An Important Personality Trait Varies with Blood and Plumage Metal Concentrations in a Free-Living Songbird.

Metal pollution is a global problem, which threatens to seriously disrupt behavioral patterns and health in humans and wildlife. Nonetheless, little is known regarding how exposure to metal pollution affects animal personalities, as defined by repeatable among individual differences in behavior. We used a large dataset to investigate the relationship between individual blood and feather metal concentrations and three personality traits (exploration behavior, territorial aggressiveness, and aggressiveness during nest defense) in great tits (Parus major), a model species for animal personality research. We previously demonstrated slower exploration behavior at highly polluted study sites, where exposure to lead, cadmium, and arsenic is high. Here, we demonstrate the across-year repeatability of exploration behavior and aggressiveness during nest defense, providing strong evidence for the existence of personalities in our populations. Furthermore, we demonstrate that individuals with high blood lead concentrations and high concentrations of multiple metals in the feathers exhibit slower exploration behavior but no differences in territorial aggressiveness or nest defense relative to less exposed birds. The mechanism underlying the relationship between metal exposure and exploration behavior remains to be determined but could involve neurotoxic effects. Our study highlights that metal pollution could have underappreciated effects on animal personalities, with implications for individual fitness and societal function.

Effects of interspecific coexistence on laying date and clutch size in two closely related species of hole-nesting birds.

Coexistence between great tits Parus major and blue tits Cyanistes caeruleus, but also other hole-nesting taxa, constitutes a classic example of species co-occurrence resulting in potential interference and exploitation competition for food and for breeding and roosting sites. However, the spatial and temporal variations in coexistence and its consequences for competition remain poorly understood. We used an extensive database on reproduction in nest boxes by great and blue tits based on 87 study plots across Europe and Northern Africa during 1957-2012 for a total of 19,075 great tit and 16,729 blue tit clutches to assess correlative evidence for a relationship between laying date and clutch size, respectively, and density consistent with effects of intraspecific and interspecific competition. In an initial set of analyses, we statistically controlled for a suite of site-specific variables. We found evidence for an effect of intraspecific competition on blue tit laying date (later laying at higher density) and clutch size (smaller clutch size at higher density), but no evidence of significant effects of intraspecific competition in great tits, nor effects of interspecific competition for either species. To further control for site-specific variation caused by a range of potentially confounding variables, we compared means and variances in laying date and clutch size of great and blue tits among three categories of difference in density between the two species. We exploited the fact that means and variances are generally positively correlated. If interspecific competition occurs, we predicted a reduction in mean and an increase in variance in clutch size in great tit and blue tit when density of heterospecifics is higher than the density of conspecifics, and for intraspecific competition, this reduction would occur when density of conspecifics is higher than the density of heterospecifics. Such comparisons of temporal patterns of means and variances revealed evidence, for both species, consistent with intraspecific competition and to a smaller extent with interspecific competition. These findings suggest that competition associated with reproductive behaviour between blue and great tits is widespread, but also varies across large spatial and temporal scales.

Neither artificial light at night, anthropogenic noise nor distance from roads are associated with oxidative status of nestlings in an urban population of songbirds.

Increasing urbanization is responsible for road-related pollutants and causes an unprecedented increase in light and noise pollution, with potential detrimental effects for individual animals, communities and ecosystems. These stressors rarely act in isolation but studies dissecting the effects of these multiple stressors are lacking. Moreover, studies on urban stressors have mainly focused on adults, while exposure in early-life may be detrimental but is largely ignored. To fill this important knowledge gap, we studied if artificial light at night, anthropogenic noise and road-related pollution (using distance from roads as a proxy) explain variation in oxidative status in great tit nestlings (Parus major) in an urban population. Artificial light at night, anthropogenic noise and distance from roads were not associated with variation of the nine studied metrics of oxidative status (superoxide dismutase-SOD-, glutathione peroxidase-GPX, catalase-CAT-, non-enzymatic total antioxidant capacity-TAC-, reduced glutathione-GSH-, oxidized glutathione-GSSG-, ratio GSH/GSSG, protein carbonyls and thiobarbituric acid reactive substances-TBARS). Interestingly, for all oxidative status metrics, we found that there was more variation in oxidative status among individuals of the same nest compared to between different nests. We also showed an increase in protein carbonyls and a decrease of the ratio GSH/GSSG as the day advanced, and an increase of GPX when weather conditions deteriorated. Our study suggests that anthropogenic noise, artificial light at night and road-related pollution are not the most important sources of variation in oxidative status in great tit nestlings. It also highlights the importance of considering bleeding time and weather conditions in studies with free-living animals.

