CHAPTER 1

INTRODUCTION

1.1

BACKGROUND OF STUDIES

This experimental based studies is aim to evaluate potential of Golden Apple Snail (GAS), Pomacea canaliculata as basic ingredient in formulation of fish pellet. This experiment will divide into 3 phase. First phase is proximate analysis of GAS meal to evaluate nutritional value of GAS. In this phase determination of moisture, ash, crude protein, crude lipid, crude fiber and digestible carbohydrates in GAS meal would be evaluate. Second phase is pellet production to partially replace or replace the fish meal using GAS meal. In this phase 5 different pellets would be formulated using 0%, 25%, 50%, 100% and 100% of GAS meal to replace the fish meal in pellet ingredient . Third phase is feeding test of different pellet formulated to the Iridescent Shark, Pangasius sutchi. This feeding test will done in one month period and in this phase the determination of Mean weight gain, Percentage body weight gain, Specific growth rate, and Feed conversion ratio of this will be determine.

1.2

STATEMENT OF PROBLEM

It is known that GAS is pest for the rice ( Sison J.A 1985) . GAS bring a lot of problem to farmers not only Malaysia but nowadays globally. Farmers cultured the snail indoors but when market response was poor many snail projects were abandoned and in many instances the snail escaped and ravaged the rice crop with losses running into millions of dollar (Naylor, 1996). Golden Apple Snail (GAS) (Pomacea spp) is considered a major pest in the rice ecosystems of Laos, especially in lowland rice fields (Douangbupha et al, 1998). According to Philippines Rice Research Institute GAS bring to agricultural economic losses $ 1 billion losses to Philippine rice crops in 1980s and 55-248 billion/year (globally). In Malaysia GAS spread rapidly following its occurrence in Keningau in 1992. To date the districts infested with the pest include Keningau, Tenom, Sook, Nabawan, Tambunan, Papar, Penampang, Kota Kinabalu, Beaufort, Kuala Penyu, Tuaran, Sipitang, Kota Belud and Kota Marudu covering an area of about 5,000 ha (Teo et al. 2002).

The protein source of choice for most fish and crustaceans is fishmeal or 'trash fish' (small fish forming the low-value component of commercial catches). However, supplies are declining and prices are increasing. There is also an increasing conflict between use of trash fish/fishmeal for aquaculture, or for human consumption (particularly for low-income indigenous people). One reason for insufficient supply of trash fish for fish meal manufacture is because about one third of the fishery catch is thrown overboard. Fishers need improved technology so a higher percentage of the catch can be landed (Peter Edwards et al. 2004). There is growing awareness of the danger and unpleasantness of air and water pollution, and it is likely that these
2

issues will continue to be of great concern to the fish meal industry (M. L. WINDSOR 2001). Every year price of fish pellet was increase because increasing price of raw material. Rising global demand for fishs trash has increased the pressures on wild fisheries with catches no longer able to satisfy demand. This is resulting in declining catch numbers and more juvenile fish being removed before they have bred.

1.3

SIGNIFICANCE OF STUDY These studies will perhaps a foundation of resolving matter for help paddy planter and

aquaculturist. This is due to the decreasing attack of GAS will be reduce by increasing demand and gives them side income for selling the GAS from their paddy farm. Aquaculturist also get benefit because their can get fish pellet with a low price. This is because decreasing of raw material price in using GAS meal in fish pellet production.

1.4

OBJECTIVE The aim of this study is to determine the potential of GAS as protein sources for fish

pellet production. This project will be dividing into two phase: first phase will be focuses in determination of moisture and dry matter, ash, crude lipid, crude protein, crude fiber. Second phase will stress in determination of perfect ratio in fish pellet production .This experiment would done to Pangasius sutchi fingerling to determine Growth performance, Feed Conversion Ratio (FCR) ,and food efficiency of all fingerling to determine the best ratio of formulated pellet.

To determine the nutritional composition in Golden apple snail powder. To determine the optimum proportion of a mixture of Golden apple snails powder in fish pellet.

