The Biology of Stress
Lance Baumgard, Sara Stoakes and Rob Rhoads
Iowa State University
Department of Animal Science
Hypothesis

Leaky gut explains the negative
consequences of heat stress and off-feed
events (all farm animals), and ketosis
(dairy cows) problems
Heat Stress: Economics and Food Security

Cost: (lost productivity, mortality, product quality, health care etc.)
American Agriculture: > $4 billion/year
 Global Agriculture: > $150 billion/year


It will get worse in the future if:
 Climate change continues as predicted
 Genetic selection continues to emphasize milk
synthesis, lean tissue accretion, piglets/sow etc..
 Heat
producing processes
St. Pierre et al., 2003; Baumgard and Rhoads, 2013
Temperature
Humidity Index (THI)
• Easy way to measure and
evaluate heat stress
• Based on cows only under
shade….solar radiation is
incredibly potent
• 72 thought to be when cows
become susceptible
• Based on 60 year old data
when cows were producing
20-30 lb/d
• Modern cows are stressed at
THI of 68
Heat Stress
Heat Stress Questions??
• Does the decrease in feed intake explain the
reduced milk yield during heat stress?
Indirect vs. direct effects of heat
• If we have a better understanding of the
biological reasons WHY heat stress reduces
production, we’ll have a better idea of how
to alleviate it.
DMI (kg/d)
Lactation:
Effects of Heat Stress on Feed Intake
20
19
18
17
16
15
14
13
12
11
10
Heat
Heat Stress
-stressed
Underfed
Pair-fed
Heat stress  feed intake by ~30 %
1
2
3
4
5
6
7
8
9
Day
Rhoads et al., 2009
Effects of Heat Stress on Milk Yield
Milk Yield (kg/d)
50
Heat-stressed
45
Pair-fed
40
35
30
25
Heat stress  yield ~45%
Pair-feeding  yield by ~19%
20
0
1
2
3
4
5
6
7
8
9
Thus,  feed intake only accounts forDay
~50% of the reductions
yield
Rhoadsinetmilk
al., 2009
Wheelock et al., 2010
Baumgard et al., 2011
Body Weight Loss
0
-10
kg
-20
-30
UF
Pair-fed
Heat Stress
HS
-40
-50
-60
Rhoads et al., 2009
Heat Stress Reduces Back-fat Mobilization
600
NEFA (uEq/l)
500
400
Heat-stressed
Pair-fed
300
200
100
0
1
2
3
4
5
Day
6
7
8
9
Rhoads et al., 2009
Wheelock et al., 2010
Baumgard and Rhoads, 2013
Milk Sugar Output
0
-50
g/d
-100
Heat Stress Cows
-150
Secrete
-200
~400 g less lactose/day
-250
than Pair-Fed Thermal
-300
Neutral Controls
-350
Something
other
than
the
mammary
gland
is
-400
utilizing ~400 g more/day?
-450
Rhoads et al., 2009
Wheelock et al., 2010
Gastro-Intestinal Tract (GIT) Review
Guts
Reminder: Intestinal Functions

GIT is a tube running from the mouth to the
anus


Digest and absorb nutrients


Everything inside of the tube is technically “outside” of
the body
GIT lumen is a inhospitable environment
Prevent parasites, pathogens, enzymes, acids,
toxins etc.. From infiltrating “self”

Barrier function
Human GIT Surface Area:
Skin 2
m
That’s an enormous~2
amount
of area to “defend”!
Lungs
No wonder 70% of the ~50X
immune system resides in GIT
GIT
~150 X
Biology of Heat Stress Symptoms
Heat Stress and Gut Health

Diversion of blood flow to skin and extremities

Coordinated vasoconstriction in intestinal
tissues
Reduced nutrient and oxygen delivery to enterocytes
 Hypoxia increases reactive oxygen species (ROS)


Reduced nutrient uptake increases rumen and
intestinal osmolarity in the intestinal lumen

Multiple reasons for increased osmotic stress
Baumgard and Rhoads, 2013
Intestinal Morphology
Thermal Neutral
Pearce et al., 2011
Heat Stress
Pair-fed
Heat Stress and Gut Health


Lipopolysaccharide (LPS) stimulates the
immune system
LPS promotes inflammation
production….catabolic condition
 TNF,
IL-1 etc..
 Reduced
appetite
 Stimulates fever
 Causes muscle breakdown
 Induces lethargy
 ....reduces productivity

LPS can cause liver damage
Healthy TJs
Compromised
TJs
Lumen
Hypoxia
Actin
Myosin
P
MLCK
TLR4
TJs
Blood stream
Submucosa
MLCK
PGE2
TNFα
IL-1β
IL-16
INFᵞ
APP
IkB
P60 P65
NFkB
HIF-1α
The effects are rapid!
Plasma LPS & LBP
18
Endotoxin
LBP
a
7
6
Plasma LBP, ug/mL
b
b
z
12
5
bc
4
c
9
3
6
y
xy
3
Serum Endotoxin, AU
15
2
xy
1
x
0
0
0
6
4
2
Time of heat stress (h)
12
Pearce et al., 2015
Heat Stress Summary

Direct and indirect effects
 ↓DMI




only accounts for 50% of reduced milk yield
Hyperinsulinemia
Blunted adipose mobilization
Liver remains sensitive to catabolic signals
Leaky gut
 Inflammation

and acute phase protein response
Unknown whereabouts of 400 g of glucose
Leaky Gut and Ketosis?
Transition Period Disorders:
Mediated Largely by NEFA ?

