Non-Invasive Methods in Endocrinology Flashcards

(69 cards)

1
Q

why use non-invasive methods?

A

Practicality

Animal welfare

Scientific validity

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2
Q

why use non-invasive methods? - Practicality

A
  • broadly applicable (comparative perspective)
  • only possibility for long-term studies in field
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3
Q

why use non-invasive methods? - Animal welfare

A
  • avoids health risks and stress associated with capture/restraint
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4
Q

why use non-invasive methods? - Scientific validity

A
  • allows studies in social context
  • reduces effects of stress response on physiology (and behaviour)
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5
Q

Types of non-invasive methods

A

faeces
saliva
urine
hair/nail/feathers
water
milk
(… and others)

extraction methods
bioassays (e.g. enzyme immunoassays)

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6
Q

What does the suitability / choice of sample type depend on?

A
  • feasibility of sample collection
  • target hormone
  • scientific question
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7
Q

what does choosing the ‘wrong’ sample type cause?

A
  • costly
  • time consuming
  • lead to a mismatch of hormones and behaviour or masked hormone-behaviour relationships
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8
Q

Time lag between hormone secretion & appearances in matrices
1. saliva
2. urine
3. faecal
4. hair or wool
5. feathers

A
  1. saliva to study acute changes in hormone concentrations - changes within minutes of hormones being in blood
  2. urine samples take hours
  3. faecal samples take hours or days
  4. hair or wool samples - can quantify cumulative of the samples - days/weeks/months
  5. feathers - weeks/months - once the feather is fully grown no hormones get sent there
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9
Q

Choice between urine or faeces samples depends on?

A
  • feasibility of sample collection - raining during urine sample, or urine sample in desert (may just soak into the ground)
  • hormone of interest (e.g. peptide hormones only present in urine)
  • species of interest - variation in the route of hormone excretion and metabolism
  • storage/transport options
  • research questions
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10
Q

(Christensen et al., 2022)
Urine or faeces for studying HPA activity?
Time lag of hormone secretion

A

differs between urine + faeces -> implications for their suitability for studying effects of different temporal nature on HPA-axis activity

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11
Q

(Christensen et al., 2022)
Urine or faeces for studying HPA activity?
what is lacking?

A

simultaneous comparisons of predictors of faecal and urinary GC metabolites (fGCs and uGCs) are lacking

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12
Q

(Christensen et al., 2022)
Urine or faeces for studying HPA activity?
what was investigated?

A

correlates of fGCs and uGCs in wild chacma baboons (Papio ursinus), including long-term (dominance rank, season, female reproductive state) and short-term (time of day, daily weather conditions) factors

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13
Q

(Christensen et al., 2022)
Urine or faeces for studying HPA activity?
results?

A
  • correlated with increasing day length, fGCs gradually decreased from winter to summer
  • no seasonal effect on uGCs but ‘rain days’ associated with increased uGCs
  • pregnant females had significantly higher fGCs compared to cycling and lactating females; no effect of reproductive state on uGC
  • Circadian effect for uGCs but not fGCs
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14
Q

(Christensen et al., 2022)
Urine or faeces for studying HPA activity?
1. what does the study highlight?
2. what uGCs offer?
3. where possible, what should be monitored? to obtain what?
4. shows importance of what?

A
  1. difference in inherent fluctuation between uGCs and fGCs and its potential consequences for HPA-axis activity monitoring
  2. uGCs offer the opportunity to study short-term effects but undergo more pronounced fluctuations, reducing their ability to capture long-term effects
  3. where possible, uGCs and fGCs should be monitored in tandem to obtain a comprehensive understanding of short- and long-term drivers of HPA-axis activity
  4. importance of choice of sampling
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15
Q

Factors Affecting Hormone Data Interpretation

A
  • delayed excretion
  • metabolic rate
  • diurnal patterns
  • effects of diet
  • storage effects
  • reproductive state
  • sex
  • disease
  • season
  • etc…
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16
Q

Delayed excretion (affecting hormone data interpretation)
urine vs faeces

A

faeces has delayed excretion (Wasser et al., 1998)

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17
Q

Delayed excretion (affecting hormone data interpretation)
Faecal cortisol metabolites in non-human primates: species and time lag (h)
1. Marmoset
2. Long-tail macaque
3. Barbary macaque
4. Baboon
5. Chimpanzee
6. Sifaka
7. Gibbon

