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Plasma metabolite indices are robust to extrinsic variation and useful indicators of foraging habitat quality in Lesser Scaup

Plasma metabolite indices are robust to extrinsic variation and useful indicators of foraging... Abstract Energy acquisition and storage are important for survival and fecundity of birds during resource-limited periods such as spring migration. Plasma-lipid metabolites (i.e. triglyceride [TRIG], β-hydroxybutyrate [BOHB]) have been used to index changes in lipid stores and, thus, have utility for assessing foraging habitat quality during migration. However, such an index may be affected by energetic maintenance costs, diet, and other factors, and further validation under experimental conditions is needed to understand potential sources of variation and verify existing indices. We evaluated a plasma-lipid metabolite index using 30 female and 28 male wild Lesser Scaup (Aythya affinis; hereafter scaup) held in short-term captivity (~24 hr) during spring migration. Similar to previous observational studies, BOHB was negatively associated and TRIG was positively associated with mass change (R2 = 0.68). BOHB estimates were nearly identical to those published on free-living scaup, but TRIG estimates differed from free-living scaup and varied by sex, with females having a greater rate of predicted mass change than captive and free-living males. Our results suggest TRIG may be a better measure of energy income than deposition because lipid deposition likely varies with energetic maintenance costs, stress, and underlying physiological processes while TRIG relates primarily to energy income. In contrast, BOHB was a reliable predictor of negative mass change across sexes. The sex-based differences in apparent lipid deposition rates warrant further research before a generalizable model is advisable for comparing mass change predictions across studies. However, if predictions are standardized, this technique is generally robust to variations in energy income vs. lipid deposition across sexes. Accordingly, our evaluation provides verification for the utility of plasma-lipid metabolites as an indicator of foraging habitat quality during migration. RESUMEN La adquisición y el almacenamiento de energía son importantes para la supervivencia y la fecundidad de las aves durante los períodos de recursos limitados, como la migración de primavera. Los metabolitos lipídicos del plasma (i.e. triglicéridos [TRIG], β-hidroxibutirato [BOHB]) han sido usados para indexar cambios en las reservas de lípidos y, por ende, tienen utilidad para evaluar la calidad del hábitat de forrajeo durante la migración. Sin embargo, tal índice puede verse afectado por los costos de mantenimiento energético, la dieta y otros factores, y se necesita la validación adicional bajo condiciones experimentales para entender las fuentes potenciales de variación y verificar los índices existentes. Evaluamos un índice de metabolitos lipídicos del plasma usando 30 hembras y 28 machos silvestres de Aythya affinis retenidos en cautiverio por un breve período (~24 h) durante la migración de primavera. De modo similar a estudios observacionales previos, el BOHB estuvo negativamente asociado y los TRIG estuvieron positivamente asociados con el cambio de masa (R2 = 0.68). Las estimaciones de BOHB fueron casi idénticas a aquellas publicadas para individuos libres de A. affinis, pero las estimaciones de TRIG fueron diferentes a las de los individuos libres de A. affinis y variaron según el sexo, con las hembras presentando una tasa mayor de cambio de masa predicho que los machos cautivos y libres. Nuestros resultados sugieren que los TRIG pueden ser una mejor medida de ingreso que de depósito energético debido a que el depósito de lípidos varía probablemente con los costos de mantenimiento energético, el estrés y los procesos fisiológicos subyacentes, mientras que los TRIG se relacionan principalmente con el ingreso energético. En contraste, el BOHB fue un predictor confiable del cambio de masa negativo para ambos sexos. Las diferencias basadas en el sexo en las tasas de depósito aparente de lípidos justifican más investigaciones antes de proponer un modelo generalizable para comparar las predicciones de cambio de masa entre estudios. Sin embargo, si se estandarizan los predictores, esta técnica es generalmente robusta a las variaciones en ingreso energético vs. los depósitos de lípidos para ambos sexos. De este modo, nuestra evaluación brinda una verificación sobre la utilidad de los metabolitos lipídicos del plasma como un indicador de la calidad del hábitat de forrajeo durante la migración. Lay Summary The past several decades have seen a growing body of literature on the utility of plasma-lipid metabolites to assess habitat quality for a multitude of migratory bird species. Research in this area is often focused on 2 key metabolites, β-hydroxybutyrate and triglyceride and their associated relationships with mass change. Triglyceride has a positive relationship with mass change and is often referred to as an indicator of lipid deposition or fattening rates. However, energetic maintenance costs may confound the relationship of triglycerides and lipid deposition. To the best of our knowledge, no studies have attempted to control for potential variation in energy expenditure in conjunction with metabolite sampling during migration. Thus, we believe our paper will be a useful addition to the scientific literature on these metabolites and will be of interest to a broad audience of national and international wildlife managers and avian researchers. INTRODUCTION Wetland loss and degradation have negative consequences for wetland-dependent birds (Czech and Parsons 2002, Ma et al. 2010). Changes in climate, land-use practices, and drainage have contributed to decreases in wetland abundance and quality throughout the upper Midwest and the Prairie Pothole Region (PPR; Dahl 1990, Anteau and Afton 2008a; Anteau et al. 2011, McCauley et al. 2015, McKenna et al. 2017). Wetland degradation can lead to declining food resources (e.g., aquatic vegetation, invertebrates) at important migratory sites, which in turn have direct implications for wetland-obligate species that rely on such habitat resources for