Fish, laboratory conditions and feeding trials

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CHAPTER 3

MATERIALS AND METHODS

3.1 Experimental diets

The commercial floating tilapia pellets (Cargill’s premium extruded starter/grower tilapia feeds) containing no added OrgacidsTM-AQUA (Control) and 2% OrgacidsTM-AQUA (Treatment) which manufactured and purchased by Cargill Sdn. Bhd., Malaysia were applied in the present study. The proximate composition of both experimental diets was summarized in Table 4.1 which indicated that formulation method was exactly the same apart from the inclusion of 2% microencapsulated organic acids (2% MOAB). To avoid feed spoilage, all commercial feed pellets which stored in air-tight polyethylene zipped bags (Plate 3.1) were kept in a refrigerator to suppress the growth of mycobiota.

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Plate 3.1 Commercial floating pellets from Cargill Sdn. Bhd.

3.2 Fish, laboratory conditions and feeding trials

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A total of 500 GST fingerlings were obtained from hatchery of Trapia Malaysia Sdn. Bhd. in Banding, Perak (Plate 3.2). Fingerlings (Plate 3.3) were fed with commercial tilapia starter feed twice daily and acclimatized in an indoor 1000-L rectangular fiberglass tank for 2 weeks before initiating the feeding trials.

After acclimatization period, group of 15 apparent healthy GST fingerlings with almost similar size (15.96 ± 0.03g) were starved for 24 hours, weighted and allotted randomly into each of 8 aquaria (n=15 fish/aquarium). Plate 3.4 demonstrates the glass-made, rectangular-shaped, 95-L aquaria (61.0cm x 30.5cm x 50.8cm) set-up with water flow-through system that used in present study. An air-stone was placed into each aquarium to provide constant aeration and de-chlorinated water was delivered to each aquarium with an average flow rate of 22.4Lh-1. A photoperiod of 14 hours in illumination and 10 hours in darkness from fluorescent light was maintained daily. The water quality parameters such as temperature, dissolved oxygen and pH in the culture system were maintained at desired conditions.

Each experimental diet was performed in 4 replicates and the fish were cultured for 7 weeks at Fish Nutrition Laboratory, USM. GST were hand-fed twice daily in a regular time interval (09:00 and 17:00) to apparent satiation. Amount of feed offered in each aquarium was recorded daily except on sampling day as there was no feed given to GST. GST from each aquarium were batch-weighed weekly to determine the growth performance. Throughout the feeding trials, some of the GST in certain aquariums were fighting with each other due to their aggressive behavior. Hence, the upper lips of the GST were removed to avoid injuries or mortality to other fingerlings.

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Plate 3.2 Nursery cage of Trapia hatchery in Temengor lake.

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Plate 3.3 ?? generation of GST fingerlings

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Plate 3.4 Aquaria set-up used in present study

3.3 Feces collection

After four weeks into the feeding trial, feces were collected once daily from each aquarium. Before collecting the feces, all aquariums were siphoned thoroughly to discharge the overnight feed and feces residues. Plastic nets were placed at the bottom of each aquarium to prevent feces disturbed or eaten by GST (Plate 3.5). Within 6 hours, the long and intact fecal strands were carefully siphoned into a fine mesh net and collected into falcon tube. The falcon tubes were then stored in the refrigerator for subsequent apparent digestibility analysis (Plate 3.6). Fecal samples collected from each aquarium were pooled, oven dried and finely ground before analysis.

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Plate 3.5 Plastic nets were placed at the bottom of each aquarium to avoid feces disturbed or eaten by GST.

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Plate 3.6 Feces that collected in falcon tubes with labels were stored in refrigerator.

3.4 Samples collection

3.4.1 Initial samples

30 GST fingerlings were randomly picked, killed and pooled for initial whole body composition at the beginning of feeding trials. The samples were then kept in a freezer (-20°C) to carry out proximate analysis subsequently (Section 3.6).

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3.4.2 Final samples

At the end of feeding trial, GST were staggered-fed at 4 hours before the final sampling started to limit the time variations. All GST in each aquarium were anaesthetized with methane sulphonate (MS222) to measure body weight and length individually. 2 to 3 GST from each aquarium were randomly selected to collect blood for hematocrit determination. After the blood collection, the same GST were subsequently killed to excise liver and viscera to determine pH and body composition. Another 2 GST from each aquarium were randomly picked, anaesthetized and killed for microbial analysis. The sex of each GST was determined after dissection.

