funds available near the end of the project should be used to convert this area into a laboratory room.
4.1 The TOR for Hardy and for Wilson involve several areas of overlap which were used to advantage. Since Dr. Hardy and I both work with GLC and HPLC equipment it was possible to complement training of staff and for demonstrations. Suggestions for improvement or interpretation of an assay procedure enhanced the effectiveness of both consultants in several areas of lipid, amino acid or vitamin assay techniques. In addition both Hardy and Halver have worked in feeds formulation, quality assurance, and in feed manufacturing techniques. Hence we all three could interact and improve feed technology activities when Wilson developed a series of test diet formulations and subsequent feed manufacturing techniques.
4.2 Dr. Hardy brought with him a standard set of menhaden oil methyl esters which had been vigorously assayed in the NMFS laboratories in Seattle, and use of these as standards enabled Dr. Supis to quantitate exactly on the NIFI GLC the essential fatty acids of the W-6 and W-3 types present in the NIFI test oils.
4.3 Dr. Halver brought vitamin C1 and C2 standards together with 5 Kg of C2 provided by Fizer Inc. for diet stability tests.
4.4 Dr. Hardy, Halver, Supis and Wilson designed together one standard type commercial macrobrachium diet made from materials available in Thailand.
4.5 Dr. Hardy and Halver test ran the HPLC system for standard amino acid mixtures and together got the equipment functioning normally. Dr. Wimol operated the HPLC in a practical manner and will soon develop skills in sample preparation from representative sample selection, then hydrolysis, PICO TAG preparation and final HPLC quantitative analysis.
4.6 Wilson and Wimol designed two soybean type diet formulations at two crude protein levels which were nearly isonitrogenous and isocaleric at appropriate formulations for an expanded catfish feed. Halver and Hardy assisted in evaluating the high temperature high pressure experimental extruder and tunnel dryer used. Changes suggested by Wilson, Hardy and Halver expedited production of an acceptable, expanded, partially floating finished feed. See annex J.
4.7 In compliance with TOR Hardy and Halver evaluated supplies and minor equipment needs for the nutrition laboratory and submitted this list to NPM to use as justification for purchase with monies recovered from the deleted larval feed expert budgeted funds. See annex K.
5.1 In response to a request from Prasert Sitasit, NPM THA/89/003 a letter of justification for the 6 months extension through 30 June 1992 was prepared and submitted to his office. Discussion with FIOD, FAO, Rome, with UNDP, Bangkok and with RAPA all supported this proposal. See annex L.
5.2 The NPM requested a briefing on the dietary mineral requirements of shrimp and fish was submitted. Sources and constraints for use of some mineral compounds was included. This material will be used as part of the reference materials for general review courses and inservice training courses in fish/shrimp nutrition and diet development given by NIFI. See annex M.
5.3 Interrelationships between vitamin C intake in fish and general fish health, resistance to certain diseases, and resistance to stress or trouma was discussed with Dr. Ron Roberts on his site visit on Ulcerdisease research in the fish disease section of NIFI.
5.4 A report on efficacy of vitamins C1, C2 and C3 TP in rainbow trout fed in seawater was presented at the aquaculture conference organized by the American Soybean Association.
5.5 General advice and suggestions for test diets for catfish and freshwater shrimp, and for design of experiments to measure nutritional response to various diet treatments was discussed with the NPM and staff.
5.6 SAFETY in the laboratories and in assay procedures with volatile solvents or compressed gases was reviewed with the NIFI staff. A temporary hood to confine and remove harmful vapors during crude fat assays in the quality control laboratory was installed. Safety chains are still not installed to fix compressed gas cylinders and the hydrogen gas reducing valve is often left open overnight.
5.7 General advice to Wimol and feed mill staff on record keeping for each formula and each manufacturing run. Established a record book for various diets made. See annex O.
6.1 Use funds saved by deletion of larval feed consultancy to purchase immediately the list of glassware, supplies and minor expendable equipment listed in Annex K.
6.2 Remodel the current lipid, amino acid, and vitamin sample preparation areas into a standard laboratory room equiped with adequate work benches, tables, utilities and make area rat and vermin free.
6.3 Adopt permanent laboratory procedures and records filing system for each sample prepared and run on equipment. Keep permanent files tracings and computer print outs of runs for fatty acids, amino acids, and vitamins assayed.
6.4 Wire and reactivate standby generator for feed mill operations.
6.5 Replumb boiler into pellet mills, obtain new parts for inactive mill and put both mills in repaired and operating condition for fish and shrimp feed manufacture.
6.6 Reverse airflow in tunnel dryer to remove moisture more efficiently and reduce drying costs.
6.7 Adopt scheduled planning meetings for weekly operations in laboratory and mill.
6.8 Develop proforma and work units for each area of research and development together with budgets for each as basis for long term planning and operations.
6.9 Emphasize SAFETY in all laboratory and milling operations to protect personnel.
| Mr. Prasert Sitasit | Government Project Manager, THA/89/003, NIFI |
| Mr. Imre Csavas | Regional Aquaculture Officer, FAO/RAPA. |
| Dr. Y. Kato | Director, FIO, FAO, Rome. |
| Mr. Chen Foo Yan | NACA |
| Mr. Sompong Hiranwat | Director, NIFI |
| Ms. Norma Bethke | Programme Officer FAO/RAPA |
| Dr. Noi Susangkorn | U. W. Virginia, Martinsville, W.VA, USA. |
| Dr. Mario Labadan | Agrispecialist, Inc. Manila. |
| Dr. Dean Akiyama | Am. Soybeans Association, Singapore. |
| Dr. Ian Portridge | Hoffmann La Roche, Sydney, Australia. |
| Dr. Ron Roberts | Stirling University, Stirling, Scottland. |
| Dr. Norbert Albers | BASF, Ludwigshafen, Germany. |
| Dr. Thierry Hatscha | Hoffman La Roche, Basel, Switzerland. |
| Dr. Bernard Ryan | Pfizer, Inc., Stores, Connecticut, USA. |
| Dr. Toshiro Tsuji | Takeda, (Thailand) Ltd. |
| Mr. Johann Stuyt | Programme Officer, UNDP, Thailand. |
| Mr. Sunil Saigal | Assistant Regional Representative, UNDP, Bangkok. |
| Dr. Mali Boonyaratpalin | NICA, Songkhla. |
