Download Nitrogen lectures (Part 2)

ANIMAL AGRICULTURE’S CONTRIBUTION TO
N LOADING OF THE ENVIRONMENT
• Gaseous emissions
Total agriculture
Animal agriculture
% of emissions in the US
NH3
N 2O
NO
80
50
6
47-73
25
1
• Contribution of different species to atmospheric
ammonia
• Contributions to total N in watersheds
– Distribution of total manure N in relation to ability to apply at
agronomic rates in US
% of farms w/ % of manure N % of counties w/ % of manure N
adequate land produced on
adequate land
produce in
land to apply farms with
land to apply
counties with
produced N
adequate land
produced N
adequate land
at agronomic to apply manure at agronomic
to apply manure
rates
N produced
rates
N produced____
78
40
95
80
– Distribution on excess manure N by species in the US
% of manure N
produced in excess
of land available
% of
% of manure N
for application at
Species
AFOs
excreted
agronomic rates
Beef
9
48
18
Dairy
49
22
7
Swine
27
9
15
Poultry
15
20
60
ROLE OF PROTEIN NUTRITION IN N
MANAGEMENT OF LIVESTOCK
• Proteins are the basic unit of life
• Average composition of protein
Carbon
Hydrogen
Oxygen
Nitrogen
Possibly sulfur and phosphorus
%
53
7
23
16
1
PROTEIN STRUCTURE
• Primary structure
– Chains of amino acids linked by a peptide linkage
– Amino acids are organic acids having an amino group on
the alpha-carbon
O
H2N
C
OH
C
H
R
– The side chain ( R) is different for each amino acid and
determines the properties of the amino acid and protein
– There are 200 amino acids of which 22 are commonly found
in proteins in varying amounts
AMINO ACIDS FOUND IN PROTEINS
• Neutral amino acids (No
special group)
–
–
–
–
–
–
–
Glycine
Alanine
Serine
Valine
Leucine
Isoleucine
Threonine
• Acidic amino acids (Have an
extra COOH group)
– Aspartic acid
– Asparagine
– Glutamic acid
• Basic amino acid (Have an
extra NH2)
–
–
–
–
Lysine
Arginine
Histidine
Glutamine
• Sulfur-containing amino
acids (Contain S)
– Methionine
– Cysteine
– Cystine
• Aromatic amino acids
(Contain a benzene group)
– Phenylalanine
– Tysosine
– Tryptophan
• Imino acids (Heterocyclic
amino acids)
– Proline
– Hydroxyproline
– Peptide linkage is the bond that hold amino acids together
in proteins
H
CH2
N H
O
+
HO
COOH
H O
C
CH2
CH2
NH2
N C
COOH
CH2
NH2
• Most proteins will have chains of 350 to 5000 amino acids
• Amino acid chains with less than 100 amino acids are called
peptides
• Secondary structure
– Hydrogen bonding that occurs with a chain producing
helixes or sheets
• Tertiary structure
– Folding and twisting of protein chains held together by
disulfide linkages between and within chains
• Quaternary structure
– Aggregates of two or more amino acid chains
IMPORTANCE OF PROTEIN STRUCTURE
• Determines biological activity
• Determines digestibility
LOSS OF PROTEIN STRUCTURE
• Denaturation
– Loss of secondary, tertiary, and quaternary structure
– Caused by heat, UV light, alkali or organic solvents
– Effects
• Loss of biological activity
• May increase digestibility
• Browning or Maillard reaction
– Results from overheating of feeds
– Causes
• Over-cooking of feeds
• Over-drying of grains
• Over-heating of feeds during storage
– Mechanism
Protein + Carbohydrate
Indigestible protein-carbohydrate
complex (Acid detergent insoluble N;
ADIN)
PROTEIN ANALYSIS
• In applied nutrition, protein content of feeds is normally
determined as crude protein
• Crude protein
– Calculated from the N content of the feed determined by the
Kjeldahl analysis
Digested in H2SO4
Sample
Distill into boric acid
(NH4)2SO4
(NH4)borate
Titrate with acid
– CP% = N% x 6.25
• The factor 6.25 comes from the fact that most proteins contain 16%
N or 16 gm N/100 gm protein
– 100 gm protein/16 gm N = 6.25
• Limitations of CP determination
– Nitrogen in feeds may come from true protein on nonprotein
nitrogen sources
• True protein
– Example
» Chains of amino acids
– Only source of protein that can be used by nonruminant
