The largest operating cost in a livestock production enterprise is the feed bill.

To keep this cost

low, one must supply the right amount of feed to the animals. Overfeeding is wasteful.
Underfeeding will decrease animal performance and profitability. Therefore, proper animal
feeding and nutrition are crucial to the profitability of the livestock enterprise.

Laboratory analyses of the composition of feed or forage are used to assess their nutritive value
(Figure 1). A typical feed analysis includes measurements of some important quality attributes
or parameters (e.g., crude protein, fiber, digestibility, etc.) used to define nutritive value. Other
parameters are analyzed under some special circumstances. For example, acid detergent
insoluble crude protein (ADICP) is usually only measured if heat damage to the feed is
suspected.

https://extension.uga.edu/publications/detail.html?number=B1367

Ash

The residue containing inorganic mineral elements of a feed sample, determined in a

laboratory by burning the sample at a high temperature (removing the organic matter)
and weighing the residue (i.e., ash).

Carbohydrates
Carbohydrates are biochemical compounds composed only of the elements carbon,
hydrogen and oxygen, and are the main source of energy for animals. Animals get the
majority of their required energy from the carbohydrates in feeds. Carbohydrates are
polymers made of basic sugar units, such as glucose (the most abundant), fructose,
galactose, etc. The two major classes of carbohydrates in plants are known as non-
structural and structural. Those that serve as storage and energy reserves and that are
available for more rapid metabolism to supply energy (e.g., sugars, starch, and pectin)
are referred to as non-structural carbohydrates. Those carbohydrate fractions that are
not used for energy storage and provide fiber and anatomical features for rigidity and
even water transport are known as structural carbohydrates (e.g., fibrous cellulose and
hemi-cellulose). Non-structural carbohydrates are more available for energy metabolism
than the structural carbohydrates.

Cellulose

Cellulose is a major structural carbohydrate that is present in plant cell walls. Cellulose
is an unbranched chain of 7,000 to 15,000 glucose molecules that are linked together
by ß-1,4 bonds. Cellulose is a major part of the structural fiber in forages and can be
utilized by microorganisms in the rumen. When utilizing the chemistry associated with
the Van Soest Detergent Fiber Fractions, cellulose is estimated as follows:

 Cellulose = ADF ? (ADL + Ash)

Where ADF is acid detergent fiber and ADL is acid detergent lignin.

Concentrates

Concentrates refer to animal feeds that are rich in energy and/or protein but low in fiber,
such as corn, soybean meal, oats, wheat, molasses, etc.

Crude Fat

Crude fat is an estimate of the total fat content of feeds taken from older collection of
methods known as proximate methodology. The crude fat is estimated using ether
extraction. Crude fat contains true fat (triglycerides) as well as alcohols, waxes,
terpenes, steroids, pigments, ester, aldehydes and other lipids. See Ether
Extract and Fat.

Crude Fiber (CF)

This older proximate method was used to divide carbohydrates into digestible and
indigestible fractions. When CF content is higher, the energy content of the feed is
lower because crude fiber is considered indigestible. Measuring crude fiber was one
part of the original system of analyzing the “digestible” fraction in feedstuffs. This
method uses sequential acid and alkali extraction. It was developed by Henneberg and
Sttohmann during the 1860s at the Weende Experiment Station in Germany, and is
often referred to as the Weende System of proximate analysis. The CF extract was
once used as a standard analysis for fibrous parts or the indigestible portion of
carbohydrates in feeds. However, some of these substances are partially digestible by
microorganisms in the rumen. Crude fiber accounts for most of the cellulose but only a
portion of the lignin and no ash, so it underestimates true fiber and is less than acid
detergent fiber (ADF). Thus, CF is not a good indicator of digestibility in ruminant
animals, and the use of this parameter in feeds for ruminants is declining.

