In Vitro, in sacco, in vivo studies of

feeds

Department of Animal Science, Faculty of Agriculture

Khon Kaen University

Prof. Dr. Metha Wanapat

Dr. Anusorn Cherdthong

130740 Tropical Feed Resources and Feeding Technology

The First Step

Know the nutrient composition of your feed ingredients! Discuss the way we determine nutrient concentration in feedstuffs

Key to Nutrient Analysis

The analysis is only as good as the sample you take !!!

1 quart sample has to represent several tons of feed/feedstuff “representative sample”

Sampling Feedstuffs

ID = label containers with your name, address, date, and feed type, etc.

Sampling: Grain or mixed feeds

Sacks: 2 handfuls from 5-7 bags Bulk: 12-15 samples from different areas

Random samples placed in bucket & mixed

Obtain uniform sub-sample

Sampling Feedstuffs

Hay Use a hay probe & take 12-15 samples

from all locations/depths Cut samples into 1-2” lengths & mix in

clean bucket Haylage or Silage

Collect samples during the entire loading process for new

For old, take series of samples (not spoiled)

Sampling Feedstuffs

Grain: send in at least 1 pt Hay: send in at least ½ lb Silage (Wet Feedstuffs): 2 qts in

an airtight container, preferably freeze or refrigerate, or deliver immediately

Samples must arrive at lab in same condition they left your farm!

Why analyze rations or feedstuffs??

Nutrient Analysis

Book values are averages over many locations Your region may differ in the nutrient

density of the feedstuffs it produces Example: Book value for SBM = 48%

CPYour SBM from SD =

46.5% CP Overfeeding/underfeeding nutrients

Contaminants in feedstuffs Toxins, chemical residues, or other

harmful compounds

Nutrient Analysis How often should you analyze your

feedstuffs/rations? Every time you change batches/loads

of feedstuffs When you change feedstuffs in your

rations Every time you mix a new batch of feed

Monthly samples of forages/silages In a perfect world Generally, take sample after harvest

Analysis Systems

Analysis Methods1. Chemical

e.g. titration, chromatography (chemistry) No estimate of utilization, lab errors

2. Biological Animals; Expensive & tedious Difficult to obtain individual nutrient

effects

3. Microbiological Microorganisms; estimations Accurate quantification difficult

Proximate Analysis

Traditional standard of the industry Developed in Germany more than a

century ago Most generally used chemical

scheme for describing feedstuffs Limitations for today’s diet

formulation systems Information is of uncertain nutritional

significance May result in misleading results

Proximate Analysis Fraction Nutrient

Dry Matter (DM) Water

Crude Protein (CP) Protein

Crude Fiber (CF) CHO—Fiber

Nitrogen Free Extract (NFE)

CHO—easily digested

Ether Extract (EE) Lipids

Ash Minerals

Missing? Vitamins

Dry Matter Weigh a sample Heat to 100 – 105 C Re-weigh the sample Difference in 2 weights is

water loss % DM = 100% - % water loss

Ash Weigh a sample Burn for 2 hrs at 600 °C

(1112 °F) Weight remaining is ash Individual minerals not

determined Use atomic absorption,

spectrophotometry to get individual minerals

Ashing Oven

Crude Protein

Kjehdahl Method: Digest a dry sample in concentrated

sulfuric acid Converts N to ammonium

During distillation ammonium is converted to ammonia

mL of acid used to bring ammonia solution to neutral pH = amount of N in sample

Total N x 6.25 = % CP

Digestion Process

Distillation Process

Kjehdahl Method

Important Point: Analysis does not distinguish

between N sources Protein Synthetic amino acids Non-protein N (urea, NH4,

biuret)

Crude Protein Combustion Method (LECO)

N is released at high temperature in presence of pure O2

N determined by thermal conductivity within the instrument

EXPENSIVE equipment!

