Animal Nitrogen

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Animal Nitrogen Overview of N cycling farm
animals a few unfortunate songbirds road-kill
down under
Nitrogen Isotopes in Mammalian Herbivores Hair
?15N Values from a Controlled Feeding
Study (Sponheimer et al., 2003) Effects of
elemental composition on the incorporation of
dietary nitrogen and carbon isotopic signatures
in an omnivorous songbird (Pearson et al.,
2003) Kangaroo metabolism does not cause the
relationship between bone collagen d15N and water
availability (Murphy et al., 2006)
2
N Cycle(human)

• protein turnover
• Some proteins turnover faster than others
• some tagged (oxidized or other means)

• amino acid pool
• throughout body
• significant mixing

3
N Cycle(human)

• Dietary protein
• Low
• deficiency of essential aas

• High
• conversion to fat/glucose
• Ammonia/urea excretion

4
Deamination
First transfer amine group to carrier Ketoglutara
te ? Glutamate
in liver or kidney
Then deaminate Glutamate to produce ammonia
5
Synthesis
First transfer amine group to carrier Ketoglutara
te ? Glutamate
Then to amino acid
in liver or kidney
6
Deamination
Synthesis
Direction of these reactions controlled by
of Glutamate Ketoglutarate Ammonia ratio of
oxidized to reduced enzymes
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Urea cycle
Urea cycle controlled by acetyl CoA and
glutamate increase in after protein rich meal
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Nitrogen excretion animals

• Ammonia NH3
• Simplest form, but toxic
• fully aquatic animals
• Urea (NH2)2CO
• Still toxic more complex than ammonia
• mammals some herps (frogs), cartilaginous fish
• Uric Acid C5H4N4O3
• Least toxic
• egg layers (bird, reptiles, insects)
• precipitates from egg

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A few things

• animals assimilate dietary components with
  varying efficiencies
• animal tissues fractionate the isotopes in their
  diet
• animals allocate nutrients in their diet
  differentially to specific tissues isotopic
  routing
• animals retain d15N, excreting d14N (6)
• protein balance is a key to fractionation
• low dietary protein protein sparing reserve
  dietary protein for tissue maintenance rather
  than catabolizing it for energy (Castellini and
  Rea 1992).
• high dietary protein use diet protein for
  tissue synthesis and catabolize excess

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Nitrogen Isotopes in Mammalian Herbivores Hair
?15N Values from a Controlled Feeding
Study(Sponheimer et al., 2003)

• Goals
• Determine the importance of
• hindgut vs foregut fermentors
• dietary protein levels
• on herbivore d15N values.

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Nitrogen uptake herbivores

• Hindgut
• Horses, rabbits, birds, iguanas, green turtle
• Limited cycling of urea nitrogen
• fermentation, N cycling, protein balance
• Foregut
• Ruminants (can synthesize proteins from inorganic
  nitrogen compounds)
• multi compartmental stomachs
• cows, llamas
• Ruminant-like
• kangaroos, wallabies, hoatzin
• cycle/mix N from diet and self
• deamination and de novo protein syntheses

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Diet-Hair Fractionation
Same diet, fair bit of variation rabbits and
alpaca vary 3.6, gt 1 trophic level! Foregut
fermenters are enriched vs hindgut fermenting
rabbits But not to horses
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High Protein vs Low Protein Herbivores
? dietary protein (9-19) causes enrichment d15N
(1.5-2.8) Not what they expected! This refutes
N cycling hypo (states that low protein group
?d15N) feces explanation poor feces is d15N
enriched (0.5-3.0), low protein less urine
loss and greater relative (not absolute) N loss
via feces, ?d15N loss
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Effects of elemental composition on the
incorporation of dietary nitrogen and carbon
isotopic signatures in an omnivorous
songbird. (Pearson et al., 2003)

• Goals
• Determine turnover rates of d15N and
• d13C in whole blood and plasma.
• d15N and d13C diet-tissue fractionation factors
  for plasma, whole blood, and feathers.
• Influence of high protein (N) and low protein
  (C) concentrations on fractionation factors.

yellow-rumped warbler
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Materials and methods

• 32 captive wild-caught migratory birds
• controlled for age sex
• Acclimation diet 32 insect
• Experimental diet
• 20,49,73, 97 insects
• Sampled
• 21 days, mass, blood (plasma, wb), feathers
  (entire)
• Determined
• CN d values of different diets
• turnover rates
• Discrimination
• Isotopic signatures of diet on different tissues

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Diets Insect, Isotopes, Concentrations
Attempted to created diets along a linear
continuum of increasing a) isotopic signature
(didnt quite work for ?15N) b) elemental
concentration by increasing the insect protein
in diet
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Diets Insect, Isotopes, Concentrations
Only 0.12 difference in d15N values among
diets. Diet containing most insects did not have
highest d15N value (diet with lowest proportion
of insects did not have the lowest d15N
value) Banana Effect (d15N 0.5 - 5.3)
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Turnover Rates
Isotope incorporation kinetics model (OBrien et.
Al 2000)
?dt discrimination factor r fractional
turnover rate
Half-life
19
Turnover Rates Half-life Plasma Blood
Half-life estimates plasma d13C 0.4-0.7
days d15N 0.5-1.7 days Half-life whole blood
d13C 4-6 days (diet 133 days!) d15N
7.45-27.7 days Whole blood is variable!
20
Discrimination Plasma, Feather, and Blood
?15N values plasma whole blood enriched 1.7 to
3.0 Apparent fractionation factor for
feathers ?15N enriched (3.2-3.6) Fractionation
factors increased linearly with elemental
concentration in diet for N
21

