Protein Digestion and Absorption

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Protein Digestion and Absorption

• Dietary proteins, with few exceptions, are not
  absorbed.

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Protein Digestion and Absorption

• Dietary proteins, with few exceptions, are not
  absorbed.
• They must be digested first into amino acids or
  di- and tri-peptides.

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Protein Digestion and Absorption

• Dietary proteins, with few exceptions, are not
  absorbed.
• They must be digested first into amino acids or
  di- and tri-peptides.
• Through the action of gastric and pancreatic
  proteases, proteins are digested within the lumen
  into medium and small peptides
• (oligopeptides).

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Digestion of protein - hydrolysis
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Protein digestion begins in stomach Pepsin -
inactive precursor pepsinogen Active _at_ pH 2-3,
inactive pHgt5 Secretion stimulated by
acetylcholine or acid Only protease which can
break down collagen Action terminated by
neutralisation by bicarbonate in duodenum. N.B.
All proteases (stomach pancreatic) secreted
as inactive precursors. Most protein digestion
occurs in the duodenum/jejunum
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Activation of pancreatic proteases
Active proteases inactivated by trypsin
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peptidases aminopolypeptidase
Amino acids
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Pancreatic enzymes
Essential for digestion ? essential for life
Acinar cells
Lipases Amylases ? Active enzymes
Proteases ? Inactive form ? Activated in gut
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Pancreatic Enzymes

• The bulk of protein
• digestion occurs within
• the intestine due to the action of pancreatic
• proteases.

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Pancreatic Proteases

• The two primary pancreatic proteases are trypsin
  and chymotrypsin.

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Pancreatic Proteases

• The two primary pancreatic proteases are trypsin
  and chymotrypsin.
• They are synthesized and packaged within
  secretory vesicles as inactive proenzymes
• trypsinogen chymotrypsin

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Pancreatic Proteases

• The two primary pancreatic proteases are trypsin
  and chymotrypsin.
• They are synthesized and packaged within
  secretory vesicles as inactive proenzymes
• trypsinogen chymotrypsin
• The secretory vesicles also contain a trypsin
  inhibitor to serve as a safeguard against
  trypsinogen converted to trypsin.

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Other Pancreatic Proteases

• Procarboxypeptidase ? carboxypeptidase
• Proelastase ? elastase

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Trypsin

• Trypsinogen is converted to trypsin by the enzyme
  enterokinase (enteropeptidase) secreted by cells
  lining duodenum.

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Trypsin

• Trypsinogen is converted to trypsin by the enzyme
  enterokinase (enteropeptidase) secreted by cells
  lining duodenum.
• Trypsin then activates the conversion of other
  zymogens from their inactive to active forms.

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Trypsin

• Trypsinogen is converted to trypsin by the enzyme
  enterokinase (enteropeptidase) secreted by cells
  lining duodenum.
• Trypsin then activates the conversion of other
  zymogens from their inactive to active forms.
• Inhibition of trypsin will slow activation of
  other proteases.

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Trypsin contd

• Trypsin catalyzes the splitting of peptide bonds
  on the carboxyl side of lysine and arginine
  residues.
• It has a pH optimum of 7.6 to 8.0 (alkaline).
• Classified as a serine protease (serine and
  histidine at the active site.

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Trypsin, Chymotrypsin

• Similar chemical compositions
• Chief differences are specificity of action
• trypsin lysine, arginine
• chymotrypsin tyrosine, phenylalanine,
  tryptophan, methionine,leucine
• (aromatic or large hydrophobic side chains)

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Lock and Key Model of Enzyme Activity
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Visualization of the Lock and Key Model of Enzyme
Function
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Lock Key Enzyme Catalysis

• The Active Site contains
• A shape that fits a specific substrate(s)
• Side chains that attract (chemically) the
  substrate
• Side chains that are positioned to speed the
  reaction

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Enzyme Catalysis

• -OH of serine 195 attacks CO of peptide bond.
  Histidine 57 donates a proton to the N of the
  peptide bond leading to cleavage and acylation of
  the enzyme. Asp-102 is also involved.

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Carboxypeptidase COO- terminal peptide bond

• Hydrolysis occurs most readily if the COO-
  terminal residue has an aromatic or bulky
  aliphatic side chain.
• Binding of a typical substrate results in a
  rearrangement of the active site (induce fit).
  Glutamate-270, Arginine-145, Arginine-127,
  Tyrosine-248 are important at the active site.