(monogastric) animals
• Nonprotein nitrogen (NPN)
– Examples
» Free amino acids
» Nucleic acids
» Ammonia
» Urea
» Biuret
– NPN may be utilized to meet the protein needs of ruminant
animals
– Nonruminants can not utilize NPN
– Crude protein says nothing about the amino acid composition
of a feed
– Crude protein says nothing about the digestibility of the protein
• Individual amino acids can be analyzed by
chromatographic procedures
– Expensive
– Slow
• Near infrared reflectance spectroscopy may have the
potential for real-time amino acid analysis
– Procedures still need to be developed
PROTEIN DIGESTION IN NONRUMINANTS
• Digestion
Proteins
•
In stomach
– HCl secreted by gastric glands
• Denatures protein
• Converts pepsinogen into pepsin
– Pepsinogen secreted
• Converted into pepsin by HCl
• Pepsin is an endopeptidase that preferentially degrades
peptide linkages next to aromatic amino acids
Peptides
Peptides
Amino acids
•
In small intestine
– Pancreatic proteases
Proenzyme Active enzyme Activated by Bonds hydrolyzed
Trypsinogen Trypsin
Enterokinase Bonds next to
lysine and arginine
ChymoChymotrypsin Trypsin
Bonds next to
trypsinogen
phenyalanine,
tyrosine, leucine,
histidine, methionine
and tryptophan
Procarboxy Carboxypeptidase Trypsin Amino acids w/
peptidase
free carboxyl group
– Small intestinal peptidases
– Digestion is normally high, but variable
Corn
Soybean meal
Wheat
Wheat bran
Meat and bone meal
Poultry byproduct meal
Protein digestion, %
(swine)
85
84-87
89
75
84
77
• Amino acid absorption
– Most protein is absorbed as amino acids, but small amounts of
peptides are absorbed
– Amino acid absorption occurs by active transport
• Requires energy and a carriers on the intestinal membranes
• Carrier systems
– Neutral amino acids (Leucine, Isoleucine, Valine)
– Neutral amino acids (Glycine, Alanine, Serine)
– Basic amino acids (Lysine, Arginine)
– Imino acids (Proline, Hydroxyproline)
• Amino acids compete for carrier systems
– Excess leucine inhibits absorption of valine and isoleucine
absorption
– Excess lysine inhibits arginine absorption
– Excess methionine inhibits lysine absorption
• Requires vitamin B6
– Amino acid can be transported in blood plasma without a
carrier
THE RUMINANT STOMACH
PROTEIN DIGESTION IN RUMINANTS
• Rumen
Total protein
NPN
Undegraded
Small intestine
Metabolizable
protein
Degraded
Recycled via
saliva
(20% of dietary N)
NH3
Microbial
protein
NH3
Liver
Urea
Kidney
Excreted
• Ruminal degradation of true protein
– By ruminal bacteria and protozoa
– Not totally desirable
• There is always some loss of NH3
– Reduces efficiency
– Increases N excretion
• Valuable to have protein escape ruminal degradation in animals
with high protein requirements
– Factors affecting ruminal protein degradation
• Protein source
% degraded in 24 hours
Fish meal
51
Corn
50
Cottonseed meal
78
Soybean meal
89
Alfalfa
90
• Increasing rate of passage decreases ruminal protein degradation
• Heat treatments
100 C for 4 hours
Soybean meal
Reduced protein degradation
• Tannins in feeds reduce protein degradation
– Example: Birdsfoot trefoil
• Coating proteins with materials resistant to degradation
• Factors affecting microbial protein production in the
rumen
– Ruminal NH3-N concentration
Microbial
protein
(% of Max)
Ruminal NH3-N
5 mg%
12%
Crude protein in diet, %
– Rate of ammonia release
Urea
[NH3]
Treshold
Biuret
2
Time after feeding, hours
– Energy level of the diet
• Energy and C-skeletons needed by rumen bacteria to produce
microbial protein from ruminal NH3
– Sulfur level of diet
• Sulfur needed to synthesize S-containing amino acids
• Particularly important when NPN fed
• Required N:S ratio
– 10:1
• Advantage of ruminant CP digestion
– Ability to utilize NPN
• Limitations of ruminant CP digestion
– Loss of ruminal NH3
• Inefficient
• Increases environmental N loading
– Urea toxicity
• Occurs when blood NH3>60 mg%
• Causes
