1. In Vitro, in sacco, in vivo studies of feeds
Prof. Dr. Metha Wanapat
Dr. Anusorn Cherdthong
Tropical Feed Resources and Feeding Technology
Department of Animal Science, Faculty of Agriculture
Khon Kaen University

2. The First Step Know the nutrient composition of your feed ingredients!
Discuss the way we determine nutrient concentration in feedstuffs

3. 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”

4. 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: samples from different areas
Random samples placed in bucket & mixed
Obtain uniform sub-sample

5. Sampling Feedstuffs Haylage or Silage Hay
Use a hay probe & take 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)

6. 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!

7. Why analyze rations or feedstuffs??

8. 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% CP
Your SBM from SD = 46.5% CP
Overfeeding/underfeeding nutrients
Contaminants in feedstuffs
Toxins, chemical residues, or other harmful compounds

9. 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

10. Analysis Systems

11. Analysis Methods 1. 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

12. 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

13. 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

14. 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

15. 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

16. Ashing Oven

17. 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

18. Digestion Process

19. Distillation Process

20. Kjehdahl Method Important Point:
Analysis does not distinguish between N sources
Protein
Synthetic amino acids
Non-protein N (urea, NH4, biuret)

21. 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!

22. LECO analyzer

23. Ether Extract Fat determination
Boil sample in ether alcohol to extract lipid fraction of sample

24. 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]

25. Van Soest Method of Forage Determination
Replaces CF Analysis

26. Van Soest Fiber Determination
Used to determine the insoluble cell wall matrix & the major subcomponents:
1. Hemicellulose
2. Cellulose
3. 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

27. Detergent System Ground forage sample ND solubles (cell contents)
Digest with neutral detergent (ND)
ND solubles
(cell contents)
ND insoluble fiber (NDF)
(cell wall components)
Digest in acid detergent (AD)
AD solubles
(hemicellulose, cell wall N)
AD insoluble fiber (ADF)
(cellulose, lignin)
Digest with 72% H2SO4
Solubles
(cellulose)
Acid insoluble lignin
Lignin by loss of ignition

28. Detergent Digestion System

29. Summary NDF = hemicellulose + cellulose + lignin
ADF = cellulose + lignin
ADL = lignin

30. Others Vitamins Minerals Individual assays for each vitamin
Chemical/biological assays using chromatography
Minerals
Assays to obtain concentration of individual minerals
Using Atomic Absorption Spectrophotometry

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

32. How to do
research
experiment ?

33. 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

34. 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

35. 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 hours
Rough estimate of digestibility

36. 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

37. In vitro methodology

38. In Vitro Gas Technique
Menke and Steingass, 1988)

39. 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

40. 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

41. Rumen fluid preparation :
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.

42. 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.

43. 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

44. Preparation of artificial saliva :
volume (ml)
Artificial saliva-final volume
Distilled water
Macromineral solution
Buffer solution
Micromineral solution
Resazurin
Reducing Solution
Distilled water
1M NaOH
Na2S9H2O (mg)

45. 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

46. Type of Feeding Experiment
Feeding trials ---> Growth, Production, reproduction
Slaughter experiment --> meat component, market value
Digestion trials --> Intake, digestibility
Balance trials ---> measure nutrients retention

47. 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

48. 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.

49. 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

50. 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

51. 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

52. 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 ?”

53. 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

54. Slaughter Experiments
Measures of market value: carcass, dressing percentages carcass quality, quality of product, selling price
Meat quality, color, vitamin), fat thickness

55. 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

56. Feeding Trials

57. 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

58. Metabolism Trial

59. In vivo methodology Metabolism Trial
Calculation: [(In – Out)/In] * 100
Nutrient retention = Nutrient intake –Nutrient excretion (Urine + Feces) x 100
Nutrient intake

60. 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

61. Cannulated Animals

62. Cannulated Animals

64. Cannulated Animals

65. In vivo methodology Digestibility studies
Effluent from small intestine or rumen or feces is collected and analyzed for nutrient(s) being studied

66. In vivo methodology Digestibility studies
Collection at terminal SI is referred to as ileal digestibility
Collection of feces determines total tract digestibility

67. 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

68. In vivo methodology Determination of 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

69. In vivo methodology Determine IV. 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

71. The use of indigestible marker in nutrition studies
Disadvantage of conventional trials
Time consuming
expensive
animal condition e.g. grazing & stall
grazing : select feeding

72. 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)

73. 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

74. 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

75. 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

76. 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)?

77. Use of marker to estimate digestibility
Total tract vs. specific site digestibility
Total tract = (intake - fecal) x100 / intake
(Marker) Nutrient digestibility, %
= x % marker in feed x nutrient in feces
% marker in feces % nutrient in feed

78. Digestibility in specific sites of digestive tract
“Slight modification of total tract digestion”
e.g..
Nutrient digestibility in rumen, %
= x % marker in feed x % nutrient in duodenum
% marker in duodenum % nutrient in feed

79. 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

80. How to estimate digestibility?
Use of marker techniques
Conventional digestion trial
In vitro DM digestibility

81. 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)

82. 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

83. 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)

84. 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

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

86. Marker for the measurement of microbial protein synthesis
Diaminopimelic acid
RNA
Purines

87. Good Luck!!