Apparent digestibility in fish nutrition Serrano.pdf

7/21/2019 Apparent digestibility in fish nutrition Serrano.pdf

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Apparent digestibility assessment in aquaculture species: measurement of Ulva spp. as examples

Augusto E. Serrano Jr., Ph.D.

Introduction

Modern aquaculture diets are routinely formulated based on digestible nutrient and energy

specifications. As a result, it is important to measure the digestible energy and nutrient content of

prospective ingredients. Essentially this means measuring the amount of the energy or nutrient from

a diet that is not excreted in faeces. Also, aquaculture nutritionists are always concerned with

efficiencies of nutrient conversion and thus also concerned with wastes. The largest proportion of

wastes, more often than not, is fecal wastes especially in omnivorous and herbivorous species. The

latter species usually digest only as much as 60% of the natural plant materials. To be able to

formulate a diet that will result in high growth and conversion efficiency, one needs to know the

digestibility (i.e. availability) of the major ingredients. It will also allow effective substitution of one

ingredient for another.

Learning Objectives

By the end of this topic, you should be able to:

• Describe digestibility determination procedures in fish.

• Determine digestibility of diets and individual ingredients in fish.

Apparent and true digestibility

Normally, some protein in the digestive tract is shed off by fish in the normal course of things as

a way of renewing cells. This could occur together with the protein contained in food. The digestion

of these endogenous proteins is not complete and a portion is excreted through feces together with

proteins coming from food (called metabolic fecal proteins). This causes a bias in the measurement

of digestibility coefficients making it lower than it usually is. Measurements without correction for

the metabolic fecal protein is called Apparent Digestibility Coefficients (ADC) while that with

correction is called the True Digestibility. In spite of its importance, true digestibility has only been

determined in fish in very few studies in fish and the variations in the two methods of expression are

usually small. Thus, ADC is sufficient for feed formulation purposes.

Estimation of Digestibility Coefficients.

Digestibility coefficient

Not all food that passes through the digestive tract is digested and absorbed. The undigested portion

is excreted as faeces. That which is absorbed is determined by difference between the ingested and

excreted nutrients, and usually expressed as a percentage of the amount ingested as 'apparent

digestibility coefficient' or ADC:



Digestibility coefficient can be determined for the food dry matter or determined separately for each

of protein, carbohydrate, and lipid.

Determining food digestibility

1. In vivo

The energy of the amount digested and absorbed (i.e. available energy) is based on accurate

determination of the amounts of ingested and excreted nutrients. This procedure is called the balance

method or direct method and is commonly used in farm animals. The formula is as follows:

% DE = ((Feed energy – Fecal energy)/Feed energy) x 100

In feeding aquatic animals, the food is given in water and faeces are excreted into water; as a

result, nutrients of both food and feces may be dispersed and dissolved in water. Since the lost

nutrients are assumed as if they are absorbed by the fish, ADC measurements are almost always

overestimated (i.e. biased toward higher values).

To remedy this problem, 'inert indicator' (indirect) method is used. In this method a certain

amount of inert substance, which is not digested by the fish, is introduced into the test diet. A number

of inert substances can serve this purpose such as lignin, chromic oxide (Cr2O3), and various radio-

isotopes. The most common inert indicator is chromic oxide which has been used in most studies on

fish. The concentrations of the nutrient and the inert indicator are determined in both the food and

the faeces and the (ADC) of the nutrient is calculated according to the following formula:

In the inert indicator or indirect method, there is no need to determine the amount of food

ingested nor the amount of faeces excreted and this is definitely an advantage over the balance or

direct method. It is sufficient to analyze a sample of both and determine the percentage contents of

nutrient and indicator. Despite this advantage, it still has its constraints and drawbacks. If the faeces

are to be sampled from the water, after being excreted, the analysis is subject to the same errors as

the balance method due to dispersion and solution of nutrients in water. However, for this method

to be reliable it must meet the following conditions:

(a) the indicator must be evenly distributed in the feed and in the faeces, so that samples will be

representative;

(b) the indicator must not be absorbed through or adsorbed onto the intestinal mucosa;

(c) the rate of passage of the indicator through the digestive tract must be equal to that of the

food nutrients.



These conditions are not always met. In spite of the constraints and drawbacks of the inert

indicator method, most researchers used this method for measuring digestibility in fish as it seems

to reduce the difficulties associated with the balance method. A reliable method for in vivo

determination of digestibility in fish has yet to be developed.

2. In vitro

For a rapid determination of digestibility of various feedstuffs for farm animals, the in vitro test is

a very common method. This is done under standard conditions using pure enzymes extracted from

the animals, such as pepsin for protein digestion. This, of course, requires a prior correlation between

the in vitro and in vivo digestibility determinations. Such tests can increase considerably the amount

of information on the digestibility of various feedstuffs for formulating fish diets.