3.4.2.1 Blood collection and hematocrit determination

Blood samples were collected from randomly sampled fish into heparinized tubes by severing the caudal peduncle. Micro-hematocrit centrifuge was used to micro-centrifuged the capillary tubes at 4,000g for 5 minutes. Relative volume of packed red blood cells was eventually measured to determine hematocrit percentage.

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Plate 3.7 One end of the heparinized tube was filled with clay sealant.

3.4.2.2 Fish tissue sampling

After blood collection, GST were dissected and their liver and viscera was excised (Plate 3.8). Weight measurement of the liver, viscera and intraperitonial fat (after removal of digesta) was recorded to obtain the hepato-somatic index (HSI), viscera-somatic index (VSI) and intraperitonial index (IPF). Plate 3.9 demonstrates the process of removal of disgesta. Body indices (percentage of organ or tissue to the whole-body weight of individual fish) were then determined by using calculations stated in Section 3.8. GST carcass and excised organ was then pooled, kept inside a polyethylene zipped bag and stored in freezer for subsequent whole-body composition analysis.

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Plate 3.8 Liver and viscera of GST was excised.

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Plate 3.9 Digesta was removed before liver and viscera weight measurement was taken.

3.4.2.3 pH determination

Digestive content of stomach, hepatic loop (HL) and terminal segment (TS) (both fluids and solids) was then extracted, pooled and weighed (Picture 3.11). The HL and TS were determined as described by Smith et al. (2000).The HL is the post-stomach segment that surrounding the liver edge whereas the TS is the only lineal part of the intestine tract that exited from the spiral region until anus.

The digesta contents (~0.5g) of stomach, HL and TS were transferred into 10ml test tubes, mixed with 4.5ml of deionized waterand their respective pH were measured with a digital pH meter (Delta 320, Mettler-Toledo Instruments Ltd., China).

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Plate 3.11 Pooled digestive content of stomach, HL and TS

3.5. Total cultivable bacterial count and Lactic acid bacterial count

One week before final sampling, freshly peeled feces samples were aseptically collected to carry out bacterial counts. The feces were weighed and made into a 10% (w/v) homogenate using sterile distilled water and serially diluted 10-fold with sterilize-distilled water until 10-8.

During GST final sampling, 2 GST were randomly picked, anaesthetized and killed to excise HL and TS. The contents of HL and TS were aseptically removed. The samples (~1g) were homogenized, suspended in 9mL of sterilize-distilled water and serially diluted 10-fold with sterilize-distilled water until 10-6. Spread plate method was implemented to determine bacteria count (colony-forming units, CFU). 0.1mL aliquots of each dilution were spread onto duplicate nutrient agar (NA) plates and deMan, Rogosa, and Sharpe (MRS) agar plates for total viable bacterial (TVB) and lactic acid bacterial, respectively. Plates were incubated for 24 to 48 hours at room temperature. TVB and lactic acid bacteria was counted if the plate containing 30 to 300 CFU per plate.

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Plate 3.21 Operating table must sterilize with ethanol to prevent contamination.

3.6 Chemical analyses

3.6.1 Proximate analysis

Proximate analysis (% dry weight basis) was carried out to determine the chemical composition (crude lipid, crude protein, ash and moisture) of experimental diets, feces and whole body of GST. Appendix B indicated procedures to carry out this analysis by referring standard Association of Official Analytic Chemists protocols (AOAC, 2000).

3.6.2 Acid-insoluble ash (AIA) determination

2N HCI method described by Van Keulen and Young (1977) was used to determine the AIA content of feed and feces. The procedures used to conduct this analysis were attached in Appendix C.

3.6.3 Phosphorus determination

Phosphorus content of experimental diets and feces were estimated using methods stated in Appendix D (AOAC, 2000).