Effects of experimentally sustained elevated testosterone on incubation behaviour and reproductive success in female great tits (Parus major).

In many seasonally breeding birds, female and male testosterone (T) levels peak at the start of the breeding season, coinciding with pair bonding and nesting activities. Shortly after the onset of egg laying, T levels slowly decline to baseline levels in both sexes, but more rapidly so in females. During this period, T in males may still function to facilitate territorial behaviour, mate guarding and extra pair copulations, either via short lasting peaks or elevated basal levels of the hormone. In some species, however, males become insensitive to increased T after the onset of egg laying. It has been postulated that in these species bi-parental care is essential for offspring survival, as T is known to inhibit paternal care. However, only very few studies have analysed this for females. As females are heavily involved in parental care, they too might become insensitive to T after egg laying. Alternatively, because territorial defence, mate guarding and extra pair copulations are expected to be less important for females than for males, they may not have had the need to evolve a mechanism to become insensitive to T during the period of maternal care, because their natural T levels are never elevated during this part of the breeding season anyway. We tested these alternative hypotheses in female great tits (Parus major). Male great tits have previously been shown to be insensitive to T after egg laying with regard to nestling feeding behaviour (but not song rate). When females had started nest building, we experimentally elevated their T levels up to the nestling feeding phase, and measured incubation behaviour (only females incubate) and reproductive success. T did not significantly affect nest building or egg laying behaviour, although egg laying tended to be delayed in T females. Females with experimentally enhanced T maintained lower temperature during incubation but did not spend less time incubating. This might explain the reduced hatching success of their eggs, smaller brood size and lower number of fledglings we found in this study. As in this species T-dependent behaviour by females during the phase of parental care is not needed, the results support the hypothesis that in this species the need for selection in favour of T-insensitivity did not occur.

Influence of mate preference and laying order on maternal allocation in a monogamous parrot species with extreme hatching asynchrony.

It is well established that in many avian species, prenatal maternal resource allocation varies both between and within clutches and may affect offspring fitness. Differential allocation of maternal resources, in terms of egg weight and yolk composition, may therefore allow the female to adjust brood reduction and to fine-tune reproductive investment in accordance with the expected fitness returns. The adaptive value of such maternal resource allocation is thought to be context-dependent as well as species-specific. We investigated the effects of female preference for her mate on the allocation of prenatal maternal resources in the budgerigar, Melopsittacus undulatus, a monogamous species of parrot that shows an extreme hatching asynchrony. We assessed mate preferences in a two-way preference test and allowed females two breeding rounds: one with the preferred and one with the non-preferred partner. We found no effect of preference on either latency to lay or clutch size, but females mated with the preferred partner laid eggs that contained significantly more yolk. Their eggs also contained significantly more androstenedione but not testosterone. Our results suggest that in this species, female preference may influence maternal resource allocation, and that the functional roles of each androgen in the yolk should be considered separately. In addition, we found a significant effect of laying order on egg and yolk weight as well as on yolk testosterone and androstenedione levels. These measures, however, did not change linearly with the laying order and render it unlikely that female budgerigars compensate for the extreme hatching asynchrony by adjusting within-clutch allocation of prenatal maternal resources.

Females Are More Aggressive Than Males towards Same- and Opposite-Sex Intruders in the Blue Tit (Cyanistes caeruleus).

During the breeding season, aggression is expressed to gain access to resources such as territories and mates and protect offspring. Female aggressiveness has received much less attention than male aggressiveness, and few studies have examined female and male aggressiveness towards intruders of both sexes in the same species. We compared female and male aggressiveness towards same- and opposite-sex intruders during the egg-laying period in blue tits (Cyanistes caeruleus) using simulated territorial intrusions. For each sex, we examined the occurrence of different behavioural responses during agonistic encounters, and compared the intensity and individual consistency of intra- and inter-sexual aggression using same- and opposite-sex taxidermy mounts. Our results show that females are the more aggressive sex. Both sexes showed similar behaviours during simulated intrusions, although females were never observed singing and males never entered the nest box. In females, aggression was predominantly independent of the sex of the intruder, while males sang more from a distance during male-male encounters. The relative levels of aggression (pecking and perching on the mounts) during intra- and intersexual conflicts were consistent for females, but not for males. Females might be under stronger selection for aggressive phenotypes due to nest-hole competition and larger reproductive investments.