Transition Period

Metabolic shift
 NEBAL
 Negative effects on
future production





Only 50% of cows in
North America
complete the transition
period without
experiencing one of
these problems




Dystocia
Milk fever
Retained placenta
Metritis
Ketosis
Displaced
abomasum
Fatty liver
Lameness
Death
Drackley, 1999
Dogma

Excess adipose tissue mobilization causes
fatty liver and ketosis

This is worse in high producing cows

Industry Goal: Reduce blood NEFA
Correlation Studies

Many studies associate NEFA and BHBA with:
 Increased
risk of ketosis, decreased milk yield, LDA,
metritis, retained placenta, laminitis, or poor
reproduction

Chapinal et al., 2011; Huzzey et al., 2011; Ospina et al., 2010a, 2010c;
Duffield et al., 2009; LeBlanc et al., 2005
 Plasma
NEFA are markedly increased (>700 mEq/L)
following calving in almost all cows
 ~15-20%
get clinical ketosis
 What makes these cows more susceptible to ketosis?

Predisposition to developing fatty liver?
Gut
Lumen
LPS/LBP
Complex
TL
R4
LPS
Immune
Cell
Liver
LBP
Portal
Circulation
↑ Inflammatory response
↑ Acute Phase Proteins:
•Serum Amyloid A
•Haptoglobin
•LBP
Sara Stoakes
Objectives

Measure biomarkers of leaky gut in cows that
were retrospectively classified as ketotic (only
diagnosed problem) and healthy herd mates
n
= 8 ketotic cows
 n = 8 “healthy” cows

Initial experiment had non-ketotic objectives
 Nayeri
et al., 2015
Increased LBP in Ketotic Cows
Healthy vs. Ketotic Transition Cows
Lipopolysaccharide Binding Protein (LBP)
*
40000
Healthy
35000
ng/ml
30000
Ketotic
*
25000
Trt: P = 0.047
DIM: P < 0.01
Trt X DIM: P < 0.01
20000
15000
10000
5000
0
-21
3
10
DIM
21
56
Nayeri et al., 2013
Objectives

Confirm that the biomarkers of leaky gut
increase during the transition period for
clinically ketotic cows

A compromised GIT barrier and subsequent
endotoxin (LPS) infiltration may play a
causative key role in ketosis development
4000
3500
Trt: P = 0.02
LPS
Trt: P=0.40
Day: P=0.26
3000
Healthy
Ketotic
EU/ml
2500
2000
1500
1000
500
0
-7
3
7
10
Days relative to calving
14
Abuajamieh et al., 2015
7000
LBP
Trt: P=0.06
Day: P<0.01
6000
ng/ml
5000
4000
3000
2000
Healthy
Ketotic
1000
-7
3
7
10
Days relative to calving
14
Abuajamieh et al., 2015
Transition Cow Problems are Associated
with Biomarkers of Leaky Gut

Intestinal barrier becomes leaky

Endotoxin induced immune activation

Immune system has energetic cost

Reprioritization of nutrients away from milk
synthesis……………..$$$ problem
Objectives:
Determine Feed Restriction’s Impact on Leaky Gut
“Out of Feed Events” are common in EVERY
animal agriculture industry
12 hours of feed restriction (40% of ad libitum intake)
causes leaky gut in the pig
Lipopolysaccharide (LPS)
LPS-BP
7
18
b
5
16
a,c
2
a,b
12
4
3
a
a
14
b,c
a
a
ug/ml
Serum Endotxon, AU
6
10
b
8
6
4
1
2
0
12 TN
12 PF
12 HS
12 HS-Zn
0
12 TN
12 PF
12 HS
12 HS-Zn
Pearce et al., 2015
Haptoglobin
Treatment: P=0.06
Linear: P=0.04
AL40 v AL40G: P=0.01
600
500
400
300
200
100
0
Linear: P=0.04
AL40 v AL40G: P=0.11
AL100 AL80 AL60 AL40 AL40G AL20
AL100 AL80 AL60 AL40 AL40G AL20
Linear: P=0.08
LPS-Binding Protein
x 103/µL
12
10
8
6
4
2
0
SAA
µg/mL
1400
1200
1000
800
600
400
200
0
µg/mL
µg/mL
The Effects of 7 day Feed Restriction on Inflammation in the cow
AL100 AL80 AL60 AL40 AL40G AL20
6
5
4
3
2
1
0
Lymphocytes
Linear: P=0.05
AL100 AL80 AL60 AL40 AL40G AL20
Effects of Feed Withdrawal (100%) on Milk Yield
12
a
Milk Yield (kg)
10
22%
b
39%
8
c
6
4
2
0
0
Pre-Trt
6
Fasting Time (h)
12
Stoakes et al., 2015
Could 12 h of FR causes leaky gut in cattle?