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Carboxypeptidase

• H H O H O
• I I II I II
• N - C - C - N - C - C
• I I I I
• R H CH2 O
• I
• AROMATIC
• SIDE CHAIN

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Induced Fit Model of Enzyme Function

• Zinc near the active site
• Arg 145, Tyr 248, and Glu 270 near the active site

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Induced Fit Model of Enzyme Function

• The active site is in the induced conformation

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Trypsin Inhibitors

• Trypsin (protease) inhibitors are found naturally
  in many seeds, particularly legumes such as soy,
  peas, other beans.
• heat labile, heat stable
• Osborne and Mendel (1917) soybeans need to be
  heated to support growth in rats

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Trypsin Inhibitors

• Kunitz inhibitor, Bowman-Birk inhibitor
• Both are inactivated during moist heat treatment.
  
• Protease inhibitors are proteins which bind to
  the enzyme, rendering them inactive.
• Symptoms include pancreatic hypertrophy due to
  stimulated secretory activity.

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Absorption of peptides and amino acids
Transport at the brush border 1. Active transport
by carrier. 2. Mostly dependent on Na gradient -
co-transport similar to that for glucose 3. Some
amino acids (basic, and neutral with hydrophobic
side chains) are absorbed by facilitated diffusion
Protein assimilation affected by - Pancreatitis,
congenital protease deficiencies, deficiencies of
specific transporters
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Absorption of Amino Acids

• The transporters bind amino acids only after
  binding sodium.
• The fully loaded transporter undergoes a
  conformational change that dumps Na and the
  amino acid into the cytoplasm. The transporter
  then reorients back to its original form.

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Absorption of Amino Acids

• Absorption of amino acids is dependent on the
  electrochemical gradient of Na across the
  epithelium.
• The basolateral membrane of the enterocyte
  contains additional transporters which export
  amino acids from the cell into the blood (not
  dependent on sodium gradients).

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Absorption of Peptides

• There is virtually no absorption of peptides
  longer than three amino acids but there is
  abundant absorption of di- and tri-peptides,
  probably by a single transport molecule.
• The vast bulk of di- and tri-peptides are
  digested into amino acids by cytoplasmic
  peptidases.

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Absorption of Intact Proteins

• Absorption of intact proteins occurs rarely.
• Very few proteins can get through the gauntlet of
  soluble (lumen) and membrane-bound proteases
  intact.
• Normal enterocytes do not have the transporters
  neededt to carry proteins across the plasma
  membrane and they cant permeate tight junctions.

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Absorption of Intact Proteins

• Shortly after birth, neonates can absorb intact
  proteins.

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Absorption of Intact Proteins

• Shortly after birth, neonates can absorb intact
  proteins.
• Most of these intact proteins are immunoglobulins
  which can be absorbed from the very first milk
  (colostrum) and this imparts early neonatal
  passive immunity.

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Absorption of Intact Proteins

• Shortly after birth, neonates can absorb intact
  proteins.
• Most of these intact proteins are immunoglobulins
  which can be absorbed from the very first milk
  (colostrum) and this imparts early neonatal
  passive immunity.
• Closure is when the small intestine loses the
  capacity to absorb intact proteins.

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Protein Requirements

• Maintenance nutritional requirements to stay
  alive (does not require positive BW gain)

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Protein Requirements

• Maintenance nutritional requirements to stay
  alive (does not require positive BW gain)
• Growth positive tissue accretion

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Protein Requirements

• Maintenance nutritional requirements to stay
  alive (does not require positive BW gain)
• Growth positive tissue accretion
• Reproduction tissue specific growth related to
  reproduction, reproductive function (milk, eggs,
  reproductive tissue)

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How do you express a protein requirement ?

• Protein percent of the diet

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How do you express a protein requirement ?

• Protein percent of the diet
• Amino acid percent of the diet

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Growth Will Dictate Feed Intake
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Intake Will Dictate Actual Requirement
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How do you express a protein requirement ?

• Protein percent of the diet
• Amino acid percent of the diet
• Amino acid percent of total protein

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How do you express a protein requirement ?

