Habitat Urbanization, Circulating Glucose and Carotenoid Levels, and Body Condition Predict Variation in Blood Ketone Levels in House Finches (Haemorhous mexicanus) from the American Southwest

Simple Summary

Finding better ways to assess the health of wild animals is important for understanding their overall condition and their vulnerability to diseases and other threats. New portable tools now allow scientists to quickly measure important nutrients and energy sources in animals directly in the field. One type of energy source, called ketones, shows when animals are using fat for fuel, but little is known about ketones in wild birds. In this study, we measured blood ketone levels in male House Finches, a common bird across North America, during summer and winter. We explored how ketone levels were related to body condition, blood sugar, vitamins, pigments, and habitat type (urban/suburban/rural). We found that ketone levels were linked to blood sugar and habitat type, and that birds with higher pigment levels had lower ketone levels in winter. Our results show that measuring ketones can help assess bird health, providing important information about their nutrition and susceptibility to pathogens and other stressors in different environments.

Abstract

Real-time health assessment is crucial for diagnosing emerging threats to wildlife. Point-of-care instruments now allow detailed, affordable measurements of blood metabolites (e.g., glucose, triglycerides, ketones) in free-ranging animals. Ketones, however, remain understudied, especially in relation to environmental and life-history traits. Here, we assessed blood ketone variation in male House Finches (Haemorhous mexicanus) across two seasons (summer and winter) as a function of body condition, circulating glucose, carotenoids, lipid-soluble vitamins, and habitat urbanization (urban/suburban/rural). In both seasons, the interaction between capture site and glucose concentration predicted ketone levels: urban and suburban birds showed a negative relationship, while in summer, rural birds showed a positive one. Additionally, in winter, ketone levels were negatively associated with plasma carotenoids, indicating birds with higher carotenoid levels had lower ketone concentrations. These findings suggest that similar to patterns seen in biomedical research and our previous work on carotenoids and health, ketone status can serve as a valuable indicator of nutritional condition and fat metabolism in wild birds, particularly in the context of urbanization.

1. Introduction

Human activities and population growth continue to threaten wildlife health and biodiversity across the globe [1]. Assessing the health state of organisms in real time—to diagnose emerging maladies and threats before populations and species disappear—has thus become increasingly important to conservation biologists [2]. There are a variety of metrics traditionally used in health assessment of wild animals [3], some of which are challenging or expensive (e.g., immunological or disease diagnoses) to employ. However, point-of-care devices have been used recently as a relatively inexpensive, accessible means of monitoring the dynamic nutritional and health state of free-ranging organisms [4,5]. The measurement of circulating biochemicals like glucose [6,7,8], ketones [9,10], uric acid [11,12], cholesterol [13,14], and triglycerides [4], for example, can provide a wealth of real-time information about the nutritional physiology and overall well-being of wild animals.

Past research on these plasma biochemical markers has focused on correlations among the nutritional–physiological parameters, as well as tracked impacts of rapid environmental changes on circulating levels. For instance, in House Finches (Haemorhous mexicanus), blood glucose concentration has been positively linked to poxvirus infection [8], a common avian disease that may increase energetic demand and alter glucose metabolism as part of an inflammatory immune response. Blood glucose concentration has also been positively linked to triglyceride concentration [4] and negatively related to ketone concentration in Pale-Bellied Tyrant-Manakins [15]. Body condition also positively reveals glucose levels in some songbird species, such as the House Finch (Haemorhous mexicanus) and the Pale-bellied Tyrant-Manakin (Neopelma pallescens) [8,15,16], with a diet-dependent relationship observed in wild rodents [e.g., Apodemus sylvaticus] and laboratory strains of Mus musculus, and the absence of a relationship observed in amphibians [e.g., Xenopus laevis, Rana catesbeiana], lizards [e.g., Tropidurus catalanensis] [17], and tropical frogs [e.g., Boana bischoffi, Scinax fuscovarius] [18]. In addition, the intensity of urban development in an area was an important predictor of lower glucose concentrations [18,19], and of genes related to breakdown of lipids and carbohydrates in White-Footed Mice [20]. Also, it was found that disturbed environments (sewage inputs in marshes) depleted triglyceride storage in marsh frogs [21]. These results underscore the significant links among different physiological parameters and the role that environmental variation can have in shaping nutritional and metabolic profiles. Overall, we still need more information on how different nutritional or metabolic pathways are perturbed by specific human-induced environmental modifications [22].