Untangling behaviours: independent expressions of female-female aggression and snake-like hissing in the blue tit (Cyanistes caeruleus).

Aggression plays a crucial role in deterring predators and securing resources to promote fitness. Nevertheless, studies focussing on female aggression remain scarce. In songbirds, aggression is prevalent during the breeding season, when same-sex individuals compete for limited resources. Additionally, females of some bird species exhibit snake-like hissing behaviour during incubation presumably to lower predation rates and improve fitness. Such behaviours may co-vary, forming a behavioural syndrome that could constrain trait expression. Here, we investigated a resident population of blue tits (Cyanistes caeruleus), to examine the repeatability and covariation of female-female aggression and hissing behaviour, aiming to determine if these constitute a behavioural syndrome. We quantified female-female aggression during simulated territorial intrusions and measured number of hissing calls in response to a simulated predator intrusion into the nest box. We found that both female-female aggression and hissing behaviour were repeatable traits, and that older females approached the intruder less. However, we found no evidence of covariation between female-female aggression and hissing behaviour. Thus, our findings suggest that female-female aggression and hissing behaviour, although both displayed in a nest defence context, are evolutionarily independent traits in the blue tit.

Anthropogenic noise and light pollution additively affect sleep behaviour in free-living birds in sex- and season-dependent fashions.

Rapid anthropogenic transformation of environments exposes organisms to diverse disturbance factors, including anthropogenic noise pollution and artificial light at night (ALAN). These sensory pollutants interfere with acquisition of, and response to, environmental cues and can be perceived as stressors. Noise pollution and ALAN are often experienced simultaneously, and are thus likely to jointly affect organisms, either additively or interactively. Yet, combined effects of noise pollution and ALAN remain poorly elucidated. We studied combined effects of noise pollution and ALAN on the sleep behaviour of a free-living songbird, the great tit (Parus major). Sleep is widely conserved across animal taxa and fulfils essential functions, and research has demonstrated independent effects of both noise and ALAN on sleep. We measured noise and light levels at nest boxes and used infrared video-recording to assess sleep behaviour. Results did not support interactive effects of noise and ALAN. However, noise pollution and ALAN were both independently related to variation in sleep behaviour, in sex- and season-dependent fashions. Males, but not females, woke up and left the nest box ∼20 min later in the noisiest as compared to quietest environments (range: 44.2-79.4 dB), perhaps because males are more sensitive to acoustical cues that are masked by noise. Furthermore, as the season progressed from November to early March, birds woke up and left the nest box ∼35 min earlier relative to sunrise on territories with the lowest, but not the highest, light levels (range: 0.01-8.5 lux). Thus, the seasonal difference in sleep duration was dampened on light polluted territories. These effects could arise if ALAN interferes with birds' ability to sense and respond to increasing daylength, and could have fitness ramifications. Our study suggests that noise pollution and ALAN exert additive effects on sleep behaviour, and that these effects can be sex- and season-dependent.

Temperature synchronizes temporal variation in laying dates across European hole-nesting passerines.

Identifying the environmental drivers of variation in fitness-related traits is a central objective in ecology and evolutionary biology. Temporal fluctuations of these environmental drivers are often synchronized at large spatial scales. Yet, whether synchronous environmental conditions can generate spatial synchrony in fitness-related trait values (i.e., correlated temporal trait fluctuations across populations) is poorly understood. Using data from long-term monitored populations of blue tits (Cyanistes caeruleus, n = 31), great tits (Parus major, n = 35), and pied flycatchers (Ficedula hypoleuca, n = 20) across Europe, we assessed the influence of two local climatic variables (mean temperature and mean precipitation in February-May) on spatial synchrony in three fitness-related traits: laying date, clutch size, and fledgling number. We found a high degree of spatial synchrony in laying date but a lower degree in clutch size and fledgling number for each species. Temperature strongly influenced spatial synchrony in laying date for resident blue tits and great tits but not for migratory pied flycatchers. This is a relevant finding in the context of environmental impacts on populations because spatial synchrony in fitness-related trait values among populations may influence fluctuations in vital rates or population abundances. If environmentally induced spatial synchrony in fitness-related traits increases the spatial synchrony in vital rates or population abundances, this will ultimately increase the risk of extinction for populations and species. Assessing how environmental conditions influence spatiotemporal variation in trait values improves our mechanistic understanding of environmental impacts on populations.