Even though CF is not a very useful parameter for quantifying forage fiber where lignin
content is substantial, the CF is a reasonable estimate of the fiber in grains because of
their low lignin content. Thus, it is still commonly used for analysis of feeds for non-
ruminants or monogastric animals (i.e., those that do not have a chambered stomach or
rumen; for example, horses and pigs). Crude fiber is still used today as the legal
measurement of fiber in grains and finished feeds. See Acid Detergent Fiber
(ADF) and Neutral Detergent Fiber (NDF) for contrast.

Crude Protein (CP)

Proteins are organic compounds composed of building blocks called amino acids. They
are a major component of vital organs, tissue, muscle, hair, skin, milk and enzymes.
Protein is required on a daily basis for maintenance, lactation, growth and reproduction.

The crude protein content of a feed sample represents the total nitrogen (N) in the diet,
which includes not only true protein but also non-protein nitrogen (e.g., urea and
ammonia in a feed; nitrate is not included in non-protein nitrogen). Because N is an
integral part of any amino acid, non-protein nitrogen has the potential to be utilized for
protein synthesis by rumen microorganisms. In laboratory analysis, total N present in a
feed sample is first determined and then the total amount of protein is calculated by
multiplying the total N by a factor. This factor is 6.25 for forages because leaf and stem
tissue proteins generally contain 16 percent nitrogen, or one part nitrogen to 6.25 parts
protein. For seeds, this factor is different (e.g., 5.70 for wheat and 5.90 for other cereal
grains). Unless otherwise stated, protein values given in lab reports, feed tables and
feed tags are crude protein.

Because the protein content of forages, silages or grains used in animal feeding are
sometimes inadequate to meet the needs of the animal class, protein supplements
become essential. Consequently, analysis for total protein or crude protein in a feed
sample is important.

Crude protein in feeds for ruminants can be further fractionated according to their rate
of breakdown in the rumen, as discussed below for neutral detergent fiber insoluble
crude protein (NDFICP) and discussed previously for acid detergent fiber insoluble
crude protein (ADFICP).

No doubt, CP is an important indicator of the protein content of a forage crop, and even
estimates of non-protein nitrogen are important in evaluating nutritive value. However, it
is a false perception that protein is always the most limiting nutrient in the animal?s diet
and CP is the ultimate measure of a forage quality. In fact, the energy value of forages
is often the most limiting attribute for meeting an animal?s requirements in most forage-
based feeding. An overemphasis on CP may cause one to fail to pay due attention to
meeting energy requirements. Furthermore, CP is merely an estimate of nitrogen
content (N, % × 6.25 = CP, %) and must be considered in context of plant maturity,
species, fertilization rate and many other characteristics. For example, a high nitrate
concentration in the forage will result in an artificially high CP level.

Degradable Intake Protein (DIP)

The DIP, also called Rumen Degradable Protein (RDP), represents the portion of intake
crude protein (CP) that can be digested or degraded to ammonia and amino acids in the
rumen by microbes. This fraction of CP consists of non-protein nitrogen (e.g., urea and
ammonia in treated silage) plus the true proteins that are soluble and those having
intermediate ruminal degradability. They are used to synthesize microbial protein in the
rumen. The RDP or DIP is expressed as a percentage of CP, where DIP = NPN +
Soluble True Protein + True Protein of Intermediate Degradability.

Detergent Fiber Analysis

Since crude fiber (CF) has been found to have an unsatisfactory relationship with
animal performance, it has limited value in ruminant nutrition. Most feed analysis
laboratories do not use the proximate analysis system (of which CF was a part) and
have replaced it with the Van Soest detergent fiber analysis system. The technique of
using detergents to separate digestible and indigestible parts of plant tissues was
originally proposed by Van Soest in 1963. The concept behind the detergent fiber
analysis is that plant cell substances can be divided into less digestible cell walls (made
of hemicellulose, cellulose and lignin) and the highly digestible cell contents (containing
starch and sugars). These two components are successfully separated by using two
different detergent systems:

 A neutral detergent solution of sodium-lauryl sulfate (C12H25NaO4S) in disodium

ethylenediaminetetraacetate (C10H14N2Na2O8) and sodium borate (Na2B4O7) with pH
=7 .0 (Van Soest, 1963a); and
 An acid detergent solution of cetyl-trimethyl-ammonium-bromide (C19H42BrN) in
1N sulfuric acid (Van Soest, 1963b; Van Soest and Wine, 1967).