Ketones are informative yet underused biomarkers in ecological studies of wildlife nutrition and metabolism. Unlike glucose and triglycerides, which reflect short-term energy intake or storage, ketones indicate how animals respond to energetic deficits from food scarcity, fasting, or carbohydrate-poor diets, common in urban settings [23]. Triggered when glucose is depleted, ketogenesis supports energy demands through lipid catabolism and serves as a marker of nutritional stress [22]. Urban animals often face unpredictable and imbalanced diets, rich in carbohydrates but low in protein and micronutrients [24], which can destabilize glucose regulation and increase ketone production. Elevated ketones have been documented in urban birds like Great Tits dependent on feeders [24], suggesting altered foraging can shift metabolic strategies.

Consistent with this, broader research in urban ecology has demonstrated that access to anthropogenic food can influence body condition, oxidative balance, and reproductive output across a variety of species [25,26,27,28], reinforcing the metabolic consequences of urban living. Additionally, fasting intervals between meals in cities may further drive ketone use. Despite their ecological relevance, ketones remain underexamined in free-ranging birds. These compounds—ß-hydroxybutyrate, acetoacetate, and acetone—are produced in the liver from fatty acids [23] and support metabolism during glucose scarcity, as shown in wintering tits and hibernating ground squirrels [10,24]. However, chronic elevation may impair metabolic balance, potentially leading to harmful ketosis [22]. Although early studies suggest links to life-history and environmental variation [29,30], more research is needed. This study explores ketone variation in songbirds to better understand their metabolic responses under natural conditions.

Previous studies on avian ketone bodies found a negative association between blood ketone and glucose levels, and that plasma total antioxidant activity was negatively related to higher levels of ketones in chickens [31], suggesting that ketogenesis may play an important role in health. In addition, higher plasma ß-hydroxybutyrate was found in several migratory passerines and shorebirds [32,33,34,35] before and during migration seasons. Also, Great Tits that ate frequently from bird-seed feeders had higher blood ketone concentrations compared to sites with sparsely and less abundant distributed feeders [24], providing support for the idea that the nutritional physiology of birds in urban and suburban locations is significantly shaped by human-provided food. This phenomenon has been widely documented across species, with studies showing that access to anthropogenic food sources can alter metabolism, oxidative stress, and reproductive output [28,29,30,31,32]. However, the direction and magnitude of these effects appear to be species- and context-dependent.

Here, we investigated a series of predictors of variation in blood ketone levels in House Finches, a passerine species found freely ranging in both natural and human-modified landscapes across Mexico and the United States of America [36]. Previous studies have shown that urban male and female House Finches exhibit drabber carotenoid-containing plumage compared to their non-urban counterparts [37]. Urban birds were also found to have higher carotenoid levels circulating through blood, suggesting they may be more efficient at absorbing carotenoids from their diet or require fewer carotenoids for free-radical scavenging [38]. In the present study, we focused exclusively on males to control for potential sex-related differences in physiology, hormonal status, and carotenoid allocation during reproduction. Female birds may invest carotenoids differently depending on reproductive stage, which could introduce variability unrelated to our focal ecological or nutritional gradients. However, prior studies on ketones in finches from Arizona, USA, found a lack of association between ketones and male plumage hue as well as several health/environmental variables, including glucose, uric acid, triglycerides, degree of urbanization, and poxvirus infection [4]. Several factors may explain this absence of detectable associations. First, previous work may have occurred during periods of low physiological stress, such as times of food abundance, when metabolic differences between habitats were minimized. Second, prior sampling may have lacked sufficient spatial or environmental contrast (e.g., subtle urban gradients or limited rural representation) to detect metabolic divergence. Third, some confounding variables—such as diet composition, reproductive state, or time since last feeding—may not have been adequately controlled or accounted for. In this study, we aimed to improve upon these limitations by sampling birds across a broader urban–rural gradient and integrating a wider array of physiological variables, including antioxidant vitamins and body condition, to better capture ecological and metabolic variation.