• Protein percent of the diet
• Amino acid percent of the diet
• Amino acid percent of total protein
• Digestible protein percent of the diet

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Digestible Protein Estimates

• Digestible protein intake output
• (amino acids)

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Digestible Protein Estimates

• Digestible protein intake output
• (amino acids)
• Diet formulated to .90 TSAA
• (methionine cystine)

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Digestible Protein Estimates

• Digestible protein intake output
• (amino acids)
• Diet formulated to .90 TSAA
• (methionine cystine)
• Feathermeal 70 digestible methionine
• Fishmeal 90 digestible methionine

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Digestible Protein Estimates

• Digestible protein intake output
• (amino acids)
• Intestinal microbes will modify amino acid
  composition of digesta

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Digestible Protein Estimates

• Digestible protein intake output
• (amino acids)
• Intestinal microbes will modify amino acid
  composition of digesta
• Excreta will reflect microbial as well as de
  novo dietary amino acid availability

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Digestible Amino Acid Estimates

• Ceacectomized roosters precision feeding
• - total excreta collection, amino acid
  determination

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Digestible Amino Acid Estimates

• Ceacectomized roosters precision feeding,
  Collect all excreta,amino acid determination
• Ileal Digesta collect digesta from terminal
  small intestine, non-digestible dietary marker
  (cannula for pigs, terminal collection for
  poultry)

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Digestible Amino Acid Determination

• Non-digestible marker in the feed
• acid insoluble ash (Celite), chromic oxide
• marker (celite) 1.5 in feed
• digesta (celite) 4.0

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Digestible Amino Acid Determination

• Digestibility
• AA / AIA (feed) AA/AIA (digesta)
• ______________________________
• AA / AIA (feed)

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Digestible Amino Acid Determination

• Digestibility
• AIA 1.5 feed, 4.0 digesta
• methionine .50 feed, .25 digesta
• Av. Meth 0.5/1.5 - .25/4.0
• 0.5/1.5

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Digestible Amino Acid Determination

• Digestibility
• AIA 1.5 feed, 4.0 digesta
• methionine .50 feed, .25 digesta
• Av. Meth 0.5/1.5 - .25/4.0 .33 - .0625
• 0.5/1.5
  .33

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Digestible Amino Acid Determination

• Digestibility
• AIA 1.5 feed, 4.0 digesta
• methionine .50 feed, .25 digesta
• Av. Meth 0.5/1.5 - .25/4.0 .33 - .0625
• 0.5/1.5
  .33
• 81 Digestible
  Methionine

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How do you express a protein requirement ?

• Protein percent of the diet
• Amino acid percent of the diet
• Amino acid percent of total protein
• Digestible protein percent of the diet
• Ideal Protein ratios (relationships among amino
  acids)

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How do you express a protein requirement ?

• Protein percent of the diet
• Amino acid percent of the diet
• Amino acid percent of total protein
• Digestible protein percent of the diet
• Ideal Protein ratios (relationships among amino
  acids)
• Protein or amino acid intake/day (gms)

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Economics of Protein Nutrition

• Animals have requirements for amino acids, not
  intact crude protein. Formulating for total
  amino acid needs using intact protein sources is
  prohibitively expensive.

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Economics of Protein Nutrition

• Animals have requirements for amino acids, not
  intact crude protein. Formulating for total
  amino acid needs using intact protein sources is
  prohibitively expensive.
• Commercially available amino acids (methionine,
  lysine, threonine) allow for lower protein diets
  with similar amino acid specifications.

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Caloric cost of protein deposition

• There is a genetic limit to protein accretion.

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Caloric cost of protein deposition

• There is a genetic limit to protein accretion.
• The goal is to maximize muscle accretion without
  feeding protein as an energy source.

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Caloric cost of protein deposition

• There is a genetic limit to protein accretion.
• The goal is to maximize muscle accretion without
  feeding protein as an energy source.
• Carbohydrate C,H,O (4 calories)
• Fat C,H (9 calories)
• Protein C,H,N (4 calories)

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Caloric cost of protein deposition

• Balancing diets, research and real world
• For a given set of growing conditions, there is
  an energetic mix of caloric sources that is
  optimum

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Caloric cost of protein deposition

• For a given number of calories consumed
• Converting protein to energy is energetically
  inefficient and results in metabolic heat
  production
• (environmental considerations)

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Caloric cost of protein deposition

• For a given number of calories consumed
• Excess calories relative to the animals genetic
  capacity to synthesize protein will increase
  carcass fat deposition (CP ratio)

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Caloric cost of protein deposition

• Concept of caloric density
• What is the proportion of total calories coming
  from protein, fat, carbohydrate.
• Isocaloric diets - high fat, low fat

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Amino Acid Balance

• How would an optimum balance of amino acids be
  defined ?