To determine effect of Golden apple snail powder in Pangasius sutchi fingerling.

CHAPTER 2

LITERATURE REVIEW

2.1

INTRODUCTION TO Pomacea canaliculata.

2.1.1 TAXONOMY Taxonomy of Pomacea canaliculata(Lamarck, 1819) is below: Kingdom : Animalia Phylum : Mollusca Class : Gastropoda Order : Caenogastropoda Superfamily : Ampullarioidea Family : Ampullariidae Genus: Pomacea canaliculata(Lamarck, 1819)

2.1.2 MORPHOLOGY OF GAS.

This species is known to have a globular shell in shape. Normal coloration typically includes bands of brown, black, and yellowish-tan; color patterns are extremely variable. Albino and gold color variations exist. The size of the shell is up to 150 mm in length (Howells, R.2008). Apple snails have a dextral shell except the apple snails from the genus Lanistes who have a sinistral shell (shell opening at the left), although there are reports of sinistral animals in the other genera (CAZZANIGA, N. J. & ESTEBENET, A. L. 1990) The surface of the shell can be smooth or rough, depending of the species and the environment. Malleation (hammered surface) occurs mainly when the snail grows fast. In such cases the anorganic periostracum, which is first created, is very thin and wrinkles before it becomes enforced with the rigid ostracum and hipostracum. Stria (growth lines) are formed at slower growth. New shell material is deposited bit by bit at the shell opening, creating the a new small ridge or stria with every new piece added. The shell lip (the margin of the shell at the shell opening) is often thickened in mature snails, while it is sharp in younger snails. The lip is curved at the outside, while rather straight near the umbilicus. The operculum (the shell door) is a corneous plate (except in the genus Pila, where the operculum is calcified at the inside during the life of the snail), with a concentric structure and a nucleus near the parietal margin (close to the umbilicus). With this shell door, the snail is able to close of its shell to survive periods of drought and as protection against predators (KEAWJAM, R. S.& Upatham, E.S. 1990.).

2.1.3 DISTRIBUTION OF GAS.

Apple snails inhabit a wide range of ecosystems from swamps, ditches and ponds to lakes and rivers. Not every species has similar preferences. However, most apple snails prefer lentic waters above turbulent water (rivers) (PERERA, GLORIA & WALLS, JERRY 1996). The

golden apple snail (Pomacea canaliculata Lamarck) is a freshwater prosobranch indigenous to South America. In the 1980s it was brought in from Argentina to Taiwan for commercial production as a food source (Mochida, 1991). From Taiwan the snail was distributed to the third world countries for backyard rearings to generate side-incomes (Anderson, 1993). The estimated snail-infested areas were 171,425 ha in Taiwan in 1986, 16,195 ha in Japan in 1989 and 400,000 ha in the Philippines in 1989 (Mochida, 1991).

2.1.4 POTENTIAL OF GAS. The GAS flesh has a high protein content, which means it can be useful as a locally available feed resource for monogastric livestock (Lampheuy Kaensombath and Brian Ogle 2006). Ensiled and fresh Golden Apple Snail (GAS) flesh as replacement for fish meal had nonegative effects for fattening pigs in terms of daily weight gains and feed conversion ratios. However, feed dry matter intakes were lower when GAS meal replaced fish meal. The cost of the diet with ensiled GAS flesh was lower than of the diets with fish meal and fresh GAS, including the labor cost of processing the snails. The snails in fresh or ensiled form in diets for growing pigs can be profitable if the farmers collect and process the snails themselves, and there should be an additional benefit of higher rice yields (Lampheuy Kaensombath and Brian Ogle 2006). Apple snails are well edible and are often considered a protein rich delicacy. Consuming these snails is therefore an interesting option in those areas where they have become a pest and treat for the rice and taro production. In such cases, eating these snails has two benefits that is collecting the snails is encouraged and the diet of the farmers (especially in thirth world countries) is enriched with a protein source. (Stijn A. I. Ghesquiere 1998). GAS appear in large numbers and are a potential feedstuff for on farm feed preparation but not for industrial manufacturing of aquaculture feed. This snail used as fish feed traditionally in Korea, Indonesia, Thailand (Ravindra C. Joshi). Feeding trial on Nile Tilapia in aquaria showed that GAS meat meal at 75-100% of the diet mixed with rice bran was beneficial (Cagauan and Doria, 1989) GAS has a high nutritive value and could be used to completely replace fish meal in African catfish production (O Phonekhampheng 2008).