Testosterone increases repertoire size in an open-ended learner: an experimental study using adult male European starlings (Sturnus vulgaris).

Song in songbirds is a learned secondary sexual behavior, first acquired during a sensitive phase of juvenile development, which is affected by hormones such as testosterone (T). While the latter has received much attention, the potential involvement of T in the adult repertoire changes observed in a number of species is much less understood. Yet, this may prove essential to understand the role of song as a sexually selected trait. We therefore performed a T-implantation experiment during the non-breeding season (when T is basal), using adult male European starlings (Sturnus vulgaris), a songbird species in which song repertoire size (and composition) changes seasonally and increases with age. Repertoire size increased rapidly in T-males, but not in control males, indicating a role for T in repertoire size changes. This increase resulted from a lower proportion of dropped song types in T-males than in control males, while the proportion of added song types did not differ between both groups. Interestingly, the observed repertoire turnover (adding and removing song types from the repertoire) in both groups, suggests that elevated plasma T levels were not essential for changes in repertoire composition (contrary to repertoire size). Finally, T-males (but not control males) significantly increased their song rate, while neither group showed a significant change in their song bout length and phrase repetition rate. Taken together, our results suggest a role for T in adult song learning and provide new insights into the information content of repertoire size and song bout length as sexually selected traits.

Testosterone stimulates the expression of male-typical socio-sexual and song behaviors in female budgerigars (Melopsittacus undulatus): An experimental study.

The hormonal control of sex differences in behavior has been extensively studied, particularly in mammals and birds. Studies have shown that the activational potential of the androgenic sex steroid testosterone (T) on male-typical behaviors in females seems to be species- as well as behavior-specific in birds. It is therefore important to study the activational effects of T in a great variety of bird species and on a wide range of behaviors, preferably in social conditions that favor their expression. Here, we investigated the activational effects of T on vocal, socio-sexual (i.e. affiliative and non-vocal courtship behaviors), aggressive and approach behavior in females of the budgerigar, Melopsittacus undulatus, a highly social monogamous parrot species. We experimentally supplemented T-females with male-like plasma T levels compared to controls. First, we observed females when they were individually housed. We found that T-females performed male-like levels of warbling song, sang significantly longer, but not more song bouts and produced more socio-sexual behaviors than controls. Then, we consecutively confronted females with a female, a dummy, and a male conspecific. T-females showed a significantly shorter latency to interact in all three social contexts. In both intrasexual and intersexual contexts, T-females performed significantly higher levels of approach and socio-sexual behavior, including "mounting (attempts)", a strictly male behavior, which was not observed in control females. Aggression in a non-reproductive context did not appear to be sensitive to T supplementation. Our data indicate that in the budgerigar even marked sex differences in socio-sexual behavior may depend on the activational effects of T, while this is generally not the case in other species.

An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Research.

Animals have evolved with natural patterns of light and darkness. However, artificial light is being increasingly introduced into the environment from human infrastructure and recreational activity. Artificial light at night (ALAN) has the potential to have widespread effects on animal behavior, physiology, and fitness, which can translate into broader-scale effects on populations and communities. Understanding the effects of ALAN on free-ranging animals is non-trivial due to challenges such as measuring levels of light encountered by mobile organisms and separating the effects of ALAN from those of other anthropogenic disturbance factors. Here we describe an approach that allows us to isolate the effects of artificial light exposure on individual animals by experimentally manipulating light levels inside nest boxes. To this end, a system can be used consisting of light-emitting diode (LED) light(s) adhered to a plate and connected to a battery and timer system. The setup allows exposure of individuals inside nest boxes to varying intensities and durations of ALAN while simultaneously obtaining video recordings, which also include audio. The system has been used in studies on free-ranging great tits (Parus major) and blue tits (Cyanistes caeruleus) to gain insight into how ALAN affects sleep and activity patterns in adults and physiology and telomere dynamics in developing nestlings. The system, or an adaptation thereof, could be used to answer many other intriguing research questions, such as how ALAN interacts with other disturbance factors and affects bioenergetic balance. Furthermore, similar systems could be installed in or near the nest boxes, nests or burrows of a variety of species to manipulate levels of ALAN, evaluate biological responses, and work towards building an interspecific perspective. Especially when combined with other advanced approaches for monitoring the behavior and movement of free-living animals, this approach promises to yield ongoing contributions to our understanding of the biological implications of ALAN.

