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In vitro ileal digestibility
In vitro gas production
Water soluble solid
These instruction are for the use in the LABORATORY and should be seen as Manual for every - day work. The equipments and technique use in this manual very simple and easy to use. Supply for any one who want to research.
Table of contents
In vitro ileal digestibility
In vitro gas production
Water soluble solids
Preparation ofthe sample
The sample preparation serves several purpose
1/ To get the sample ready for grinding
2/ to get it in the proper physical state standardized particel size
3/ To get the sample suitable for storage
It is a big advantage to pre - dry all samples because the grinding will be much easier and the samples will not deteriorate during storage.
The first step of the sample preparation is the mixing of the whole sample brought to the laboratory. This includes in some cases chopping. Take care not to get any separation of the fine particels during this mixing step.
Determination of dry matter (DM)
The principle of the method is that, by heating a certain sample, it is possible to eliminate all its water content, and then dry material is obtained. This technique necessitates a balance and a heating device, usually an oven.
1/ Weight a dry and clean container.
2/ Add the original, fresh sample to the container, and weight both of them together.This
operation is made at least in duplicates of the same sample.
3/ Dry the sample to constant weight, which implies that all the water has been eliminated.
4/ Weight the dry sample in the container.
% DM = (DS/OS) x 100
Taking into account the weight of the container (C), then
OS = Weight of the original sample, and (OS + C)- C = OS
DS = Weight of the dry sample, and (DS + C) - C = DS
The weigh of a sample of fresh leaves plus a container was 15.12g and 13.40g in the fresh (original) and dry state, whereas the weight of the container was 12.88 g. Then :
OS = 15.12 - 12.88 = 2.24 g
DS = 13.40 - 12.88 = 0.52 g
Therefore, % DM = (0.52/ 2,24) x 100 - 23.21 %
The DM content from a duplicate of the same sample was 22.8%. Then the average DM content of the sample of leaves was.
Average % DM = (23.21 + 22.87)/2 = 23.04.
Determination of HCN
Weigh of the samples ( fresh leaf about 20 grams, dry leaf about 5 grams)and then put in to flask kieldalh add 250 - 300 ml distill water and add8 ml Chloroformafter that boiling them.
Put 8 ml potassium hydroxyt 0.1 N into a 250 ml E - flask and place it on the upper shelf with receive tube dipped in the potassium. When there is 100 ml in the E - flask it OK.
Titration with Nitrate silver 0.1 N the colour from white in to chase white colour
V * 0.005204
% HCN =----------------------* 100
V: ml silver nitrate titration
0.005204 : 1 ml silver nitrate equivalent0.005204 g HCN
Determination of Amoniac
- Weight of the samples and then put in to the kieldalh
add about 5 g Oxyt magie add 300 ml distill water after that boiling them.
- Using 250 ml triangle bottleadd 50 ml boric acid 4% collect amoniac until 200 - 250 mland then titration with sulfuric acid 0.1 N. The colour from white in to initial pink
V * 0.0017
% Amoniac =----------------* 100
V : ml acid titration
0.0017 : constant 1 ml acid equivalent 0.0017 gamoniac
Determination of Nitrogen
- Salt mixture : K2SO4 : CuSO4. 5H2O in to 10 : 1 proportion
Take 1000g K2SO4 and mix well with 100g CuSO4.5H2O
- Sulphuric acid for digestion : Concentrated, ind. Grade is enough
- Sodium hydroxide : 40% NaOH in tap water = 10 N
Dissolve 2kg NaOH in tap water and make up to 5 litter
- Mix indication : Methylenblue and Methyl red
Dissolve 0.12g Methylen blue in 100 ml Ethanol
Dissolve 0.270g Methyl red in 200 ml Ethanol
Mix the two!
*Boric acid + indicator
2% boric acid in water + mix indicator
Dissolve 100 g of boric acid in water and make up to 5 litter.Add 25 ml of mixed indicator.
* Sulphuric acid for titration 0.1 N
Prepare a 1,00 N (0.5 M) H2SO4 from aspulle if possible.
Dilute it 10 times. Take 100 ml and make up to 1000 ml
Weigh accurately samples and put samples in to kieldaldflask add 10 - 15 g of this salt mixture and add 25 ml acid sulphuric concentration and then put the flask on the heatersand boiling them.