In a sequential analysis, the feed sample is initially boiled in the neutral detergent
solution to separate the neutral detergent soluble fraction (cell contents) from the
neutral detergent insoluble fraction (cell walls). The cell contents are highly digestible
(about 98 percent) and include various sugars, starches, pectins and other soluble
carbohydrates, proteins, non-protein nitrogenous compounds, lipids, water-soluble
minerals and vitamins. The remaining dry matter is estimated and the proportion gives
the neutral detergent fiber (NDF).

In sequential analysis, the NDF is then further fractionated by boiling in the acid
detergent solution. Hemicellulose is solubilized during this procedure while lignin and
cellulose remain insoluble. The residue remaining after boiling NDF in acid detergent
solution is called acid detergent fiber (ADF). Cellulose is then separated (i.e.,
solubilized) by adding sulfuric acid. Only lignin and acid insoluble ash remain after this
step. The residue is then combusted in a furnace, and the difference of the weights
before and after ashing yields the amount of lignin that was present in the sample.
Generally,

 NDF = Hemicellulose + Cellulose + Lignin + Ash

 ADF = Cellulose + Lignin + Ash

Hemicellulose, cellulose and lignin are indigestible in non-ruminants, while

hemicellulose and cellulose are partially digestible in ruminants. NDF is a good indicator
of the "bulk" fiber and has been used to predict feed intake. In contrast, ADF is a good
indicator of digestibility (negatively correlated) and thus energy intake.

The detergent fiber analysis system is the most widely accepted method for forage
analysis. However, many agencies still base part of their regulations on terms in the
proximate. As a result, both methods are used in most laboratories, including the
University of Georgia?s Feed and Environmental Water Laboratory.

Digestibility

Digestibility refers to the extent to which a feedstuff is absorbed in the animal body as it
passes through an animal?s digestive tract. It varies greatly with the type of feedstuff
and type of animal concerned.

Digestible Dry Matter (DDM) or Dry Matter Digestibility

(DMD)

DDM (or DMD) is the portion of the dry matter in a feed that is digested by animals at a
specified level of feed intake. There is no direct laboratory method for measuring
DDM/DMD. It is often estimated by measuring in vitro or in situ digestibility. Both of
these analyses are rather expensive and laborious. So, in vitro digestibility is frequently
estimated by near infrared reflectance (NIR) analysis and/or estimated from the acid
detergent fiber. The DDM can be calculated as follows:

 %DDM = 88.9 – [0.779 × %ADF (on a dry matter basis)].

Digestible Energy (DE)

Digestible energy provides an indication of the actual amount of energy from a feed that
can be available for use by the animal. It is estimated by subtracting energy lost in the
feces (fecal energy or FE) from the gross intake energy (GE), (i.e., DE = GE ? FE).
Digestible energy is commonly used to evaluate poultry and horse feed. For poultry
feed, DE is considered as an appropriate measure of feed quality, because FE is almost
the sole form of energy loss during digestion. However, in horses, given that FE only
partially accounts for the energy losses (considerable losses also occur via urine and
gases) in the process of the utilization of nutrients, DE may over estimate low quality
feeds relative to high quality feeds.

Digestible Neutral Detergent Fiber (dNDF)

The 48-hour in vitro digestible fraction of Neutral Detergent Fiber (NDF) is expressed as
a percentage of the dry matter content of a feed sample. Contrast with Neutral
Detergent Fiber Digestiblity (NDFD) below.