nutrient acquisition (Anteau and Afton 2011). Given the myriad of anthropogenic threats to ecological services that wetlands provide, there is a growing need for quick and efficient tools for assessing wetland quality. Avian researchers often employ various measures of body condition as a proxy to quantify fitness of individuals and to make inferences about habitat quality and population health (Brown 1996). The acquisition of energy at migratory stopover areas is paramount for survival and reproduction of many species of migratory birds (Alisauskas and Ankney 1992, McWilliams et al. 2004, Newton 2006, Sedinger and Alisauskas 2014). Many migratory bird species undergo periods of hyperphagia during migration and require high-quality habitat resources at stopover sites to replenish or accumulate lipid reserves (Bairlein 1990, Ramenofsky 1990, Stafford et al. 2014). Plasma-lipid metabolites can be useful for indexing lipid accumulation and catabolism, where free triglycerides (hereafter TRIG) have a positive relationship to mass change and β-hydroxybutyrate (hereafter BOHB) has a negative relationship to mass change (Jenni-Eiermann and Jenni 1994, Williams et al. 1999, Guglielmo et al. 2005, Smith et al. 2007, Anteau and Afton 2008b). Further, plasma-lipid metabolite concentrations provide a quantitative approach to assess spatial and temporal changes in lipid stores and offer utility in assessing foraging habitat quality during migration (Schaub and Jenni 2001, Guglielmo et al. 2002, 2005, Anteau and Afton 2011, Evans-Ogden et al. 2013, Thomas and Swanson 2013). Several researchers have previously used free-living birds to demonstrate that lipid metabolite concentrations vary logically in response to presumed habitat quality (Guglielmo et al. 2005, Seaman et al. 2006, Williams et al. 2007, Lyons et al. 2008). In previous studies, TRIG concentrations have been described as indicators of body mass, mass change, lipid deposition, and fattening rates (Jenni-Eiermann and Jenni 1994, Schwilch and Jenni 2001, Guglielmo et al. 2002, 2005, Owen et al. 2005, Anteau and Afton 2008b, Thomas and Swanson 2013). However, environmental variation in foraging efficiency, maintenance costs, weather events, and disturbance likely influence the allocation of energy income deposited into lipid reserves vs. that which is metabolized outright (Madsen 1995, Biebach 1996, Palm et al. 2013). Because TRIG is present in blood plasma and used during energetically costly activities (Jenni and Jenni-Eiermann 1998, McWilliams et al. 2004), not all TRIG acquired from exogenous sources will be deposited as lipid stores. This is not a trivial concern because during spring migration, environmental conditions or behavioral changes (e.g., pair bonding, maintenance) could change the proportion of free TRIG that gets catabolized or deposited as lipid reserves. Few studies have been able to obtain and track changes in body mass concurrent with plasma metabolite concentrations within a short time period in free-living birds (e.g., 24 hr; Jenni-Eiermann and Jenni 1994, Williams et al. 1999, Anteau and Afton 2008b). Metabolite concentrations can change rapidly (i.e. 10–20 min) in blood plasma of some bird species in response to feeding rates, specifically TRIG and BOHB (Zajac et al. 2006). A primary challenge of this type of study with free-living birds is attaining an adequate sample size of birds recaptured after a short period during which metabolite profiles should reflect mass changes (Guglielmo et al. 2005). For example, Anteau and Afton (2008b) captured, weighed, and marked >3,800 Lesser Scaup (Aythya affinis; hereafter scaup) during 2 spring migration periods and were only able to obtain 22 corresponding metabolite and mass change samples from individuals recaptured within a 24-hr period. Another challenge of studies using free-living specimens is that variation among individuals in feeding rates, diets, energy expenditure (i.e. pairing behaviors, foraging efficiency), and habituation to bait at trap sites may confound the relationship of lipid metabolites to daily mass changes (DMC). Although several evaluations of metabolite changes over time and in response to nutrient acquisition have been conducted using migratory birds held in captivity, none have included waterfowl despite this taxa underpinning conservation planning efforts for wetland and upland habitats throughout North America (North American Waterfowl Management Plan (NAWMP) 2012). Using captive scaup to evaluate TRIG and BOHB relationships with DMC would control for potentially confounding factors such as diet composition and energy expenditures. Moreover, this approach would provide a comparison of energy income and lipid deposition given captive scaup likely have lower energy requirements than free-living scaup (Anteau and Afton 2008b). Such a comparison should provide information regarding the potential utility of TRIG to index lipid deposition rates despite varying environmental conditions that could affect energetic maintenance costs. Additionally, such a comparison could allow for analyses of intrinsic factors (e.g., sex) that may influence plasma-lipid metabolite indices and may clarify an assumption made by Anteau and Afton (2008b) that male and female scaup would exhibit similar metabolite profiles because they consume the same forage items during spring migration. If sex-based differences exist in the relationships of TRIG or BOHB concentrations and mass change, then the index developed based on male-only information may provide biased results in females (Anteau and Afton 2011). Our primary objective was to validate a previously developed plasma metabolite index generated from free-living male Lesser Scaup by controlling for energy expenditure and potential diet effects and testing for differences between sex using captive birds (Anteau and Afton 2008b). We examined the relationship between TRIG, BOHB, and DMC using wild-captured scaup held in short-term captivity and subjected to feeding and fasting treatments (Anteau and Afton 2008b). A metabolite index robust to variation in intrinsic and extrinsic factors would allow a direct evaluation of foraging habitat quality without measuring indirect indicators such as food density, which can be resource-intensive and may not be