2.2

INTRODUCTION OF Pangasius sutchi

2.2.1 TAXONOMY OF Pangasius sutchi Kingdom : Animalia

Phylum

: Chordata

Class

: Actinopterygii

Order

: Siluriformes

Family

: Pangasiidae

Genus

: Pangasius

Species:

P. hypophthalmus

Binomial name Pangasius hypophthalmus(Sauvage, 1878)

Synonyms Pangasius sutchi Fowler, 1937

10

2.2.2 Pangasius sutchi MORPHOLOGY. The sutchi catfish is a popular fish for aquaculture; it is cultured widely in Asia in countries such as Vietnam, Malaysia, Indonesia, Laos, Cambodia, and China. This fish have long body, latterly flattened with no scales. Head relatively small. Mouth broad with small sharp teeth on jaw, vornerine and palatal bones. Eyes relatively large. Two pairs of barbels, upper shorter than the lower. Fins dark grey or black. Six branched dorsal-fin rays. Gill rakers normally developed. Young fish have black stripe along lateral line and another long black stripe below lateral line; large adults uniformly grey but sometimes with greenish tint and sides silvery. Dark stripe on middle of anal fin; dark stripe in each caudal lobe; small gill rakers regularly interspersed with larger ones (Gustiano, R. 2003). 2.2.3 DIET OF Pangasius sutchi P. sutchi is omnivorous, feeding on algae, higher plants, zooplankton, and insects, while larger specimens also take fruit, crustaceans and fish (Griffiths et al.2010). The farm made diet for Pangasius sutchi is primarily compose of several kind of locally available by-product. Farmer also use formulated pellet when local by-product was not available (C D Webster 2002). Ingredient % Fish meal 15 Soybean meal 15 Groundnut 24 Ricebran 30 Broken rice 15 Vitamin and mineral premix 1 Table 1: Model diet formulae for Pangasius (C D Webster 2002).

11

2.2.4 POTENTIAL OF Pangasius sutchi P. hypophthalmus, are important targets of commercial fisheries, such activities are considered to have led to the decline of stocks in recent years (Allan et al. 2005). P. hypophthalmus is presently cultured in several countries, including Thailand (Na-Nakorn et al. 2006) and Vietnam (Phuong et al. 2008). iet Nam exports P. hypophthalmus to over 80 countries, including several in Europe (especially Poland and Spain), Asian countries, Mexico, Australia, the United States of America, and the Middle East. New markets such as Russia are emerging. The European Union remains the most significant market (35 percent by volume, 40 percent by value).Viet Nam has the capacity to process 3 500 tonnes of aquatic product daily and the capacity is increasing. There are presently 405 industrial-scale processing plants in Viet Nam, of which 301 are certified for export to Europe and 30 are certified to export to the Russian Federation;16 percent are currently ISO certified (Griffiths,2010).

FIGURE 1: Global aquaculture production of Pangasius hypophthalmus (FAO Fishery Statistic)

12

CHAPTER 3

MATERIAL AND METHODS 3.1 STUDY LOCATION

The study was conducted at the freshwater hatcheries of the Universiti Malaysia Terengganu and labrarotary (UMT) between July 2010 and February 2011.