Distillers Grains

Distillers grains are residual grains or byproducts remaining after the starch from grains
has been fermented to alcohol. Traditionally, alcohol was produced mainly for
beverages by the liquor industry. However, in the last 25 years its use as an alternative
fuel has increased significantly. This increased demand has led to the development of
ethanol production plants in various places in the U.S. With increasing ethanol
production, the opportunity currently exists for using a substantial quantity of distillers
grains as feed in livestock industry.

Dry Matter (DM)

Dry matter represents everything contained in a feed sample except water; this includes
protein, fiber, fat, minerals, etc. In practice, it is the total weight of feed minus the weight
of water in the feed, expressed as a percentage. It is determined by drying the feed
sample in an oven until the sample reaches a stable weight. This is normally a simple
analysis. However, estimates of the DM of fermented materials such as silage are
complicated by the presence of volatile fatty acids. These acids are removed in the
drying process but they are part of the dry matter and are digestible. This introduces a
variable amount of error. Analysis of the fodder without ensiling provides a more
accurate estimate of fiber fractions and digestibility contained in the silage.

Dry Matter Basis

Dry matter basis indicates the nutrient levels in a feed sample based on its dry matter
content (i.e., excluding its water content). This is also referred to as “Dry Basis,” “Dry
Results” or “Moisture-free Basis.” As there is considerable variation in the water content
of forages, excluding the water or expressing the nutrient levels on a dry matter basis
eliminates the dilution effect of the water, thereby providing the essential common basis
for direct comparison of the nutrient contents across different forages.
Comparison of essential nutrients, feed chemical composition, and analytical testing
procedures.

Chemical
Essential Nutrients Analytical Procedures
Components

Fatty acids, Fat-soluable Lipids, pigments,

Ether Extract
vitamins sterols

Nitrogen-containing
compounds - Kjeldahl Procedure
Protein, amino acids
Protein, Nonprotein (Crude Protein)
nitrogen

Inorganic minerals Ash Ashing (complete combustion)

Sugars
Nonstructural
Glucose
Carbohydrates ** Nonfiber
Starches
Carbohydrates +
Soluable Fiber
Carbohydrates
Dietary Hemicellulose

Fiber Neutral
Cellulose Acid Detergent Detergent Fiber
Fiber
Lignin*
*Lignin is not truly a carbohydrate compound but is so intimately associated with cell
wall carbohydrates that it is often included as such.
**Newer methods are being used to measure starch content.
+Determined by difference (100 - CP - EE - NDF - Ash).

https://extension.psu.edu/determining-forage-quality-understanding-feed-analysis

Determinants of Forage Quality

As previously described, forage quality reflects the ability of a given forage to meet
the nutrient needs of the consuming animal. Forage fiber content is the primary
detractor to high intake and nutrient availability. Relative to assessing forage quality,
fiber tests are our single best method, though additional tests for protein and
moisture can help to further characterize the forage. The following are brief
descriptions of forage analysis tests and their interpretation relative to forage quality.

Dry Matter (DM)

Dry matter is defined as the non- moisture portion of a feed ingredient or diet. The
sum of moisture and dry matter content of a feed on a percent of total will always
equal 100. Dry matter contains the essential nutrients within a given feed ingredient
or forage. Feeds, and thereby diets, vary widely in their moisture content. Pastures
and liquid feeds have moisture content between 75 and 90% (10-25% DM). Dried
feeds usually have less than 15% moisture (>85% DM). Moisture or dry matter
content of a feed is determined by heating a weighed sample of feed in a convection
drying oven until a constant weight is reached (24-48 hours). Dry weight is expressed
as a ratio to original sample weight (moisture + DM) or converted to a percent. For
example, a feed sample weighs 150 g wet and 50 g dry. The DM ratio would be 0.33
(50/150) and percent DM 33.3% (50/150 x 100). The moisture content of this feed
would be 66.7% (100-33.3 or [150-50]/150 x 100).

