PART II TECHNICAL PAPERS (Cont.)

PRELIMINARY OBSERVATIONS ON THE NUTRITIONAL EFFECTS OF
A BETAINE/AMINO ACID MIXTURE: SURVIVAL, GROWTH AND
FOOD CONVERSION OF JUVENILE PENAEUS MONODON
FED WITH FINNSTIM¹

by

Eng-Huan Ung² M.Sc. and Matti Junilla³ D.V.M., M.S.c

ABSTRACT

A commercially available betaine (trimethylglycine)/amino acid mixture was tested on juvenile Penaeus monodon housed in individual test chambers so as to rule out death due to cannibalism for 60 days. Three pelleted diets were used using a commercial basal formulation. The FINNSTIM mixture was included in at 0 percent, 1 percent and 2 percent inclusion rates. Enhanced growth, food conversion and survival was observed. The control gave a 99.45 percent weight gain while the 1 percent and 2 percent regimes gave weight gains of 116.3 and 119.3 percent, respectively. Food conversion ratios of 3.74 and 3.94 were obtained for the 2 percent and 1 percent regimes, respectively, while the control performed less well at 4.64. Survival rates appeared to be enhanced at the 2 percent level only giving 88.8 percent survival while. the 1 percent and 0 percent diets gave 72.5 percent and 70 percent survival rates. Possible physiological reasons explaining the observed results are discussed. Both choline chloride and DL-methionine which are also methyl group donors were not included in the diet.

1. INTRODUCTION

Betaine or trimethylglycine functions as a methyl group donor which may be used to replace methionine and choline (Albers, et. al., 1984) (Figure 1). It occurs naturally in sugar beet and Finnish Sugar Co., Ltd. has been a leading producer of betaine. The company produces a commercial aquafeed additive by the trade name of FINNSTIM of which betaine accounts for more than half of its constituents. The other amino acids are glycine, alanine, isoleucine, leucine and valine. The composition is proprietary and is said to be the result of empirical experimentation.

Research carried out on a 1.5 percent FINNSTIM inclusion on arctic char Salvelinus alpinus gave a 68 percent survival rate against a control giving only 54 percent survival after a 30-week growth period at 35 ppt and temperature of 1–10 Celcius. Since S. alpinus is not a marine teleost, the betaine/amino acid mixture apparently had some osmoregulatory function to bring about survival enhancement. (Personal communication, Finnish Sugar Co., Ltd.).

In another experiment with 1.0 mg mean body weight white fish (Coregonus sp.) larvae, a 1.5 percent FINNSTIM inclusion gave a 39.9 percent survival against a 0 percent FINNSTIM control diet giving only 11.4 percent survival. A live zooplankton fed control gave a 37.2 percent survival showing that the FINNSTIM diet surpassed slightly the live food control. Reports from Spanish Penaeus japonicus farm trials with 1.5 percent FINNSTIM inclusion showed that survival rates could be improved from this betaine/amino acid mixture. (Personal communication, Finnish Sugar Co., Ltd.). The present experiment is to test its effect on P. monodon which is very widely cultured in Asia to see if FINNSTIM had any enhancement effects.

¹ The views expressed in this paper are those of the authors and do not necessarily reflect the views of FAO.
² Aqua-Ace Sdn. Bhd., 44, Jln. Camar 1, Johore Bahru, Malaysia
³ EWOS AB., P.O. Box 618, S-15127, Södertälje, Sweden

2. OBJECTIVE

The present experiment is intended to examine the nutritional effects of this betaine/amino acid mixture on P. monodon in an experimental set-up that would eliminate losses due to cannibalism that are normally encountered in any feeding trial. This is accomplished by using individual compartments to house the test animals.

Figure 1. Metabolic pathway for betaine

3. DIET

A local commercial diet formulation was used as the basal diet (Table 1). It should be pointed out that choline and DL-methionine which are also methyl donors were excluded from the diet in order to test the effect of betaine alone. There was also no lecithin inclusion as this is also a potential methyl donor.

Table 1. Test diets (37 percent protein, 4 percent lipids)

4. METHOD

Sibling Penaeus monodon were hatchery reared and placed at postlarva 20 stage into a small 0.1 ha outdoor nursery pond till about 1.4 grammes. They were then transported using oxygen to the laboratory where they were acclimated in 5 000 liter circular fiberglass tanks and fed on pelleted feeds. They were placed in individual compartments at about 1.6 grammes to prevent cannibalism during the experiment. Each compartment measured 18 × 15 × 22 cm or about 5.94 liters volume. These compartments were made using sterilized PVC baskets each subdivided into two by a central 1.0 mm HDPE netting screen.