Digest until the colour has turned green it Ok
Allow the flask to cool down for at least 30 min
Add 250 ml of distill water
Put 50 ml of boric acid + indicator solution in to a 250 - 300 ml E - flask and place it on the upper shelf with the receiver tube dipped in the acid
Pour careful 90 - 100 ml of NaOH 40% in the flask
connect the flask to the distiller and then shake it carefully. This has to be done as quickly as possible to avoid lossed of nitrogen
When there is 150 ml in the E-flask, lift it down to the lower shelf, continue the distillation until there is 175 ml in the E-flask
Wait for 5 - 10 minute so the last distillate can come down into the E-flask
Titration and calculation
Titration with 0.1 N H2SO4 until the colour is between green and purle
correct the titer with the blank - value
Two blanks should be run every time a new preparation of boric acid is used and also when you start using a new bulk of any of the chemicals.
(Titer - blank x 0.875)
% CP =----------------------------
Sample weight (g)
(Titer - blank x 0.14)
% Nitrogen =--------------------------
Sample weight (g)
0.1 x 0.014 x 6.25 x 100
* 0.875 =-------------------------------
* 0.1= The strength of the acid for titration
* 14=Formula weight for nitrogen
* 6.25= Factor to transfer from nitrogen to protein on most feed samples
* 1000 = Conversion from liters to milli - liters
* 100= To get %
Note : Always use protectives like lab, coats, gloves and spectacles when working with
strong acids and alkalines.
Determination of NDF
Prepare chemicals for NDF
Bottle 1 : 150 g sodium dodecyl sulfate + 50 ml Tri - ethylenglycol + 2 litter water
Bottle 2 : 93.05g EDTA + 34.05g sodium tetraborate + 1 litter water
Bottle 3 : 22.8g Disodium hydrogen phosphate + 1 litter water gently boiling and cool down
Bottle 4 = put 2 and 3 into 1 adiust until 5 litters and correct pH 6.9 - 7.0 it OK
Weigh accurate 1 - 1.5g of the grinded sample into a beaker (600ml) or flask. Add 100 ml of NDF - solution and 2 ml of decalin.
Heat to boil and reflux for 60 minute
Place a previously tared glassfillter - crucible on the filtermaniflold. Transfer contents of beaker to the crucible while using low vacuum from the beginning. Rinse beaker with hot water and also the residue in the crucible with hit water. Rinse finally twice with acetone. Dry crucible at 105oC over night. Weigh out after cooling down in a desiccator. Ash at 500oC for 2 - 3 hours. Cool down in desiccator and weigh again.
OS - DR
% WL (DM) =-------------- x 100
Weigh after drying - Tare weight
NDF (INCL ASH) % =----------------------- x 100
Weigh after drying - weigh after ASH
NDF (EXCL ASH) % =------------------------ x 100
Weigh accurately ( 1 mg) 1 - 5 grams of sample into a tare porcelain crucible. Ash the material until greyish ash results in an electric furnace at 550oC - 600oC . The time it takes depends on the sample and the furnace, normally 3 - 4 hours. Cool the crucible and its contents in a desiccator and weigh. If the ash is not greyish you could treat it with hot water, filter through, ashfree filterpaper, wash with hot water and transfer the filterpaper to a crucible and ash again for 5 - 10 min, then cool in a desiccator and weigh
Weight of sample after ashing
ASH % =----------------------------------------- x 100
Weight of sample before ashing
Determination of dissolved oxygen
The oxygen which is dissolved in water is determined by a portable disolved oxygen meter (DO meter) from Hanna instruments (HI 8543). The principle of the determination is based on polarography. The DO meter has a disolved oxygen probe consisting of a membrane covering the polarographic sensor and a built-in thermistor for temparature measurements and compensation. Dissolved oxygen can be estimated in the range of 0.0 to 19.9 mg/l with a resolution of 0.1mg/l and an accuracy of 0.2 mg/l.
1/ Connect the DOmeter with the DO probe and switch on. Wait about 10 minutes to ensure
the probe has been polarized.
2/ Prepare the electrolyte solution by adding 15 to 20 drops of HI 7041 solution into 20 ml of distilled water.
3/ Wet the sensor by soaking the bottom of the probe in the dilute electrolyte solution for 5 minutes.
This is done by unscrewing the protective red and black plastic cap. Ensure the o-ring is properly placed inside the membrane. Rinse the membrane cap with electrolyte while shaking it gently. Refill with fresh electrolyte. Gently tap the sides of the membrane with the finger tip to ensure that no air bubbles remain. To avoid damage, do no tap the membrane directly on the bottom. With the sensor facing down, firmly screw the membrane cap clock -wise to the end of the threads. Some electrolyte will overflow.
4/ For slope calibration, rinse the probe throughly (particularly after the zero calibration) with
some water and blot dry in a manner as not to damage the membrane (use cloth or softpaper). Place the probe in vertical position in air. Wait a few minutes until themeasurement stabilizes.
