1. Experimental diet 2. Dose 3.

Supplementation level vs analyzed values

Methodology in Fish Nutrition

From Prof. Shi-Yen Shiau from Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan . Only for teaching in East China Normal University.

4. Control group 5. Acclimation 6. Replicates 7. Test fish 8. Fish number

9. Feeding rate 10. Feeding frequency 11. Rearing system 12. Duration 13. Measuring response 14. Statistical analysis 15. Others

1. Experimental diet

Experimental diet
Experimental diets for evaluating nutrient requirements should be prepared from highly purified ingredients to allow maximum control over the nutrient being tested. Preferably, all diets in an experiment should be 1. alike in all respects except the variable being tested. 2. palatable 3. feedable (water stable, optimum particle size and texture). 4. nutritionally complete (except for the nutrient being tested). 5. as far as possible, made from highly purified ingredients.

Experimental diet (Cont)

Completed diet formulation weighing Accurate weighing is essential mixing
Careful attention need to be exercised in the mixing of dietary ingredients. The order in which ingredients are added greatly affects mixing efficiency. Materials such as vitamin mixes, which are added at low inclusion rates, should not put into an empty mixer. Put one of the major ingredients in first. Vitamins and vitamin premixes should be kept either in the manufacturers or in air-tight light-proof containers. They should be kept in the coolest place available. Stocks should be turned over at least 6 months.

Experimental diet (Cont)

mixing (cont)
1). Mix the dry ingredients well before adding oil, then add oil and mix. Efficient mixing is the key to good feed production.

Experimental diet (Cont)

Binder
Leaching rates of water-soluble nutrients were binder specific (Goldblatt et al., 1979)

(at least 15 min after the last ingredient has been added to the mixer)
2). Add approximately 400-500 ml water/kg diet mixture and stir, the moist mixture should have a stiff, plastic consistency when compressed. If it does not stick together, add more water, but too much water will cause sticking in the food grinder. 3). Extrude thru a food grinder with proper diameter holes. 4). Break extrusions into short lengths. 5). Care should be exercised in the drying of diets. To avoid destruction or reduction of labile nutrients, air drying, if completed overnight, rather than heated drying should be performed. 6). Freeze stored.

Interactions
When formulating experimental diets for nutrition research it is important to be aware of the possible effects of each ingredient on the bioavailability of other nutrients in that diet. Both alginate and phytic acid bind and reduce the bioavailability of divalent metal ions (such as Ca). Bioavailability of Zn to fish is reduced by Ca, P and phytate. Alginate and guar gum reduced the digestibility of protein and lipid in rainbow trout. Dietary glucose reduced the apparent digestibility of protein and dry matter in grass shrimp.

1. Experimental diet 2. Dose

EX:
Estimation of the dietary vitamin A requirement of juvenile grass shrimp, Penaeus monodon
( 2000)
- In the Exp.1, dose level: 0, 1,500, 3,000, 15,000, 30,000, 45,000, and 60,000 RE/kg - In the Exp.2, dose level: 0, 600, 1,200, 1,800, 2,400, 3,000, 3,600 and 4,500 RE/kg - Requirement derived with the broken-line method for weight gain of grass shrimp in Exp. 2 is 2,511 RE/kg diet.

EX:
M. japonicus requires 2000 mg inositol/kg diet for maximal growth Myo-inositol inclusion level: 200, 400, 1,000 and 2,000 mg inositol/kg diet ( 1976)

EX:
M. japonicus requires 600 mg choline/kg diet for maximal growth Choline inclusion level: 24, 120, 600 and 3,000 mg choline/kg diet ( 1976)

EX:
Weight gain (g/fish)

35

30

He et al. (1992) used 4,800 IU of vitamin A/kg in their investigation with P. vannamei. Notes: He et al. (1992) was qualitatively evaluate of dietary essentiality of fat-soluble vitamins, A, D, E and K. There was only one dietary vitamin A inclusion level in that study (i.e. 4,800 IU/kg). It did not provide formal estimates of dietary requirement.