A
  1. 8
  2. 28
  3. 46
  4. 36
  5. 26
  6. 26
  7. 27
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18
Q

(Goymann, 2012)
Metabolic rate (affecting hormone data interpretation)

A

high metabolic rate of bird = bigger faeces (more hormones)

hormone measurement on faecal samples depends on size of faeces

can effect sample measurements

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19
Q

(Sousa & Ziegler, 1998) (Smith & French, 1997)
Diurnal patterns (affecting hormone data interpretation)

urinary samples of Callithrix jacchus (Marmoset)

A

females:
- higher urinary cortisol at around 11:00 (highest peak during the day)
- lowest urinary cortisol at 18:00

males:
- higher urinary cortisol = 10:00/11:00
- lowest urinary cortisol = 13:00/15:00

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20
Q

(Higham et al., 2007)
effects of diet (affecting hormone data interpretation)
Olive baboons (Vitex doniana)
1. what have lab analyses show?

A
  1. show fruit contains more progesterone substances than leaves

fruit affects faecal progesterone (higher)

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21
Q

(Goymann, 2012)
Sex differences in hormone metabolism (affecting hormone data interpretation)

blood and faeces & droppings - male vs female

A
  • males and females had equal hormone concentrations in their bloods
  • males had a higher metabolic concentration than females in faeces & droppings
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22
Q

(Khan et al., 2002)
storage effects (affecting hormone data interpretation)

A
  • best to freeze soon after collection
  • samples split and stored at frozen (-20 degrees - lower faecal estrogen and glucocorticoids) or room temperature (higher faecal estrogen and glucocorticoids) - measured hormones concentrations at different times
  • reason - activity in faecal sample effects hormones concentrations if not frozen
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23
Q

Reproductive state (affecting hormone data interpretation)

  1. (Fürtbauer et al., 2014, PNEC) - Macaca assamensis, glucocorticoid hormone
  2. (Christensen et al., 2022) - fGCs and uGCs
A
  1. pregnant = highest glucocorticoids, then lactating, then cyclic, then acyclic
  2. Pregnant = highest fGCs/uGCs, then lactating, then cycling
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24
Q

Validation:
Laboratory validation

A

demonstrate that the target hormone is precisely measured (specificity, sensitivity, accuracy and precision of measurements)