Why is knowing moisture content important? One important aspect is our ability to
compare nutrient content of different feeds on an equal basis. Nutrient content of a
feed can be determined on an "As Fed" (AF; moisture included), or dry matter
(moisture excluded) basis. Intuitively, nutrient content will always be higher on a DM
compared to AF basis for any feed. Feeds having more water content (i.e., pastures)
will have much lower nutrient content than dry hay when compared on an as fed
basis. From Table 1, it can be seen the pasture has much lower nutrient content on an
AF basis; however, when corrected for water content, both pasture and hay have
equal nutrient content. To appropriately compare these two feeds equally, nutrient
content needs to be converted to a DM basis. Feed moisture determinations also
facilitate calculations and monitoring of animal DM intake. Finally, DM determinations
can be used to evaluate whether or not feed moisture content is within expected
ranges. For hay or any dry feed, moisture content should not exceed 15%, as this
amount of moisture is necessary to promote mold growth.

Table 1. Comparison of nutrient content expressed on As Fed (AF) or Dry Matter (DM)
basis for generic grass pasture and hay.

Nutrient Density % Nutrient Content

Basis* DM Protein NDF ADF Calcium

*Conversion formula: As Fed nutrient content = DM nutrient content x DM ratio or DM

nutrient content = As Fed nutrient content/DM ratio. DM ratio is 0.2 for pasture and
0.9 for hay in this example.

AF 20 2.2 11.0 8.0 0.12

Grass Pasture
DM 100 11.0 55.0 40.0 0.60

AF 90 9.9 49.5 36.0 0.54

Grass Hay
DM 100 11.0 55.0 40.0 0.60
Fiber
The detergent feed analysis system is used to characterize fiber or total cell wall
content of a forage or feed. That portion of a forage or feed sample insoluble in neutral
detergent is termed neutral detergent fiber (NDF), which contains the primary
components of the plant cell wall, namely, hemicellulose, cellulose, and lignin. As cell
wall production increases, as occurs in advancing plant maturity, NDF content will
increase. As NDF content of a feed increases, dry matter intake will decrease and
chewing activity will increase. Within a given feed, NDF is a good measure of feed
quality and plant maturity. For legume forages, NDF content below 40% would be
considered good quality, while above 50% would be considered poor. For gras s
forages, NDF < 50% would be considered high quality and > 60% as low quality.
Another measure of fiber is acid detergent fiber (ADF), a subset of NDF. Acid detergent
fiber contains the poorly digestible cell wall components, namely, cellulose, lignin, and
other very resistant substances. Due to its nature, ADF is often used to predict energy
content of feeds. Like NDF, ADF is a good indicator of feed quality; higher values
within a feed suggest lower-quality feed. A goal would be to have < 35% ADF in either
legume or grass forages. Refer back to table 1 shown in the first column (page 33 in
June 2006 issue) for the changes in NDF and ADF with forage maturity.

Crude Protein (CP)

Feed protein content is often considered a good determinant of quality. In a ctuality
protein cannot be directly measured, it is estimated from feed sample nitrogen (N)
content. On average all biological proteins contain 16% N, therefore protein content is
estimated by multiplying N% by 6.25 (6.25 = 1 ) 0.16). Thus, crude protein d oes not
differentiate between N in feed samples coming from true protein or other nonprotein
nitrogen (NPN) compounds, nor does it differentiate between available and
unavailable protein.

Although issues have been raised concerning application of crude protein as a feed
measure, it continues to be a commonly used measure of feed quality. Crude protein
content is very different across feeds, but within a feed, higher protein is usually
associated with higher quality. This certainly is true in forages. As forages mature,
their crude protein is diluted with increasing fiber content. Forage fertilization
practices can alter this relationship, suggesting crude protein should not be solely
used as a quality criterion without evaluating fiber content.