25

20

15

10

0 0 1 2

Level of phenylalanine (%)

( 1993)

1. Experimental diet 2. Dose 3. Supplementation level vs analyzed values

EX:
Vitamin B6 requirement of grass shrimp Quantification of the requirement based on dietary B6 supplementation level 102-113 mg PN/kg diet. Quantification of the requirement based on analyzed dietary B6 level 72-89 mg PN/kg diet. ( 2003)

1. Experimental diet 2. Dose 3. Supplementation level vs analyzed values 4. Control group

antibiotic

EX:
K, B12, inositol

Acclimation 1. Experimental diet 2. Dose 3. Supplementation level vs analyzed values 4. Control group 5. Acclimation
The fish should be conditioned to the rearing environment for 1 to 2 weeks prior to beginning the experiment Sometimes it is necessary to feed all the experimental diets for a preliminary period to be sure there is no difference in diet acceptability. (such as grouper study) Fish should be sampled at the beginning of the experiment for analysis for initial condition or composition of the fish.

1. Experimental diet 2. Dose 3. Supplementation level vs analyzed values 4. Control group 5. Acclimation 6. Replicates

EX:
Vitamin B6 level: 0, 60, 120 and 240 mg B6/kg diet Weight gain: 169, 287, 314 and 200% n=1, no statistical analysis ( 1979) For statistical purposes each treatment should be replicated in a minimum of 3 rearing tanks.

Test fish 1. Experimental diet 2. Dose 3. Supplementation level vs analyzed values 4. Control group 5. Acclimation 6. Replicates 7. Test fish
Small fish respond faster than large fish to nutritional variables. Also small are more sensitive to diet differences. Higher numbers of small fish per rearing tank can be used. However, where nutrient requirements will change with size, different fish size groups should be evaluated.

Fish number 1. Experimental diet 2. Dose 3. Supplementation level vs analyzed values 4. Control group 5. Acclimation 6. Replicates 7. Test fish 8. Fish number
Size of rearing thanks should be large enough to accommodate the originally stocked population after a 500 1000% weight increase.

Feeding rate 9. Feeding rate

ad libitum Feeding a steady percentage of fish weight The percentage of feed to be fed is not a fixed amount. It should decrease as the fish grow, to reflect their decreasing metabolic rate.
Thus the ratio of weight of feed per day to fish weight (biomass) is high at the start of the growing period and lower towards the time when the fish reach marketable size.

Feeding a steady percentage of biomass throughout the growing cycle usually results in underfeeding when the fish are small, depressing growth and survival rates, and overfeeding when the animals are larger.

Feeding rate (Cont) 9. Feeding rate

Biweekly weighing will allow comparison of weight gain over the course of the experiment. This will also be useful for adjusting feed allowance. However, with some species, sampling stresses the fish and makes them more sensitive to disease and causes them to stop eating.

10. Feeding frequency

Feeding frequency
frequent feeding reduces starvation and stunting of small fish; thus the group has better uniformity. infrequent feeding rate results in feed wastage, poor FCR, water quality problems, and the leaching out of water soluble nutrients. There is a good deal of controversy about the optimum time and feeding frequency for marine shrimp and freshwater prawns. Some species burrow during the day and feed most actively at night. Others feed in the shallower parts of the pond but avoid these areas in daylight when temperatures are highest. For these species it would seem best to feed in the late afternoon or early evening.

9. Feeding rate 10. Feeding frequency 11. Rearing system

Rearing system
A flow-through system generally assures constancy of the chemical composition of the water and also minimizes the probability of episodic occurrence of poor water quality. A recirculating system is acceptable but careful and more frequent monitoring of water quality is essential.