LECO analyzer

Ether Extract

Fat determination

Boil sample in ether alcohol to extract lipid fraction of sample

Crude Fiber Industry method for fiber

determination BUT--80% of hemicelluloses, 60%

of lignin, and as much as 50% of celluloses can be lost CF value lower than actual amount

of fiber in feedstuff Lignin can attach to N

Overestimated [lignin]

Van Soest Method of Forage Determination

Replaces CF Analysis

Van Soest Fiber Determination

Used to determine the insoluble cell wall matrix & the major subcomponents:

1. Hemicellulose2. Cellulose3. Lignin

Able to determine heat-damaged protein Maillard Products N content of ADF fraction

(ADIN=indigestible N) Tells you the amount of N in a sample

that is actually AVAILABLE to the animal for use

Detergent System

Ground forage sample

ND insoluble fiber (NDF)(cell wall components)

ND solubles(cell contents)

AD insoluble fiber (ADF)(cellulose, lignin)

AD solubles(hemicellulose, cell wall N)

Acid insoluble lignin Solubles (cellulose)

Lignin by loss of ignition

Digest with neutral detergent (ND)

Digest in acid detergent (AD)

Digest with 72% H2SO4

Detergent Digestion System

Summary

NDF = hemicellulose + cellulose + lignin

ADF = cellulose + lignin

ADL = lignin

Others Vitamins

Individual assays for each vitamin Chemical/biological assays using

chromatography

Minerals Assays to obtain concentration of

individual minerals Using Atomic Absorption

Spectrophotometry

Energy Determination

Total digestible nutrients (TDN) vs. Bomb Calorimetry Explained in “Energy Systems”

How to do research experiment ?

Feedstuff Evaluation

Remember—Chemical analysis is the starting point for determining the nutritive value of feeds

The actual value of ingested feedstuffs is dependant upon the ability of the body to make use of the nutrients in the feedstuff

Feedstuff Evaluation

Two general classifications of methods In vitro methodology: Simulate

digestion in a test tube to estimate nutrient digestibility

In vivo methodology: Feed animal and measure response criteria

Growth Retention/Excretion Digestibility

A. In vitro methodology

Method to estimate digestibility of feedstuffs Uses enzymes and (or) microorganisms in

a test tube to simulate GIT environment

Method is cheap, with results in about 24 - 48 hours

Rough estimate of digestibility

In vitro methodology

Use enzymes to simulate digestion in upper GIT Mouth Stomach Small Intestine

Use fecal inoculant to simulate fermentation in lower GIT Large Intestine

In vitro methodology

In Vitro Gas TechniqueMenke and Steingass, 1988)

Sample preparation :

• All substrate should be milled using a 1 mm screen

• Weigh 200 mg substrate into each syringe

• Blank (RF + artificial saliva)

• Sample should be done in duplicate or triplicate

Artificial Saliva preparation :

• add distilled water, buffer solution, macro- and micro-

mineral solution, resazurin solution into round flat-

bottomed flask.

• warm to 39 oC then add reducing solution

• place water bath set at 39 oC on magnetic stirrer

• put magnet into flask and gentle bubble CO2 into

solution until blue color turns to pink then clear-

• provide a stream of CO2 and an O2 free atmosphere,

buffer should be pH 7.0-7.3

• collect RF from animal, strain RF through three layers

of gauze, final ratio of artificial saliva:RF (2:1).

• pour the SRF into the artificial saliva, make sure the

magnet is stirring properly during the whole process of

dispensing the RF/artificial saliva into the syringe.

• add 30 ml of solution to each syringe using a dispenser.

• fill the syringe, then open the clip and gentle push the

syringe’s plunger so that all the air is removed.

• record the volume and place in water bath.

Rumen fluid preparation :

Reading taken :• Forage 3, 6, 12, 24, 48, 72 and 96 hr.

• Concentrate it may be necessary to take more reading

in the first24 hrs.

• It is advisable to gentle mix each syringe 2-3 times

during the first day as well as each time a reading is

taken.