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25
Validation: Biological validation
demonstrate that hormone measures reflect the physiological events in question
26
Biological validation - how to ensure?
1. comparing circulating and excreted hormones, e.g: - Ovarian function in the saddle-back tamarin - faecal, urinary, and plasma (Heistermann et al., 1993) 2. measuring a predicted change/difference in hormone levels, e.g: - Progestogen / Reproductive stage (high faecal progestogen in pregnant non human primate) (Barelli & Heistermann, 2009)
27
faecal hormone collection and storage 1. Collection without? 2. what measure of hormone concentrations (steroid hormones)? 3. what needs to be considered with this type of sampling? 4. collection using what? and what first occurs with the sample? 5. what temperature to keep it at? 6. extraction prior to analysis? 7. what if no freezer available?
1. collected without capture or restraint 2. cumulative measure of hormone concentrations (steroid hormones) 3.consider diurnal variation/delay in hormone secretion 4. collection wearing gloves, homogenise if needed, remove large seeds/plant material 5. keep cool (e.g. cool packs) until freezing Freeze at -20°C (avoid multiple freeze-thaw cycles) 6. time consuming extraction prior to analysis (lyophilize, grind and sieve, weigh and add methanol to extract hormones, centrifuge and collect supernatant) 7. samples can be extracted in the field if no freezer available (Nugraha et al., 2017)
28
Faecal hormone collection and storage
- wear gloves - homogenise - remove large seeds/plant material - leak-proof vials - labels (ID, Date, Time, Collector) - Cool packs - Freeze at -20°C - avoid multiple freeze-thaw cycles
29
Faecal hormone sample preparation
- lyophilise to remove water content - mortar & pestle - grind poo - sieve - weigh - add methanol
30
Faecal hormone extraction/analysis
- shake for 10 minutes - centrifuge for 10 minutes - collect supernatant - dilute - analyse
31
(Fürtbauer et al., in prep) Habitat fragmentation and HPA-axis activity in nocturnal lemurs 1. background info? 2. two locations - one fragmented, one not? 3. results?
1. Northern Giant Mouse Lemur (Mirza zaza) + faecal samples - ~95% of species classified as threatened (IUCN) - Over 80% of their forest habitat destroyed - Remaining forest heavily fragmented 2. Ankarafa = fragmented (lots of edge) Anabohazo = not fragmented (less edge) 3. higher cortisol in Ankarafa than Anabohazo higher cortisol at core than edge Ank Core = highest cortisol then Anab Core then Ank Edge then Anab Edge
32
Benefits of hormone measurements from urine
- collected without capture or restraint - various collection methods: pipette, Salivette, filter paper - contains various classes of hormones (steroid and peptide hormones) - cumulative measure of hormone concentrations - relatively small volumes are sufficient - can be used to measure other physiological markers (e.g. C-Peptide)
33
what is important to consider with urine samples?
consider diurnal variation/delay in hormone secretion
34
what must occur with storage of urine samples?
MUST be stored frozen (avoid thawing and re-freezing)
35
Extraction in urine samples?
may or may not be required
36
what has to be corrected for urine concentration?
hormone measurements have to be corrected for urine concentration (creatinine, specific gravity)
37
what are salivary hormones sensitive to?
acute changes ('real-time assessment')
38
in salivary hormones, measurements are highly variable within ...?
within short time periods
39
how to collect salivary hormones?
passive drool collection (e.g. using saliva collection aid) oral swab saliva collection
40
what are salivary hormones strongly affected by?
diurnal fluctuations in hormone secretion
41
when are salivary hormones difficult to obtain?
from wild animals (easier from domestic animals, humans)
42
what should occur with salivary hormone samples?
stored frozen until analysis
43
is extraction needed in salivary hormones samples?
microcentrifuge/analysis (no extraction step needed)
44
what is hormone measurements from hair useful for?
long-term assessment of sex steroids, glucocorticoids (chronic HPA-axis activity), thyroid hormones etc.
45
what is hair samples for hormones not suitable for?
time point analyses or the assessment of acute changes in hormone concentrations
46
hair collection using?
clippers or scissors (or collars, trees, traps, etc.)
47
where is collected hair stored?
in foil and/or paper envelope at room temperature
48
how should hair samples be stored?
not frozen
49
what needs to occur to hair samples prior to analysis?
time consuming extraction procedure prior to analysis
50
(Stalder & Kirschbaum, 2012) unique characteristics of the analysis of cortisol in scalp hair 1. covering an extended secretion period 2. small amount of hair and non-invasive sampling 3. easy storage 4. lack of situational confounding
1. analysis of 3m hair = cortisol secretion over 3-month-period (only poss w/ hair - not urine or faeces etc.) 2. only 10 mg of hair/segment required for analyses, hair sampling not leave visible marks + mostly well tolerated by participants 3. samples stored at room temperature over long periods of time (suited for field research) 4. due to retrospective nature of hair sampling + extended detection window, HCC (hair cortisol concentration) should not be affected by situational characteristics
51
Hormone incorporation in hair
- passive diffusion from blood in root of hair - sweat scalp to surface - Sebum on surface - naturally oily surface - external sources - locally produced - at root hair