Fecal collection

The method of collecting feces in the water during digestibility trial affect estimates of feed

digestibility values for fish. There are three major ways of collecting fecal material tested and all of

them have advantages and disadvantages in terms of leaching of nutrients. These include:

(a) Collecting from the water by netting, pipetting, mechanical filtration and settling

(b) Collection by stripping the fish by applying slight pressure upon its ventral surface between

the ventral fin and the anus.

(c) Collection of fecal material by dissecting into several parts of the digestive tract of fish.

Diet vs. individual ingredient digestibility assessment

The above procedures are meant to determine digestibility of a diet. However, there may be need to

determine the digestibility of an ingredient. The following formula is used when determining

digestibility coefficients of an ingredient of interest.

%AD = (ADtd-0.7ADrd)/0.3

Where:

AD is apparent digestibility

ADtd is apparent digestibility of test diet

ADrd is apparent digestibility of reference diet

Factors affecting digestibility

There are a number of factors that affect digestibility in fish. The following are some of the importantfactors that should be known:

• Feed composition

• Ration composition

• Feed preparation

• Enzyme supplementation in feed

• Fish species (animal factors)

• Level of feeding



• Temperature of water

• Salinity of water

Examples of measuring ADC of the seaweeds Ulva intestinalis and Ulva lactuca in Oreochromis

niloticus and Penaeus monodon, respectively

Example 1. Study on digestibility of Enteromorpha intestinalis (protein concentrate and raw dried

forms) in Oreochromis niloticus

Table 1 Composition of the reference diet and the test diets in g kg-1

Ingredients Reference

diet

Test Diet

UPC

Test Diet

RDUP

Sardine meal 280.0

Defatted soybean meal 270.0

Seaweeds UPC 300.0

Seaweeds RDUP 300.0

Danish meal 20.0

Wheat Pollard 50.0

Copra Meal 50.0

Bread Flour 180.0

Rice Bran 64.8

Chromic Oxidea

10.0Soybean Oil 30.0

Vitamin Mix b 30.0

Mineral Mix c 15.0

Vitamin C 0.2

Reference Diet 700.0 700.0

Total 1000.0 1000.0 1000.0

a Sigma- Aldrich Chromic (III) oxideb Vitamin mix (mg kg-1 dry diet unless otherwise stated): Vitamin A

1 200 000 IU; Vitamin D3 200 000 IU; Vitamin E 20 000 IU; Vitamin

B1 8000; Vitamin B2 8000; Vitamin B6 5000; Vitamin B12 1% 2000mcg; Niacin 40 000; Calcium Pantothenate 20 000; Biotin 40; Folic

acid 1 800; Ethoxyquin 500; Carrier q.s ad to make 1 kg.c Mineral mix (mg kg-1 dry diet unless otherwise stated): Iron 40

000; Manganese 10 000; Zinc 40 000; Copper 4 000; Iodine 1 800;

Cobalt 20; Selenium; Carrier q.s ad to make 1 kg.



Feeding regime. Fish were starved on stocking day and were acclimated to the diets for 4 days before

faecal collections started (Koprucu and Ozdemir 2005). The reference or experimental diets (Table 1)

were fed to juvenile Nile tilapia (22.0 ± 0.7 g) twice daily (0800 and 1400 h) for 30 days at a rate of 4%

body weight. There was no observed abnormality in feed intake of fish throughout the experimental

period.

Faecal collection. Faeces and other residues were siphoned 1 h before feeding. Faeces were siphoned

using a 5 ml medicine dropper between 0900 and 1300 h. To minimize leaching, faeces were collected

immediately after excretion, pooled by treatment and stored at -20oC. At the end of the trial, faeces

were oven dried at 60 to 80 oC for 24 h and were subjected to analysis for moisture, ash and chromic

oxide contents.

Chromium Analysis. Feeds, feces and corresponding blanks were done in 3 replicates. Fecal samples

were thawed, dried, ground using mortar and pestle and thoroughly mixed prior to analysis. One g of

each sample set aside for moisture and ash determination prior to digestion. One ml distilled water was

added to the sample, mixed, dried at 60-80°C and placed in a furnace for 1 h at 550°C. To the resultingresidue was added 5 ml of 6 M HCl, heated to dryness and the resulting residue was dissolved in warm

30 ml of 0.1N N2O3. The resulting solution was filtered, made up to 50 ml with distilled water and was

subjected to flame atomic absorption spectrophotometry following the method of Monteiro et al

(2002).

