Transcript Challenge Protocol

Principal Research Question:
Why do mammals get old and die?
How is this affected by:
a. Reproduction
b. Natural Stressors
(competition, predators, etc.)
c. Maternal and Cohort Effects
But physiological evidence
obtained can be used to examine
other areas simultaneously:
1. Genotypic and phenotypic variation.
2. Role of territory quality on variation in
physiology.
3. Impact of reproductive effort on
physiology.
4. Cohort and year effects.
Senescence
the process of decline
in physiological
functioning that results
in increasing mortality
rates with increasing
age after some point in
the lifespan
Theory of Senescence
1. The age at which senescence is first expressed
depends on how much mortality occurs
independent of the effects of senescence.
2. Senescence should not be expressed until
after the age at first reproduction.
Principal Research Approach:
Target the key physiological system - the
STRESS AXIS - that is crucial for
survival, allows animals to cope with
challenges, and integrates and reflects
the past
Examine at 2 levels:
a. Changes in stress response.
b. Changes in regulation of this
response at the level of the Brain.
Stress
Stress Response - the set of responses by
birds and mammals by the stress axis to
potentially harmful environmental challenges


Stressor - anything that upsets the
homeostatic balance within an animal
Environmental
 Physical
 Psychological

Response to Stressor is
Crucial and Changes with Age,
Condition, Experience, etc.
Crucial components:
1. Response to the stressor how rapid is it and how
intense?
2. Negative Feedback - how
rapidly is it terminated?
HIPPOCAMPUS
STRESS
HYPOTHALAMUS
CRF
PITUITARY
ACTH
ACTH
ADRENALS
Cortisol
BLOOD
Negative
Feedback
GR
Hippocampus
Hypothalamus
MR
PVN
CRH AVP
Anterior
Pituitary
ACTH
Adrenal
Cortex
Glucocorticoids
Mobilization
Of Energy
Immunosuppression
Suppression
Suppression
Of Growth Reproductive Of Digestion
Suppression
Stress Response
Good: if short term = Acute Response
[Classic Flight or Fight Response]
Bad: if long term = Chronic Response
[short term effects are prolonged,
with potential permanent
consequences - Brain changes,etc.]
LIVER
GLUCOSE
GLUCONEOGENESIS
GLYCOGEN
ENERGY
SUPPLY
FATTY
ACIDS
Cortisol has
both
these effects:
ANABOLIC
MUSCLE
GLYCEROL AMINO
ACIDS
GLUCOSE
LYMPHOID
SKIN
CONNECTIVE
ADIPOSE
CATABOLIC
Stress Response is not Static
1. May be modulated over annual cycle to
optimize reproduction, survival, or
both
2. Modified during development:
Programming of the Brain.
3. Modified by experience.
Methods to study stress response
in Natural Populations
 Before and After assessment (crude)
Measurement in feces and urine (rough
and limited utility)


Challenge Protocol
 Capture
 Hormonal - Will Use this
ACTH Stimulation Test
Inject ACTH
 Measure glucocorticoid levels


Moderate increase is normal

Excessive increase or reduced
response (species dependent)
indicative of chronic stress.
HIPPOCAMPUS
ACTH
HYPOTHALAMUS
CRF
PITUITARY
ACTH
ACTH
ADRENALS
Cortisol /
Corticosterone
BLOOD
Hormonal Challenge Protocol
Units
Time 0
BASE Bleed
ACTH Injection
30
60
ACTH Bleeds
120
Poor Condition
Free
Cortisol
Good Condition
ACTH
Time
Hormonal Challenge Protocol
1.
2.
3.
4.
Capture squirrel
Bring to standard location
Bleed four times with suborbital sinus bleeds
Anesthetize each time with isofluorane and portable
anesthetic device.
5. Bleeds : collect about 300 ul each time. Thus about
1.2 mls (note that a 250 g squirrel has about 25 ml
blood; thus no problem here)
6. Bleeds: first = Baseline.
Then inject intramuscular into Thigh approximately
100 ul ACTH. This Stimulates adrenals to mobilize
cortisol
7. ACTH response: serial bleeds at 30, 60 and 120 min to
observe ability to handle challenge
Blood Samples will be analyzed for
1. Cortisol and carrier protein - CBG.
Will allow us to see rapidity and extent of response.
2. Testosterone (in males)
2. Measures of Energy mobilization:
a. Glucose (principal fuel)
b. Free fatty acids (secondary fuel)
3. Other measures of CONDITION
a. hematocrit (packed cell volume - more is better)
b. complete white blood cell profile: concentration
and ratio of lymphocytes, neutrophils, and albumin
etc. Will only do for BASE bleed.
This must be done in Veterinary lab in Edmonton or
Toronto
Assessment of Immune System and its ability
to respond to a challenge is also be a good
indication of physiological condition and
status of the squirrel.
Question - Does an animal’s ability to handle a
immunocompetence challenge vary with:
1.
2.
3.
4.
5.
Age
Condition of Territory (e.g.Fed vs Nonfed)
Investment in Reproduction
Cohort & Maternal Effects
Year Effects
Immunocompetence
Challenge Protocol
Measure: Cell Mediated Immunity Only
1. Day 1: capture animal, inject standardized
antigen (sheep erythrocytes), release.
2. Day 7: recapture animal, collect blood sample,
take sample to lab, do agglutination
tests against antigen to assess animal’s
ability to mount a immune response.
3. Simple, fast, and direct.
Possible Effects of Protocols
on Red Squirrels
1. Have done Hormonal Challenge
Protocol on about 50 Lloyd animals. All
released alive, many called on release,
and immediately went on with lives.
2. Is there a survival cost to hormonal
challenge protocol?
Don’t think so. But can test will Lloyd
animals. In hares, animals challenged
with a similar procedure in winter and
released had similar survival of animals
not challenge.
Sex to be examined

Nonbreeding season: both sexes

Breeding Season: restrict tests to
MALE red squirrels only to avoid any
pregnancy or lactation effects on
young.
When?
Early August at or before food
gathering. Will allow examination of
relationship to overwinter survival
 Late February or early March before
breeding season. Will allow
examination of relationship between
reproductive fitness and
physiological condition.
