This is a novel method for the assessment of the glucocorticoid hormone, cortisol, from koala fur. It provides a novel biomarker for the assessment of chronic stress in koalas. The main advantage of this technique is that it is a highly sensitive and reproducible method with a fairly quick turnaround time between sample preparation and hormone assay.
This technique allows for the quantitative assessment of chronic stress in koalas, which is a highly useful clinical management tool. Helping me with this procedure is Dylan Fox, a grad student from my laboratory. To begin this procedure retrieve the fur from storage at minus 80 degrees Celsius and let it thaw at room temperature.
Then, weigh the fur on a laboratory analytical precision balance. Place 60 milligrams of the fur into a pre-weighed and labeled 1.5-milliliter centrifuge tube and repeat this until 18 tubes are filled. Using a pipette, add one milliliter of 100%HPLC grade isopropanol to each tube.
Vortex the samples for 30 seconds. Strain each sample with a 0.5-milliliter micro-precision sieve so as to achieve separation of liquid and fur, and discard the liquid into a waste container. Place each fur sample into a labeled plastic weighing boat.
Then place the weighing boats into a vacuum desiccator and leave for three days to let the fur dry. Once the fur is completely dry, place each sample into a labeled 1.5-milliliter microcentrifuge tube. Place each sample in a bead mill with three chrome steel beads and pulverize for two minutes at 30 shakes per second.
Repeat this transfer with 100%analytical grade methanol and 100%analytical grade isopropanol until 18 total tubes have been filled. After this, use a pipette to transfer one milliliter of the first extraction technique into six 1.5-milliliter centrifuge tubes containing the fur sample. Cap each tube, and use a shaker to incubate them at room temperature with constant pulsating for at least 12 hours.
Then strain the samples with a 0.5-milliliter micro-precision sieve. Use a pipette to transfer the liquid into a new labeled 1.5-milliliter microcentrifuge tube, while ensuring that the fur is discarded appropriately. In a fume hood, completely try the solvent extract under a stream of nitrogen vapor.
Then, reconstitute the dried sample with 400 microliters of assay buffer and 100 microliters of 100%analytical grade ethanol. To make the controls, first select samples from animals with known exposure to the stressor. To make an extract pool, take 20 microliters of extract from each sample until a total volume of 200 microliters is obtained.
Store the extract pool at minus 80 degrees Celsius until ready to run assays. When ready, obtain the dilution factor for the 30%and 70%binding points from the parallelism graph for the extract against the cortisol standard, and use assay buffer to dilute the sample pool appropriately to create the high and low controls. Using a commercial cortisol kit set up a 96-well strip plate including the samples, controls, cortisol standards, nonspecific-binding wells, and maximum-binding wells according to the supplier's instructions.
Use the plate layout sheet included in the kit booklet to list the positions of the samples, controls and standards. Next, follow the fur hormone extraction process described previously to obtain 100%methanol-extracted koala fur. Prepare the cortisol kit's reagents according to manufacturer's instructions.
Pipette 50 microliters of samples or standards into the wells of the plate. Pipette 75 microliters of assay buffer into the nonspecific-binding wells, and 50 microliters of assay buffer into the maximum-binding wells. Using a repeater pipette add 25 microliters of the cortisol conjugate to each well.
Then, pipette 25 microliters of the cortisol antibody into each well, except the nonspecific-binding wells. Gently tap the sides of the plate to ensure that the reagents are well mixed. Cover the plate with the plate sealer, and use an orbital shaker to shake at room temperature for one hour.
After this, remove the plate seal, and aspirate the well plate by washing each well with 300 microliters of wash buffer four times. Then dry the plate by tapping it on clean absorbent towels. Pipette 100 microliters of tetramethylbenzidine substrate to each well.
Place the plate sealer on the well plate, and incubate at room temperature for 30 minutes. After this, pipette 50 microliters of stop solution into each well. Transfer the plate into a plate reader capable to reading 450 nanometers, and calculate the final hormone concentration as outlined in the text protocol.
In this study, the essay detection of hormone metabolites of interest is determined using parallelism. On the parallelism curve, the 50%binding point determines the sample dilution factor on the standard curve. As can be seen, the 100%ethanol and 100%isopropanol extracts did not provide parallel displacement against the cortisol standard.
However, the 100%methanol extract does provide parallel displacement against the cortisol standard. Intra-and inter-assay coefficients of variation are determined from high-and low-binding sample extracts run in all the assays. The low-binding internal controls are seen to be neat koala extract pool, while the high-binding internal controls are seen to be one-to-two diluted koala extract pool.
The association between each solvent extract and cortisol standard are then determined using a regression plot. The 100%methanol extract provides the best line of regression with the highest R-squared value compared to the 100%ethanol and 100%isopropanol extracts. It is important to remember that the sample wash step must be performed before grinding the samples to avoid the loss of steroid hormones.
Following this procedure, we can measure other steroidal hormones, such as reproductive hormones to look at the interactions between stress and reproduction in koalas. This technique provides a new tool for the emerging field of conservation physiology, enabling the non-invasive assessment of cortisol in koala fur. The commercial cortisol kit provides reagents in minute quantity.
However, it is important to wear personal protective equipment to minimize injury to oneself whilst conducting this laboratory procedure.