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Amino Acid Balance

• How would an optimum balance of amino acids be
  defined.
• This question is outcome dependent.

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Amino Acid Balance

• How would an amino acid optimum be defined ?
• Order of limitation influencing growth.
• Diets selected amino acids.

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Amino Acid Balance

• How would an amino acid optimum be defined ?
• Order of limitation influencing growth.
• Composition of carcass protein depots.

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Amino Acid Balance

• Minimizing ammonia production (N excretion)

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Amino Acid Balance

• Minimizing ammonia production (N excretion).
• Minimizing total manure production
• (15 ? 17 CP diets, 4?6 SBM, increased excreta
  production 12-15)

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Dietary Protein/Amino Acid Balance

• Minimizing ammonia production (N excretion).
• Minimizing total manure production
• (15 ? 17 CP diets, 4?6 SBM, increased excreta
  production 12-15)
• Microbial management within the gut in the new
  world of no antibiotics

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Protein Quality Evaluation

• The capacity of an intact protein source to
  support growth

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Protein Quality Evaluation

• The capacity of an intact protein source to
  promote body weight gain
• Protein Efficiency Ratio
• Body weight response to protein intake

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Protein Quality Evaluation

• Protein Efficiency Ratio
• Growth response to a single source of dietary
  protein over a range of protein intakes.

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Protein Quality Evaluation

• Protein Efficiency Ratio
• Growth response to a single source of dietary
  protein over a range of protein intakes.
• The response needs to be within the deficiency
  range of the target species.

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Protein Quality Evaluation

• Protein Efficiency Ratio
• Body weight response to a single source of
  dietary protein over a range of protein intakes.
• PER Body Weight Gain (g)
• Protein Intake (g)

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Protein Efficiency Ratio
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Protein Efficiency Ratio

• A comparison of slopes (multiple CP levels)
• 12 CP 100 gm intake, 36 gm gain
• 36 g gain / 12 g protein
  intake

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Protein Efficiency Ratio

• A comparison of slopes (multiple CP levels)
• 12 CP 100 gm intake, 36 gm gain
• 36 g gain / 12 g protein
  intake
• 15 CP 100 gm intake, 45 gm gain
• 45 g gain / 15 g protein intake

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Protein Efficiency Ratio

• A comparison of slopes (multiple CP levels)
• 12 CP 100 gm intake, 36 gm gain
• 36 g gain / 12 g intake
• 15 CP 100 gm intake, 45 gm gain
• 45 g gain / 15 g intake
• 18 CP 100 gm intake, 54 g gain
  54 g gain / 18 g protein intake
• PER 3.0

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Comparison of Protein Sources
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Net Protein Ratio

• The PER does not allow for any consideration of
  the maintenance protein requirement.

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Net Protein Ratio

• The PER does not allow for any consideration of
  the maintenance protein requirement.
• The maintenance protein requirement for body
  weight gain is represented by BW loss from a test
  group fed a protein free diet.

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Protein Efficiency Ratio

• A comparison of slopes (multiple CP levels)
• 12 CP 100 gm intake, 36 gm gain
• 36 g gain / 12 g protein
  intake
• BW loss by 0 CP treatment 6 gms

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Net Protein Ratio/Net Protein Utilization

• A comparison of slopes (multiple CP levels)
• 12 CP 100 gm intake, 36 gm gain
• 36 g gain / 12 g protein
  intake
• BW loss by 0 CP treatment 6 gms
• 36 gm gain (6 gm BW loss)/ 12 g protein
• NPR 42 / 12 3.5
• NPU carcass protein gain / protein consumed

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Commercial Application of PER

• Some companies will use the PER assay in quality
  control assays for incoming ingredients.

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Commercial Application of PER

• Some companies will use the PER assay in quality
  control assays for incoming ingredients.
• These assays will often incorporate chicks and
  utilize one level of CP, usually 6 or 9.
• IAMS uses this assay to monitor their incoming
  poultry byproduct meal.