3.7 Evaluation of growth and feed efficiency

  • Weight gain (%)

= 100 x [Final mean weight (g) Initial mean weight (g)]

Initial mean weight (g)

  • Specific growth rate (SGR, % day-1)

= 100 x (ln final ln initial fish weight)

Number of days

  • Feed conversion ratio (FCR)

""= Total dry diet fed (g)

Total wet weight gain (g)

  • Protein efficiency ratio (PER)

""= Wet weight gain (g)

Total protein intake (g)

  • Survival rate (%)

= 100 x Final fish number

Initial fish number

3.8 Evaluation of fish body indices

  • Hepatosomatic index (HSI, %)

= 100 x Liver weight (g)

Body weight (g)

  • Viscerosomatic index (VSI, %)

= 100 x Viscera weight (g)

Body weight (g)

  • Intraperitoneal fat index (IPF, %)

= 100 x Intraperitoneal fat weight (g)

Body weight (g)

  • Hematocrit (%)

""= 100 x Volume of packed red blood cell (mm)

Volume of packed red blood cells and plasma (mm)

  • Condition factor (K)

""=100 x Weight of the fish (g)

[Total length of fish (cm)] 3

3.9 Evaluation of nutrient digestibility

  • Apparent digestibility (%) in dry weight basis

= 100 – [100 x (Di/ Fi) x (F/D)]

Where, Di = Cr2O3 concentration in feed

Fi = Cr2O3 concentration in feces

D = nutrient concentration in feed

F = nutrient concentration in feces

3.10 Statistical analysis

One way analysis of variance (ANOVA) was used to analyze all the data obtained in this study while independent samples t-test was used to compare mean variations among samples. Both of the analysis tests were conducted by using SPSS software (SPSS version 14.0 for Windows) with the statistical significance set at P<0.05.

CHAPTER 4

RESULTS

4.1 Experimental diets

The analyzed proximate composition, pH, phosphorus and AIA content of two experimental diets were summarized in Table 4.1.

Table 4.1 Analyzed proximate composition (g/100g dry weight), pH, phosphorus and AIA of experimental diets.

Parameters

Experimental diets

Control

2% MOAB

Moisture

8.61 ± 0.05

7.04 ± 0.01

Crude protein

36.71 ± 0.12

38.01 ± 0.06

Crude lipid

5.66 ± 0.04

5.60 ± 0.03

Ash

9.11 ± 0.01

8.57 ± 0.01

Fiber

3.09 ± 0.04

2.87 ± 0.10

NFE[a]

45.43

44.95

Gross energy[b]

11.43

11.68

pH

5.99 ± 0.00

5.56 ± 0.00

Phosphorus

1.38 ± 0.01

1.19 ± 0.01

AIA

0.31 ± 0.02

0.58 ± 0.02

4.2 Growth performance and feed efficiency

After 7 weeks of feeding trials, all GST indicated excellent growth performance and low FCR. The mean body weight gain of GST per week was presented in Figure 4.1. Generally, GST fed with control diet showed numerically better (P>0.05) growth performance and feed utilization than GST fed with 2% MOAB (Table 4.2). No significant difference was observed in growth performance and feed utilization across treatments with the exception of PER. GST fed control diet indicated significantly higher PER (3.31 ± 0.03) than GST fed 2% MOAB diet (3.13 ± 0.04).

""

Figure 4.1 Mean body weight gain of GST fed with two different experimental diets after 7 weeks of feeding trials. No significance difference was observed at P<0.05.

Table 4.2 Effect of MOAB on growth performance and feed efficiency parameters of GST after 7 weeks of feeding trials[c].

Parameters

Dietary treatments

Control

2% MOAB

Initial weight (g)

15.95 ± 0.02

15.96 ± 0.03

Final weight (g)

115.21 ± 3.42

109.20 ± 2.06

Survival rate (%)

100.00 ± 0.00

96.67 ± 1.92

Weight gain (%)

622.35 ± 21.23

584.34 ± 12.54

SGR (%)

4.03 ± 0.06

3.92 ± 0.04

FCR

0.83 ± 0.01

0.84 ± 0.01

PER

3.31 ± 0.03*

3.13 ± 0.04

4.3 Body indices

All data of body indices was not significant difference across treatments (Table 4.3). GST fed 2% MOAB supplemented diet showed numerical higher VSI and IPF whereas GST fed the control diet showed numerical higher HIS and condition factor. In addition, percentage of hematocrit of GST was not significant affected by the effect of MOAB as both diets yielded almost similar values.