indicative of nutrient acquisition by foragers. METHODS AND MATERIALS Study Species Scaup are a migratory diving duck species that primarily winter in waters along the Gulf of Mexico, migrates through the Midwest, and breeds from the PPR to the boreal forest of Alaska and Canada (Anteau et al. 2020). Scaup were an ideal study species for this experiment because, as wetland obligates, they derive all their energy from wetland habitat resources (Anteau and Afton 2011). Furthermore, they have exhibited declining population trends and previous investigations have identified female body condition (i.e. low lipid reserves) during spring migration as a likely contributing factor to this decline (Austin et al. 2000, 2006, 2014, Afton and Anderson 2001). Further, a plasma-lipid metabolite index was developed (Anteau and Afton 2008b) and applied to scaup migrating across the landscape during spring migration (Anteau and Afton 2011). Study Area During spring migration 2016 and 2017 (February 20 to March 31), we captured wild scaup at navigation Pool 19 of the Mississippi River (hereafter, Pool 19) near Nauvoo, Illinois. Pool 19 is the largest impounded reservoir on the Mississippi River and extends 70 km from the lock and dam at Keokuk, Iowa northward to lock and dam 18 at Gladstone, Illinois, USA. The lower portion of the pool (i.e. Nauvoo, Illinois southward to Keokuk, Iowa) is wider, has less flow, and is characterized by a substrate that historically supported aquatic vegetation (e.g., Heteranthera dubia, Potamogeton pectinatus, Ceratophyllum demersum, Vallisneria americana, Nelumbo lutea, Sagittaria sp.; Steffeck et al. 1985), but declines in aquatic vegetation communities make benthic invertebrates (e.g., bivalves, gastropods, insects) the primary food of diving ducks on Pool 19 (Thompson 1973, Moore et al. 2010, Hagy et al. 2015). Pool 19 is an important mid-latitude stopover site for scaup, especially during spring migration, and has been a resource for capturing and banding large numbers of scaup since the 1980s (Havera et al. 1992, Anteau and Afton 2009, Arnold et al. 2017). Field Methods We located areas with high numbers of scaup and established bait sites by placing whole kernel corn daily near a post in ~1 m of water until scaup readily fed nearby the site. We deployed modified swim-in/dive-in traps (i.e. funnel traps) baited with corn (Haramis et al. 1982, Anteau and Afton 2008b), monitored traps, and removed birds 2–3 times daily dependent upon trapping success (~0900, 1400, 1800 hr). Following removal from traps, ducks were weighed on a digital balance (±1 g), banded with a U.S. Geological Survey leg band, and transported to outdoor holding pens at Kibbe Field Station in Warsaw, Illinois, USA. To minimize the potential for variation among individuals and unknown food items, we excluded birds from the experiments that had palpable amounts of ingesta in their crop/esophagus at time of capture. Captive Methods We held wild scaup (31 females, 30 males) in captivity for ~2 hr prior to the experiment to further minimize the potential for variation in the amount and type of ingesta present in their guts from wild origins. Our experiment ran for a 24-hr duration, during which time we controlled diet and feeding rates. Following capture, we randomly assigned individuals to 1 of the 2 treatment groups (i.e. fasting or feeding). The goal of the fasting and feeding treatments was to elicit a range of mass loss and gain, respectively, that was similar to that observed from free-living scaup (see Anteau and Afton 2008b). We recognized from pilot work that birds would lose and gain mass at different rates based on individual variation. Scaup selected for fasting treatment were given access to water ad libitum and did not receive food for the duration of stay in captivity. Scaup selected for feeding treatments were provided water ad libitum and manually fed a slurry of food and water with a 60-cc syringe fitted with 30 cm of 4.8-mm diameter, vinyl tubing by inserting the tubing ~3 cm down the esophagus. The slurry was a standardized mixture (gross energy mean = 3,959.42 ± 48.61 cal g–1) of 1.0-part layer crumble (Nutrena Co., Minneapolis, Minnesota, USA), 0.5-part scratch grain (Nutrena Co.), and 0.25-part meal worms (Happy Hen Treats Co., Boerne, Texas, USA). We added water to the food mixture as needed until it flowed through tubing without resistance. We administered food mixture until the esophagus/crop was approximately full as determined by palpation; each feeding bout took ~2 min. Our pilot efforts indicated that feeding to near the point of refusal at 4-hr intervals was required to reasonably ensure that birds in the feeding treatments gained mass during the experiment. To ensure mass gain, we repeated feeding procedures at 4-hr intervals for the duration of the captivity period. Free-living scaup can feed throughout the daily cycle, including nocturnally (Custer et al. 1996, Herring and Collazo 2006). While the volume of food administered was not standardized among individuals, this procedure did standardize the feeding interval and the volume of food relative to the bird’s esophagus, crop, and proventriculus capacity. This procedure simulated foraging bouts to the point of satiation of free-living birds. Prior to entering treatments, we obtained baseline blood samples by extracting 0.5 mL of blood from the tibiotarsal vein anterior the intertarsal joint using a heparinized 1-cc syringe affixed with a 25-ga needle. Approximately 24 hr after recording initial weight and 4-hr post-experimental treatment, we recorded final mass measurements and extracted 0.5 mL of blood as described prior to treatment. We transferred blood to 1.5-mL heparinized micro-centrifuge tubes and centrifuged at 6,000 rpm (2,000× g) for 7 min within 30 min of collection. We extracted plasma using a micropipette, transferred to a sterile vial, and froze within 20 min at –20°C until analysis (Guglielmo et al. 2002, Anteau and Afton 2008b, Janke 2016). Upon completion of experimental procedures, we released scaup near the initial capture site. Animal capture and handling methods used during our study were approved by the Institutional Animal Care and Use Committee at Western Illinois University (#16-06), University of Illinois (#16020), and