Hence, the main objective of this study was to analyze if and how ketone levels in male house finches may relate to other nutritional–physiological variables, such as glucose, vitamins—specifically alpha-tocopherol (vitamin E), a lipid-soluble antioxidant that protects cells from oxidative damage, and retinol (vitamin A), which plays key roles in immunity and epithelial health—circulating levels of carotenoids (diet-derived pigments with antioxidant and immunomodulatory functions), and degree of habitat urbanization. Each of these physiological markers reflects distinct nutritional and metabolic pathways that may interact with ketone metabolism. Glucose provides immediate energy, and its scarcity can trigger ketogenesis. Carotenoids serve as dietary antioxidants and immune modulators, often reflecting diet quality and absorption efficiency. Tocopherol (vitamin E) and retinol (vitamin A) are lipid-soluble vitamins involved in oxidative stress regulation and immune function. Lower availability or higher usage of these antioxidants may be associated with increased oxidative burden, potentially leading to altered energy allocation and reliance on ketone-based energy pathways. We hypothesized that male finches in urban environments would exhibit higher ketone levels due to increased nutritional stress and altered metabolic conditions, primarily related to food and glucose availability. Urban-specific challenges such as potentially limited access to diverse food sources and inconsistent glucose intake could drive these metabolic shifts, setting urban finches apart from their suburban and rural counterparts. Also, we expected that urban birds with a lower body condition, as well as lower antioxidant compounds, may show a higher ketone concentration due to greater breakdown and utilization because of lower glucose availability or lower ketone concentrations if ketones have already been extensively utilized.

2. Materials and Methods

2.1. Data Collection

In summer 2020 (3–7 August), we captured 43 hatch-year male House Finches, and in winter 2020–2021 (25–30 January 2021), we trapped 87 after-hatch-year males, sexed by plumage differences. We sampled hatch-year individuals during summer and after-hatch-year individuals during winter, reflecting the natural age class availability in each season; this approach also allowed us to capture representative metabolic responses typical of each seasonal cohort. In both seasons, we used live traps surrounding sunflower seed feeders at the following sites: Arizona State University Tempe Campus (latitude 33°25′11.4″ N, longitude 111°55′49.5″ W), South Mountain Regional Park (latitude 33°21′01.9″ N, longitude 111°55′49.5″ W), and a residential backyard in Tempe, AZ (latitude 33°20′41.1″ N, longitude 111°55′49.5″ W). We selected these three sampling locations because, based on our prior studies, they are representative of urban, rural, and suburban study sites [39,40]. At capture, we measured body mass (recorded to the nearest 0.1 g using a digital balance) and tarsus length (measured to the nearest 0.1 mm with digital calipers). We estimated body condition (sensu [8]) separately in each season, using a generalized linear mixed model (using the glmer function from the lme4 package [41]) to extract the residuals of a body mass–tarsus length regression. We also entered capture location (urban, suburban, and rural) as a random term and found that, for individuals captured in both summer 2020 (Estimate = 0.70, se = 0.30, p-value = 0.02, r² = 0.11) and winter 2020–2021 (Estimate = 0.55, se = 0.16, p-value < 0.01, r² = 0.11), the mass–tarsus regressions were positive and statistically significant. We included capture location as a random effect in the body condition index model to account for potential site-level variation in the relationship between body mass and tarsus length, as our aim was to control for spatial structure in condition estimation. Also, at capture, we used a 26-gauge needle to draw fresh blood from the brachial vein of all birds and collected approximately 80–100 μL (<1% of bird weight) whole blood into heparinized capillary tubes, and used a drop of blood each to estimate circulating glucose (in mg/dL, AccuChek^® Guide blood glucose meter, Roche Diabetes Care Inc., Indianapolis, IN, USA) and ketone (in mmol/L, β-hydroxybutyrate, Precision Xtra^® blood glucose, and ketone monitoring system, Abbott Laboratories, Abbott Park, IL, USA) concentrations. In our previous work, we demonstrated that these blood–nutritional parameters can be consistently measured using point-of-care (POC) devices, highlighting their reliability and cost-effectiveness as tools for monitoring real-time nutritional physiology in wild birds [4]. Time elapsed between trapping and blood collection was consistent across individuals, with all trapping conducted in the morning between sunrise and 10 AM. The remainder of the blood sample was centrifuged to obtain plasma that was frozen at −80 °C to later measure circulating carotenoids and lipid-soluble vitamins (see more below). We measured glucose levels twice in succession for 22 individuals in August (Estimate = 0.96, se = 0.06, p-value > 0.01, r² = 0.91), and 17 individuals in the winter (Estimate = 0.94, se = 0.08, p-value > 0.01, r² = 0.87), and found that intra-individual measurements were highly repeatable. Due to the high costs of ketone strips, we did not conduct multiple measurements on any individual birds, but in prior work we showed high repeatability of the blood ketone assay for house finches as well [4]. After processing, all individuals were released at their capture site.