Press and hold the red button between the two calibration screws. The decimal point shoulddisappear from the display. Using a small screwdriver, turn the slope trimmer until the
display reads 100. Release the red button and the display will show the saturation value at
that ambient temperature. Saturation value is the maximum dissolvable oxygen value in a
liquid at that temperature.
5/ Dissolved oxygen is determined in the samples after slope calibration. All the
measurementsare referred to sea level and zero salinity..
In vitro ileal digestibility of dry matter (DM), N andorganic matter (OM) in pigs.
The in vitro estimation of ileal digestibility permits the evaluation of the quality of feeds, specially N and amino acids for pigs. It has been well established the straight correlation which does exits between in vitro ileal and in vitro (pepsin/ pancreatin), and in turn, the interdependence between in vitro ileal N digestibility and performance traits of economic importance in pigs.
The methods herein described is that proposed by Dierick et al (1985) fully developed in tropical non conventional feeds at the swine Research Institute at Havana by the research team of Ly (see for example, Dominguez et al 1996).
1/ Weight an amount of dry and ground sample containing 150 mg of protein (N x 6.25) and put it into a 100 ml erlenmeyer flask. Add 20 ml of a pepsin solution ( 1mg enzyme/`ml HCL 0.075 N) plus a drop of timerosal.
2/ Incubate the sample in a water bath at 37oC (preferably 80 strockes per minute) during 4 hours.
3/ Neutralize until pH = 7.5 with NaOH (or KOH) 0.2N (about 7.5 ml).
4/ Add 20 ml of a solution of 15% pancreatin in a phosphate buffer. The phosphate buffer
can be prepare by mixing 10 of a solution of 31.50g KH2PO4.3H2O/ L and 90 ml of
another solution of 45.6g K2HPO4/ L. The solution is adjusted to pH = 7.5 and made up to
a L with distilled water. Repeat the incubation in a water bath at 37oC as in 2.
5/ Add a solution of sodium tungstate (5%) to precipitate the undigested protein.
6/ Transfer the solution quantitatively to a 120 ml centrifuging tube and centrifugue the
sample during 10 minutes at 1250 rpm. As alternative, filter quantitatively, in a previously
weighed filter paper.
7/ Dry to constant weight to determine the dry insoluble residue
8/ Determine the ash content in other dry insoluble residue to calculate the insoluble organic
9/ Determine the N content by the Kjeldhal procedure in another dry insoluble residue.
OS - DR
% IVDDM =--------------- x 100
* OS = Weight of the original sample, in dry basis
* DR = Weight of the dry residue
When the in vitro digestibility of organic matter (IVDOM) or in vitro digestibility of N (IVDM) is to be estimated, calculate by the same procedure for example, the IVDN should be calculated by the following equation.
NOS -N DR
% IVDN =-------------------x 100
* NOS = mg of N in the original sample
* NDR = mg of in the dry residue.
Dierick, N, Vervaeke, I., Decuypere, J. and Henderickx, H. 1985. Protein digestion in pig measured in vitro and in vitro. In : Proc, 3rd Int. Seminar Dig. Physiol. Pig (A. Just, H. Jogensen and J. A. Fernandez, ed.) Kobenhavn p 329 - 332.
Dominguez, P. L., Molinet, Y. and Ly, J. 1997. Ileal and in vitro digestibility in the pig of three floating aquatic macrophytes. Livest. Res. Rural Devel. 8(4) : 37 - 44.
Determination of the in vitro gas production.
The in vitro gas production (IVDP) technique measures the evolution of gases (methane and carbon dioxyde) which are produced as end products of fermentation in the rumen. Production of carbon dioxyde is partly from the fermentation and partly as result of formation of short chain fatty acids which expels carbon dioxyde from the bicarbonate buffer solution.
The method is important for feed evaluation, particularly to predict animal performance such as feed digestibility and intake in the ruminant. It has the advantage of the in vitro technique a great number of samples can be evaluate at the same time, it is time saving and cheaper than those methods requiring the use of animals, thus becoming expensives by feed cost.
The IVGP technique provides a great advantage in that the fermentation take place in a glass syringe which allow for several measurements to be made in the same by measuring the gas volume at different intervals of time. This means that not only the possible extent of fermentation can be measured.
The procedure herein described is that of Menke and Steingass (1988)
1/ All substrates should e milled using a 1mm screen. Weigh 200 mg substrate into each
(numbered) syringe and record actual weight. Include a blank (for example. Rumen fluid/
buffer mixture on its own) at the beginning, in the middle of the set, and at the end. A
sample of hay can be milled and used as a control by including a syringe with the hay at the beginning and the end of each rum. Samples should be done in duplicate or triplicate.