Exactly 20 baskets i.e. 40 compartments were fitted into each of six fiberglass tanks. Each tank had a false bottom with coral sand, polyester wool and activated carbon to act as an in-situ bottom filter. The baskets were rested on the sand and overhead PVC water delivery pipes were installed. Water was jetted in under gravity pressure from a central overhead reservoir at the rate of 0.4 liters/min/compartment of 5.94 liters. Thus, there would be a complete water change every 15 minutes approximately through the compartment via the bottom filter.

The six fiberglass tanks used for the experiment were all connected on the bottom with PVC pipes with control valves attached. The pipes fed into a central drain pipe which in turn fed into a centrifugal pump (type DAB KP30/16M, Italy) which pumped water up into the overhead reservoir. This had a 1.25 m hydraulic head to facilitate gravity feeding.

Animals were fed in excess of satiation about six times daily with some pellets left over after each feeding. These uneaten pellets were removed using a wide-bore syringe at the next feeding time and their weights recorded. One diet was fed per tank so that there would be a duplicate trial for each of the three diets.

Animals were weighed every 15 days until the end of the experiment using a Whatmans 100 (UK) electronic balance (Table 2 and Figure 2).

Table 2. Growth (As percentage increase of body weight)

All weights given are expressed in grammes and are wet body weights.

Initial represents day 0 and final represents day 60 of experiment.

All weights measured using Whatmans (UK) 100 electronic balance.

Experimental feed was manufactured at a commercial prawn feed manufacturing plant using a Buhler-Maig pelleter in 5 mt lots and samples were taken from mid of processing. Steam pressure was 2.0 bar under steam conditioning and die temperature was kept at 90 Celcius. Three feeds were made: F O — a control without FINNSTIM, F 1 with 1 percent FINNSTIM and F 2 with 2 percent FINNSTIM. Feeds were stored under normal room temperature for about 20 days prior to first feeding. Room temperature conditions ranged from 25 to 30 Celcius. Once removed from the 25 kg bags which had three paper and one plastic layer, they were stored in small air-tight plastic bottles. Water samples were constantly monitored for pH, nitrate, nitrite, ammonia and phosphate. The experiment was carried out at 27 ppt salinity, pH 7.7 and the water temperature was thermostatically controlled to 27 Celcius ± 1.0 Celcius.

Figure 2. Growth (in percentage)

5. PRELIMINARY OBSERVATIONS

Penaeid growth under experimental tank conditions is always a fraction of what is normally achievable in a grow-out pond. In this case, due to the size of the experimental chamber of about 5.9 liters, the animals would not be able to swim around normally and may have affected the growth of the tail muscle which accounts for 65 percent of the body weight. The objective of ruling out death by cannibalism required individual compartments that have a practical size limit for a 240 chamber test. Additional stress may be from handling and low water depth which in the wild, penaeids are not fond of especially in the daytime. Nevertheless, all animals were subject to identical experimental conditions and this feeding trial is believed to be experimentally unbiased.

Our preliminary observations centre around three critical issues of concern to nutritionists, feedmillers and farmers alike. They are growth, survival and food conversion. These three factors will determine the commercial viability of FINNSTIM as a penaeid feed additive.

Growth as expressed in percentage increase in body weight revealed that FINNSTIM gave superior growth when added into a standard formulation commercial feed (Tables 3, 4, 5, 6, 7, and 8). The control gave a 99.45 percent body weight increase while the 1 percent FINNSTIM inclusion gave a 116.3 percent increase. The 2 percent inclusion gave a slightly higher increase of 119.3 percent. FINNSTIM is shown here to promote growth but whether it functions as an appetite stimulant or as an anti-stress agent is not known.

Even at commercial application rates of 1 percent FINNSTIM, a mean size of prawn about 9 percent better than a non-FINNSTIM feed can be expected. This premise, however, should now be pond tested where experimental laboratory stress is absent.

Survival was shown to be markedly better for the 2 percent FINNSTIM inclusion. The control and the 1 percent FINNSTIM inclusion were not significantly different at 70 and 72.5 percent, respectively. The two percent FINNSTIM inclusion gave a very superior 88.8 percent survival which was excellent for a 60 day experiment. It appears that FINNSTIM at the 2 percent level greatly enhances survival although its effects are not observable at the 1 percent level. FINNSTIM may be a good anti-stress agent like Vitamin C which would be good for prawn populations recovering or experiencing any form of environmental stress. Food conversions in laboratory scale experiments are often a factor of 2.0 higher than normal field trials from our experience (Table 9). FCR based on biomass increase showed that the 1 percent inclusion gave a FCR of 3.94 compared to a control of 4.64 (Figure 3). The 2 percent inclusion gave a slightly better FCR of 3.74.