• ? N

in
? tissue d15N
out
? urine w/ ? 14N
22
Importance of Elemental Concentrations
Both isotopic signature of diet and
fractionation factors influence the ultimate
isotopic signature of tissues (at least
plasma). Supports the importance of using
concentration-dependent mixing models when
reconstructing diet.
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Results

• Discrimination factors depend on diet and tissue
• Fractionation factors to reconstruct diet
  requires an estimate of elemental concentrations
  in the diet.
• Turnover rates
• Plasma 1 day (short) Whole blood 1 wk (longer)
• Carbon and nitrogen fractionation factors
  increase linearly with elemental concentration in
  the diet.
• Relationship between the isotopic signature of
  the diet and the sum of a given tissues (at
  least plasma) isotopic signature fractionation
  factor was also positive linear.
• USE CONCENTRATION-DEPENDENT MIXING MODELS WHEN
  ATTEMPTING TO ESTIMATE THE RELATIVE CONTRIBUTION
  OF DIFFERENT FOOD SOURCES TO AN ANIMALS DIET!!!

24
Kangaroo metabolism does not cause the
relationship between bone collagen d15N and water
availability (Murphy Bowman, 2006)

• Goals
• Evaluate importance of water availability and
  dietary d15N in determining d15N values in
  herbivore bone collagen
• Indirectly determine if ? d15N linked to animal
  metabolism
• Assessed if d15N in grass and Kangaroo bone
  collagen are constant with respect to a Water
  Availability Index
• Examine other factors influencing
• d15N in herbivore bone collagen

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Does ? Water availability? d15N in Animal Tissue?

• Plants enriched in arid environs
• openness N cycle theory (Austin Vitousek
  1998)
• ? water in system ? in ratio of N loss to
  intrasystem N turnover
• Cryptobiotic crusts
• Why ? animal d15N when in water limited systems?
• Metabolic enrichment theories
• ? Urea osmolarity, urine excreted is more
  nitrogen (d15N) concentrated (Ambrose Deniro
  1986, Sealy 1987)
• excrete more d15N deplete urea when arid
  (Sponheimer 2003)
• not experimentally shown for rats (Ambrose 2000)
• not tested rigorously
• BUT can ? d15N be explained by herbivore diet
  alone?

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Methods

• 173 grass collections (3-4 primary
  spp/collection)
• 779 road killed roos
• macropus sp, grazers
• Water Availability Index estimated from mean
  annual actual and potential evapotranspiration
• Akaikes Information Criterion (AIC)
• Big study!

27
?
28

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Results
Found relationship of d15N and WAI similar
between grass and kangaroo bone collagen 4.74
to 4.79 enrichment 0.05 variation over
entire range of data
30

• When plotted against annual rainfall Murphy
  Bowmans d15N relationship fits with
• Previous Kangaroo work
• Eutherian herbivores
• North America Africa
• matches
• Sealey et al 1987 follows similar pattern

31

• What about C3 vs C4 grasses?

• d13C of bone collagen as proxy
• negative and weak relationship
• Found lower d15N in C4 plants (1.1)
• C4 diet (high d13C, low protein) lower consumer
  d15N

C4
C3
C3

• Model gave little support for other variables
• slope
• chenopod

C4
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Summary

• Strong negative relationship of herbivore d15N
  bone collagen and water availability.
• Near identical negative pattern of d15N in grass
  and kangaroo bone collagen with water
  availability (near constant offset in slopes)
• Suggest dietary d15N is main cause of negative
  relationship between d15N of kangaroo bone
  collagen, with water availability and metabolic
  factors having little discernible effect.

33
Importance

• Ties water availability directly to plant d15N to
  animal d15N values, with little animal affect
• Huge support for historic trophic ecology and
  past climate change data that rely on direct
  relationship between herbivores and plants which
  not confounded by animal metabolism

34
Marine food webs are enriched in d15N
(Kelly 2000)
35
Trophic Systems
Marine systems 3-4/trophic level Herbivores
3.2 Carnivores 5
(Hobson Welch 1992)
36
Trophic Systems

• Marine food chains tend to have longer food webs
• Diet affects, as ascend trophic chain, ? N in
  diet
• expect more catabolism discrimination _at_ high
  trophic level
• Trophic enrichment commonly produces 31 slope
  for d15N and d13C ratios

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• N15 to get at rainfall abundance

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Diet-Hair d15N Equilibration
Hair
Dietary ?15N values changed from 2.5 to
7.8. Dietary equilibration took 8-10 weeks
42
Kelly
43
d15N in top consumers in C3, C4 and Marine food
chains
(Kelly 2000)
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Discrimination

• Tissue effects
• Feathers more enriched than plasma or wb

45
Diet Tissue Relationship

• C N signatures linearly related with tissue
  signatures discrimination factors

46
Turnover Rates

• Correlated linearly with metabolic rate of tissue
• Different species have different turnover rates
  for same tissues
• Likely related to size, mass specific metabolic
  rates, life history factors
• half-life for wb C in bear gt crow gt quail gt
  warbler

47
Turnover Rates

• Plasma (1-5 days)
• Whole Blood (5-35 days)
• Feces (
• Feathers, Hair, Nails, Hoof (time when grown,
  maybe a lag here)
• Bone
• Teeth

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Importance of Elemental Concentrations
Phillips Koch 2002
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Implications

• CO2

WUE
N demand
NPP
openness of N cycle
d15N in plants
d15N in herbivores
50

• Pearson
• Funk/questions
• variability in initial mass and mass change
  following dietary switch among treatment groups
  (shows they like carbs
• Diets did not have ? d15N values w/
• ? insects
• Fractionation vs. discrimination

51
CO2 effects on d15N
(Coltrain et. Al. 2004)