Towards more physiological manipulations of hormones in field studies: comparing the release dynamics of three kinds of testosterone implants, silastic tubing, time-release pellets and beeswax.

Hormone manipulations are of increasing interest in the areas of physiological ecology and evolution, because hormones are mediators of complex phenotypic changes. Often, however, hormone manipulations in field settings follow the approaches that have been used in classical endocrinology, potentially using supra-physiological doses. To answer ecological and evolutionary questions, it may be important to manipulate hormones within their physiological range. We compare the release dynamics of three kinds of implants, silastic tubing, time-release pellets, and beeswax pellets, each containing 3mg of testosterone. These implants were placed into female Japanese quail, and plasma levels of testosterone measured over a period of 30 days. Testosterone in silastic tubing led to supraphysiological levels. Also, testosterone concentrations were highly variable between individuals. Time-release pellets led to levels of testosterone that were slightly supraphysiological during the first days. Over the period of 30 days, however, testosterone concentrations were more consistent. Beeswax implants led to a physiological increase in testosterone and a relatively constant release. The study demonstrated that hormone implants in 10mm silastic tubing led to a supraphysiological peak in female quail. Thus, the use of similar-sized or even larger silastic implants in males or in other smaller vertebrates needs careful assessment. Time-release pellets and beeswax implants provide a more controlled release and degrade within the body. Thus, it is not necessary to recapture the animal to remove the implant. We propose beeswax implants as an appropriate procedure to manipulate testosterone levels within the physiological range. Hence, such implants may be an effective alternative for field studies.

Seasonal and diel variation of hormone metabolites in European stonechats: on the importance of high signal-to-noise ratios in noninvasive hormone studies.

Most vertebrates living in seasonal environments show seasonal reproductive cycles and diel rhythms. The rhythmicity in behavior and morphology is accompanied by diel and seasonal patterns of hormone secretions. In small animals, the investigation of diel patterns of hormones has been hampered because repeated blood sampling is difficult and may influence subsequent measurements. A possibility to avoid these caveats is to investigate excreted hormone metabolites instead. Here, we describe the diel excretion patterns of testosterone and corticosterone metabolites in a small captive songbird during 4 seasons: winter, early spring (Zugunruhe), summer, and autumn molt. Our approach is quite unique, because the diel patterns of steroids have rarely--if at all--been investigated in the same individuals within several seasons. Small birds should be ideal to investigate diel patterns of hormone metabolites, because they defecate frequently enough to establish a diel profile. However, concentration measurements of hormone metabolites rely on the assumptions that hormone metabolites are placed into droppings of similar mass (the "dropping amount assumption") and are excreted in constant time intervals (the "constant interval assumption"). These assumptions were clearly violated in our study, as the dropping mass per time interval and the defecation intervals varied depending on the time of day and season. We thus used the rate of hormone metabolite excretion as an alternative measure to concentration. Both testosterone and corticosterone metabolites showed diel and seasonal rhythmicity. Furthermore, the diel pattern varied depending on season. Concentration and rate measurements gave similar results when the differences between hormone metabolite levels were large-for example, when testosterone metabolites were compared across seasons. When the differences were more subtle, though, the 2 measures did not always correspond well, indicating that the violation of the 2 basic assumptions affected the comparability of concentration measurements. We conclude that diel and seasonal comparisons of hormone metabolites potentially give biologically meaningful results, especially when rates instead of concentrations are measured. However, such studies require awareness of the limitations and pitfalls of noninvasive hormone measurements, a carefully designed experiment, and very cautious interpretation of the data.

Use of ethanol for preserving steroid and indoleamine hormones in bird plasma.

Endocrinological research on wild animals inhabiting remote areas has been hampered by the need to store plasma samples at subzero temperatures. In an attempt to remedy this logistical issue, we here investigate the use of ethanol as an alternative to freezing for the preservation of steroid and indoleamine hormones in avian plasma. Known quantities of the steroids 5alpha-dihydrotestosterone (DHT), testosterone, 17beta-estradiol, corticosterone, and the indoleamine melatonin were added to a stripped pool of chicken plasma. Samples were either immediately frozen at -40 degrees C, or treated with pure ethanol. Ethanol-treated samples were either immediately frozen, or-to simulate storage conditions at various field locations-left sitting at room temperature for one to two months, or incubated at 36 degrees C for one month before all treatment groups were frozen at -40 degrees C. All samples were then analyzed by radioimmunoassay. For DHT and estradiol there were no differences among treatment groups suggesting that ethanol-treatment is as effective as immediate freezing in preserving plasma steroid concentrations. For testosterone, corticosterone and melatonin ethanol-treated samples differed significantly from immediately frozen samples suggesting that caution is needed when comparing absolute concentrations of hormones between samples preserved in different ways. However, differences among ethanol-treated samples in general were small, demonstrating the feasibility of this preservation method in the field at remote locations.

Low ambient temperature increases food intake and dropping production, leading to incorrect estimates of hormone metabolite concentrations in European stonechats.

Non-invasive methods to measure steroid hormone metabolites in bird droppings or mammalian feces have become very popular. However, the accuracy of these measurements may be affected by many factors. Here, we use the stonechat (Saxicola torquata) as a passerine bird model to test whether differences in ambient temperature affect food intake and dropping production and whether these changes lead to measurement artefacts in hormone metabolite concentrations. In addition, we tested for diurnal patterns in hormone metabolites. We held European stonechats in climate chambers and subjected them to two different long-term ambient temperature regimes, +5 degrees C and +22 degrees C. As expected, food intake and dropping production was higher at +5 degrees C than at +22 degrees C. Plasma concentrations of corticosterone and testosterone did not differ between different ambient temperature regimes. However, corticosterone and testosterone metabolite concentrations (in ng/g) were significantly lower at +5 degrees C than at +22 degrees C. When we measured the rate of hormone metabolite excretion (in picogram per hour) instead of the concentration, there was no difference between treatment groups. Thus, the measurement of hormone metabolite concentrations can be flawed because, depending on the treatment, similar amounts of hormone metabolites can be excreted into very different amounts of droppings. In conclusion, hormone metabolite concentration measurements are sensitive to changes in ambient temperature and probably any other factor that alters metabolic rates. Any study involving systematic changes in metabolism--i.e., during molt, migration, hibernation, egg production, or seasonal comparisons--needs to take these caveats into account.