3.2

GAS MEAL PREPARATION GAS were collected from FELCRA oil palm plantation from Kg. Kuala Tahan , Jerantut ,

Pahang, Malaysia. There are many methods to process GAS meal first is: Snail was put in clean water without feeding for 48 hour to make sure intestine is emptied. GAS boiled in water for 1520 minutes. The flesh was separate from it shell minced and dried at temperature not more than 0 (Basa S.S 1988). A second method is the shell was broken and removed, and the remaining

cover and flesh cleaned with water before chopping and grinding (O Phonekhampheng et al 2008). The third method is the shells and cover of the snails were removed and then the flesh washed with clean water and drained. The flesh was then chopped into small pieces of 0.51.0 cm in size. Then both were weighed and mixed in the proportion of 10% of molasses to 90% of rice bran, on a fresh basis (Lampheuy Kaensombath et al 2004).

13

3.3 PROXIMATE ANALYSIS The proximate chemical composition of feed ingredients were estimated by the methods described by the (AOAC, 1984), to determine the crude protein content. Proximate analysis would done to 6 different sample such as GAS meal, pellet GAS0, GAS25, GAS50, GAS100, GAS100B. This phase would focuses in determination of moisture and dry matter, ash, crude lipid, crude protein, crude fiber.

3.4

PELLET PRODUCTION. Five type of experimental fish pellet (GAS0 (control), GAS25, GAS50 ,GAS100

,GAS100B ) were formulated (Table 2). The diets were prepared by mixing all dry ingredients in a mixer and subsequently adding the wet ingredients to form moist dough. A dough steaming for 15 minutes and will put to forming machine to form a favorable shape to feed. Feed were dry inside oven under temperature 60C for 12-24 hour. Dried feed were cut using hand to make it smaller.

Ingredient/ratio (fish meal : GAS meal Fish meal GAS meal Soy bean meal Rice bran Wheat flour Fish oil Mix vitamin Mineral

0%(control)

25%

50%

100%

100+ shell% 0g 140 g 466.9 g 37.1 g 21 g 21 g 7g 7g

140 g 0g 466.9 g 37.1 g 21 g 21 g 7g 7g

105 g 35 g 466.9 g 37.1 g 21 g 21 g 7g 7g

70 g 70 g 466.9 g 37.1 g 21 g 21 g 7g 7g

0g 140 g 466.9 g 37.1 g 21 g 21 g 7g 7g

Table 2: pellet ingredient

14

3.5 3.5.1

FISH MANAGEMENT TANK DESIGN

Tank: A (Control) Fish: 20 Feeding : pellet GAS0%

Tank: B Fish: 20 Feeding : pellet GAS25%

Tank: C Fish: 20 Feeding : pellet GAS50%

Tank: D Fish: 20 Feeding : pellet GAS100%

Tank: D2 Fish: 20 Feeding: pelletGAS100%s

Aeration : 24 hour Tank size : 150l

Figure 1: Culture system of Pangasius sutchi

3.5.2 CULTURE SYSTEM

Pangasius sutchi fingerlings were obtained from a freshwater hatchery UMT. Fish from same brood stock will culture in 5 different tanks. Each tank contains 20 Pangasius sutchi fingerlings. Each tank will supply with aeration. Water quality parameters (temperature, salinity, pH, and dissolved oxygen) during the experimental period will be observed.

15

3.6

FEEDING AND GROW RATES DETERMINATION. Fish in each tank will be give different formulation of pellet GAS0, GAS25,

GAS50, GAS100, GAS100B twice per day in each different tank. Fish will be measure every week until 1 month duration of experiment. The following calculations were made on collected data to describe and evaluate fish performance: (i) Mean weight gain (MWG) = Wf -Wi Where, Wf is the mean final fish weight and Wi the mean initial fish weight.

(ii) Percentage body weight gain (PWG) = (MWG x 100)/Wi

(iii) Specific growth rate (SGR, %) (Brown 1957) = [(log Wf log Wi) x 100)]/D

(iv) Feed conversion ratio (FCR) = feed consumed (dry weight)/fish weight gain

3.7 DATA ANALYSIS The data were subjected to analysis of variance (ANOVA) by using the General Linear Model (GLM) procedure of Minitab version 14 (2000). When the F test was significant (p<0.05) the Turkeys test for paired comparisons was used to compare means.

