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PROXIMATE ANALYSIS OF FEEDSTUFF
Sampling and Preparation for Analysis
Before undertaking an analysis the results of which are to be used to represent the composition of
a consignment of a feedstuff, it is important that the sample is sufficient in amount and that it is
selected properly from the bulk so as to be fairly representative of it
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The sample to be used, if necessary, is expected to be sufficiently dried to
enable it to be finely ground
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This method
partitioned nutrients in feed into 6 components: water, ash, crude protein, ether extract, crude
fibre and NFE
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About 2g of a feed sample is weighed into a silica dish previously dried and
weighed
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The drying and weighing continues until a constant weight is achieved
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%DM = 100 - %Moisture
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Soxhlet apparatus is the
equipment used for the determination of ether extract
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An extractor: comprising the thimble which holds the sample
2
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250ml flask
Procedure: about 150ml of an anhydrous diethyl ether (petroleum ether) of boiling point of 4060 0C is placed in the flask
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The thimble with content is placed into the extractor; the ether in the

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flask is then heated
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The extraction continues for at least 4 hrs
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The flask is then disconnected and placed in
an oven at 650C for 4 hrs, cool in desiccator and weighed
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Do this three times
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25% H2SO4 is added and the solution is
gently boiled for about 30mins, maintaining constant volume of acid by the addition of hot water
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The boiled acid sample mixture is then filtered hot through the funnel under sufficient
suction
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Then 200mls of pre-heated 1
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Filter under suction and wash thoroughly with hot water
and twice with ethanol
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The residue is
transferred into a crucible and placed in muffle furnace (400-6000C) and ash for 4hrs, then cool in
desiccator and weigh
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25
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Crude
protein is determined by kjeldahl method
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Digestion: weigh about 2g of the sample into kjeldahl flask and add 25mls of concentrated
sulphuric acid, 0
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Apply heat in a fume cupboard slowly at first to prevent undue frothing, continue to digest for
45mins until the digesta become clear pale green
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Rinse the digestion flask 2-3 times and add the rinsing to the bulk
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Steam up the distillation
apparatus and add about 10mls of the digest into the apparatus via a funnel and allow it to boil
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Distil
into 50mls of 2% boric acid containing screened methyl red indicator
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1N HCl
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The volume of acid used is fitted into the
formula which becomes
%N =

14 x VA x 0
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25

Ash
Ash is the inorganic residue obtained by burning off the organic matter of feedstuff at 400-6000C
in muffle furnace for 4hrs
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The crucible is
placed into muffle furnace at 400-6000C for 4hrs or until whitish-grey ash is obtained
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It is obtained by subtracting the sum of
percentages of all the nutrients already determined from 100
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ELECTROPHORESIS
Electrophoresis is a separation technique that is based on the mobility of the ions in an electric
field
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Thus electrophoresis means the techniques in which molecule are forced across a
span of gel motivated by an electric field

Electrophoresis is a technique whereby charged molecules are separated on the basis of their
speed of migration in an applied electric field
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The higher the charge, the faster will a molecule move towards the
oppositely charged electrode
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It similar to chromatography in many
ways except that separation is due to movement of charged particles through an electrolyte
(buffer when subjected to electric current
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High electric field strengths are used to separate molecules based on differences in charge, size
and hydrophobicity
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5 to 1 m in length
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Due to electroosmotic flow, all sample components migrate towards the negative
electrode
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CE detection is
similar to detectors in HPLC, and includes absorbance, fluorescence, electrochemical, and mass
spectrometry
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Separation is
accomplished as in conventional gel electrophoresis but the capillary allows higher resolution,
greater sensitivity, and on-line detection
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It is usually performed
for analytical purposes, but may be used as a preparative technique prior to use of other methods
such as mass spectrometry
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When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is
usually composed of different concentrations of acrylamide and a cross-linker, producing
different sized mesh networks of polyacrylamide
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In both cases, the gel forms a
solid, yet porous matrix
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By placing the molecules in wells
in the gel and applyng an electric current, the molecule will move through the matrix at different
at different rates usally determined by mass toward the positive (Anode) if negatively charged or
toward the negative (Cathode) if positively charged
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Ethidium bromide, silver, or coomassie blue dye may be used for this process
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If
the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under
ultraviolet lighting conditions
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If several mixtures have initially been injected next to each other, they will run parallel in
individual lanes
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Incomplete separation of the components can lead to overlapping bands, or to indistinguishable
smears representing multiple unresolved components
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There are molecular weight size markers available that contain a mixture of molecules
of known sizes
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The
distance a band travels is approximately inversely proportional to the logarithm of the size of the
molecule
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GEL
Gel is a solid, jelly-like material that can have properties ranging from soft and weak to hard and
tough
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[1] By weight, gels are mostly liquid, yet they behave like solids due to a threedimensional crosslinked network within the liquid
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Gel electrophoresis is commonly carried out on a slab of agarose
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Other media that are used as support are: paper, cellulose acetate,
starch, or polyacrylamide
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The velocity, v, will also depend on the resistance to the movement of the molecules provided by
the matrix through which the molecules are moving
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Agarose gels of various concentrations may be prepared by altering the ratio of dry
agarose to the buffer
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5%

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and 2%
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Agarose gels are normally used to separate native proteins, that is, proteins that have retained
higher orders of structure
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Separation on
native gels takes place by both charge AND size
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Two major materials used in making gels are-:
1
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It is extracted in the form of agar
from several species of red marine algae, or seaweed
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Agarose gels have a very large pore size and are used primarily to
separate molecules (large molecular mass)
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The band formed are usually far
apart
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It is usually used between
1% and 3%
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Polyacrylamide – Polyacrylamide gels can be used to provide varieties of
electrophoretic condition
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The pore size can be varied so as to produce different molecular
sieving for protein of different sizes
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Polyacrylamide gels are made by polymerising acrylamide monomer (
persulphate (

) in the presence of N,N'-methylene-bisacrylamide (

) with ammonium
) ("bis crosslinker")
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There exists a linear relationship
between the distance travelled in a given time under defined conditions and the logarithm of the
molar mass of the proteins (diagram above, left)
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The migration distance of unknown proteins is simply related to that of "markers" of
known molecular masses
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Lane B consists of a mixture three proteins: x being the
largest, and z the smallest, with y having a size intermediate between the two
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Electrophoresis Using Acetic Strips

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Cellulose acetate electrophoresis nowadays plays an important role in clinical diagnostics
routine procedures but has also helped in investigating a broad range of subjects in life
science research
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Protein migration
takes place on buffer film on the surface of the cellulose acetate paper
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Also in separation of enzyme (creatinine,
phospokinase, GOT, acidic erythrocyte, phosphatase, phosphoglucomutase etc),
mucopolysaccharide, plasma serum, cerebro-spinal fluid, urine and other body fluids
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It is an accurate
simple method of protein quantification

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