This document discusses proximate analysis, which quantitatively analyzes macronutrients in feed. It describes the Weende or proximate analysis method developed in 1860 that partitions compounds in feed into six categories based on chemical properties: moisture, ash, crude protein, crude fat, crude fiber, and nitrogen-free extracts. The document then provides procedures and calculations for determining the percentages of each component through methods like drying samples to find moisture content or ashing samples in a furnace to determine ash content.

1. Proximate analysis
• A method for the quantitative analysis of different macronutrients in feed is the
Weende or proximate analysis
• Weende analysis was developed in 1860 by Henneberg and Stohmann in
Germany.
• Proximate Analysis is a partitioning of compounds in a feed into six categories
based on the chemical properties of the compounds
• Moisture Contents
• Ash
• Crude Protein
• Crude Fat
• Crude Fibre
• Nitrogen-free extracts (digestible carbohydrates)

2. Moisture contents
• The moisture contents are very important in the sense: As moisture
content of feed varies, its proximate composition also varies and
consequently it nutritional value also varies
• As the moisture content increased the dry matter decreased, while
purchasing the feed the dry matter content is taken into consideration,
rather than gross weight of the feed
• Moisture content is important in storage of feed
• In general feed more than 11% moisture get mold and spoiled
• Moisture is determined by drying a feed sample in a hot air oven at 100 oC
for a specified length of time
• The loss of weight is the moisture content of the sample
• The residue left over after the removal of moisture is called the dry matter

3. Procedure
• Accurately weight a moisture dish of appropriate size
• Add approximately 10 g of the sample and reweight
• Place the container in a vacuum oven at 100 °C for
approximately 5 hours.
• Remove dish from the oven, cover, cool in desiccator,
and weight
• Re-dry 1 hr and repeat process until constant weight
has been achieved
• Calculate the percentage moisture (wet weight basis) as
follows:
Percent moisture= (P-a) x 100
P
• P = sample weight in g
a = dried sample weight in g

4. Ash contents
• Ash is one of the components in the proximate analysis of biological
materials
• The residues after a sample is completely burnt
• The residue from burning any biological material in furnace at 550 °C
is called ash
• The total ash content is designed to measure the total amount of
residual material after ignition
• Ashing is the first step in the preparation of a sample for specific
elemental analysis by atomic spectroscopy

5. Procedure
• We take dried sample in a crucible
• Place crucible in drying oven at 100 °C for 24 hours.
• Transfer to muffle furnace and increase the temperature step wise to 550
°C ± 5 °C.
• Maintain temperature for 6 hours.
• When the sample completely burnt and ash became white the crucible was
recovered from the furnace and cooled in desiccator.
• If white ash is not obtained after 6 hours, moisten ash with distilled water,
slowly dry on a hot plate, and re-ash at 550 °C to constant weight.
• Repeat if necessary. The ash content was weighed and percentage is
calculated as follow:
Ash: Weight of ash in sample x100
weight of the sample

6. Crude protein
• Estimated by a process developed in 1883 by a Brewer Johann Kjeldahl
• He discovered that "all protein" contains about the same amount of nitrogen (16%).
• He analysed for nitrogen, which is relatively easy, and calculated crude protein on the
basis: 100/16 = 6.25
• therefore: NITROGEN x 6.25 = CRUDE PROTEIN
• The Kjeldahl procedure can be basically divided into three parts: (1) digestion, (2)
distillation, (3) titration.
• Placing 1 gm of the sample containing protein, into a digestion flask, along with 12-15
mL of concentrated sulfuric acid (H2SO4)
• Heating the mixture in the flask until white fumes can be seen, and then continuing
the heating for about 60-90 mins.
• Cooling the flask and cautiously adding 250 ml of water.

7. • To digested mixture, 40% NaOH was added and the resultant mixture was distilled in
Micro-Kjeldahl apparatus.
• The purpose of distillation, is to separate the ammonia (that is, the nitrogen) from the
digestion mixture
• The liberated ammonia was condensed and collected in Sulphuric acid solution having
methyl orange as an indicator. This dye should turn a strong color, indicating that a
significant amount of the original trapping acid is still present.
Titration
• Putting a standard solution of NaOH (sodium hydroxide) into the buret (a long tube
with a tap at the end), and slowly, slowly adding small amounts of the sodium
hydroxide solution to the acid solution with the dye.
• watching for the point at which the dye turns orange, indicating that the "endpoint"
has been reached and that now all the acid has been neutralized by the base.
• Recording the volume of the neutralizing base (sodium hydroxide solution) that was
necessary to reach the endoint.
• Performing a calculation to find the amount of ammonia, and thus nitrogen, that came
from the original sample.

8. 0.0014 is factor for equivalence of H2SO4 to NH3
Distillation: The action of purifying a liquid by a process of heating and cooling.
Titration is a technique where a solution of known concentration is used to
determine the concentration of an unknown solution
Percentage of Nitrogen in selected sample was calculated as:
To convert to protein multiply by the appropriate factor:
x 6.25 for meat and general protein
x 6.38 for milk protein
x 5.70 for cereal protein.