Energy
Energy content is often used to compare feeds and evaluate quality. Feed energy
content is not directly measured like other nutrients but derived through regression
equations. Traditionally ADF alone or with CP were used to predict energy value of
various feeds. Most laboratories report feed energy values based on cattle equations,
reporting total digestible nutrients (TDN) and net energy (NE) values. The question is
how applicable are these predicted values to camelids? Cattle TDN values are the best
estimate we have and should reasonably reflect feed energy for llamas and alpacas
given the similarity in digestive function. In comparison, predicted cattle feed energy
availability would be inappropriate for use in swine or horse diets given anatomic and
physiologic differences in digestive capacity. However, in considering the differences
in fiber degradability between ruminants and camelids, one would anticipate that
cattle energy predictions may be too low for lower-quality forages.

In Figure 1, a large portion of feed carbohydrates, especially those associated with

higher digestibility and glucose production, are not measured. The neutral detergent
soluble carbohydrate fraction of feed is termed nonfiber carbohydrates (NFC). This
fraction is not directly measured, but determined by difference. Inherently, all
laboratory analytical method errors associated with other feed fractions will be
compiled into the NFC fraction. Although susceptible to error, NFC represents a highly
available portion of a feed and as such positively reflects on evaluation of feed quality.
More recently some laboratories have offered an enzymatic analysis for feed starch
content; helping to further define the more digestible portion of NFC,
termed nonstructural carbohydrates (NSC). Higher values for NFC and NSC would
reflect higher quality forages. For grasses and legume forages, NFC values >20 and
>30%, respectively, would be considered higher quality, especially if associated with
lower fiber values.

Other Feed Fractions

Additional analyses may be completed on a feed sample, including fat content (ether
extract) and mineral analysis. Ether extract is a chemical method by which all lipid
(fat) soluble compounds are extracted by being dissolved in ether (Figure 1). This
technique is of little value in evaluating feed quality except in the cases of comparing
feeds with high fat content.

Total feed mineral content can be measured by a procedure where the feed sample is
completely combusted into ash. This does not separate out any individual minerals
and does not separate macro- and microminerals of interest from silica and other less
important minerals. Selected macrominerals (calcium, phosphorus, magnesium,
potassium, sodium, and sulfur) and microminerals (iron, copper, zinc, manganese, and
molybdenum) can be determined using sophisticated wet chemistry atomic
absorption spectroscopy. As previously stated, NIR analyses are not very accurate in
determining feed mineral content. Mineral analysis is not always done since it is the
most expensive test. Feed mineral content has no bearing on feed quality evaluation,
but can provide insight as to the type of mineral supplement required.

Visual Assessment of Forage Quality

Although forage testing is the most definitive method of determining forage qua lity, it
often is not complete. Associated costs, lack of laboratory availability or constant
forage turnover are the most often reasons people cite for not testing their forages.
The first two reasons are not good excuses; however, the third is an issue o n many
farms that purchase small lots of hay often. One can use their various senses to
evaluate small amounts of forages, though sensory evaluation does not provide any
sense of nutrient content.
Table 2 summarizes visual and chemical analysis of forages with guidelines for
assessing quality.

Testing Method Description/Comment

Sensory Evaluation

Visual

Stage of Look for the presence of seed heads (grass forages) or flowers or
maturity seed pods (legumes), indicating more mature forages

Look at forage and determine whether the stems or leaves are

Leaf to Stem
more obvious; good-quality legume forages will have a high
ratio
proportion of leaves, and stems will be less obvious and fine

Color is not a good indicator of nutrient content, but bright green

color suggests minimal oxidation; yellow hay indicates oxidation
Color
and bleaching from sun, and hay will have lower vitamins A and E
content

Foreign Look for presence and amount of inanimate objects (twine, wire,
Objects cans, etc.), weeds, mold, or poisonous plants

Feel stiffness or coarseness of leaves and stems; see if alfalfa

Touch stems wrap around your finger without breaking; good-quality
hay will feel soft and have fine, pliable stems

Good quality hay will have a fresh mowed grass odor; no musty
Smell
or moldy odors