Table 3. Growth in grammes: Diet F 0: Tank-4

Table 4. Growth in grammes: Diet F 0: Tank-3

Table 5. Growth in grammes: Diet F 2: Tank-6

Table 6. Growth in grammes: Diet F 2: Tank-1

Table 7. Growth in grammes: Diet F 1: Tank-5

Table 8. Growth in grammes: Diet F 1: Tank-2

Table 9. Food conversion (dry weight to wet weight basis)

Biomass taken to be total population weight of live animals.
All weights measured in grammes.
Feed fed to animals that died are not included.
Uneaten food removed from the tank are also not included.
Dry weight of feed not absolute as it contains about 11 percent moisture.
Food conversion ratio calculated as feed fed divided by biomass increase.

Figure 3. Food conversion ration

6. DISCUSSION

Growth in the 1 percent and 2 percent regimes were 17 percent and 20 percent higher than in the control. Although it cannot be ascertained for sure at the moment, this may be due to the betaine/ amino acid mixture stimulating feeding responses from the test animals. Ishida and Hidaka (1987) found that four or five species they tested by recording neural responses from the facial nerve innervating the anterior pallate were responsive to glycine betaine at 10^-4 M although proline thresholds were lower at 10^-8 - 10^-6 M. Schmidt and Gnatzy (1986) found that the walking legs of the crab Carcinus maenas had broadly tuned and narrowly tuned sensory units both of which types were sensitive to betaine as well as other amino acids at 10^-6 - 10^-4 M. Studies on the spiny lobster by Ache, et. al., (1986) suggest that different odorant molecules compete for common receptor sites in the antennular olfactory pathway in a manner consistent with competitive inhibition. The betaine/amino acid mixture may have solicited additional appetite stimulation but it cannot be ruled out that increased growth could also have been due to metabolic reasons, e.g. methyl group donation. This may be the reason for superior FCRs for the FINNSTIM diets.

Survival enhancement was extraordinary at 89 percent for FINNSTIM 2 percent inclusion as compared with 70 percent and 72.5 percent for the control and the 1 percent inclusion (Table 10 and Figure 4). This data agrees with farm trial data involving P. japonicus cultured in Spain where controls gave about 60 percent survival while a 1.5 percent inclusion gave close to 90 percent survival (Personal communication, Finnish Sugar Co., Ltd.).

Table 10. Survival

Initial represents population at day 0 while final represents day 60.
Moribund animals, if any are classified as dead.
There are no cases of accidental death or cannibalism.
Handling during weight measurement and relatively low water depth may have constituted some form of stress upon the experimental animals.

In mammals, certain detoxication reactions, e.g. pyridine and nicotinic acid involve methylations and it is very possible that various methyl compounds, e.g. epinephrine derive their methyl groups from dietary choline, methionine and betaine (Albanese, 1950). It may be that increased methylation activity in the test animals may have somehow acted to reduce a form of unidentified physiological stress experienced during the experiment. Dalla Via (1986) for example showed that during hypoosmotic shock, the Free Amino Acid (FAA) pool can lower by 80 percent while during hyperosmotic shock, the FAA pool can increase by 86 percent in P. japonicus. Since glycine is the main osmoeffector, rapid demethylation from betaine which is trimethylglycine may explain its apparent osmoregulatory function in S. alpinus subjected to marine conditions as well as Spanish P. japonicus reported by Finnish Sugar Co., Ltd. The animals in the present experiment were not subject to stress in the form of fluctuating salinities but were subject to handling during weight measurements five times during the course of the experiment. This may have possibly constituted some form of osmotic stress, too. Nevertheless, the extraordinary survival enhancement shown at the 2 percent FINNSTIM inclusion may be useful for incorporation to certain larval, starter, booster and medicated feeds as well as feeds for animals that are probably going to experience wide fluctuations in salinity.

Figure 4. Survival (in percentage)

Future experiments should examine betaine against choline and methionine in relation to growth, survival, enhancement, FCR and cost efficiency.

REFERENCES

Albers, N., G. Behm, D. Dressler, W. Klaus K. Kuther, and H. Lindher. 1984 Vitamins in animal nutrition. AWT Press. Adenauerallee 170. 5300 Bonn.

Albanese, A.A. 1950 Protein and amino acid requirements of mammals. Academic Press. New York.