9. Crude fat
• Crude fat is also known as ether extract
• It is estimated by extraction with petroleum ether (boiling point 40-60 °C)
by a soxhlet extractor
• A dried, ground sample is extracted with diethyl ether which dissolves fats,
oils, pigments and other fat soluble substances.
• The ether is then evaporated from the fat solution.
• The resulting residue is weighed and referred to as ether extract or crude
fat.
• Both the ether and the samples must be free of moisture to avoid co-
extraction of water-soluble components in the sample such as
carbohydrates, urea, lactic acid, glycerol, etc.
• Low temperatures are used to evaporate the ether and remove residual
moisture to prevent oxidation of the fat.

10. • The crude fat is determined by extracting powdered sample
with petroleum ether, using a Soxhlet apparatus.
• Dried sample (5 g) was taken in a thimble and was placed in
the extraction tube of Soxhlet apparatus.
• The temperature of the heater was so adjusted that continuous
drops of ether fell on the sample in extraction tube.
• The process of extraction was carried out for 16 hours to allow
complete extraction.
• Then the sample was removed and the solvent was evaporated
under the fume hood.
• The extract was completely dried in oven for 30 minutes at
105oC. The weight of extract was recorded after cooling in
desiccator. Crude fat in the samples were calculated by using
following formula.
• Crude fat (%) = Weight of fat in sample (g) × 100
Weight of sample (g) Soxhlet Extractor

11. Crude Fiber
• In a 1000 mL beaker, 3 g of dried and fat free sample was taken.
• 200 mL of 1.25 % H2SO4 was added to it and level of beaker was marked.
• This was allowed to boil for 30 minutes with constant stirring
• These contents were filtered while warmed giving 2 to 3 washings with hot water until it
became alkali free.
• The residue was carefully transferred in a crucible (W1) and dried in hot air oven at
100°C for 3-4 hours until smoke ceased to come out.
• Then this sample W2 (sample+crucible) was put in a muffle furnace at 550 °C for 4 hours
until grey color ash was obtained.
• This was allowed to cool in a desiccator and reweighed (W3).
% crude fiber in ground sample = Loss in weight on ignition (W2 - W1) – (W3 - W1) x 100
Weight of the sample

12. Nitrogen free extract (NFE)
• Nitrogen free extracts percentage was calculated by the formula:
%NFE = 100-(% of crude proteins + % crude fiber + % crude fat + %
crude ash)
• NFE supposedly represents the soluble carbohydrate of the feed, such
as starch and sugar.

13. Assessment of Nutritional Status
• Nutritional assessment is the interpretation of anthropometric,
biochemical (laboratory), clinical and dietary data to determine whether a
person or groups of persons are well nourished or malnourished.
• The consumption of adequate amount of food both in terms of quantity
and quality is one of the key determinants, which has a significant impact
on the nutritional status
• Eating pattern of an individual is a crucial factor that dictates the
occurrence of a disease, especially some chronic conditions such as
coronary heart disease, hypertension, stroke, diabetes mellitus, and cancer
• In addition, adverse outcomes such as low birth weight, malnutrition,
disability, poor quality of life, and mortality are also related to poor eating
pattern in both developed and under developing countries

14. METHODS FOR NUTRITIONAL
ASSESSMENT
• The assessment of the nutritional status involves two methods:
• Direct (deals with individuals and measures the objective criteria)
• These methods include ABCD, anthropometric, biochemical, clinical and
dietary measurements to cover all the phases of the disease
• Each of them has some advantages and limitations
• Utilizing all of them to assess the nutritional status gives an overall picture
of an individual’s nutritional status
• The use of any one method or a combination of methods is recommended
depending on the purpose of the nutritional assessment

15. Indirect (uses community health indices reflecting nutritional
influences)
• Ecological variables including agricultural crops production, Economic
factors e.g. household income, per capita income, population density,
food availability and prices, Cultural and social habits
• Vital health statistics: morbidity, mortality and other health indicators
e.g., infant and under-fives mortality, Utilization of maternal and child
health care services, fertility indices and sanitary conditions

16. Anthropometric measurements
• Include height, weight, skin-fold thickness, and circumference
• It is an essential component of clinical examination of infants, children &
pregnant women
• It could detect the change of body composition to assess the nutritional
status in specific population groups, including newborn, children and adults
• These measurements are compared to the reference data (standards) of
the same age and sex group, in order to evaluate the nutritional status.
• The advantage is that routine anthropometric measurements can suggest
patterns of growth and development of an individual
Biochemical and Laboratory investigations
• Extremely helpful in detecting early changes in body metabolism and
nutrition before the appearance of clinical signs
• The results obtained are precise, accurate and reproducible