16

CHAPTER 4

PROJECT SCHEDULE

Year

2010

2011

Project activities/ week 1. Preparation of research proposal. 2. Sent a research proposal 3. Preparation of golden apple snail meal 4. Lab set up. a) Lab design set up b) Hatcheries design set up. 5. Breeding and care of fish for test. 6. Proximate analysis for golden apple snail meal. 7. Formulation of fish pellet. 8. Production of fish pellet 9. Proximate analysis of each pellet 10. Determination of growth rates of fish given different pellet. a) Run experiment and data collection 11. Determination of water quality in different tank. a) Run experiment and data collection 12. Data analysis 13. Report writing 14. Send report.

M J J A S O N D J F M x x 0 x 0 x x x x x x x x x x x x x 0 x x x x 0 x x x x 0

x : Activities

0: Planned milestone

17

CHAPTER 5 MILESTONE Date 3/8/2010 Progress Completing first draft proposal Setting up experiment 1st week of August 2nd week of August 3rd week of August Preparing GAS meal. Preparing formulated diet Introduce fish to tank. Feeding experiment Water quality sampling 4th week of August 1st ,2nd week of September 22th of August 29th of August 5th September 12th September 13th September 4th week of September 1st and 2nd week of October 3rd week of October onwards Completing thesis. Proximate analysis of GAS meal and each formulated feed. 1st sampling 2nd sampling 3rd sampling 4th sampling Cleaning tank and experimental stuff. Data analysis

18

CHAPTER 6

REFERENCES

Allan JD, Abell R, Hogan Z, Revenga C, Taylor BW, Welcomme RL, Winemiller K ( 2005) Overfishing of inland waters. Bioscience 55:10411051 ANDERSON, B. 1993. The Philippines snail disaster. The Ecologist 23, 70 72. Australian Centre for International Agricultural Research ACIAR Basa S.S 1988 : COUNTRY REVIEW Caugauan,A.G, and L.S.Doria1989 golden apple snail meal as feed for Nile tilapia fingerling in aquaria.CLSU Scientific journal.9(3) CAZZANIGA, N. J. & ESTEBENET, A. L. 1990. A sinistral Pomaceacanaliculata (Gastropoda: Ampullariidae). Malacologica l Review, 13(1/2): 123-143. C D Webster, Aquaculture Research Center, Kentucky State University, USA, C E Lim, USDA-ARS, Fish Diseases and Parasites Research Laboratory, Auburn, Alabama, USA. Gustiano, R. 2003. Taxonomy and phylogeny of Pangasiidae catfishes from Asia (Ostariophysi, Siluriformes). PhD thesis. Katholieke Universiteit Leuven, Leuven, Belgium. 295 pp. Griffiths, D., Van Khanh, P., Trong, T.Q. FAO. 2010. - . Cultured Aquatic Species Information Programme. In: FAO Fisheries and Aquaculture Department [online]. Rome. Updated 16 February 2010. [Cited 3 August 2010].