Songs differing in consistency elicit differential aggressive response in territorial birds.

Acoustic signals during intrasexual interactions may help receivers to establish the cost and benefits of engaging in a confrontation versus avoiding the cost of escalation. Although birdsong repertoires have been previously suggested as providing information during agonistic encounters, the cost (time/neural resources) of assessing large repertoires may decrease the efficiency of the signal for mutual assessment. Acoustic-structural features may, therefore, be used to enable a fast and accurate assessment during this kind of encounters. Recently, it has been suggested that the consistency of songs may play a key role during intrasexual interactions in bird species. Using a playback experiment in a colour-ringed great tit population, we tested the hypothesis that songs differing in consistency may elicit a differential response, indicating that the signal is salient for the receivers. Great tit males clearly responded more aggressively towards highly consistent songs. Our findings, together with previous evidence of increased song consistency with age in the great tit, suggest that song consistency provides information on experience or dominance in this species, and this phenomenon may be more widespread than currently acknowledged.

Little parental response to anthropogenic noise in an urban songbird, but evidence for individual differences in sensitivity.

Anthropogenic noise exposure has well-documented behavioral, physiological and fitness effects on organisms. However, whether different noise regimes evoke distinct responses has rarely been investigated, despite implications for tailoring noise mitigation policies. Urban animals might display low responsiveness to certain anthropogenic noise regimes, especially consistent noise (e.g. freeway noise), but might remain more sensitive to more diverse noise regimes. Additionally, whether individuals differ in noise sensitivity is a rarely explored issue, which is important to fully understand organismal responses to noise. To address these knowledge gaps, we used a field experiment to measure how urban great tits (Parus major) altered parental behaviors in response to two noise regimes: consistent freeway noise, and a diverse anthropogenic noise regime that incorporated variability in noise type and temporal occurrence. We also evaluated whether sex, age, or a well-described personality trait, novel environment exploration behavior, were associated with responses to noise, although our power to assess individual differences in responses was somewhat limited. We found no evidence for mean population-level changes in nestling provisioning behaviors during either noise treatment. However, despite this overall canalization of behavior, there was evidence for individual differences in noise sensitivity, particularly during the diverse noise treatment. Females and birds that explored a novel environment more rapidly (fast explorers) reduced nestling provisioning rate more relative to baseline levels than males and slow explorers during the diverse urban noise, but not during the consistent freeway noise. Furthermore, first year breeders and fast explorers displayed larger increases in latency to return to the nest box relative to baseline conditions during the diverse noise only. Results suggest that urban animal populations might become overall tolerant to anthropogenic noise, but that certain individuals within these populations nonetheless remain sensitive to certain types of noise exposure. CAPSULE: In an urban songbird, we found no population-level changes in nestling provisioning behavior during noise exposure, but did find evidence for individual differences in noise sensitivity.

Variable and consistent traffic noise negatively affect the sleep behavior of a free-living songbird.

Anthropogenic noise is a ubiquitous disturbance factor, which, owing to the extensive nature of transportation networks, and ability of sound waves to penetrate distances, has wide-reaching impacts on biological communities. Research effort on biological effects of anthropogenic noise is extensive, but has focused on waking behavior, and to our knowledge, no published experimental study exists on how noise affects sleep in free-living animals. Sleep plays vital functions in processes such as cellular repair and memory consolidation. Thus, understanding the potential for noise to disrupt sleep is a critical research objective. Whether different noise regimes exert distinct effects on behavior also remains poorly understood, as does intraspecific variation in noise sensitivity. To address these knowledge gaps, we used a repeated-measures field experiment involving broad-casting traffic noise recordings at great tit (Parus major) nest boxes over a series of consecutive nights. We evaluated whether increasing the temporal variability and amplitude of traffic noise increased deleterious effects on sleep behavior in free-living great tits, and whether individuals differed in the magnitude of responses. We found that traffic noise reduced sleep duration, proportion, and bout length, and induced birds to exit nest boxes earlier in the morning. There was some support for a stronger effect of more variable noise, and relative to lower amplitude noise, higher amplitude noise resulted in less and more fragmented sleep. Effects of noise on sleep duration were stronger in older adults, and substantial, repeatable variation existed in individual responses. We demonstrate for the first time that anthropogenic noise can have strong effects on sleep in free-living animals, which may have cascading effects on waking behavior, physiology and fitness. Results suggest that reducing the amplitude of traffic noise may be an effective mitigation strategy, and that differences in individual sensitivity are important to consider when evaluating effects of noise exposure. CAPSULE: Experimental exposure to temporally variable and consistent traffic noise negatively affected sleep behavior in a free-living songbird.