Ache, B.W., R.A. Gleeson, and D.H. 1986 Thompson. Chem. Senses 11(4): 575.

Dalla Via, G.J. 1986 Salinity responses of the juvenile penaeid prawn Penaeus japonicus 2: Free Amino Acids. Aquaculture, 55 (4):307–316.

Ishida, Y. and I. Hidaka. 1987 Gustatory response profiles for amino acids, glycine betaine and nucleotides in several marine teleosts. Bull. Jap. Soc. Sci. Fish. 53(8):1391–1398.

Schmidt, M. and W. Gnatzy. 1986 Chem. Senses 11(4):657.

FORMULATION OF FEEDS FOR CAGE CULTURE OF FINFISH
IN TAAL LAKE, PHILIPPINES

by

Sofia S. Basa¹

ABSTRACT

This paper is the result of an experiment done at Taal Lake, Batangas, South of Manila where formulated feeds using local ingredients were tested. Three floating net cages each measuring 5 × 10 × 5 m were stocked with 1 800 Tilapia nilotica fingerlings 8–10 g in weight. Cage 1 was fed with test diet; Cage 2 was fed only ricebran and Cage 3 has no supplemental feed. Growth rate, feeding rate and feed conversion were determined. Mean weight gain was significant in Cage 1. Feed conversion ratio was excellent. The size of cages and stock density used were the actual size and practice of tilapia cage culture farmers.

1. INTRODUCTION

Ricebran is the most common single ingredient supplemental feed used by most fish culturists in the Philippines. This is available in local stores as D1 and D2 according to its fineness. However, to meet the nutritional requirements of the fish being cultured, formulation of supplemental dry diets to meet the protein and energy requirements for their growth and reproduction may be resorted to in case the natural food in the culture system is not sufficient. Vitamins and minerals may also be added.

Cage culture of tilapia is widely practised in the Philippines particularly in lakes. Tilapia nilotica is next to milkfish in importance for local consumption. Its market price is sometimes higher than the milkfish. The purpose of this experiment was to assist farmers formulate feeds for their fish and determine the effect on fish growth. Most farmers use ricebran by broadcasting in the cages. Much of this is lost, hence, a waste of inputs.

2. MATERIALS AND METHODS

The chemical composition as well as the physical properties of the raw materials used are known. The efficiency of the formulated feed for the growth and survival of the fish and the cost of the diet are important considerations in commercial aquaculture operations. The factors considered in formulating a practical diet were:

2.1 The nutrient requirement of the fish

Tilapia nilotica which was the species used in this experiment requires protein, energy (lipids and carbohydrates), vitamins and minerals in their diet. Table 1 shows the protein requirements for Tilapia nilotica fingerlings 3–8 g as crude protein of 20–30 percent.

2.2 Availability and cost of feed ingredients

The feed ingredients used in this experiment were: ricebran, fish meal, copra meal and ipil-ipil leaf meal. These were readily available in the market. Peruvian fish meal was preferred over the local fish meal due to very high C.P. content of 60 percent over 45 percent C. P. of local fish meal.

Feed ingredients used	Cost per kilogram ()*
Ricebran	2.50
Fish meal	17.00
Copra meal	3.00
Ipil-ipil leaf meal	1.50

* US$1.00 = 20.00

¹ Supervising Fishery Biologist and Project Leader, Feed Formulation Project, Bureau of Fisheries and Aquatic Resources, Arcadia Bldg., 860 Quezon Avenue, Quezon City, Philippines

Table 1. Protein requirements of some local species*

* After Santiago, 1986

2.3 Nutrient contents of feed ingredients used

The nutrient contents of the feed ingredients used were based on the analyses made by Castillo and Gerpacio (1979). Table 2 gives a list of some feed ingredients and their nutrient composition.

2.4 Availability of nutrients to the fish being fed

Digestibility of nutrients in cereal grains and other fibrous feedstuffs is lower for fish unlike fats and oils which are highly digestible to fish.

Fish feed are usually formulated with the presumption that one feedstuff will substitute for another. These are made strictly on the basis of nutrient content because of insufficient data on digestibility for other species of fish like tilapia.

2.5 Process or method

Formulation of feed was done by the use of meat grinder. The feed ingredients were sieved first, then weighed following the formula:

Water was added to the 10 kilograms mixture at 3.5 kg. Premix vitamins was also added.

2.6 Stability of pellets in water

To make a water stable feed, grinding the feed finely, adding oil and fiber or adding a binder which are also sources of nutrients were pelleted. The pellets were stable up to 10–12 minutes in the water before disintegrating and the tilapia readily consumed them in the cage.

