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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.

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Presentation on theme: "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."— Presentation transcript:

1 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 Tropical Feed Resources and Feeding Technology

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 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 FractionNutrient Dry Matter (DM)Water Crude Protein (CP)Protein Crude Fiber (CF)CHO — Fiber Nitrogen Free Extract (NFE)CHO — easily digested Ether Extract (EE)Lipids AshMinerals 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 O 2 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 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 Solubles(cellulose) Lignin by loss of ignition Digest with neutral detergent (ND) Digest in acid detergent (AD) Digest with 72% H 2 SO 4

28 Detergent Digestion System

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

30 Others Vitamins 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 In Vitro Gas Technique Menke and Steingass, 1988)

39 Sample preparation : All substrate should be milled using a 1 mm screen All substrate should be milled using a 1 mm screen Weigh 200 mg substrate into each syringe Weigh 200 mg substrate into each syringe Blank (RF + artificial saliva) Blank (RF + artificial saliva) Sample should be done in duplicate or triplicate Sample should be done in duplicate or triplicate

40 Artificial Saliva preparation : Artificial Saliva preparation : add distilled water, buffer solution, macro- and micro- add distilled water, buffer solution, macro- and micro- mineral solution, resazurin solution into round flat- bottomed flask. warm to 39 o C then add reducing solution warm to 39 o C then add reducing solution place water bath set at 39 o C on magnetic stirrer place water bath set at 39 o C on magnetic stirrer put magnet into flask and gentle bubble CO2 into 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, provide a stream of CO2 and an O2 free atmosphere, buffer should be pH

41 collect RF from animal, strain RF through three layers 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 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. add 30 ml of solution to each syringe using a dispenser. fill the syringe, then open the clip and gentle push the 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. record the volume and place in water bath. Rumen fluid preparation :

42 Reading taken : Forage 3, 6, 12, 24, 48, 72 and 96 hr. Forage 3, 6, 12, 24, 48, 72 and 96 hr. Concentrate it may be necessary to take more reading Concentrate it may be necessary to take more reading in the first24 hrs. It is advisable to gentle mix each syringe 2-3 times 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 solutionMicromineral solution Na 2 HPO 4 5.7gCaCl 2 2.H 2 O13.2g KH 2 PO 4 6.2gMnCl 2 4.H 2 O10.0g MgSO 4 0.6gCoCl 2 6.H 2 O1.0g make up to 1 L with distilled water FeCl 2 6.H 2 O0.8g make up to 1 L with distilled water Artificial saliva :Resazurin aqueous NaHCO335g(100mg/100ml) (NH 4 )HCO 3 4g make up to 1 L with distilled water

44 Preparation of artificial saliva : volume (ml) Artificial saliva-final volume Distilled water Macromineral solution Buffer solution Micromineral solution Resazurin Reducing Solution Distilled water M NaOH Na 2 S9H 2 O (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 Feeding trials ---> Growth, Production, reproduction Slaughter experiment --> meat component, market value Slaughter experiment --> meat component, market value Digestion trials --> Intake, digestibility Digestion trials --> Intake, digestibility Balance trials ---> measure nutrients retention Balance trials ---> measure nutrients retention

47 Feeding Trials Compare between > 2 rations Compare between > 2 rations Feed intake (input-feed cost) Feed intake (input-feed cost) Growth, milk production, reproduction, or other function Growth, milk production, reproduction, or other function efficiency of feed utilization efficiency of feed utilization ADG, weekly gain, final (weight % initial wt (%), FCR ADG, weekly gain, final (weight % initial wt (%), FCR

48 Body size: height, length, circumference etc. Body size: height, length, circumference etc. Milk production: yield ( average, lactation) persistency, peak, composition & yield Milk production: yield ( average, lactation) persistency, peak, composition & yield Egg : hen day, hen house etc. Egg : hen day, hen house etc. Draft animal : Speed, Area, time ets Draft animal : Speed, Area, time ets Experimental designs: Factorial, LS etc. Experimental designs: Factorial, LS etc.

49 Feeding Trials with Laboratory Animals Small animals e.g. Rat Small animals e.g. Rat Growth, reproduction, lactation Growth, reproduction, lactation cheap (feed, labor, short life cycle) cheap (feed, labor, short life cycle) Useful for fundamental principle of Nutrition Useful for fundamental principle of Nutrition

50 The purified-diet Feeding Trials Methods Diets contain of purified source of nutrients Diets contain of purified source of nutrients E.g. Casein as protein, urea, starch as CHO E.g. Casein as protein, urea, starch as CHO Specific nutrient interested Specific nutrient interested more completely diet --> less satisfactory on Animal more completely diet --> less satisfactory on Animal

51 Feeding Management in the Trials Group Feeding vs Individual Feeding Group Feeding vs Individual Feeding group - simplest equipment need group - simplest equipment need cheap labor cost cheap labor cost complicate in the interpretation of results complicate in the interpretation of results some animal many consume less feed some animal many consume less feed Individual Individual correlation of individual performance with food intake correlation of individual performance with food intake statistics analysis advantage statistics analysis advantage

52 Controlled vs ad libitum feeding Ad libitum is the most common in farm practice Ad libitum is the most common in farm practice Gives unbiased results of direct practical Gives unbiased results of direct practical Measure : feed required per kg gain total increase in body weight 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 ?” 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 Killing of the animal when require specific information Analysis of certain specific tissues or whole body Analysis of certain specific tissues or whole body e.g. Protein source - protein tissue & concentration e.g. Protein source - protein tissue & concentration (Initial - Final) composition of body chemical (Initial - Final) composition of body chemical Time & labor cost Time & labor cost