2.2. Circulating Levels of Carotenoids and Vitamins

We assessed plasma carotenoid and lipid-soluble vitamin concentrations (vitamin E = alpha-tocopherol, vitamin A = retinol) using high-performance liquid chromatography (HPLC) (sensu [42]). Carotenoids and vitamins were extracted using organic solvents [43], followed by the centrifugation of the solutions at 10,000 rpm for 3 min. The resulting supernatants were transferred to labeled tubes and subjected to nitrogen-streamed evaporation. Subsequently, we added 200 µL of HPLC mobile phase (composed of methanol, acetonitrile, and dichloromethane at a ratio of 42:42:16, v:v:v), vortexed the sample briefly, and snap-centrifuged to remove any residual protein. Analysis was performed using an Agilent HPLC (Alliance 2695, Waters Corp. Milford, MA, USA) and Open Lab Software version A.01.04 (Agilent Technologies, Santa Clara, CA, USA) to determine area under the curve for various detected compounds. For the summer plasma samples, we recovered alpha-tocopherol, retinol, beta-cryptoxanthin, lutein, astaxanthin, zeaxanthin, canthaxanthin, beta-carotene, 3′-hydroxy-echinenone, and two isomers of 4-oxo-rubixanthin. In winter, we recovered alpha-tocopherol, retinol, violaxanthin, antheraxanthin, lutein, zeaxanthin, beta-cryptoxanthin, and beta-carotene. We calculated concentrations (in μg/mL) of each carotenoid [44], and in both seasons summed all carotenoid levels within samples to determine total carotenoid concentrations.

2.3. Statistical Analysis

We initially visualized the distribution of all numeric variables through histograms, namely body condition and levels of plasma carotenoids, lipid-soluble vitamins, glucose, and ketones. We log-transformed glucose and total carotenoid concentrations to achieve normal distributions. We then tested for multicollinearity among predictors using a Pearson correlation matrix and found various statistically significant correlations (Supplementary Tables S1 and S2). Therefore, for plasma samples from summer 2020, we retained body condition and concentrations of glucose, lutein, and 3′-hydroxy-echinenone as uncorrelated predictors. For winter 2020–2021, we retained body condition as well as glucose and total carotenoid concentrations. We then produced two generalized linear models (GLMs, Gaussian family), one for summer and one for winter. In addition to examining the main effects of these aforementioned variables in our models, we tested interactions of each predictor with capture site in both seasons. We used the stepAIC function from the MASS package [45] to generate the best model based on the lowest AIC value, with the direction parameter set as “both”. We then used the ggplot2 package [46] to plot all statistically significant variables. All analyses were performed using the R software version 4.3 [47].

3. Results

The best-fit model for predicting blood ketone concentrations during summer included glucose, capture site, and the interaction between glucose and capture site (

Table 1. Full and best-fit models for each dataset (summer and winter seasons) separately. Here we show the r-squared, AIC, variables, estimates, t-value (t), and p-value values for each model. Statistically significant variables are marked with an asterisk.