– Feeding excess urea (> 1% of ration dry matter)
– Inadequate energy fed with urea
– Poor mixing of urea in diet
• High blood NH3 toxic to brain cells
• Protein digestion in the abomasum
– The abomasum is the fourth compartment of the ruminant
stomach
– Functionally it is similar to the stomach on nonruminant animals
– Abomasum secretes hydrochloric acid and pepsinogen
• Hydrochloric acid converts pepsinogen into the active protease,
pepsin
• Peptides hydrolyzes ruminal undegraded protein and microbial
protein into peptides
• Small intestinal protein digestion in ruminants
– Pancreas secretes proenzymes of proteases
•
•
•
•
Trypsinogen converted to the protease, Trypsin
Chymotrypsinogen converted to the protease, Chymotrypsin
Procarboxypeptidase converted to the protease, Carboxypeptidase
Pancreatic proteases degrade proteins to peptides and amino acids
– Intestinal mucosa secretes peptidases that degrade peptides to
amino acids
• Digestibility of ruminal undegraded protein and
microbial protein normally high
– 80%
TRANSPORT OF PLASMA AMINO ACIDS INTO
CELLS
• Amino acid transport into cells by active transport
• Transport enhanced by certain hormones
Tissue
Liver
Uterus
Muscle
Hormones
Epinephrine
Glucocorticoids
Estradiol
Growth hormone
Insulin
Testosterone
• Amino acid pool in cells for metabolism comes from
dietary amino acids and protein degradation in the
animal
AMINO ACID METABOLISM
• Protein synthesis
– Mechanism
• Protein synthesis controlled by DNA in the nucleus of cells
• DNA composed of chains on nucleotides composed of:
– Deoxyribose
– Phosphoric acid
– 1 of 4 purine or pyrimidine bases:
» Adenine
» Cytosine
» Guanine
» Thymine
• Three nucleotides represent the codon for one amino acid in a protein
chain
• Messenger RNA is produced from DNA
– If DNA has
mRNA will have
Adenine
Uracil
Cytosine
Guanine
Guanine
Cytosine
Thymine
Adenine
• Messenger RNA migrates to ribosomes where it acts as the
template for protein
• To be used in protein synthesis, amino acids are bound to
transfer RNA
• Transfer RNA travels along the messenger RNA to place amino
acid in chain
• If an given amino acid is not present, synthesis of this protein
stop and no more amino acids will be used
– Hormonal control
Growth hormone
Thyroxine
Amino acid
Increase IGF (Liver)
Growth hormone
Testosterone
Insulin
Transport
Amino acid
Muscle
DNA synthesis
x
Synthesis
Increase IGF (Muscle)
Protein
Estrogen
Degradation
Testosterone
• Transamination
– Transfer of an amino group from one amino acid to another
carbon chain (called a keto acid) to construct a new amino acid
• Alpha amino acid1 + keto acid2
– Importance
keto acid1 + alpha amino acid2
• A method of synthesizing amino acids from intermediates of
carbohydrate metabolism
Carbohydrate metabolism intermediate
Amino acid
Pyruvate
Alanine
Oxalacetate
Aspartic acid
Alpha-ketoglutarate
Glutamic acid
3-phosphoglycerate
Serine
– Animals can synthesize
Glycine
Alanine
Serine
Aspartic acid
Asparagine
Glutamic acid
Glutamine
Cysteine
Cystine
Tyrosine
Proline
Hydroxyproline
– Although these amino acids are needed metabolically, they
aren’t needed in the diet; called Nonessential amino acids
– Amino acids that can’t be synthesized in adequate
quantities are called ‘essential amino acids’
– Essential amino acids
Phenylalanine
Valine
Tryptophan
Threonine
Isoleucine
Methionine
Histidine
Arginine
Leucine
Lysine
• Decarboxylation
– Removal of the carboxyl producing amino
NH2
R
C
NH2
COOH
H
– Examples
Amino acid
Histidine
Tyrosine
Tryptophan
R
CH2
CO2
Amine
Histamine
Tyramine
Hydroxytryptamine
Physiological effects
Inflammation,
dilates capillaries,
depresses
blood pressure
Constricts capillaries,
Increased blood pressure
Constricts capillaries,
Increased blood pressure
• Deamination
– Releases amino group from excess amino acids
– Mechanism
NH2
R
C
O
COOH + O
R C COOH + NH3
(C skeleton)
H
– Uses of C skeleton
• Energy metabolism
• Glucose synthesis
