Non-Invasive Methods in Endocrinology Flashcards
why use non-invasive methods?
Practicality
Animal welfare
Scientific validity
why use non-invasive methods? - Practicality
- broadly applicable (comparative perspective)
- only possibility for long-term studies in field
why use non-invasive methods? - Animal welfare
- avoids health risks and stress associated with capture/restraint
why use non-invasive methods? - Scientific validity
- allows studies in social context
- reduces effects of stress response on physiology (and behaviour)
Types of non-invasive methods
faeces
saliva
urine
hair/nail/feathers
water
milk
(… and others)
extraction methods
bioassays (e.g. enzyme immunoassays)
What does the suitability / choice of sample type depend on?
- feasibility of sample collection
- target hormone
- scientific question
what does choosing the ‘wrong’ sample type cause?
- costly
- time consuming
- lead to a mismatch of hormones and behaviour or masked hormone-behaviour relationships
Time lag between hormone secretion & appearances in matrices
1. saliva
2. urine
3. faecal
4. hair or wool
5. feathers
- saliva to study acute changes in hormone concentrations - changes within minutes of hormones being in blood
- urine samples take hours
- faecal samples take hours or days
- hair or wool samples - can quantify cumulative of the samples - days/weeks/months
- feathers - weeks/months - once the feather is fully grown no hormones get sent there
Choice between urine or faeces samples depends on?
- 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
(Christensen et al., 2022)
Urine or faeces for studying HPA activity?
Time lag of hormone secretion
differs between urine + faeces -> implications for their suitability for studying effects of different temporal nature on HPA-axis activity
(Christensen et al., 2022)
Urine or faeces for studying HPA activity?
what is lacking?
simultaneous comparisons of predictors of faecal and urinary GC metabolites (fGCs and uGCs) are lacking
(Christensen et al., 2022)
Urine or faeces for studying HPA activity?
what was investigated?
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
(Christensen et al., 2022)
Urine or faeces for studying HPA activity?
results?
- 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
(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?
- difference in inherent fluctuation between uGCs and fGCs and its potential consequences for HPA-axis activity monitoring
- uGCs offer the opportunity to study short-term effects but undergo more pronounced fluctuations, reducing their ability to capture long-term effects
- 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
- importance of choice of sampling
Factors Affecting Hormone Data Interpretation
- delayed excretion
- metabolic rate
- diurnal patterns
- effects of diet
- storage effects
- reproductive state
- sex
- disease
- season
- etc…
Delayed excretion (affecting hormone data interpretation)
urine vs faeces
faeces has delayed excretion (Wasser et al., 1998)
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
- 8
- 28
- 46
- 36
- 26
- 26
- 27
(Goymann, 2012)
Metabolic rate (affecting hormone data interpretation)
high metabolic rate of bird = bigger faeces (more hormones)
hormone measurement on faecal samples depends on size of faeces
can effect sample measurements
(Sousa & Ziegler, 1998) (Smith & French, 1997)
Diurnal patterns (affecting hormone data interpretation)
faecal samples of Callithrix jacchus
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
(Higham et al., 2007)
effects of diet (affecting hormone data interpretation)
Olive baboons (Vitex doniana)
1. what have lab analyses show?
- show fruit contains more progesterone substances than leaves
fruit affects faecal progesterone (higher)
(Goymann, 2012)
Sex differences in hormone metabolism (affecting hormone data interpretation)
blood and faeces & droppings - male vs female
- males and females had equal hormone concentrations in their bloods
- males had a higher metabolic concentration than females in faeces & droppings
(Khan et al., 2002)
storage effects (affecting hormone data interpretation)
- 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
Reproductive state (affecting hormone data interpretation)
- (Fürtbauer et al., 2014, PNEC) - Macaca assamensis, glucocorticoid hormone
- (Christensen et al., 2022) - fGCs and uGCs
- pregnant = highest glucocorticoids, then lactating, then cyclic, then acyclic
- Pregnant = highest fGCs/uGCs, then lactating, then cycling
Validation:
Laboratory validation
demonstrate that the target hormone is precisely measured (specificity, sensitivity, accuracy and precision of measurements)
Validation:
Biological validation
demonstrate that hormone measures reflect the physiological events in question
Biological validation
- comparing circulating and excreted hormones, e.g:
- testosterone in Macaca fuscata (Barrett et al., 2002)
- Ovarian function in the saddle-back tamarin (Heistermann et al., 1993) - measuring a predicted change/difference in hormone levels, e.g:
- Progestogen / Reproductive stage (high faecal progestogen in pregnant non human primate) (Barelli & Heistermann, 2009)
- testosterone / reproductive condition, age, sex (high urinary androgen conc in intact, vs castr, juv, female) (Moehle et al., 2002)
- iEA - major metabolite of testosterone - faecal samples of iEA = highest in adults, then large subadult, then immature (Ostner et al., 2011)
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?
- collected without capture or restraint
- cumulative measure of hormone concentrations (steroid hormones)
3.consider diurnal variation/delay in hormone secretion
- collection wearing gloves, homogenise if needed, remove large seeds/plant material
- keep cool (e.g. cool packs) until freezing
Freeze at -20°C (avoid multiple freeze-thaw cycles) - time consuming extraction prior to analysis (lyophilize, grind and sieve, weigh and add methanol to extract hormones, centrifuge and collect supernatant)
- samples can be extracted in the field if no freezer available (Nugraha et al., 2017)
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
Faecal hormone sample preparation
- lyophilise to remove water content
- mortar & pestle
- grind poo
- sieve
- weigh
- add methanol
Faecal hormone extraction/analysis
- shake for 10 minutes
- centrifuge for 10 minutes
- collect supernatant
- dilute
- analyse
(Fürtbauer et al., in prep)
Habitat fragmentation and HPA-axis activity in nocturnal lemurs
- background info?
- two locations - one fragmented, one not?
- results?
- Northern Giant Mouse Lemur (Mirza zaza)
- Lemurs = most threatened group of mammals
- ~95% of species classified as threatened (IUCN)
- Over 80% of their forest habitat destroyed
- Remaining forest heavily fragmented
- Nocturnal lemurs particularly threatened - Ankarafa = fragmented (lots of edge)
Anabohazo = not fragmented (less edge) - 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
Lecture 9
Slide 34