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

55 Balance trials Provide more information than digestion trial Provide more information than digestion trial Measure: nutrient retention (Positive or negative) Measure: nutrient retention (Positive or negative) Needed to accurate & Precisely measurement method Needed to accurate & Precisely measurement method Use Metabolism cages-intensive care Use Metabolism cages-intensive care Example: N-retention study Example: N-retention study (N-Intake)- (Nexcretion )= N balance (N-Intake)- (Nexcretion )= N balance Short period but, useful information 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

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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 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 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

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

72 Employed of indigestible marker or reference substances Extensively used in grazing research Extensively used in grazing research Determination propose Determination propose digestibility and intake digestibility and intake rate of passage of nutrients in GI tract rate of passage of nutrients in GI tract site and extent of digestion site and extent of digestion microbial protein synthesis (e.g. in ruminant) microbial protein synthesis (e.g. in ruminant)

73 The ideal marker Inert, no toxic on animal & micro flora Inert, no toxic on animal & micro flora not be absorbed or metabolized in GI not be absorbed or metabolized in GI mixed well/ associated with feed mixed well/ associated with feed should not influence GI secretion digestion absorption or motility should not influence GI secretion digestion absorption or motility precise - quantitative analysis / not interfere with other analysis precise - quantitative analysis / not interfere with other analysis

74 Two type of marker use in nutrition studies Internal marker Internal marker component of feedstuffs component of feedstuffs e.g. lignin, AIA, indigestible ADF, NDF e.g. lignin, AIA, indigestible ADF, NDF External marker External marker indigestible substances added to a feedstuff indigestible substances added to a feedstuff e.g. Cr 2 O 3 (chromium sesquioxide) e.g. Cr 2 O 3 (chromium sesquioxide) Cerium Cerium Dysprosium Dysprosium ytterbium ytterbium ruthenium phenanthroline complex ruthenium phenanthroline complex binding marker with a specific feed. E.g. Yb labeled feed 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 maker to determine amount of microbial protein synthesis Total protein passing to the lower tract in ruminant Total protein passing to the lower tract in ruminant determine portion of total protein : microbial origin determine portion of total protein : microbial origin

76 Marker specific to bacteria Diaminopimelic acid (DAPA) Diaminopimelic acid (DAPA) found only in bacteria found only in bacteria Determine DAP content in digesta and DAP:N ratio in bacteria Determine DAP content in digesta and DAP:N ratio in bacteria therefore, estimate portion of nitrogen in digesta from microbial origin therefore, estimate portion of nitrogen in digesta from microbial origin Ribonucleic acid (RNA) Ribonucleic acid (RNA) assumed that feed RNA 100% digest in rumen assumed that feed RNA 100% digest in rumen thus, only bacterial RNA passes to lower tract thus, only bacterial RNA passes to lower tract determine RNA: N ratio in bacteria and % digesta RNA, then --> microbial protein synthesis calculated. determine RNA: N ratio in bacteria and % digesta RNA, then --> microbial protein synthesis calculated. Purine (Adenine, Guanine) and purine derivatives (urine,plasma)? Purine (Adenine, Guanine) and purine derivatives (urine,plasma)?

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

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

79 Use of marker to measure feed Intake Feed intake effect on economy & livestock production Feed intake effect on economy & livestock production Difficult to measure in take in grazing ruminant Difficult to measure in take in grazing ruminant Indicator methods to estimate intake Indicator methods to estimate intake Information: fecal excretion and digestibility Information: fecal excretion and digestibility

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

81 How to estimate fecal output? Fecal collection bags Fecal collection bags Total collection of feces Total collection of feces Indigestible marker e.g. chromic oxide Indigestible marker e.g. chromic oxide Fecal output,g = indicator consumed (g/day)/Indicator concentration in feces(g/g DM) 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 Particulate & fluid materials Fluid flow rate or fluid dilution rate(% of fluid volume leaving the rumen per hour) Fluid flow rate or fluid dilution rate(% of fluid volume leaving the rumen per hour) Faster dilution rate ==> more efficient microbial growth Faster dilution rate ==> more efficient microbial growth Increase rate of passage ==> increased voluntary feed intake 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 Marker for measure rate of fluid passage from the rumen Chromium EDTA (Cr-EDTA) Chromium EDTA (Cr-EDTA) Cobalt EDTA (Co-EDTA) Cobalt EDTA (Co-EDTA) Polyethylene glycol (PEG) Polyethylene glycol (PEG) Measurement the rate of fluid passage Measurement the rate of fluid passage Flow rate at a sampling site 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 Single dose vs.. continuous dose Calculate flow rate or volume of particulate phase Calculate flow rate or volume of particulate phase Common particulate phase marker for estimate turnover rate Common particulate phase marker for estimate turnover rate Chromic oxide Chromic oxide Ytterbium Ytterbium Dysprosium Dysprosium Cerium Cerium Ruthenium phenanthroline complex Ruthenium phenanthroline complex Internal marker e.g. indigestible ADF,NDF and AIA 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 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 Marker Marker Diaminopimelic acid Diaminopimelic acid RNA RNA Purines Purines

87 Good Luck!!


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