• New amino acids
– Removal of NH3
O
• Mammals
– Synthesis of urea
– Detoxifies NH3
H2N
C
NH2
• Poultry
– Synthesis of uric acid; excreted with feces
O
H
N
C
H
N
C
C
O
C
C
N
H
N
H
O
THE PROTEIN REQUIREMENT
• Nonruminants
– Not a requirement for protein per se, but really a
requirement for the essential amino acids
– Essential amino acids in the diet
• For growth of pigs
–
–
–
–
–
–
–
–
–
–
Phenylalanine
Valine
Tryptophan
Threonine
Isoleucine
Methionine
Histidine
Arginine
Lysine
Leucine
• Additional amino acids for poultry
– Arginine
– Glycine
• Cystine can replace ½ of the methionine
• Tyrosine can replace 1/3 of the phenyalanine
– Balance of amino acids in a diet is as important as the
amounts of individual amino acids
• Amino acids can only be used to the extent of the least abundant
amino acid relative to the animal’s requirement
– Remainder of amino acids will be deaminated and N will be
excreted
• An excess of one amino acid may cause a deficiency of another
amino acid
Excess leucine
Deficiencies of valine and isoleucine
• The term “protein quality” refers to the amino acid balance of a
protein relative to an animal’s requirement for each of the
essential amino acids
– A “high quality protein” called an “ideal protein” has the
essential amino acids present in proportions equal to an
animal’s requirements.
» It says nothing about the concentration of protein in the
diet
– A ration with a “high quality protein” may be composed from
two or more feeds if they complement each other’s
deficiencies
20 kg pig
Corn
Soybean meal
Corn/soybean meal mix
Amino acid requirements of pigs (% of protein)
Leucine Lysine S-containing AAs Tryptophan
3.8
4.4
2.8
.7
12.5
2.3
3.0
1.1
7.4
6.3
2.6
1.3
11.5
4.4
2.7
1.2
– An “ideal” protein can be synthesized by adding individual
amino acids to a diet
• Measures of protein quality
– Chemical score
» The chemical score is the percentage of the requirement of the
most limiting amino acid
» Example
Amino acid, Requirement (% of protein) Chemical
A
B
C
D
E score
Reqt.
4
10
8
12
5
Protein 1
3
12
3
8
4
% of AA reqt. 75
120
37.5
67
80
37.5
Protein 2
4
14
6
0
6
% of AA reqt. 100
140
75
0
120
0
– Biological value
» % of digested and absorbed N that is retained by the body
» Range in value
High > 70 (means good amino acid balance)
Low < 70 (means poor amino acid balance)
» Feeds
BV
Egg albumen
97
Milk
85
Beef
76
Cereal proteins
50-65
Gelatin
12
» A ration with a dietary protein with a high BV can be made from
feeds with low BV if their amino acid compositions complement
each other (or if individual amino acids)
• Ruminant protein requirements
– Ruminants have no essential amino acid requirements in their
diets
• The rumen microbes can synthesize all of the amino acids
– Ruminants require
• Degradable N up to 12% crude protein in the diet dry matter
– To meet the N needs of the rumen bacteria
• Undegraded protein above 12% crude protein
FACTORS AFFECTING PROTEIN REQUIREMENTS
• Growth
– Young, growing animals deposit more protein, but have lower
feed intakes than larger animals
Swine, kg
1-5
5-10
10-20
20-35
35-60
CP reqt. %
27
20
18
16
14
• Sex
– Males deposit more protein at a given weight than females
300 kg large frame gaining 1 kg/d
Bulls
Steers
Heifers
gm protein/day
807
804
735
• Production of milk, eggs, or wool
• Balance of protein and energy
– As energy increases, feed intake decreases
• Should increase the percentage of protein in the diet
• Environmental temperature
– As temperature increases, feed intake decreases
• Should increase the percentage of protein in the diet
PROTEIN DEFICIENCY
•
•
•
•
•
•
•
•
Reduce feed intake
Reduced growth rate
Reduced feed efficiency
Fat accumulation in liver
Reduced fertility
Reduced birthweight
Reduced production of milk or effects
Reduced fiber digestion (in ruminants)
PROTEIN EXCESS
•
•
•
•
Reduced feed intake
Reduced growth rate
Reduced fertility in cattle
Excess N excretion