Illinois Department of Natural Resources (#NH15.5864). The U.S. Geological Survey Bird Banding Laboratory approved markers, blood drawings, and handling methods (#23923). Laboratory Methods We followed established protocols that utilize endpoint assay for measuring total triglycerides (TRIG + glycerol) and glycerol. Free TRIG was calculated by subtracting glycerol from total TRIG (Guglielmo et al. 2002, Anteau 2006, Janke 2016). We measured BOHB by kinetic assay using standard protocols (Guglielmo et al. 2005, Anteau 2006, Janke 2016). Plasma-lipid metabolite assays, both endpoint and kinetic, were completed at Northern Prairie Wildlife Research Center using a microplate spectrophotometer (BioTek, Inc., model EON, Winooski, Vermont, USA). Upon completion of metabolite assays, we censored 2 males and 1 female due to irregular sample quality, thus our analysis included 28 males and 30 females. Data Analysis We used generalized linear models (R Core Team 2017) with a normal distribution and identity link function to evaluate the relationships of sex and plasma-lipid metabolites (i.e. TRIG, BOHB) on mass change of scaup. We examined residuals and determined that the relationship between BOHB and DMC was curvilinear; hence, we natural-log-transformed BOHB (Jenni-Eiermann et al. 2002, Anteau and Afton 2008b). We used backward elimination procedures (α = 0.05) to evaluate main effects and interaction terms for BOHBln, TRIG, and sex. We considered the potential for carryover effects given birds’ recent time spent in the wild and evaluated initial metabolite values as covariates. Baseline values for TRIG and BOHBln prior to entering treatments did not improve model fit (P > 0.426). We used a dataset of BOHBln and TRIG values from female scaup (n = 43) lethally collected at Pool 19 in spring 2004 and 2005 (see Anteau and Afton 2011) to compare our predictive model with that of Anteau and Afton (2008b). We compared both model-predicted values of mass change (g) and Z-standardized model-predicted values of mass change from each index to make comparisons of model correspondence (Janke 2016). We examined summary statistics for mass change predictions and standardized mass change predictions and conducted linear regressions between both indices. We considered perfect correspondence between indices to have a slope = 1, intercept = 0, and r2 = 1. RESULTS Female DMC ranged from –65 to 61 g; 92% of females in the feeding treatment gained mass and 100% of females in the fasting treatment lost mass. DMC for males ranged from –91 to 21 g; 25% of males in the feeding treatment gained mass and 100% of males in the fasting treatment lost mass. Concentrations of TRIG ranged from 0.32 to 1.97 in female scaup and 0.44 to 2.14 (mmol L–1) in male scaup (Table 1). Concentrations of BOHB ranged from 0.19 to 1.61 and 0.21 to 1.40 (mmol L–1) for female and male scaup, respectively (Table 1). Table 1. Sample size (n), minimum, maximum, mean values, and upper (UCI) and lower (LCI) 95% CIs for TRIG and BOHB concentrations and DMC (grams) for captive Lesser Scaup in the fasting (Fast) and feeding (Feed) treatments. . Captive female . . . Captive male . . . . CF alla . Fast . Feed . CM allb . Fast . Feed . n 30 17 13 28 15 13 TRIG Minimum 0.32 0.32 0.73 0.44 0.44 0.50 Maximum 1.97 1.01 1.97 2.14 1.65 2.14 Mean 0.94 0.6 1.39 0.91 0.66 1.19 95% LCI 0.76 0.50 1.15 0.72 0.50 0.90 95% UCI 1.13 0.71 1.63 1.09 0.82 1.48 BOHB Minimum 0.19 0.46 0.19 0.21 0.49 0.21 Maximum 1.61 1.61 0.40 1.40 1.40 0.57 Mean 0.58 0.78 0.31 0.64 0.89 0.34 95% LCI 0.46 0.64 0.27 0.50 0.73 0.29 95% UCI 0.69 0.92 0.34 0.77 1.05 0.40 DMC Minimum –65.00 –65.00 –28.00 –90.00 –90.00 –27.00 Maximum 61.00 –20.00 61.00 21.00 –33.00 21.00 Mean –12.97 –45.29 22.28 –34.64 –55.53 –10.54 95% LCI –27.70 –50.94 17.20 –44.80 –64.73 –18.36 95% UCI 1.73 –39.65 41.42 –24.40 –46.34 –2.72 . Captive female . . . Captive male . . . . CF alla . Fast . Feed . CM allb . Fast . Feed . n 30 17 13 28 15 13 TRIG Minimum 0.32 0.32 0.73 0.44 0.44 0.50 Maximum 1.97 1.01 1.97 2.14 1.65 2.14 Mean 0.94 0.6 1.39 0.91 0.66 1.19 95% LCI 0.76 0.50 1.15 0.72 0.50 0.90 95% UCI 1.13 0.71 1.63 1.09 0.82 1.48 BOHB Minimum 0.19 0.46 0.19 0.21 0.49 0.21 Maximum 1.61 1.61 0.40 1.40 1.40 0.57 Mean 0.58 0.78 0.31 0.64 0.89 0.34 95% LCI 0.46 0.64 0.27 0.50 0.73 0.29 95% UCI 0.69 0.92 0.34 0.77 1.05 0.40 DMC Minimum –65.00 –65.00 –28.00 –90.00 –90.00 –27.00 Maximum 61.00 –20.00 61.00 21.00 –33.00 21.00 Mean –12.97 –45.29 22.28 –34.64 –55.53 –10.54 95% LCI –27.70 –50.94 17.20 –44.80 –64.73 –18.36 95% UCI 1.73 –39.65 41.42 –24.40 –46.34 –2.72 aCombined sample of captive females (CF) from both treatments. bCombined sample of captive males (CM) from both treatments. Open in new tab Table 1. Sample size (n), minimum, maximum, mean values, and upper (UCI) and lower (LCI) 95% CIs for TRIG and BOHB concentrations and DMC (grams) for captive Lesser Scaup in the fasting (Fast) and feeding (Feed) treatments. . Captive female . . . Captive male . . . . CF alla . Fast . Feed . CM allb . Fast . Feed . n 30 17 13 28 15 13 TRIG Minimum 0.32 0.32 0.73 0.44 0.44 0.50 Maximum 1.97 1.01 1.97 2.14 1.65 2.14 Mean 0.94 0.6 1.39 0.91 0.66 1.19 95% LCI 0.76 0.50 1.15 0.72 0.50 0.90 95% UCI 1.13 0.71 1.63 1.09 0.82 1.48 BOHB Minimum 0.19 0.46 0.19 0.21 0.49 0.21 Maximum 1.61 1.61 0.40 1.40 1.40 0.57 Mean 0.58 0.78 0.31 0.64 0.89 0.34 95% LCI 0.46 0.64 0.27 0.50 0.73 0.29 95% UCI 0.69 0.92 0.34 0.77 1.05 0.40 DMC Minimum –65.00 –65.00 –28.00 –90.00 –90.00 –27.00 Maximum 61.00 –20.00 61.00 21.00 –33.00 21.00 Mean –12.97 –45.29 22.28 –34.64 –55.53 –10.54 95% LCI –27.70 –50.94 17.20 –44.80 –64.73 –18.36 95% UCI 1.73 –39.65 41.42 –24.40 –46.34 –2.72 . Captive female . . . Captive male . . . . CF alla . Fast . Feed . CM allb . Fast . Feed . n 30 17 13 28 15 13 TRIG Minimum 0.32 0.32 0.73 0.44 0.44 0.50 Maximum 1.97 1.01 1.97 2.14 1.65 2.14 Mean 0.94 0.6 1.39 0.91 0.66 1.19 95% LCI 0.76 0.50 1.15 0.72 0.50 0.90 95% UCI 1.13 0.71 1.63 1.09 0.82 1.48 BOHB Minimum 0.19 0.46 0.19 0.21 0.49 0.21 Maximum 1.61 1.61 0.40 1.40 1.40 0.57 Mean 0.58 0.78 0.31 0.64 0.89 0.34 95% LCI 0.46 0.64 0.27 0.50 0.73 0.29 95% UCI 0.69 0.92 0.34 0.77 1.05 0.40 DMC Minimum –65.00 –65.00 –28.00 –90.00 –90.00 –27.00 Maximum 61.00 –20.00 61.00 21.00 –33.00 21.00 Mean –12.97 –45.29 22.28 –34.64 –55.53 –10.54 95% LCI –27.70 –50.94 17.20 –44.80 –64.73 –18.36 95% UCI 1.73 –39.65 41.42 –24.40 –46.34 –2.72 aCombined sample of captive females (CF) from both treatments. bCombined sample of captive males (CM) from both treatments. Open in new tab Our final model for predicting mass change with metabolite concentrations included BOHBln, TRIG, and the interaction of sex and TRIG (R2 = 0.68, F3,54 = 37.79, P < 0.001; Figure 1). Triglycerides were positively associated with mass change (t57 = 5.45, P ≤ 0.001), but the relationship differed by sex (t = –3.95, P ≤ 0.001). BOHBln was negatively associated with mass change (t57 = –4.18, P ≤ 0.001) but did not differ by sex (t57 = –0.01, P = 0.99). We found no support for a main effect of sex (t57 = 0.58, P = 0.57). Therefore, we derived the following predictive equations for DMC in scaup: Figure 1. Open in new tabDownload slide Linear relationships of TRIG and natural-log transformed BOHBln to DMC of (A) male and (B) female Lesser Scaup held in short-term captivity during spring migration 2016 and 2017. Solid circles depict TRIG concentration, and open circles depict BOHBln concentration. Figure 1. Open in new tabDownload slide Linear relationships of TRIG and natural-log transformed BOHBln to DMC of (A) male and (B) female Lesser Scaup held in short-term captivity during spring migration 2016 and 2017. Solid circles depict TRIG concentration, and open circles depict BOHBln concentration. Female DMC=−69.82+41.36(TRIG)−26.73(BOHBln) Male DMC=−69.82+19.93(TRIG)−26.73 (BOHBln) Correspondence of non-standardized predictions for free-living male and captive female indices was positive (r2 = 0.79), but estimates suggested a directional bias between the 2 estimation methods (female slope = 0.65, 95% confidence interval (CI) = 0.54 to 0.75; intercept = 21.83, 95% CI = –14.10 to –29.6; Figure 2). Standardized predicted values of DMC also corresponded positively between models (r2 = 0.79), but there was no directional bias between the 2 estimation methods (female slope = 0.89, 95% CI = 0.75 to 1.03; intercept = 0; 95% CI = –0.14 to 0.14; Figure 3). Figure 2. Open in new tabDownload slide Boxplots represent the minimum, first quartile, median, third quartile, and maximum values of predicted mass change from scaup collected at Pool 19 of our the Mississippi River during springs 2004–2005 (data from Anteau and Afton 2011) and calculated from Anteau and Afton’s (2008b) free-living male (FLM) model and captive female (CF) model. Figure 2. Open in new tabDownload slide Boxplots represent the minimum, first quartile, median, third quartile, and maximum values of predicted mass change from scaup collected at Pool 19 of our the Mississippi River during springs 2004–2005 (data from Anteau and Afton 2011) and calculated from Anteau and Afton’s (2008b) free-living male (FLM) model and captive female (CF) model. Figure 3. Open in new tabDownload slide Boxplots represent the minimum, first quartile, median, third quartile, and maximum values of Z-standardized, predicted mass change from scaup collected at Pool 19 of the Mississippi River during springs 2004–2005 (data from Anteau and Afton 2011) and calculated from Anteau and Afton’s (2008b) free-living male (FLM) and our captive female (CF) regression models. Figure 3. Open in new tabDownload slide Boxplots represent the minimum, first quartile, median, third quartile, and maximum values of Z-standardized, predicted mass change from scaup collected at Pool 19 of the Mississippi River during springs 2004–2005 (data from Anteau and Afton 2011) and calculated from Anteau and Afton’s (2008b) free-living male (FLM) and our captive female (CF) regression models. DISCUSSION We observed a similar relationship between DMC and TRIG and BOHB concentrations with previous studies of migratory birds, indicating that plasma metabolites can be valuable indicators of energy income and, presumably by extension, foraging habitat quality (Jenni-Eiermann and Jenni 1994, Williams et al. 1999, Guglielmo et al. 2002, 2005, Smith et al. 2007, Anteau and Afton 2008b, 2011). Rate of mass change estimates for BOHBln (26.73) were similar to those reported by Anteau and Afton (28.65; 2008b) and did not differ by sex. Correspondingly, research with Western Sandpipers (Calidris mauri) showed that concentrations of BOHB did not differ by age or sex (Guglielmo et al. 2002). This result suggests that BOHB concentration is a reliable indicator of negative mass change because it responds to changes in energetic costs and food intake correspondingly with rates of lipolysis regardless of age and sex (Janke 2016). In contrast to previous findings, our results indicated that plasma TRIG concentrations provided an index of energy income rather than lipid deposition, per se, particularly for male scaup. In landscape-level applications of plasma metabolite indices, our findings suggest limitations where habitat and life history events are not static. Within a study site and species, raw estimates of lipid dynamics may be useful for interpreting relative habitat quality, but different metabolite concentrations across studies and species require standardization to be useful (Janke 2016). Although the direction of the relationship between TRIG and mass change is consistent with Anteau and Afton (2008b), our slope estimates for TRIG (females: 41.36, males: 19.93) were greater than previously reported for free-living male scaup (11.82; Anteau and Afton 2008b). Captive females had a similar range of mass change as previously reported for free-living male scaup (Anteau and Afton 2008b; Table 1). In contrast, captive females had lower concentrations of TRIG than did free-living males (Tables 1 and 2). This result suggests that TRIG values may be affected by energetic maintenance costs, life history events, and initial refueling rates at sites during migration. Such effects could impart a directional bias if estimates of TRIG are not standardized (Janke 2016). Table 2. Sample size (n), minimum (Min.), maximum (Max.), mean values, and upper (UCI) and lower (LCI) 95% CIs for TRIG and BOHB concentrations and DMC (grams) for male free-living Lesser Scaup (Anteau and Afton 2008b). . Free-living Male . . . . FLM alla . MLOb . MGOc . N 22 12 10 Min. TRIG 0.39 0.39 0.81 