19

Howells, R. Personal communication. Texas Parks and Wildlife Department. In: United States Geological Survey. 2008. Pomacea canaliculata.USGS Nonindigenous Aquatic Species Database, Gainesville, FL. Revision Date: 2/4/2008 KEAWJAM, R. S.& Upatham, E.S. 1990. Shell morphology, reproductive anatomy and genetic patterns of three species of apple snail of the genus Pomacea in thailand. Med. & Appl. malacol., 2: 45-57. Lampheuy Kaensombath* and Brian Ogle**Laboratory-scale ensiling of Golden Apple Snails (GAS)(Pomacea spp)Lampheuy Kaensombath* and Brian Ogle Lampheuy Kaensombath* and Brian Ogle**Faculty of Agriculture, National University of Laos, Vientiane, Lao PDR Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Box 7024, 75007 Uppsala, Sweden Peter Edwards, Le Anh Tuan, Geoff L. Allan A survey of marine trash fish and fish meal as aquaculture feed ingredients in Vietnam ACIAR Working Paper No. 57 (printed version published in 2004) M. L. WINDSOR 2001 DEPARTMENT OF TRADE AND INDUSTRY TORRY RESEARCH STATION Fish Meal. MOCHIDA, O. 1991 Spread of Freshwater Pomacea snails Pilidae Mollusca from Argentina to Asia. MICRONESICA 3: 51-62 Na-Nakorn U, Kamonrat W, Ngamsiri T (2004) Genetic diversity of walking catfish, Clarias macrocephalus, in Thailand and evidence of genetic introgression from introduced farmed C. gariepinus. Aquaculture 240:145163 OPINION 1913 Pila Rding, 1798 and Pomacea Perry, 1810 (Mollusca, Gastropoda): placed on the Official List, and AMPULLARIIDAE Gray, 1824: confirmed as the nomenclaturally valid synonym of PILIDAE Preston, 1915 O Phonekhampheng, L T Hung* and J E Lindberg( 2009). Ensiling of Golden Apple snails (Pomacea canaliculata) and growth performance of African catfish (Clarias gariepinus) fingerlings fed diets with raw and ensiled Golden Apple snails as protein source. PERERA, GLORIA & WALLS, JERRY 1996. Apple Snails in the Aquarium. 121 pages, T.F.H. Publishing Ltd Phuong NT, Sinh LX, Thinh NQ, Chau HH, Anh CT, Hau NM (2008) Economics of aquaculture feeding practices: Viet Nam. In: Hasan MR (ed) Economics of
20

aquaculture feeding practices in selected Asian countries, FAO Fisheries Technical Paper 505. FAO, Rome, pp 183205 Ravindra C. Joshi Philippine Rice Research Institute (PhilRice), Maligaya, Science City of Muoz, Nueva Ecija 3119, Philippines E-mail: rcjoshi@philrice.gov.ph Robert H. Cowie & & IUCN/SSC Invasive Species Specialist Group (ISSG). (Last Modified: Wednesday, 13 April 2005) Global Invasive Species Database, 2008. Pomacea canaliculata . Sison J.a (1985): Hand book on crisis management on feedmeal and technology for the Philippines. .Feedindex (Phills.),Quezon City. The Philippines. Stijn A. I. Ghesquiere 1998 http://www.applesnail.net/ Teo, Su Sin1 Golden Apple Snail (Pomacea canaliculata Lamarck, 1819) in Sabah, Malaysia Current Situation and Management Strategy

21

APPENDIX

1) Nutrient determination and fish pellet production. Proximate analysis: Moisture Procedure 1. Accurately weigh a moisture dish of appropriate size. 2. Add approximately 10 g of the comminuted sample and reweigh. 3. Place the container in a vacuum oven at 100 oC and less than 100 mm Hg for approximately 5 hours. 4. Remove dish from the oven, cover, cool in desiccator, and weigh. 5. Redry 1 hr and repeat process until constant weight has been achieved, i.e., change in weight between successive dryings at 1 hour intervals is < 5 mg. Calculation Calculate the percentage moisture (wet weight basis) as follows: 100 (P-a) percent moisture = P P = weight in g of sample a = weight in g of dried sample %

22

Proximate analysis: Ash Procedure 1. Accurately weight ca 5 g of sample in a crucible wich has been ignited and tared (use 2.5 g of sample in the case of products which have a tendency to swell). 2. Place crucible in drying oven at 100 oC for 24 hours. 3. Transfer to cool muffle furnace and increase the temperature step wise to 550 oC 5 oC. 4. Maintain temperature for 8 hours or until a white ash is obtained. 5. If white ash is not obtained after 8 hours, moisten ash with distilled water, slowly dry on a hot plate, and re-ash at 550 oC to constant weight. Repeat if necessary. 6. Remove crucible to a desiccator and weight soon after cool. Calculation Calculate the percentage ash content (wet weight basis) as follows: (wt. crucible and ash - wt. crucible) % ASH (wet)= (wt. crucible and sample - wt. crucible) Calculation of ash content on dry basis (when moisture content is known) as follows: % ash (wet) % ASH (dry)= (100 - % moisture) x 100 x 100