Macromineral solution Micromineral solution

Na2HPO4 5.7g CaCl22.H2O 13.2g

KH2PO4 6.2g MnCl24.H2O 10.0g

MgSO4 0.6g CoCl26.H2O 1.0g

make up to 1 L with distilled water FeCl26.H2O 0.8g

make up to 1 L with distilled water

Artificial saliva : Resazurin aqueous

NaHCO3 35g (100mg/100ml)

(NH4)HCO3 4g

make up to 1 L with distilled water

Preparation of artificial saliva :volume (ml)

Artificial saliva-final volume 500 1000 1500 2000

Distilled water 237.5475.0712.5950.0Macromineral solution 120.0240.0360.0480.0Buffer solution 120.0240.0360.0480.0Micromineral solution 0.06 0.12 0.18 0.24Resazurin 0.61 1.22 1.83 2.44

Reducing SolutionDistilled water 23.8 47.5 71.3 95.01M NaOH 1.0 2.0 3.0 4.0Na2S9H2O (mg) 168 36 504 672

B. In vivo methodology

I. Feeding trials Simply give an indication of:

Palatability of feedstuff in a ration (will the animals eat it?)

Growth response compared to another feedstuff/ration

Tells NOTHING of why different results were obtained

Type of Feeding Experiment

Feeding trials ---> Growth, Production, reproduction

Slaughter experiment --> meat component, market value

Digestion trials --> Intake, digestibility

Balance trials ---> measure nutrients retention

Feeding Trials Compare between > 2 rations Feed intake (input-feed cost) Growth, milk production ,

reproduction, or other function efficiency of feed utilization ADG, weekly gain, final (weight %

initial wt (%) , FCR

Body size: height, length, circumference etc.

Milk production: yield ( average, lactation) persistency, peak, composition & yield

Egg : hen day, hen house etc. Draft animal : Speed, Area , time

ets Experimental designs: Factorial, LS

etc.

Feeding Trials with Laboratory Animals

Small animals e.g. Rat Growth, reproduction, lactation cheap (feed, labor, short life cycle) Useful for fundamental principle of

Nutrition

The purified-diet Feeding Trials Methods

Diets contain of purified source of nutrients

E.g. Casein as protein, urea, starch as CHO

Specific nutrient interested more completely diet --> less

satisfactory on Animal

Feeding Management in the Trials Group Feeding vs Individual

Feeding group - simplest equipment need cheap labor cost complicate in the interpretation of

results some animal many consume less feed

Individual correlation of individual performance

with food intake statistics analysis advantage

Controlled vs ad libitum feeding Ad libitum is the most common in farm

practice Gives unbiased results of direct practical Measure : feed required per kg gain

total increase in body weight n Does one animal grow because it eats more

or the other fail because it eats less ?”

Slaughter Experiments

Killing of the animal when require specific information

Analysis of certain specific tissues or whole body

e.g. Protein source - protein tissue & concentration

(Initial - Final) composition of body chemical Time & labor cost

Slaughter Experiments

Measures of market value: carcass, dressing percentages carcass quality, quality of product, selling price

Meat quality, color, vitamin), fat thickness

Balance trials Provide more information than digestion trial

Measure: nutrient retention (Positive or negative)

Needed to accurate & Precisely measurement method

Use Metabolism cages-intensive care Example: N-retention study

(N-Intake)- (Nexcretion )= N balance Short period but, useful information

Feeding Trials

In vivo methodology

II. Metabolism Trial Determines nutrient

retention/excretion Complete analysis on ration Feed known amount to animals Collect urine/feces Compete analysis on urine/feces

Metabolism Trial

In vivo methodology

Metabolism Trial Calculation: [(In – Out)/In] * 100

Nutrient retention = Nutrient intake –Nutrient excretion (Urine + Feces) x 100Nutrient intake

In vivo methodology

III. Digestibility studies

Use of cannulated animals

Can determine small intestinal digestibility (hydrolytic digestion) as well as total tract digestibility (hydrolytic + fermentative digestion) of nutrients

Cannulated Animals

Cannulated Animals

Cannulated Animals

In vivo methodology

Digestibility studies

Effluent from small intestine or rumen or feces is collected and analyzed for nutrient(s) being studied