Table 4.3 Effect of MOAB on body indices, condition factor and hematocrit of GST after 7 weeks of feeding trials[d].

Parameters

Dietary treatments

Control

2% MOAB

HIS (%)

2.52 ± 0.06

2.48 ± 0.14

VSI (%)

3.17 ± 0.15

3.28 ± 0.16

IPF (%)

0.82 ± 0.15

0.88 ± 0.11

Condition factor (K)

2.05 ± 0.01

2.02 ± 0.03

Hematocrit (%)

37.29 ± 1.05

37.31 ± 0.28

4.4 Whole-body composition

Even though the initial whole-body composition indicated numerical higher moisture, crude protein, ash and phosphorus content compared to the final whole-body composition, there were no significance different was observed across treatments (Table 4.4). From the data yielded in final whole-body composition, 2% MOAB diet showed insignificant but numerical higher crude protein, crude lipid and phosphorus content than control diet.

Table 4.4 Effect of MOAB on whole-body composition (% dry weight) and phosphorus content of GST after 7 weeks of feeding trials[e].

Parameters

Initial

Dietary treatments

Control

2% MOAB

Moisture (%)

77.21 ± 0.30

72.62 ± 0.23

72.27 ± 0.29

Crude protein (%)

61.56 ± 1.43

57.65 ± 0.97

58.27 ± 0.89

Crude lipid (%)

22.27 ± 0.26

27.44 ± 0.61

27.67 ± 0.41

Ash (%)

13.55 ± 0.23

11.82 ± 0.13

11.62 ± 0.25

Phosphorus (%)

1.82 ± 0.04

1.73 ± 0.02

1.72 ± 0.04

4.5 pH of digestive content from gastrointestinal tract

2% MOAB diet indicated positive influence on pH of GST gastrointestinal tract content (Figure 4.2). Although the data was not significant different, 2% MOAB diet manifested markedly lower pH in stomach, HL and TS content compared to control diet (Appendix E).

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Figure 4.2 Effect of MOAB on pH of GST digesta of stomach, HL and TS after 7 weeks of feeding trials.

4.6 Apparent nutrient digestibility

2% MOAB diet revealed potent effect on apparent nutrient digestibility as all the data showed distinctly higher values compared to the control diet (Figure 4.3). With the exception of apparent crude lipid digestibility, all digestibility data achieved significant difference at P<0.05 among the two experimental diets. Apparent phosphorus, dry matter and ash digestibility in 2% MOAB supplemented diet was highlighted extremely significant difference from control diet (Appendix F).

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Figure 4.3 Effect of MOAB on apparent nutrient digestibility (% dry weight basis) of GST after 7 weeks of feeding trials.

4.7 Total viable bacterial count and Lactic acid bacterial count

Reduction in total viable bacterial count of both expelled feces and gut was observed in GST fed 2% MOAB diet (Figure 4.4). From the data yielded, GST fed 2% MOAB discharged significant less total bacteria per g of feces (P<0.05). In addition, number of adherent bacterial in gut was also declined but insignificantly in GST fed 2% MOAB diet compared to GST fed control diet (Appendix G). However, there were no any Lactic acid bacterial was detected in neither feces nor gut of GST fed control or 2% MOAB diets.

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Figure 4.4 Effect of MOAB on total cultivable bacterial count (x106 CFU/g) of the GST expelled feces, HL and TS with two different experimental diets


[a] Nitrogen-free extract= 100 – (crude protein + crude lipid + ash + crude fiber)

[b] Gross energy (kJg-1), calculated based on the physiological fuel values of 23.6kJg-1 protein, 39.5kJg-1 lipid and 17.2kJg-1 carbohydrate.

[c] All values are the mean ± standard deviation of four replicates from each treatment. Symbol (*) indicates the data were significantly different at P<0.05.

[d] All values are the mean ± standard deviation of four replicates from each treatment and were no significant difference at P<0.05.

[e] All values are the mean ± standard deviation of four replicates from each treatment and were no significant difference at P<0.05.