23

Proximate analysis: Crude protein Procedure 1. Accurately weigh a suitable quantity of fine-grained material (ca 1.2 g for fishmeal, ca 2.5 g for solubles or homogenized fish) and place in digestion flask. 2. Add sequentially 15 g Na2SO4, 1 g CuSO4, one or two selenized boiling granules and 25 mL of conc H2SO4 to the flask. 3. Digest until solution is almost colourless or light green (2 hrs for inorganic material) and then at least a further 30 minutes. Do not heat any part of the Kjeldahl flask above the level of the digestion mixture. 4. Cool (do not allow to solidify), and cautiously add 200 mL water. Add additional boiling granules (if necessary) to prevent bumping. 5. Pipette 100 mL 0.1 N HCl into a 500 mL erlenmeyer flask, add 1 mL Conway's indicator and place the flask under the condenser ensuring that the condenser tip is immersed in the acid solution. (volume of standardized HCl used in distillation may be varied according to the expected nitrogen content of the sample). 6. Tilt the Kjeldahl flask containing the digested sample and add 100 mL of 50% NaOH solution slowly down the side of the Kjeldahl flask so that it forms a layer underneath the digestion mixture.Immediately connect the flask to the distilling bulb of the distillation apparatus. Rotate flask to thoroughly mix contents. 7. Heat until all ammonia has passed over into the standard acid. Collect approximately 150 mL. Caution, flask will bump. Remove immediately (prolonged boiling and too rapid distillation of acid during digestion should be avoided as loss of ammonia may occur). 8. Wash tip of condenser and titrate excess standard HCl in distillate with NaOH standard solution (detailed titration procedure) .

24

Calculation Calculate the percentage nitrogen (wet weight basis) as follows: (A - B) x 1.4007 % Nitrogen (wet) = weight (g) of sample where:

x 100

A = vol. (mL) std. HCl x normality of std. HCl B = vol. (mL) std. NaOH x normality of std. NaOH

Calculate nitrogen content on dry basis (when moisture content is known) as follows: % Nitrogen (wet) % Nitrogen (dry)= (100 - % moisture) Calculate the percentage protein (wet or dry basis) as follows:

x 100

% PROTEIN = % nitrogen x 6.25

where 6.25 is the protein-nitrogen conversion factor for fish and fish by-products.

25

Proximate analysis: Crude lipid Extraction: 1. Line the fat beakers up in front of the extractor and match the thimbles with their corresponding fat beakers. 2. Slip the thimble into a thimble holder and clip the holder into position on the extractor. 3. Add about 40 mL of diethyl ether (one glass reclaiming tube full) to each fat beaker. 4. Wearing white gloves, slip the beaker into the ring clamp and tightly clamp the beaker onto the extractor. If the clamp is too loose, insert another gasket inside the ring. 5. Raise the heaters into position. Leave about a 1/4 inch gap between the beaker and the heating element. 6. Turn on the heater switch, the main power switch, and the condenser water. 7. After the ether has begun to boil, check for ether leakage. This can be detected by sniffing around the ring clamp. If there is leakage, check the tightness of the clamp and if necessary replace the gasket(s). 8. Extract for minimum of 4 hr on a Hi setting (condensation rate of 5 to 6 drops per second), or for 16 hr on a Low setting (condensation rate of 2 to 3 drops per sec). 9. After extraction, lower the heaters, shut off the power and water, and allow the ether to drain out of the thimbles (about 30 min). This is a good stopping point. Destillation and weighing 1. Remove the thimble from the holder, and rinse the holder with a small portion of diethyl ether from the washbottle. Clip an ether reclaiming tube in place and reattach the fat beaker. 2. Reposition the heaters and turn on the electricity and water. Proceed to distill the ether using a Hi setting. Watch Closely. 3. Distill until a thin layer of ether remains in the bottom of the beaker, and then lower the heater. Do not allow beakers to boil dry. Overheating will oxidize the fat. When the last beaker has finished, shut off the power and water.