In vivo methodology

Digestibility studies

Collection at terminal SI is referred to as ileal digestibility

Collection of feces determines total tract digestibility

In vivo methodology How is TRUE digestibility

determined? Usually only in monogastrics Usually only concerned with true

AA digestibility Chicken—cectomized animals

Surgically remove ceca from birds and measure digestibility

Pigs—feed diet containing no protein

In vivo methodology Determination of endogenous

losses Endogenous losses

Sloughed intestinal cells Sloughed microbial cells Enzymes Mucin

Measure AA output from protein-free diet = endogenous losses

Corrects for AA present but not of feed origin

In vivo methodologyIV. In-Situ digestibility

Digestibility within a localized area or position

rumen, abomasum, small intestine

Use cannulated animals Mesh bag to contain the feedstuff and allow

microbial action to take place

Determine Rate/extent of digestibility

The use of indigestible marker in nutrition

studies

Disadvantage of conventional trials Time consuming expensive animal condition e.g. grazing & stall

grazing : select feeding

Employed of indigestible marker or reference substances

Extensively used in grazing research

Determination propose digestibility and intake rate of passage of nutrients in GI

tract site and extent of digestion microbial protein synthesis (e.g.

in ruminant)

The ideal marker Inert, no toxic on animal & micro

flora not be absorbed or metabolized in

GI mixed well/ associated with feed should not influence GI secretion

digestion absorption or motility precise - quantitative analysis /

not interfere with other analysis

Two type of marker use in nutrition studies

Internal marker component of

feedstuffs e.g. lignin,

AIA, indigestible ADF, NDF

External marker

indigestible substances added to a feedstuff

e.g. Cr2O3 (chromium sesquioxide)

Cerium Dysprosium ytterbium ruthenium

phenanthroline complex

binding marker with a specific feed. E.g. Yb labeled feed

Other typed of marker

Measure microbial protein synthesis maker to determine amount

of microbial protein synthesis

Total protein passing to the lower tract in ruminant

determine portion of total protein : microbial origin

Marker specific to bacteria Diaminopimelic acid (DAPA)

found only in bacteria Determine DAP content in digesta and

DAP:N ratio in bacteria therefore, estimate portion of nitrogen

in digesta from microbial origin Ribonucleic acid (RNA)

assumed that feed RNA 100% digest in rumen

thus, only bacterial RNA passes to lower tract

determine RNA: N ratio in bacteria and % digesta RNA, then --> microbial protein synthesis calculated.

Purine (Adenine, Guanine) and purine derivatives (urine,plasma)?

Use of marker to estimate digestibility

Total tract vs. specific site digestibility Total tract = (intake - fecal) x100 / intake (Marker) Nutrient digestibility, %

= 100 - 100 x % marker in feed x nutrient in feces

% marker in feces % nutrient in feed

Digestibility in specific sites of digestive tract

“Slight modification of total tract digestion”

e.g..

Nutrient digestibility in rumen, %

= 100 - 100 x % marker in feed x % nutrient in duodenum

% marker in duodenum % nutrient in feed

Use of marker to measure feed Intake

Feed intake effect on economy & livestock production

Difficult to measure in take in grazing ruminant

Indicator methods to estimate intake Information: fecal excretion and

digestibility

How to estimate digestibility?

Use of marker techniques

Conventional digestion trial

In vitro DM digestibility

How to estimate fecal output?

Fecal collection bags Total collection of feces Indigestible marker e.g. chromic

oxide Fecal output,g = indicator consumed

(g/day)/Indicator concentration in feces(g/g DM)

Use of marker to measure rate of

passage Particulate & fluid materials Fluid flow rate or fluid dilution

rate(% of fluid volume leaving the rumen per hour)

Faster dilution rate ==> more efficient microbial growth

Increase rate of passage ==> increased voluntary feed intake

Use of marker to measure rate of passage(cont....)

Marker for measure rate of fluid passage from the rumen Chromium EDTA (Cr-EDTA) Cobalt EDTA (Co-EDTA) Polyethylene glycol (PEG)

Measurement the rate of fluid passage

Flow rate at a sampling site = infusion rate(g/d)/marker

concentration at sampling point(g/ml)

Measurement of particulate passage

rate Single dose vs.. continuous dose Calculate flow rate or volume of

particulate phase Common particulate phase marker for

estimate turnover rate Chromic oxide Ytterbium Dysprosium Cerium Ruthenium phenanthroline complex Internal marker e.g. indigestible ADF,NDF

and AIA

Measurement of particulate passage

rate(cont..)

Bind the marker with feedstuffs (soak, with rare earth) then fed or dosed for estimation of passage rate

Marker for the measurement of

microbial protein synthesis

Marker Diaminopimelic acid RNA Purines

Good Luck!!