9. Feeding rate 10. Feeding frequency 11. Rearing system 12. Duration 13. Measuring response

Measuring response
Weight gain is usually considered the most important measurement of the productivity of experimental diets. Other parameters

Growth performance Other parameters

Hepatosomatic index (HSI)

EX:
HSI can be used to estimate the dietary folic acid and inositol requirement of grass shrimp.
( 2001&2004)

Growth performance Other parameters

Hepatosomatic index Enzyme activity

EX:
Hepatopancreatic alkaline phophatase activity was used to estimate the vitamin D requirement of grass shrimp.
( 1994)

EX:
Gill Na+-K+ ATPase activity was used for estimating the K and Na requirements of tilapia.
( 2001&2004)

The vitamin B6 requirement of grass shrimp was estimated by hepatopancreatic GOT activity.
( 2003)

EX:
Growth performance Other parameters
Hepatosomatic index Enzyme activity Hematological assay Lipid peroxidation degree

The studies with rainbow trout (Cowey et al., 1981) and channel catfish (Wilson et al., 1984) showed that weight gain of the fish did not respond to the dietary vitamin E supplementation. Thus a requirement of vitamin E of the two species was obtained by using the ascorbic acid-stimulated lipid peroxidation assay in liver microsomes of the fish. This assay was also perfected for tilapia and the vitamin E requirement obtained from this assay agreed well with the weight gain data.
( 2001)

EX:
Growth performance Other parameters
Hepatosomatic index Enzyme activity Hematological assay Lipid peroxidation degree Immune response

The altered total haemocyte count (THC) found in grass shrimp study suggests that this variable may permit a satisfactory evaluation of vitamin E status of the shrimp.
( 2004)

Tissue concentration
- Growth is not always a good indicator - Estimation of the dietary riboflavin required to maximize tissue riboflavin concentration in juvenile shrimp. ( 1992)

Whole body balance (mineral)

EX:
K
Whole body potassium balance

Initial whole body K content mg 0.3 0.3 0.3 0.3 0.4 0.4

Total K fed

Final whole body K content mg 6.3 6.9 8.3 9.6 10.8 11.8

Final Whole initial whole body K body K retention content mg 6.0 6.6 8.0 9.3 10.4 11.4 mg 4.2 1.6 0 -2.4 -4.0 -5.9

0.19
2

g/100 g diet 0 0.1 0.2 0.3 0.4 0.5

mg 1.8 5.0 8.0 11.7 14.4 17.3

-2

Y= X 04 7. -1

-4

7 .2 +3

-6

(r= ) 99 0.

-8

-10

-12 0.5 1.0

0.7 0.4 21.1 12.5 12.1 -9.0 Whole body Se retention = Final initial whole body Se content total Se fed 1.0 0.4 26.3 13.8 13.4 -12.9

Potassium concentration (g/100g diet)

( 2001)

ymax=5.59 ymax=0.82

0.9 0.8

4
y= 0.2 40 60. 21 00 69 0

0.6 0.5 Xmax=24.11 Xmax=24.66 0.4 0.3 0.2 0.1 0.0 0 8 16 24 32 40 48 56 -0.1 -0.2

3 2 1 0 -1

Y= 0. 0

40

a broken-line response regression

polynomial

Protein level (%)

( 1989)

800

Y=-1.20x10-4X2+0.21X+587.18 ( r=0.79)
500

Weight gain percent

600 880

Weight gain (%)

400
.9 =0 8)

40.51

Y=499.030.86X (r= 0.75

300
8 26 4 .5

.8 +4

3X

(r

200

Y=

400

100

C2MP-Mg
0 0 20 40 60 80 100 120

200 0 500 1000 1500 2000 2500

Analyzed ascorbate concentration (mg/kg)

Choline chloride concentration (mg/kg diet)

( 1999)

( 2000)

380

9. Feeding rate 10. Feeding frequency

0.30

Weight gain, g/100 g initial wt

360

340

11. Rearing system

3 2

320 Y=466.98X -888.59X +403.00X+302.61 (r=0.96) 300

12. Duration 13. Evaluating fish response

280

260 0.0 0.2 0.4 0.6 0.8 1.0

Potassium concentration (g/100 g diet)

14. Statistical analysis 15. Others

( 2001)

Protein gain per fish (g)

In fish/crustacean nutrition study, a descriptive response curve has commonly been fitted to the growth data

Average weight gain per fish (g)

0.7

10.1