Models	Variables	Estimate	t	p-Value
Summer 2020
Best model	Intercept A	3.97	6.01	<0.01 *
r2 = 0.35	Glucose	−0.00	−3.07	<0.01 *
AIC = 37.71	Sampling location (suburban)	−1.04	−0.94	0.35
	Sampling location (rural)	−4.99	−3.26	<0.01 *
	Sampling location (suburban) × Glucose B	0.00	0.95	0.34
	Sampling location (rural) × Glucose B	0.01	3.04	<0.01 *
Winter 2020–2021
Best model	Intercept A	27.31	4.02	<0.01 *
r2 = 0.55	Glucose	−3.90	−3.22	<0.01 *
AIC = 221.31	Sampling location (suburban)	−2.82	−0.27	0.78
	Sampling location (rural)	−22.19	−2.51	0.01 *
	Total circulating carotenoid concentration	−0.27	−3.18	<0.01 *
	Sampling location (suburban) × Glucose C	0.16	0.08	0.92
	Sampling location (rural) × Glucose C	3.71	2.34	0.02 *

^A Reference levels: sampling location—urban. ^B Changing the reference level to suburban—sampling location (urban): estimate = −0.00, t-value = −0.95, p-value = 0.34; sampling location (rural)—estimate = 0.01, t-value = 2.23, p-value = 0.03. The reference level to rural: sampling location (suburban)—estimate = −0.01, t-value = −2.31, p-value = 0.03; sampling location (urban)—estimate = −0.01, t-value = −3.04, p-value < 0.01. ^C Changing the reference level to suburban: sampling location (urban)—estimate = −0.16, t-value = −0.08, p-value = 0.92; sampling location (rural)—estimate = 3.55, t-value = 2.07, p-value = 0.04. The reference level to rural: sampling location (urban)—estimate = −3.71, t-value = −2.34, p-value = 0.02; sampling location (suburban)—estimate = −3.55, t-value = −2.05, p-value = 0.04.

). Specifically, for urban and suburban males, we found that ketone and glucose concentrations were negatively linked, whereas this relationship was positive in rural house finches (Table 1 and Figure 1).

In winter, the best-fit model for predicting blood ketone levels contained glucose concentration, capture site, total circulating carotenoids, and the interaction between glucose and capture site (Table 1). As in summer, ketone and glucose levels were significantly negatively related in urban and suburban male House Finches, but not rural males (Table 1 and Figure 2). We also found that males with higher total plasma carotenoid concentration had lower ketone levels (Table 1 and Figure 3).

4. Discussion

We investigated the impact of several physiological and environmental factors on variation in blood ketone bodies in male House Finches during two seasons. We found that habitat urbanization significantly influenced the relationship between glucose and ketone concentrations in both summer and winter, suggesting that urbanization affects the trade-offs between different nutritional–physiological parameters. Furthermore, we observed during winter that total carotenoid concentration significantly predicted ketone levels in males, indicating a link between antioxidant stores and metabolic fuel sources in these birds.

First, we found that the relationship between ketone and glucose levels was influenced by capture site during both summer and winter, such that, unlike in rural males, urban and suburban males with higher glucose concentrations had lower ketone concentrations. These results suggest two non-mutually exclusive hypotheses: (a) urban and suburban birds struggling to find energy-rich resources in both summer and winter may need to break down fatty acids in the liver for energy, as indicated by low glucose and high ketone levels. Conversely, birds with sufficient resources used glucose as the primary energy source, resulting in lower ketone levels. This pattern aligns with findings in migratory passerines [35] and seabirds [5], suggesting that nutritional state may indicate foraging effort and success in house finches across seasons. (b) In summer, rural birds with high glucose levels also had high ketone levels (though this relationship was absent in winter), suggesting that rural birds can face greater nutritional [48] and thermoregulatory demands [49], particularly as they were captured immediately following the breeding and molt season cycles, and during one of the hottest months of the year [50]. Consequently, the higher demand for resources during the summer may compel rural birds to rely on ketones in addition to glucose to manage nutritional–physiological stressors. This reliance is particularly pronounced given that urban and suburban birds likely have greater access to feeders, providing a more consistent food supply compared to their rural desert counterparts [51]. While we did not directly measure dietary supplies, our findings suggest that, in urban and suburban locations, the relationship between ketone and glucose accumulation may be closely linked to access to energy-rich food resources.