52
3cm hair segment represents ?
past 3 months
53
hair follicle 3-5 mm beneath scalp has lagtime of...?
1-2 weeks
54
Hair collection
clippers or scissors foil and/or paper envelope
55
hair storage
at room temperature do not freeze
56
Hormone measurements from hair (step by step)
- wash hair w/ isopropanol to remove external cortisol from sweat and sebum - e.g. hormones on hands from grooming, contamination w/ faeces or urine that have the hormone in - dry hair - grind or cut dried hair into a powder/small pieces to increase surface area - ideal 1-2mm in size or tube with beads to break up hair (tube method not work for sheep but does work for coarse hair, e.g. dog hair) - extract steroid hormones with methanol - evaporate methanol and reconstitute in assay buffer - analyse cortisol using sensitive and specific enzyme immunoassay
57
where to collect hair sample from species in relation to skin?
closest to skin without damaging the skin (can damage sample) closer to skin = recent time frame cut in segments of cm - can pair hair piece with time period with e.g. temperature
58
sheep wool cortisol 1. where samples were collected? 2. type of correlation found with cortisol before and after grazing? 3. when was cortisol higher - before or after grazing (for both periods)? 4. how is body mass impacted after grazing? any change between two periods? 5. relationship between body mass and cortisol?
1. back samples n=120 (before and after grazing period 1 and 2); shoulder samples n=30 (2.5cm segments - split into A B and C, n=90 sub samples) 2. positive correlation between pre- and post-grazing cortisol levels -> higher wool cortisol before grazing tended to have higher cortisol after as well -> individual differences in baseline stress levels are consistent over time 3. Wool cortisol decreases after grazing in both periods -> grazing may be stress-reducing (possibly due to better foraging, more space, or social stability) - Grazing period 1 appears to start with slightly higher cortisol levels. 4. Body mass increases after grazing in both periods -> good food access -> weight gain -> nutritionally beneficial. 5. significant negative correlation between body mass and wool cortisol -> higher body mass = lower cortisol -> conditioned animals are less stressed -> higher body mass = lower physiological stress.
59
Anthropogenic activity + long-term HPA-axis activity 1. where? 2. methodology? 3. data collected? 4. results?
1. French Alps 2. GPS collars (ranging data) on Rupicapra rupicapra (Chamois) N=64 individuals (since 2015) 3. data on altitude, rugosity, vegetation, visibility data on human disturbance, e.g. hiking + hunting 4. more habitat roughness = more hair cortisol
60
effects of habitat fragmentation on hair cortisol concentrations (Sportive Lemur - Lepilemur sahamalazensis - critically endangered)
more habitat edges (more fragmentation) = higher hair cortisol
61
hormone measurements from feathers - collection method?
- feather collected with/without capture/restraint - wash feathers with isopropanol - air -dry feathers - grind or cut feathers into a powder/small pieces to increase surface area - extract steroid hormones with methanol - evaporate methanol and reconstitute in assay buffer - analyse cortisol using sensitive and specific enzyme immunoassay
62
Anthropogenic activity + L-T HPA-axis activity - feathers 1. sample size? 2. results?
1. N = 58 birds n = 86 feathers 2. higher fCORT (and variation) on farm than in open fields
63
waterborne hormones - steroid hormones in fish
- monitoring of steroid hormones in fresh- and sea-water fish - steroid hormones diffuse into water via the gills - waterborne hormone concentrations correlate with concentrations in the blood (e.g. Kidd et al., 2009)
64
waterborne hormone collection (not for larger fish)
1. prepare known volume of water 2. weigh fish 3. collect hormones (known amount of time) 4. collect sample
65
waterborne hormone extraction & analysis
1. defrost samples 2. Solid Phase Extraction (SPE) = concentrates samples 3. Evaporate eluates under stream of nitrogen and store/resuspend
66
Habituation to sampling procedure 1. what occurs? 2. (Fürtbauer et al., 2015) testosterone and cortisol? 3. (Wong et al., 2008) day of confinement and cortisol? 4. recommended?
1. fish in small container with limited water = cortisol is likely to change due to different conditions 2. testosterone = stable ; cortisol decrease as days go on 3. cortisol decrease as day of confinement continue on 4. expose fish to sampling procedure several times to reduce effect, and to start collecting data on acute effect of stimulus
67
Personality & Plasticity how do fish respond to predation risk in terms of: 1. activity? 2. shyness? 3. cortisol? 4. method? 5. results?
1. less active at high predation risk 2. more shyness at high predation risk 3. higher cortisol at high predation risk 4. predation risk based on makeshift predator (e.g. heron) dipped into water where stickleback fish is 5. population-level effect individuals are equally capable of responding to changes in predation risk
68
VIE tagging & Stress 1. effect of visible implant elastomer (VIE) tagging on the immediate physiological stress response?
1. physiological stress response similar to those occurring everyday situations
69
Enzyme Immunoassay (EIA)
measure the presence or concentration of a macromolecule in a solution through the use of an antibody or immunoglobulin (RIA: Radio Immunoassays -> require the use of radioactive substances)