Max. TRIG 3.77 1.71 3.77 Mean TRIG 1.56 1.02 2.20 95% LCI 1.16 0.79 1.56 95% UCI 1.95 1.26 2.84 Min. BOHB 0.13 0.24 0.13 Max. BOHB 2.06 2.06 0.36 Mean BOHB 0.45 0.64 0.23 95% LCI 0.36 0.31 0.18 95% UCI 0.54 0.96 0.28 Min. DMC –68.00 –68.00 2.00 Max. DMC 61.00 –1.00 61.00 Mean DMC –5.91 –27.25 19.7 95% LCI –19.47 –39.59 5.65 95% UCI 7.65 –14.91 33.75 . Free-living Male . . . . FLM alla . MLOb . MGOc . N 22 12 10 Min. TRIG 0.39 0.39 0.81 Max. TRIG 3.77 1.71 3.77 Mean TRIG 1.56 1.02 2.20 95% LCI 1.16 0.79 1.56 95% UCI 1.95 1.26 2.84 Min. BOHB 0.13 0.24 0.13 Max. BOHB 2.06 2.06 0.36 Mean BOHB 0.45 0.64 0.23 95% LCI 0.36 0.31 0.18 95% UCI 0.54 0.96 0.28 Min. DMC –68.00 –68.00 2.00 Max. DMC 61.00 –1.00 61.00 Mean DMC –5.91 –27.25 19.7 95% LCI –19.47 –39.59 5.65 95% UCI 7.65 –14.91 33.75 aTotal sample of free-living male (FLM) scaup (Anteau and Afton 2008b). bCategorized by mass loss observed (MLO) in free-living specimens. cCategorized by mass gain observed (MGO) in free-living specimens. Open in new tab Table 2. Sample size (n), minimum (Min.), maximum (Max.), mean values, and upper (UCI) and lower (LCI) 95% CIs for TRIG and BOHB concentrations and DMC (grams) for male free-living Lesser Scaup (Anteau and Afton 2008b). . Free-living Male . . . . FLM alla . MLOb . MGOc . N 22 12 10 Min. TRIG 0.39 0.39 0.81 Max. TRIG 3.77 1.71 3.77 Mean TRIG 1.56 1.02 2.20 95% LCI 1.16 0.79 1.56 95% UCI 1.95 1.26 2.84 Min. BOHB 0.13 0.24 0.13 Max. BOHB 2.06 2.06 0.36 Mean BOHB 0.45 0.64 0.23 95% LCI 0.36 0.31 0.18 95% UCI 0.54 0.96 0.28 Min. DMC –68.00 –68.00 2.00 Max. DMC 61.00 –1.00 61.00 Mean DMC –5.91 –27.25 19.7 95% LCI –19.47 –39.59 5.65 95% UCI 7.65 –14.91 33.75 . Free-living Male . . . . FLM alla . MLOb . MGOc . N 22 12 10 Min. TRIG 0.39 0.39 0.81 Max. TRIG 3.77 1.71 3.77 Mean TRIG 1.56 1.02 2.20 95% LCI 1.16 0.79 1.56 95% UCI 1.95 1.26 2.84 Min. BOHB 0.13 0.24 0.13 Max. BOHB 2.06 2.06 0.36 Mean BOHB 0.45 0.64 0.23 95% LCI 0.36 0.31 0.18 95% UCI 0.54 0.96 0.28 Min. DMC –68.00 –68.00 2.00 Max. DMC 61.00 –1.00 61.00 Mean DMC –5.91 –27.25 19.7 95% LCI –19.47 –39.59 5.65 95% UCI 7.65 –14.91 33.75 aTotal sample of free-living male (FLM) scaup (Anteau and Afton 2008b). bCategorized by mass loss observed (MLO) in free-living specimens. cCategorized by mass gain observed (MGO) in free-living specimens. Open in new tab Our comparison of predictions for DMC between our captive scaup model and the model from free-living scaup published by Anteau and Afton (2008b) suggests that there was a directional bias between the 2 studies. Specifically, TRIG estimates for captive female scaup were greater than those of free-living males. Therefore, predictions made about mass change for free-living male scaup would be inflated when using a captive female-derived model. Possible explanations for this difference in directionality include variation in energetic maintenance costs, diets, stress levels, and other physiological effects between captive and free-living scaup. For example, diets of birds held in captivity may have been nutritionally different than wild birds (i.e. high energy and digestibility), and diet composition has potential to influence metabolite concentrations independent of mass changes (Seaman et al. 2005, Smith et al. 2007). Further, it is possible that male scaup in captivity had different rates of absorption than females given the same diet, and that could have potential to impact plasma TRIG values. However, birds analyzed by Anteau and Afton (2008b) were captured in traps baited with corn and may have had similar dietary effects, especially on TRIG concentrations of birds that gained mass (Seaman et al. 2005). Although we attempted to minimize stress in captive scaup by limiting handling time due to potential effects of stress on lipid metabolite concentrations (Jenni and Jenni-Eiermann 1996, Jenni-Eiermann and Jenni 1997, Dietz et al. 2009), wild birds held in captivity likely experienced greater short-term stress responses than free-living birds analyzed by Anteau and Afton (2008b) and it has been suggested that stress associated with capture may influence metabolite concentrations (Guglielmo et al. 2002). However, Schwilch and Jenni (2001) reported no difference in TRIG or BOHB concentrations for Eurasian Reed Warblers (Acrocephalus scirpaceus) that had been handled when compared with newly arrived individuals and TRIG levels did not change in response to prolonged and repeated stress in European Starlings (Sturnus vulgaris; Cyr et al. 2007). The impact of stress was not clearly evident on metabolite concentrations, but it may have affected the dietary energy absorption rates in the group that was fed, particularly males. Stress levels likely differed across studies and should be evaluated further as a potential mechanism affecting these metabolite indices. Despite potential differences between species and studies, standardizing predictions from metabolite indices removed the directional bias among the estimation methods. Thus, the directionality and general relationship of lipid metabolites with DMC appears consistent, and that there is utility with these indices (Janke 2016). Similarly, standardized model predictions of mass change derived from scaup and Garden Warblers (Sylvia borin) were similar (Janke 2016). Accordingly, it appears that standardized predictions of DMC are generally robust to variation in intrinsic and extrinsic factors (e.g., energetic maintenance costs, stress, etc.), but we urge that researchers be cognizant that the relationship between TRIG concentration and lipid deposition likely varies by these extrinsic factors. At first glance, our finding of sex-based differences in the relationship of TRIG and DMC for spring-migrating scaup calls into question the assumption made by Anteau and Afton (2008b) that TRIG would correspond to mass gain similarly between sexes. Dombrowski et al. (2003) noted that female Northern Pintail (Anas acuta) exhibited higher concentrations of TRIG in blood serum compared to males at the same stopover sites during spring. Guglielmo et al. (2002) documented substantial differences in the relationship between TRIG and body mass among male and female Western Sandpipers. Among scaup in our feeding treatment, 92% of females gained mass, but only 25% of males gained mass, even