Serotonin transporter (SERT) polymorphisms, personality and problem-solving in urban great tits.

Understanding underlying genetic variation can elucidate how diversity in behavioral phenotypes evolves and is maintained. Genes in the serotonergic signaling pathway, including the serotonin transporter gene (SERT), are candidates for affecting animal personality, cognition and fitness. In a model species, the great tit (Parus major), we reevaluated previous findings suggesting relationships between SERT polymorphisms, neophobia, exploratory behavior and fitness parameters, and performed a first test of the relationship between single nucleotide polymorphisms (SNPs) in SERT and problem-solving in birds. We found some evidence for associations between SERT SNPs and neophobia, exploratory behavior and laying date. Furthermore, several SNPs were associated with behavioral patterns and success rates during obstacle removal problem-solving tests performed at nest boxes. In females, minor allele homozygotes (AA) for nonsynonymous SNP226 in exon 1 made fewer incorrect attempts and were more likely to problem-solve. In both sexes, there was some evidence that minor allele homozygotes (CC) for SNP84 in exon 9 were more likely to problem-solve. Only one SNP-behavior relationship was statistically significant after correcting for multiple comparisons, but several were associated with substantial effect sizes. Our study provides a foundation for future research on the genetic basis of behavioral and cognitive variation in wild animal populations.

The serotonin transporter gene and female personality variation in a free-living passerine.

Quantifying variation in behaviour-related genes provides insight into the evolutionary potential of repeatable among-individual variation in behaviour (i.e. personality). Yet, individuals typically also plastically adjust their behaviour in response to environmental conditions and/or age, thereby complicating the detection of genotype-phenotype associations. Here, using a population of free-living great tits (Parus major), we assessed the association between single nucleotide polymorphisms (SNPs) in the serotonin transporter gene (SERT) and two repeatable behavioural traits, i.e. female-female aggression and female hissing behaviour. For female-female aggression, a trait showing age-related plasticity, we found no evidence for associations with SERT SNPs, even when assessing potential age-dependent effects of SERT genotype on aggression. We also found no strong support for associations between SERT SNPs and hissing behaviour, yet we identified two synonymous polymorphisms (exon 13 SNP66 and exon 12 SNP144) of particular interest, each explaining about 1.3% of the total variation in hissing behaviour. Overall, our results contribute to the general understanding of the biological underpinning of complex behavioural traits and will facilitate further (meta-analytic) research on behaviour-related genes. Moreover, we emphasize that future molecular genetic studies should consider age-dependent genotype-phenotype associations for behavioural trait (co)variation, as this will vastly improve our understanding of the proximate causes and ultimate consequences of personality variation in natural populations.

Acute stress induces a rapid increase of testosterone in a songbird: implications for plasma testosterone sampling.

Testosterone (T) and glucocorticoids, such as corticosterone, have both been shown to be important for the way vertebrates in general, and birds specifically, react to their immediate environment. For both corticosterone and T, many sources of variation in plasma levels have been demonstrated. Interestingly, a small number of studies on bird species have indicated that acute stress can have a positive effect on plasma T levels, analogous to what has been observed for plasma corticosterone levels. Using captive male European starlings (Sturnus vulgaris), we provide the first evidence in songbirds of a (twofold) increase in plasma T levels after the onset of acute stress, as elicited with a capture-handling-restraint method, when comparing plasma T levels during the first 4 min with plasma T levels between 12 and 33 min. Furthermore, no significant change in plasma T levels was observed within the first 4 min after the onset of acute stress. Notably, although plasma corticosterone was also significantly elevated, the stress-induced change in levels of corticosterone and T were not significantly correlated. Our findings indicate that, when measuring plasma T levels, it may be essential to use a standardized method with fast capture and blood sampling (i.e. within 3-4 min), similar to the method for corticosterone sampling. Furthermore, it is necessary for future studies to examine the effect of using different capture techniques on measured plasma T levels, which may be of particular importance when interpreting T samples gathered in field studies.