Chemical Testing

Measures amount of moisture in forage; moisture content will

Moisture/Dry
determine how well the forage will store without molding; Goal
Matter
for any hay <15% moisture (>85% dry matter)

Measures total cell wall content of plant and indicates maturity;

Neutral
the higher the value, the more mature and lower quality the
Detergent Fiber
forage; Goal < 40% Alfalfa and < 55% Grasses

Measures the more indigestible portion of cell wall and reflects

Acid Detergent
degree of lignification; Higher values indicate more mature,
Fiber
lower quality forages; Goal: < 35% Alfalfa and < 35% Grasses
Testing Method Description/Comment

Crude protein content reflects maturity of forage as well as

Crude Protein fertilization amount; Good-quality forages generally will have
higher protein content; Goal > 9% Grasses and >15% Alfalfa
Although the general idea is that forages grown in North America are of better quality
than South American forages, forage quality can not be ignored in our feeding
programs. Many factors influence forage quality, the most critical being plant
maturity. Feeding programs consisting of low quality forage and limited variety of
feedstuffs can potentially result in protein- energy malnutrition, failure to thrive and
hepatic lipidosis disease problems. There is no best single feeding program that fits all
situations, but extremes of only high quality alfalfa hay or low quality grass hay are
not appropriate. Feeding programs with pasture access may have the greatest
application, allowing the animals to best express their selective feeding behaviors.
When hay is the primary forage in a feeding program, critical assessment of nutrient
content via laboratory analysis is highly recommended. Further questions or
comments about forage quality and nutrition are welcomed.
http://www.fao.org/livestock/agap/frg/AHPP126/Ch9.pdf
Chemical analysis of feed and faeces
Preparation of samples
Samples of material to be analyzed must be oven-dried at 65EC and then ground to pass through
a 1 mm screen. Further drying to constant weight may be necessary to remove residual moisture.
Dried material may be stored at room temperature in sealed vessels or plastic bags, preferably
under nitrogen gas. Moisture A sample containing the equivalent of about 2 g dry matter is dried
to constant weight at 95-100EC for 24 hr. Use an aluminium dish or porcelain crucible.
Calculate percentage moisture from the loss in weight (to first decimal place).

Ash
Weigh a 2 g sample into a weighed porcelain crucible and place in a temperature-controlled
furnace preheated to 600EC. Take care to avoid loss of material by convection currents. Hold at
this temperature for 4 hr. Transfer crucible directly to desiccator, cool and weigh immediately.
Calculate percentage ash (to first decimal place).