though both sexes were given the same high-carbohydrate diet and fed in excess of their presumed daily energy needs. Despite males losing mass in the feeding trial, their TRIG concentrations were similar to those of females in the feeding trial (Table 1). The lack of mass gain was not likely attributable to males being at or above optimal mass at the start of the experiment; initial mass of males that lost mass averaged 732 g while those that gained mass averaged 788 g. Moreover, calorimetry of the diet we used indicated that it exceeded estimated daily energy requirements for scaup such that both sexes should have been energetically satiated (Miller and McA Eadie 2006, Gross et al. 2020). There are 2 non-competing mechanisms we can propose to explain the sex-based difference in the relationship between mass change and TRIG. First, differing energy demands between sexes results in morphological and physiological differences during spring that resulted in males depositing far less lipids than females, despite lipid income in excess of their daily energetic requirements (see Heitmeyer 1988, Loesch et al. 1992). Based on the optimal body mass hypothesis, there are mobility disadvantages for increasing lipid reserves beyond what is needed (Lima 1986, Rogers 1987). Accordingly, males may not increase body lipids during spring migration due to relatively lower energetic demands of males relative to females during migration and pre-breeding (Anteau and Afton 2004). Additionally, if females have greater energetic demands than males, then females may have invested more in gut morphology than males to handle increased throughput, making females better morphologically equipped to take advantage of the feeding treatment. Second, behavioral differences between sexes resulted in differing levels of activity and (or) stress that increased maintenance costs and (or) decreased dietary nutrient absorption of males. Male scaup are driven to form pair bonds during spring migration, particularly because of the male-biased sex ratio and breeding chronology of the species (Weller 1965, Afton 1985, Afton and Anderson 2001). Perhaps, males being able to see but not interact with the females caused an increase in activity and stress of males which increased maintenance costs and (or) dietary absorption rates resulting in lower lipid deposition rates. Canvasbacks (Aythya valisineria) are a similar species that is considered to rely more on stored energy during breeding relative to Lesser Scaup. This suggests that sex-based differences in the need for mass gain on spring stopovers should be greater for Canvasbacks given that female Canvasbacks likely carry greater lipid reserves than female scaup during migration. However, a captive experiment on Canvasbacks, with the same procedures as used here, did not find a sex-based difference in the relationship of mass change and TRIG (Bouton 2018). It remains an open question whether the sex-based difference we observed in the relationship of mass change and TRIG would translate to free-living scaup. This difference would not apply to free-living scaup if it was caused more exclusively by our second mechanism (sex-based differences in captive activity and stress), which we suspect is more likely. Regardless of the mechanism, this result also provides evidence that TRIG is more indicative of energy income than deposition. As plasma-lipid metabolites can change rapidly in migrating birds in response to foraging bouts (Zajac et al. 2006), we attempted to minimize potential sources of variation in metabolite responses by standardizing diet and maintenance costs of scaup. By restricting movements, manually feeding birds to the point of satiation, and restricting energy income through fasting, we controlled for a presumably large amount of variation in energy income and expenditure occurring in free-living populations. Thus, fasting birds should have catabolized lipid reserves and produced BOHB resulting from lipolysis (Ramenofsky 1990, Boismenu et al. 1992), whereas force-fed birds should have deposited lipids as indicated by higher TRIG levels produced from energy income. Despite these controls and a substantially larger sample size, our model fit (R2 = 0.68) was slightly less than for free-living populations (R2 = 0.75) reported previously by Anteau and Afton 2008b. There was considerable individual variation in mass change within each treatment (feed and fasted), especially for males. We suspect that underlying physiological differences between male and female scaup during spring, potentially in combination with an interacting effect of stress, is the best explanation for these results. In spite of these differences, our results indicate that using lipid metabolites to broadly indicate mass change and/or food acquisition in Lesser Scaup to predict foraging habitat quality is robust to the issues of forage timing, abundance, and diet composition as questioned by Anteau and Afton (2008b). However, the considerable individual variation in mass change response within our treatments, even in females, suggests the need for robust sample sizes for studies using this technique to index stopover site quality. CONCLUSIONS Our results provide information on the utility of TRIG as a measure of energy income and BOHB as a reliable predictor of negative mass change in birds. When predictions of mass change are standardized, it appears that metabolite indices are robust to variations in energy expenditure and should be useful indicators of daily lipid dynamics. Consequently, our findings should be useful for interpretation of TRIG and BOHB concentrations in landscape-level inferences about habitat quality and refueling performance for migratory birds (Seaman et al. 2006, Lyons et al. 2008, Anteau and Afton 2011). We suggest that future research explore the potential mechanism(s) for sex-based differences in the relationship of TRIG and mass change, evaluate temporal variation in TRIG concentrations to better understand rates of change, and test effects of diet on metabolite concentrations for scaup. ACKNOWLEDGMENTS We thank R. Smith (Illinois Department of Natural Resources); C. Hine, A. Yetter, and others (Illinois Natural History Survey); A. Afton (Louisiana State University); R. Klaver (Iowa State University); and J. Lamer and S. Jenkins (Western Illinois University) for contributions to the study design, in-kind contributions, and logistical support for this project. We thank A. Janke for providing support with metabolite assays and helpful comments on earlier drafts of our manuscript. We thank A. Bouton, K. Farinosi, S. Lynch, J. Norman, M. Ryckman, and numerous volunteers for field assistance. We extend a sincere thank you to R. Leonard for his hospitality and care for field crews during scaup capture and banding operations. