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Contents
Dry
matter
Nitrogen
Ammonia
Ash
HCN
NDF
Disolved
oxygen
In
vitro ileal digestibility
In
vitro gas production
pH
Wash
value
Water
soluble solid
Preface
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
Dry
matter
HCN
Amoniac
Nitrogen
NDF
ASH
Disolved
oxygen
In
vitro ileal digestibility
In
vitro gas production
pH
Wash
value
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.
Instructions
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.
Calculations
%
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
Example
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
Principle
:
Procedure
:
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
Calculation
:
V
* 0.005204
%
HCN =----------------------*
100
Weight
V:
ml silver nitrate titration
0.005204
: 1 ml silver nitrate equivalent0.005204
g HCN
Determination
of Amoniac
Principle
:
Procedure
:
-
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
Calculation
:
V
* 0.0017
%
Amoniac =----------------*
100
Weight
V
: ml acid titration
0.0017
: constant 1 ml acid equivalent 0.0017 gamoniac
Determination
of Nitrogen
Prepare
chemical
-
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
Principles
Procedure
Digestion
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
Distillation
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.
Calculation
(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 =-------------------------------
1000
*
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
Principle
Procedure
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
OS
Weigh
after drying - Tare weight
NDF
(INCL ASH) % =-----------------------
x 100
Sample
weight
Weigh
after drying - weigh after ASH
NDF
(EXCL ASH) % =------------------------
x 100
Sample
weight
Determination
oftotalASH
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
Calculation
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.
Instructions
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.
Principle
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).
Procedure
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 matter.
9/
Determine the N content by the Kjeldhal procedure in another dry insoluble
residue.
Calculation
OS
- DR
%
IVDDM =--------------- x 100
OS
Where
:
*
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
N
OS
Where
:
*
NOS = mg of N in the original sample
*
NDR = mg of in the dry residue.
References
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)
Instroduction
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.
Na2HPO45.7
g
KH2PO46.2
g
MgSO4.
7H2O0.6 g
Make
up to one liter with distilled water
2/
Buffer solution
NaHCO335
g
(NH4)HCO34
g
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
NaOH
1N2ml
Distilled
water47.5 ml
Na2.7H2O0.285
g
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.
Instructions
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.
Instructions
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
rubber
band.
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
calculations.
Calculations
OS
- DR
%
WL (DM) =-------------- x 100
OS
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
NS
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%)
Calibration
procedure
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
prism
gently.
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.
Instructions.
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 |