Additionally, our study revealed that, in winter, birds with higher total carotenoid concentrations had lower ketone levels. Since birds with higher carotenoid levels are often in better overall health [52,53,54,55], they may also exhibit more efficient metabolic processes, allowing them to rely less on ketones, which are energy reserves produced from fatty acids when glucose is low. Carotenoids are known to modulate oxidative stress and immune responses, which may indirectly support metabolic efficiency under environmental challenges. For example, in broiler chickens, high levels of stress (cyclophosphamide and lipopolysaccharide) had a lower jejunal digestion metabolism [56]. Our findings thus suggest for the first time that birds with higher carotenoid concentrations may rely less on fatty acids as their primary energy source, potentially due to their superior overall condition and foraging capacity. This novel insight highlights the intricate link between nutritional status, antioxidant capacity, and energy metabolism in birds, and underscores the potential role of carotenoids in mediating energy metabolism and stress responses. From a conservation perspective, these insights emphasize the importance of maintaining access to high-quality, carotenoid-rich diets—particularly in urban habitats where nutritional imbalance is common. Future work could build on these results by incorporating experimental manipulations of diet quality or oxidative stress, and by expanding sampling to include females, whose reproductive physiology may influence these relationships in distinct ways.

We found no evidence that body condition was a significant predictor of ketone levels, consistent with studies on Pale-Bellied Tyrant-Manakins [15], Wood Thrushes (Hylocichla mustelina [57]), and Pied Flycatchers (Ficedula hypoleuca [58]) during the breeding season. Additionally, we did not find that vitamins (tocopherol and retinol) or any individually measured circulating carotenoid directly predicted ketone concentrations in house finches, either in summer or winter. These findings suggest that overall accumulation of carotenoids, rather than specific carotenoids or vitamins, equips individuals to utilize less lipid as an energy reserve. However, because this is a correlational study, further research is needed during breeding and molting seasons to understand these nutritional–physiological trade-offs and potential directionality of whether or not carotenoids, or ketones, causally influence one another.

We also offer several considerations for future studies of ketones in finches and other birds. Although we did not examine them here, variations in handling stress [59,60,61] and in stages of molt (due to changes in energy demands associated with feather growth [33,62,63,64] will be worth tracking to determine potential influences on blood nutritional –physiological parameters. Increasing the number of urban, suburban, and rural study sites would also strengthen the reliability of findings and provide deeper insights into the effects of urbanization on avian nutritional physiology. As a potential limitation, the seasonal differences in detected carotenoids may reflect biological variation in diet or metabolic turnover, but we cannot fully rule out methodological factors such as detection thresholds; we summed all identified carotenoids to generate a composite measure, which we interpret as a general proxy for antioxidant capacity and dietary quality.

In summary, our study revealed that habitat urbanization affected the relationship between glucose and ketone levels in males from a common species of free-ranging songbird. Urban and suburban male House Finches with higher glucose concentrations had lower ketone levels, whereas in summer, rural birds with higher glucose concentrations also had higher ketone levels. Additionally, higher total carotenoid concentrations in winter predicted lower ketone levels, indicating superior nutritional and physiological condition of these birds, such that they did not require catabolic lipid fuel. Overall, these findings suggest that urbanization influences nutritional and physiological trade-offs, with carotenoids playing a key role in energy metabolism and stress response. Understanding these nutritional and metabolic strategies broadly for avian species is crucial for conservation efforts, as our work highlights the significant impact of rapid environmental change on different aspects of wildlife condition and health. This information, now more readily available using inexpensive and accessible point-of-care devices, can guide more effective management and conservation practices to support wildlife across many different landscapes across the globe.