Don’t know….depends upon mechanism
Decreased
Continues
luminal nutrient delivery….then likely not
out-flow of ruminal nutrients
Psychological
stress
Hunger-induced ACTH-Cortisol
Cortisol

action…..then possible
causes leaky gut in multiple models
Intestinal Mast cells are responsive to nerves
Upon
CRF stimulation they release proteases and TNF
 Both proteases and TNF cause tight junction breakdown
Can “leaky gut” explain suboptimal production
frequently observed in animal agriculture?


Heat Stress
Inadequate feed intake
 “off-feed
event”
 The
negative effects on growth and milk yield are
bioenergetically unexplainable by reduced feed intake
 Transition
 Cause
period
of ketosis?
 Weaning
 Shipping
 Overcrowding
 Unpalatable
feed
$$ Billion Dollar Question $$

Can the Feed or Animal Health Industry do
anything about leaky gut????

Targets:
 Direct
action at intestine
 Indirect via:
 Increased
feed intake
 Rumen acidosis prevention

Hind gut acidosis prevention
 Improved
immune function
Potential nutritional strategies to ameliorate
intestinal permeability
Supplement
Bicarbonate
Glutamine
Zinc
Dairy Products
Vitamin A
Vitamin C
Vitamin E
Selenium
Dexamethasone
Betaine
Conjugated Linoleic Acid
Chromium
Yeast, yeast extract/DFM
Ionophores
β-glucan
Mannanoligosaccharide
Rehydration therapy
Butyrate
Mycotoxin binders
OmniGen-AF
Presumed Mechanism of Action
Acidosis prevention
↑ intestine integrity
↑ intestine integrity, antioxidant
↑ intestine integrity
Antioxidant
Antioxidant
Antioxidant
Antioxidant
↑ intestine integrity
Osmotic regulation; CH3 donor
↑ Energy balance
↑ Feed Intake, Increase neutrophil #
Acidosis prevention & ↑ Feed Intake
Acidosis prevention
Immune modulation
↑ intestine integrity
↑ intestine integrity & ↑ Feed Intake
↑ intestine integrity
↑ intestine integrity
Immune modulation
Baumgard et al., 2014
Lactating Dairy Cow Metabolic
Adaptation to Heat Stress and
Maladaptation to Lactation
Summary
Successful Transition
Metabolic Flexibility:
Decreased Insulin Sensitivity
Baumgard and Rhoads, 2013
Unsuccessful Transition
Fatty Liver, Excessive Ketone Synthesis
LPS
Metabolic Flexibility:
Decreased Insulin Sensitivity
Glucose redirected to immune system
Baumgard and Rhoads, 2013
LPS
Metabolic Inflexibility
Remains Insulin Sensitive
Baumgard and Rhoads, 2013
Ketosis: When to intervene?

Treat:
High ketones
 Not coming into milk
 Not aggressively eating
 Looks sick
 Has a mild fever


Don’t mess with
High ketones….but she’s
 Eating like a champ
 Milking like a world-record holder
 Looks great
 No fever

Summary

Heat stress and ketotic cows have a similar
metabolic and endocrine fingerprint
 Leaky
gut is a common denominator in both

The activated immune system utilizes an
enormous amount of glucose.

Dietary Strategies
Conclusions

Leaky gut may play an important role in suboptimal
milk yield commonly observed during “stress”


Strategies that can improve intestinal integrity need
to be researched…in a “stressed model”

If leaky gut is the fundamental cause of many typical
on-farm problems….then it is a financial problem
that dwarfs all others combined
Acknowledgments
Funding Support
• USDA NRI/AFRI
• # 2005-35203-16041
• # 2008-35206-18817
• # 2010-65206-20644
• # 2011-67003-30007
• # 2014-67015-21627
• Zinpro Inc.
• Elanco Animal Health
• Kemin Inc.
• Dr. Victoria Sanz-Fernandez
• Dr. Sarah Pearce
• Dr. Jay Johnson
• Amir Nayeri
• Nathan Upah
• Sam Lei
• Erin Laughlin
• Erin Nolan
• David Valko
• Dr. Pat Gorden
• Mohannad Abumajieh
• Mohammad MalQaisi
• Johanna Mayorga Lozano
• Jake Siebert
• Mr. Howard Green
• Dr. Chel Moore
• Dr. Mark McGuire
• Dr. Arnaldo Burgos
• Dr. Howard Green
• Dr. Matt Waldron
• Dr. Lynn Davis
• Dr. Jeff DeFrain
• Dr. Mark Wilson
• Dr. Mike Socha