After the weighings are completed, grease the plungers with vaseline, and place inincubator at 38oC. This is normally done the day before the run.
2/ Measure distilled water, buffer solution, macromineral solution, micromineral solution
and resazurin solution into a round, flat bottomed, flask - warm to 38oC. Then add
reducing solution of sodium sulphide. Place it in a small water bath on magnetic stirrer,
put magnet into flask and gently bubble carbon dioxyde through the solution until the blue
color turn to pink, then clear. This means the buffer solution is now reduced. Raise the
carbon dioxyde tube so that it will be above the level of the buffer/ rumen fluid mixture,
but providing a stream of carbon dioxyde into the flask throughout the dispensing
procedure. The pH of buffer should be 7 - 7.3.
3/ Collect rumen fluid from the donnor animal, strain through gauze into a warm beaker, the
final ratio of rumen buffer fluid should be 1:2 Mix rumen fluid in beaker and transfer to
flask with buffer solution. Make sure the magnet is mixing properly during the whole
process of dispensing the buffer/ rumen fluid mixture into the syringes. Add 30 ml to each
syring using the dispenser (do 2 - 3 times 30 ml amounts into a beaker at the beginning to
be sure the dispenser is properly charger). Fill the syringe, then open the clip and gently
push the plunger of the syringe so that all the gas is removed. Record the level in each
syringe and place it in the water bath.
Times of reading can be chosen to suit the type of substrate in the syringes. For forages 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours and 96 hours are suitable but for concentrate type substrate it may be necessary to take more reading in the first 24 hours. It is advisable to gently mix each syringe2 - 3 the lectures made at 24 hours and 48 hours.
Gas production is estimated in ml/ 0.2 g of sample per 48 hours.
Preparation of solution
1/ Macromineral solution.
MgSO4. 7H2O0.6 g
Make up to one liter with distilled water
2/ Buffer solution
Make up to one liter with distilled water
3/ Micromineral solution
CaCL2. 2H2O13.2 g
MnCL2. 4H2O10.0 g
CoCL2. 6H2O1.0 g
FeCL2. 6H2O0.8 g
Make up to 100 ml with distilled water
4/ Reducing solution
Distilled water47.5 ml
5/ Resazurin solution
Resazurin 0.100 g
Make up to 100 ml with distilled water
6/ Preparation of the buffer solution
Distilled water474 ml
Macromineral solution (number 1)237 ml
Buffer solution(number 2)237 ml
Micromineral(number 3 )0.12 ml
Resazurin solution (number 5)1.22 ml
Warm to 38oC, then add reducing solution (number 4), prepared fresh for each run.
Determination of pH
The pH value in different types of samples is determined by a portable digital pHmeter. The principle of the determination is based on potentiometry. Thee pHmeter has a pH combination electrode and pH values can be estimated in the range of 0.00 to 14.00 with a resolution of 0.01 and accuracy of 0.01.
1/ Connect the pHmeter with the pH electrode and switch. Set the mode switch to thr pH
position. This will activate the liquid cristal display. Condition the electrode in buffer 7.00
until the display is fully stable (about 30 minutes). Give a stirring action to the electrode
each time it is placed in a new solution.
2/ Adjust the calibration control to make the display read the value of buffer 7.00. Rinse the
electrode with distilled water and blot dry.
3/ Inmerse the electrode in buffer 4.00. When the display is stable (30 seconds), adjust the
slope control to make the display read the value of buffer 4.00. The pH value are 6.99 and
4.01 for buffer 7.00 and 4.00 respectively at 30oC. Error may be minimum in the case that
pH value are not adjust to the temperature of the solution to be tested.
The apparatus is now calibration to read samples with pH 0 - 14.
4/ Rinse the electrode with distill water, blot dry and inmerse in the saple. The sample must
be homogenous. When the display is stable (30 seconds), it will read the pH of the sample
Repeat this step for continued samples. Periodically check calibration, at least one per day.
5/ When all samples are completed, keep the electrode inmerse in distilled water. The
electrode shall never be kept dry, otherwise electrode damage shall be irreversible.
Determination of the wash value
Washing losses or a wash value of a sample are considered to be the amount of water soluble substances readily utilizable by the animal. This technique was first applied to ruminant studies concerning the digestibility of roughages.It now has been suggested to be directly related to the cell contents (100 - NDF) of a sample, which in turn, could be of application in non ruminant nutrition studies too.