26

4. Wipe the exterior of the beaker clean with a Kimwipe as it is being removed from the extractor. 5. Empty the reclaiming tubes into the "USED" diethyl ether container. 6. Place the tray of beakers in an operating hood to finish evaporating the ether. If there is no hurry, air moving through the hood will be sufficient without heat. A steambath may be used to speed up the evaporation. Beakers should remain in the hood until all traces of ether are gone. Carefully sniff each beaker to determine if any ether remains. 7. Place the beakers in a 102C gravity convection oven. Warning: If a beaker containing ether is placed in the oven an explosion may occur. 8. Dry for 1/2 hr. No longer. Excessive drying may oxidize the fat and give high results. 9. Cool in a desiccator and weigh and record weight to the nearest 0.1 mg (W2). The fat beakers are best cleaned by warming on a steambath or on a hot plate on a low setting. Add some used ether to dissolve the fat. The use of a rubber policeman is helpful. 10. After soaking the beakers in Alconox detergent, wash them using hot water and vigorous brushing. The thimbles are best cleaned by blowing out with air. If doing a proximate analysis, the residue left in the thimble may be used to determine crude fiber. Calculation Percent Crude Fat (Ether Extract), DM basis: (AWres - Wta) % Crude fat (wet) = weight (g) of sample

x DM (%)

Wta = tare weight of beaker in grams Wres = weight of beaker and fat residue in gram

27

Proximate analysis: Crude fiber Procedure 1. Determine separately the sample moisture by heating in an oven at 105 C to constant weight. Cool in a desiccator. 2. Weight accurately 1 g about of grinded sample (1 mm about) approximately with 1 mg. ==> W1 3. Add 1.25% sulfuric acid up to the 150 ml notch, after preheating by the hot plate in order to reduce the time required for boiling. 4. Add 3-5 drops of n-octanol as antifoam agent. 5. Boil 30 minutes exactly from the onset of boiling. 6. Connect to vacuum for draining sulfuric acid. 7. Wash three times with 30 ml (crucible filled up to the top) of hot deionized water, connecting each time to compressed air for stirring the content of crucible. 8. After draining the last wash, add 150 ml of preheated potassium hydroxide (KOH) 1.25% and 3-5 drops of antifoam. 9. Boil 30 minutes. 10. Filter and wash as point 7. 11. Perform a last washing with cold deionized water aimed to cool the crucibles and then wash three times the crucible content with 25 ml of acetone, stirring each time by compressed air. 12. Remove the crucibles and determine the dry weight after drying in an oven at 105 C for an hour or up to constant weight. Let cool in a desiccator. This weight (W2)represents the crude fiber plus ash content in comparison to initial weight.

Calculation Calculate the percentage crude fiber (wet weight basis)as follows:

28

(W2 - W1) % Crude fiber (wet) = W1 x 100

Proximate analysis: Nitrogen Free Extract (NFE)

% NFE = % DM - (% EE + % CP + % ash + % CF) where: NFE = nitrogen free extract DM = dry matter EE = ether extract or crude lipid CP = crude protein CF = crude fiber

2) GROWTH PERFOMANCES The following calculations were made on collected data to describe and evaluate fish performance: (i) Mean weight gain (MWG) = Wf -Wi Where, Wf is the mean final fish weight and Wi the mean initial fish weight. (ii) Percentage body weight gain (PWG) = (MWG x 100)/Wi (iii) Specific growth rate (SGR, %) (Brown 1957) = [(log Wf log Wi) x 100)]/D (iv) Feed conversion ratio (FCR) = feed consumed (dry weight)/fish weight gain

29

30