The apparent dry matter digestibility coefficient (ADMD) of the experimental diets were computed using

the following formula (Maynard et al 1981):

ADMD = 100− [100 *(Cdiet/DMdiet)*(DMfeces/Cfeces)]

Where:

C = Chromic oxide content

DM = dry matter

The ingredients apparent dry matter digestibility of the ingredients (ADI) was calculated using the

following equation (Bureau, D. P. & Hua 2006):

ADI = ADMtest diet + [(ADMtest diet – ADMref diet) x (0.7 x DMref diet) / (0.3 x DMtest ingredient)]



Results

Table 2 Derived apparent digestibility (%) of dry matter of U. intestinalis protein concentrate (UPC) and

raw dried powder (RDUP) diets and ingredients for Nile tilapia, Oreochromis niloticus

Treatments Apparent Dry

Matter Digestibility(ADMD)

Apparent Dry Matter

Digestibilityingredient (ADMDi)

Reference Diet 98.3±0.1 a

UPC Diet 97.2±0.3 ab 94.7±1.0

RDUP Diet 96.8±0.4 b 92.8±1.5

Values are expressed as means ± S.E.M. Means in the same column with different

superscripts are significantly different (P < 0. 05).

Example 2. Study on the digestibility of Ulva lactuca (protein concentrate and raw dried forms) in

Penaeus monodon

Table 3. Composition of reference and test diets used for in vivo digestibility experiment in giant tiger

shrimp, P. monodon (g/kg).

Ingredients Reference

Diet

Test Diet 1

(70%Ref:30%UPC)

Test Diet 2

(70%Ref:30%

Danish fish meal 320.0 224.0 224.0Ascetes 100.0 70.0 70.0

Squid Meal 160.0 112.0 112.0

Soybean Meal 240.0 168.0 168.0

Bread flour 24.5 17.2 17.2

Rice bran 10.0 7.0 7.0

Cholesterol 5.0 3.5 3.5

Cod liver oil 97.0 67.9 67.9

Lecithin 3.0 2.1 2.1

Ligno bond 10.0 7.0 7.0

Vit. mix 10.0 7.00 7.00

Min. mix 10.0 7.00 7.00

BHT 0.5 0.4 0.4

Cr2O3 10.0 7.0 7.0

Ulva conc. - 300.0 -

Ulva raw - - 300.0

Total 1000.0 1000.0 1000.0



Experimental Animal, Acclimation and Management. Thirty shrimps Penaeus monodon (average body

weight of 17.28 ± 1.67 g) were stocked randomly in nine 60-L plastic boxes. Four shrimps were assigned

to each box in three replicates. The boxes were provided with continuous aeration and salinity was kept

at 28 ppt. Prior to the commencement of the digestibility trial, P. monodon were acclimatized and fed

the reference diet 3 times a day for 5 days. In the morning, uneaten feeds and fecal maters were

siphoned off before feeding. Water exchange was done daily at a rate of 30-40% of the total watervolume. Water temperature, salinity, dissolved oxygen, pH, nitrite and total ammonia nitrogen (TAN)

were monitored and exhibited ranges of 27-30°C, 28-30 ppt, 7-8 mg l-1, 8-8.4, 0.1-0.2ppm and 0.1-

0.15ppm, respectively.

Feeding and Fecal Collection. Shrimps were fed thrice a day (0800, 1200, and 1600) with the test diets at

3% body weight. They were allowed to feed for 45 min after which uneaten feeds were siphoned off.

Fecal collection was done by manually by the use of a long pipette (Bautista-Teruel et al 2003, Smith &

Tabrett 2004) between 1 and 2 h after feeding. Feces were collected until sufficient amount for analysis

was reached, pooled, weighed and stored at -20°C until analysis.

Calculation of ADC and ADI and chromium analysis were the same as in example 1.

Results

Figure 1. Apparent Digestibility Coefficients of dry matter of diets (ADMD). Bars with the same letters

are significantly different (P<0.05). UPC, Ulva protein concentrate; RDUP, raw dried Ulva powder

0

20

40

60

80

100

120

Ref. Diet RDUP Diet UPC Diet

A D M

D ( % )

Experimental Diets



Figure 2. Apparent digestibility coefficients for dry matter of test ingredients (i.e. ADIof Ulva protein concentrate and raw dried Ulva powder). Bars with same letters are not

significantly different (P ≥ 0.05). ADI, apparent digestibility coefficient of test ingredient;

UPC, Ulva Protein Concentrate; RDUP, Raw Ulva Dried Powder.

References

Glencross B.D., Booth M., Allan G.L. 2007. A feed is only as good as its ingredients – A review

of ingredient evaluation for aquaculture feeds. Aquacult Nutr:13, 17–34.

Hepher, B. 1988 Nutrition of Pond Fishes. Cambridge Univ. Press, UK. pp. 48-63.

0

20

40

60

80

100

120

RDUP UPC

A D I ( %

)

Ingredient

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