The method herein described it that proposed by Ly and Preston (1997) which studies some detail of the analytical technique, not taken into consideration previously. This technique requires a balance, an oven and a comercial semiautomatic washing machine. On the other hand, it is necessary to use nylon bags of 50 x 50 mm. This bag are made of nylon filter cloth with a pore size of 45 to 55 micrometers, and are available from some catalogue list.
1/ Weight a dry and clean bag
2/ Add to the bag, very carefully, 1 g f air - dried sample or 5 g of fresh sample. Weight again
this operation is made at least in triplicate of the same sample. The mouth of the bag must
be tightly close, such as to avoid losses during washing. This procedure can be afford by a
3/ Wash the bag in a comercial, semiautomatic washing macbine of 15 min - cycle, during 90
min, discarding the water every two cycle. Use tap water at room temperature. The ratio of
water per bag is of 3 litters.
4/ After washing, let the bag to drain and dry it in an oven to constant weight
5/ Determine the wash value taking into account the original dry matter of the sample.
6/ The sample is followed for determining the wash value of nitrogen in the sample. In this
opportunity, the N content in the original and in the washed sample should be used for
OS - DR
% WL (DM) =-------------- x 100
Where OS is the weight in g of the original sample, in dry basis, and DR is the weight in g of the dry residue.
If it is desire to determine the wash value of N in the sample, then the calculation will be as follows
NS - NR
% WL (N) =-------------- x 100
Where NS and NR in the nitrogen content determined in the original sample and in the residue obtain after washing. The nitrogen content is usually expressed in percent in dry basis, in both cases.
Determination of water soluble solids
Water soluble solids (WSS) are estimated by refractometry, taking into account that a straight interdependence exists between brix and refractive indices in certains solutions. A hand refractometer (Fisher Scientific, catalogue No. 13 - 946 - 60A) will be used for WSS determination in leaves and sugar cane juice. The measuring range of the instrument is 0 - 32% (accuracy, 0.2%)
1/ Keep distilled water and instrument at room temperature (20oC) for 1 hours.
2/ Open the prism cover and put one or two drops of distilled water on the prism. Close the
3/ Direct the prism towards a light source and look at the scale through the eyepiece.
4/ Rotate the view control to a clean image of the scale. Real the scale. It the scale is correct,
the boundary line meets with ?0" position. No calibration is needed. IF the boundary line is
out of ?0" position, calibration the boundary line to indicate ?0" position by turning the
calibration screw with a screwdriver.
5/ Calibration is completed wipe off the distilled water from the prism with a soft clean cloth
or tissue paper.
1/ Open the prism cover and put one or two drops of the sample in the prism.
2/ Close the prism cover gently
3/ The sample volume must cover the prism surface entirely
4/ Direct the prism towards a light source and look at the scale through the eyepiece
5/ Rotate the view control to focus a clean image of the scale. Real the scale where the
boundary line interrupts it.
6/ After every measurement, wipe the sample from the prism with a soft cloth of tissue paper
and water. Keep the prism clean and dry to get the correct reading.
7/ Calculate WSS content (Brix %) from the determined refractive indice (nD), see table 1
Table 1 : Brix (%) vs refractive index (nD)
Brix (%) Refractive indices (nD)Brix (%)Refractive indices (nD)
0 1.33299 17 1.35890
1 1.33423 18 1.36053
2 1.33588 19 1.36218
3 1.33733 20 1.36348
4 1.33880 21 1.36551
5 1.34027 22 1.36719
6 1.34176 23 1.36888
7 1.34027 24 1.37059
8 1.34477 25 1.3723
9 1.34629 26 1.3740
10 1.34783 27 1.3758
11 1.34927 28 1.3775
12 1.35093 29 1.3793
13 1.35250 30 1.3811
14 1.35408 31 1.3829
15 1.35567 32 1.3847
The hand refractometer is designed to be used at 20oC, and when used above 20oC, the reading value (brix %) have to be correcting referring to table 2 by adding the following value
Table 2 : Temperature compensation table
Reading (brix %)
Temp (0oC) 0 5 10 15 20 25 30 35 25 0.33 0.35 0.36 0.37 0.38 0.38 0.39 0.39 26 0.40 0.42 0.43 0.44 0.45 0.46 0.47 0.47 27 0.48 0.50 0.52 0.53 0.54 0.55 0.55 0.55 28 0.56 0.57 0.60 0.61 0.62 0.63 0.63 0.63 29 0.64 0.66 0.68 0.69 0.72 0.72 0.72 0.72 30 0.72 0.74 0.77 0.78 0.79 0.80 0.80 0.81