Kjeldahl
Nitrogen determination The Kjeldahl technique can be divided into three basic steps:
-Digestion of the sample in concentrated sulphuric acid during which all organic compounds are
broken down, and organic N is converted to ammonia.
-Over-neutralization of the solution with a caustic soda solution and distillation and collection of
the ammonia.
-Titration of the ammonia.
Reagents
50% Sodium Hydroxide: Dissolve 600 g of NaOH in distilled water and make up to a volume of
1 litre. When the pellets of sodium hydroxide are added to water, stir with a glass rod. This is
necessary to prevent NaOH from fusing to the bottom of the beaker. Keep in a rubber- or plastic-
stoppered bottle. Digestion mixture: Mix 8 g selenium with 400 g potassium sulphate, add the
mixture into 2 litres concentrated sulphuric acid and heat until all reagents are dissolved. Note:
When the chemicals are mixed the Se and potassium sulphate set solid so it is easier to put the
chemicals into the digestion flask and then add the acid. Alternatively, Se catalyst tablets can be
purchased and concentrated sulphuric acid is used as the digestion mixture. Sample size
Determination of sample size assumes some prior knowledge of the material under investigation.
For maximum accuracy, a sample size should be taken which will require 10-20 ml of the
standard acid. The amount of titrant can also be varied by changing the normality of the standard
acid. Some feeds may be low in protein, and it may be difficult with small samples to obtain
truly representative samples. Consequently, a considerable amount of dry material must be
digested. Using 0.1 M acid as titrant for the ammonia that had been distilled, it is recommended
that the following sample sizes are used: C Dry feed samples 300 mg C Milk, except colostrum 1
ml (or 1 g) C Colostrum 300 mg C Plasma and serum 0.5 ml C Urine 0.2 ml Very dilute samples
(e.g., rumen fluid) may require use of a 0.01M standard acid for titration. Because of the
sensitivity of the analysis, high accuracy cannot be obtained without thorough mixing of the
material to be analyzed prior to sampling. This is especially true with materials which have been
frozen and allowed to thaw.
Digestion To the 100 ml Kjeldahl digestion flask, add: C Sample (approximately 150 mg DM) C
One glass bead to prevent bumping C 5 ml conc. H2SO4 C 1 Se catalyst tablet Heat the mixture
on the digestion rack in an area with air extraction. If foaming occurs, the early part of the
digestion can be carried out at a lower temperature. Silicone antifoam agents should never be
used (contrary to several current texts). The silicone spray coats the sides of the digestion tube
producing a non-wetting surface. Large water droplets collect, and when sufficiently large, drop
into the superheated anhydrous digestion mixtures, with violent consequences. Following
removal of all the water, white sulphur dioxide fumes will be evolved. These fumes are irritating
and toxic and must be exhausted in a hood with sufficient capacity to prevent transfer into the
laboratory. During the digestion, charred material can be washed down into the digestion
mixture by swirling the digestion flask. If swirling does not flush all charred material into the
digestion mixture, let the mixture cool completely and wash the charred material down with a
fine stream of water. Then redigest until the mixture clears. After white fumes are no longer
evolved and the boiling mixture is clear, allow the digestion to proceed for a further 30 minutes.
Then allow the flasks to cool. Add about 20 ml of deionized water and mix immediately to
prevent crystallization of the sodium sulphate. Distillation This is the same as for ammonia
estimation. Turn on the heater under the steam generator and increase the heat to boil the water
steadily (not violently) and turn on water to condenser. Put the empty digestion flasks on the
collector tubes and, with the alkali stopcock closed and steam directed into the apparatus, run
steam through the assembly and collect the condensates in 100 ml beakers for several minutes.
This serves to warm up the apparatus, and flush out any residual alkali
When the apparatus is preheated, open the alkali stopcock and direct the steam into a water
drain. Place samples in the distillation apparatus and place 100 ml flasks containing 20 ml 2%
(w/v) boric acid (containing indicator) under the condenser stem. Be sure the tip of the condenser
stems are below the surface of the boric acid solution. Admit alkali solution through the alkali
stopcock (about 5 ml alkali for 1 ml of H2SO4 used in the original digestion) and close the alkali
stopcock. Then turn steam on through the apparatus and allow steam distillation to proceed for 6
min. Near the end of this period, lower the receiving beaker so that the distillate washes any
remaining ammonia solution from the tip of the condensing units. When the distillation is
completed, turn steam stopcock into the position which diverts the steam to sink waste and
another opens the distillation flasks to atmospheric pressure. Remove distillation flasks and turn
steam stopcock to the off position. Quantification of the ammonia Titrate the ammonia-boric
acid solution to the pink end-point with standardized acid (0.1N HCl or 0.05N H2SO4 ).
Appropriate blanks must be run and their values subtracted from the sample titration values.
Calculations There is a direct mole-per-mole relationship between ammonia released, the acid
needed to titrate that ammonia, and the total N originally present. The number of ml of acid
multiplied by its molarity gives the millimoles of ammonia. Since the average protein is 16% N,
multiplication of per cent N by the factor 6.25 gives per cent crude protein (some factor other
than 6.25 may be used for particular proteins). Precautions Care must be taken when working
with hot concentrated acid and alkali. Take normal precautions: safety goggles must be worn
when starting distillations. In each step where water is added to acid and alkali to acid, the
solutions must be cool, otherwise the reactions can be quite violent.