17. • Initial Laboratory Assessment includes Laboratory tests based on blood
and urine can be important indicators of nutritional status, but they are
influenced by non-nutritional factors as well
• Lab results can be altered by medications, hydration status, and disease
states or other metabolic processes, such as stress
• Hemoglobin estimation is the most important test & useful index of the
overall state of nutrition. Beside anemia it also tells about protein & trace
element nutrition

18. Dietary assessment
Nutritional intake of humans is assessed by five different methods
1. 24 Hours Dietary Recall
2. Food Frequency Questionnaire
3. Diet history
4. Food Diary
5. Observed Food Consumption
Interpretation of Dietary Data
 Qualitative Method
Using the food pyramid & the basic food groups method
• Different nutrients are classified into 5 groups (fat & oils, bread & cereals, milk
products, meat-fish-poultry, vegetables & fruits)
• Determine the number of servings from each group & compare it with minimum
requirements
Quantitative Method
• The amount of energy & specific nutrients in each food consumed can be calculated
using food composition tables & then compare it with the recommended daily intake

19. Functional indicators
• These indicators for nutritional status are emerging as an important category of
diagnostic tools and supporting the biochemical investigations
• Some of the commonly used functional indices are for hemostasis and nerve
conduction
Hemostasis or haemostasis is a process to prevent and stop bleeding, meaning to keep
blood within a damaged blood vessel (the opposite of hemostasis is hemorrhage). It is
the first stage of wound healing. This involves coagulation, blood changing from a liquid
to a gel
A nerve conduction velocity (NCV) test — also called a nerve conduction study (NCS) —
measures how fast an electrical impulse moves through your nerve. NCV can
identify nerve damage
Vital statistics
• All the data collected from different countries will present an overall picture of the
nutritional status for that population of interest to help the government-making
policy decisions.
• For example, analysis of morbidity and mortality data can be used in estimating the
prevalence of the disease in the community and identifying the high-risk groups

20. Ecological factors
• Different ecological factors such as socioeconomic factors, quality,
accessibility, availability of health care services, and diseases effect on
occurrence of malnutrition
• It is extremely important to make an “ecological diagnosis” to identify,
which factors will affect the nutrition status of the community

21. Protein Quality Assessment
• Protein quality is the digestibility and quantity of essential amino acids for
providing the proteins in correct ratios for human consumption
• Protein quality can be measured by calculating the nitrogen used for tissue
formation divided by the nitrogen absorbed from food. This product is
multiplied by 100 and expressed as a percentage of nitrogen utilized.
• A complete protein or whole protein is a food source of protein that
contains an adequate proportion of each of the nine essential amino acids
necessary in the human diet. Examples of single-source complete proteins
are red meat, poultry, fish, eggs, milk, cheese and yogurt
• A general recommendation for healthy adults is to eat at least 0.36 grams
of protein per pound of your body weight each day — but you
may need more depending on your activity level and general health

22. Factors Effecting Protein quality
There are a number of factors which may affect the protein quality from food
• The amino acid profile of the protein
• The structure of the protein
• The digestibility of the protein
• The amount of protein consumed in one meal
• Other nutrients and food constituents present in the meal, e.g. fiber,
carbohydrate
• How the food has been prepared
• Recent intake of protein
• The metabolic state of the individual, e.g. illness, exercise, sleep
• The individual's age, weight, sex, and general health

23. Methods of Determining Protein Quality
1. Amino Acid Scoring (AAS)
• AAS is fast and cheap. It measures the essential amino acids (EAAs) – also
known as indispensable amino acids (IAAs) – present in a protein and
compares the values with a reference protein
2. Protein Efficiency Ratio (PER)
• PER measures the ability of a protein to support the growth. It represents
the ratio of weight gain to the amount of protein consumed
3. Nitrogen Protein Utilization (NPU)
• This is the ratio of the nitrogen used for tissue formation versus the amount
of nitrogen digested. This method doesn’t account for the amino acid profile
4. Biological Value (BV)
• BV is the most commonly used and well-known protein scoring system. It
measures the amount of nitrogen retained in comparison to the amount of
nitrogen absorbed. It looks at how similar the amino acid profile is to that of
human requirements. Proteins are grouped into those of high BV (HBV) and
low BV (LBV)

24. 6. Protein Digestibility-Corrected Amino Acid Score (PDCAAS)
• The protein digestibility-corrected amino acid score (PDCAAS) has been
adopted by FAO/WHO as the preferred method for the measurement of the
protein value in human nutrition
• PDCAAS takes into account the profile of IAAs of the protein in question, as well
as its digestibility in humans; it is the AAS with an added digestibility
component. Scores are from 0.1 to 1.0, with 1.0 being a high-quality protein
7. Digestible Indispensable Amino Acid Score (DIAAS)
DIAAS is much like PDCAAS in that it determines the digestibility of the amino
acids as well as the protein’s contribution to human amino acid and nitrogen
requirements.
DIASS is shown as a percentage and doesn’t limit to scoring the protein to a
maximum value; instead, proteins can have a higher score
DIAAS hasn’t been adopted as the preferred protein scoring method US FAO