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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 radioisotopes. 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 important
factors 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
a
Chromic Oxide
10.0
Soybean Oil
30.0
b
Vitamin Mix
30.0
c
Mineral Mix
15.0
Vitamin C
0.2
Reference Diet
700.0
700.0
Total
1000.0
1000.0
1000.0
a
Sigma- Aldrich Chromic (III) oxide
b
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% 2000
mcg; 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 60-80C and placed in a furnace for 1 h at 550C. To the resulting
residue 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


Digestibility
ingredient (ADMDi)

Reference Diet
UPC Diet
RDUP Diet

98.30.1 a
97.20.3 ab
96.80.4 b

94.71.0
92.81.5

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

Test Diet 1

Test Diet 2

Danish fish meal


Ascetes
Squid Meal
Soybean Meal
Bread flour
Rice bran
Cholesterol
Cod liver oil
Lecithin
Ligno bond
Vit. mix
Min. mix
BHT
Cr2O3
Ulva conc.
Ulva raw

Diet
320.0
100.0
320
160.0
240.0
24.5
10.0
5.0
97.0
3.0
10.0
10.0
10.0
0.5
10.0
-

(70%Ref:30%UPC)
224.0
70.0
224
112.0
168.0
17.2
7.0
3.5
67.9
2.1
7.0
7.00
7.00
0.4
7.0
300.0
-

(70%Ref:30%
RDUP)
224.0
70.0
224
112.0
168.0
17.2
7.0
3.5
67.9
2.1
7.0
7.00
7.00
0.4
7.0
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 water
volume. Water temperature, salinity, dissolved oxygen, pH, nitrite and total ammonia nitrogen (TAN)
were monitored and exhibited ranges of 27-30C, 28-30 ppt, 7-8 mg l-1, 8-8.4, 0.1-0.2ppm and 0.10.15ppm, 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 -20C until analysis.
Calculation of ADC and ADI and chromium analysis were the same as in example 1.
Results

120

ADMD (%)

100
80
60
40
20
0
Ref. Diet

RDUP Diet

UPC Diet

Experimental Diets

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

120

ADI (%)

100
80
60
40
20
0
RDUP

UPC
Ingredient

Figure 2. Apparent digestibility coefficients for dry matter of test ingredients (i.e. ADI
of 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, 1734.
Hepher, B. 1988 Nutrition of Pond Fishes. Cambridge Univ. Press, UK. pp. 48-63.