Table 2. Proximate analyses of some feedstuffs

Source: Castillo, L.S. and A.L. Gerpacio, 1979. Nutrient composition of some Philippine feedstuffs. UPLBTech. Bull. 21. 4th ed. 117p.

2.7 Size of pellets

The optimum particle size of pellet was based on what the tilapia can readily eat. The smaller particles have, however, greater surface area exposed to water allowing leaching of nutrients and are more likely not eaten except perhaps by other filter feeders. Uneaten food will just disintegrate in water. Tilapia was found to consume unpelleted feed almost as efficiently as pelleted feed.

2.8 Density

The fish feed was made to stay in the water column long enough for the tilapia to eat them.

2.9 Attractability

The fish should be attracted to either its taste, color and shape. Feed ingredients like fish meal, shrimp meal and fish oils are used not only for their nutritional value but also for their palatability to fish.

2.10 Texture

The texture of pellets is inversely related to the amount of fines produced in a packaged feed. Fines should not exceed 10 percent of the total weight.

3. RESULTS AND DISCUSSIONS

Three fish cages measuring 5 × 10 × 5 m located in Taal Lake, Talisay, Batangas were stocked each with 1 800 Tilapia nilotica fingerlings (8–10 g wt each). Cage 1 was fed with formulated feeds, Cage 2 was fed with commercial ricebran only and Cage 3 served as the control.

Feed ration per day at 6 percent of fish biomass daily was given. Feeding rate was reduced after the third month to 3 percent of biomass (Table 3).

From the table data there was an increase of 17.60 g on the average weight of the fish fed with formulated feeds compared to the average weight of the fish fed with ricebran. A 2.47 g increase was recorded from the average weight of the fish without supplementary feed.

The initial mean body weight of 19 656 g of the fish in Cage 1 given formulated feeds increased to 116 802 with a total weight gain of 97 146 g (97.146) or an average of 10.92 g compared to the total weight increment of 76 428 g (76.428) or an average weight of 4.83 g in Cage 2 for those fed with ricebran alone (Table 4).

The feed conversion ratio is 1.028 for Cage 1 and 0.591 for Cage 2 is within the normal range of feeding tilapia in cages (Table 5).

During the month of October, the lake nutrient was highest at 7.5 mg/l (Viliamater, 1987). Also observations during the culture period from July to November, the lake water becomes fertile which is favourable for growth of the fish. A 90 percent rate of survival was recorded for each of the three cages.

4. SUMMARY

The FCR of 1:028 was excellent since the cost of the formulated fish diet was only 6.00 per kilogram. A monthly growth increment of 4.94 g was recorded. Although the fish only attained a weight of 53.97 g per piece, we were able to sell the harvest at P20.00 (US$1.00) per kilogram.

Table 3. Total and average data from the three cages

Table 4. Total weight gained after 104 days of feeding experiment

Table 5. Food conversion ratio (FCR) of tilapia reared in three cages

Another retrial is being done this year using a 30 percent crude protein formulated fish diet. Tilapia nilotica fingerlings 5–8 g initial mean body weight will be fed with 3 percent of fish biomass for Cage 1; 5 percent of fish biomass for Cage 2; 8 percent of fish biomass for Cage 3 and Cage 4 as control. The four cages each measuring 5 × 10 × 5 m will be stocked with 1 500 Tilapia nilotica fingerlings.

ACKNOWLEDGMENT

The author would like to acknowledge the Project staff for their invaluable services. She is also grateful for the assistance extended by Mrs. Medina N. Delmendo, Project Coordinator, ASEAN/UNDP/FAO Regional Small-Scale Coastal Fisheries Development Project.

LITERATURE CITED

Mok, T.K. 1982 Feeds and feeding of fishes in cages. SCS/GEN/82/34. Manila, South China Sea Fisheries Programme, 1982.

Natividad, J.M. 1982 Fish diet formulation. Fisheries Newsletter ISSN 0115-2459, Vol. XI, No. 4, October-December. pp. 67–71.

Pascual, F.P. 1983 Nutrition and feeding of Penaeus monodon. ISSN-0115-5369, Extension Manual, No. 2, Third Edition. June 1983.

Santiago, C.B. 1983 Feed formulation, nutrition and feeding. LP/T/I/HO-2. Mimeographed.

Santiago, C.B., et.al. 1986 An evaluation of formulated diets for Nile tilapia fingerlings. Fish. Res. Jour. of the Phil. Vol. 11, Nos. 1 and 2, Jan-Dec. 1986.