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Data used in this paper are owned, held, and managed by Western Illinois University. The findings and conclusions in this article are those of the author(s) and the U.S. Geological Survey and do not necessarily represent the views of the U.S. Fish and Wildlife Service. Funding statement: We thank the Mississippi Flyway Council and Department of Biological Sciences and Kibbe Field Station at Western Illinois University for funding. We thank Illinois Natural History Survey and Northern Prairie Wildlife Research Center for in-kind support. Ethics statement: All methods were in compliance with the Western Illinois University Institutional Animal Care and Use Committee (Protocol # 16-06), U.S. Geological Survey Bird Banding Laboratory (Permit # 23923), and Illinois Department of Natural Resources (Permit #NH15.5864). Author contributions: E.J.S., H.M.H., M.J.A., and C.N.J. designed the study; E.J.S. collected the data; E.J.S., H.M.H., M.J.A., and C.N.J. analyzed the data; and all authors wrote or substantially edited the paper. Data depository: Analyses reported in this article can be reproduced using the data provided by Smith et al. (2021). LITERATURE CITED Afton , A. D . ( 1985 ). 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Plasma metabolite indices are robust to extrinsic variation and useful indicators of foraging habitat quality in Lesser Scaup

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Copyright © 2021 American Ornithological Society
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Abstract

Abstract Energy acquisition and storage are important for survival and fecundity of birds during resource-limited periods such as spring migration. Plasma-lipid metabolites (i.e. triglyceride [TRIG], β-hydroxybutyrate [BOHB]) have been used to index changes in lipid stores and, thus, have utility for assessing foraging habitat quality during migration. However, such an index may be affected by energetic maintenance costs, diet, and other factors, and further validation under experimental conditions is needed to understand potential sources of variation and verify existing indices. We evaluated a plasma-lipid metabolite index using 30 female and 28 male wild Lesser Scaup (Aythya affinis; hereafter scaup) held in short-term captivity (~24 hr) during spring migration. Similar to previous observational studies, BOHB was negatively associated and TRIG was positively associated with mass change (R2 = 0.68). BOHB estimates were nearly identical to those published on free-living scaup, but TRIG estimates differed from free-living scaup and varied by sex, with females having a greater rate of predicted mass change than captive and free-living males. Our results suggest TRIG may be a better measure of energy income than deposition because lipid deposition likely varies with energetic maintenance costs, stress, and underlying physiological processes while TRIG relates primarily to energy income. In contrast, BOHB was a reliable predictor of negative mass change across sexes. The sex-based differences in apparent lipid deposition rates warrant further research before a generalizable model is advisable for comparing mass change predictions across studies. However, if predictions are standardized, this technique is generally robust to variations in energy income vs. lipid deposition across sexes. Accordingly, our evaluation provides verification for the utility of plasma-lipid metabolites as an indicator of foraging habitat quality during migration. RESUMEN La adquisición y el almacenamiento de energía son importantes para la supervivencia y la fecundidad de las aves durante los períodos de recursos limitados, como la migración de primavera. Los metabolitos lipídicos del plasma (i.e. triglicéridos [TRIG], β-hidroxibutirato [BOHB]) han sido usados para indexar cambios en las reservas de lípidos y, por ende, tienen utilidad para evaluar la calidad del hábitat de forrajeo durante la migración. Sin embargo, tal índice puede verse afectado por los costos de mantenimiento energético, la dieta y otros factores, y se necesita la validación adicional bajo condiciones experimentales para entender las fuentes potenciales de variación y verificar los índices existentes. Evaluamos un índice de metabolitos lipídicos del plasma usando 30 hembras y 28 machos silvestres de Aythya affinis retenidos en cautiverio por un breve período (~24 h) durante la migración de primavera. De modo similar a estudios observacionales previos, el BOHB estuvo negativamente asociado y los TRIG estuvieron positivamente asociados con el cambio de masa (R2 = 0.68). Las estimaciones de BOHB fueron casi idénticas a aquellas publicadas para individuos libres de A. affinis, pero las estimaciones de TRIG fueron diferentes a las de los individuos libres de A. affinis y variaron según el sexo, con las hembras presentando una tasa mayor de cambio de masa predicho que los machos cautivos y libres. Nuestros resultados sugieren que los TRIG pueden ser una mejor medida de ingreso que de depósito energético debido a que el depósito de lípidos varía probablemente con los costos de mantenimiento energético, el estrés y los procesos fisiológicos subyacentes, mientras que los TRIG se relacionan principalmente con el ingreso energético. En contraste, el BOHB fue un predictor confiable del cambio de masa negativo para ambos sexos. Las diferencias basadas en el sexo en las tasas de depósito aparente de lípidos justifican más investigaciones antes de proponer un modelo generalizable para comparar las predicciones de cambio de masa entre estudios. Sin embargo, si se estandarizan los predictores, esta técnica es generalmente robusta a las variaciones en ingreso energético vs. los depósitos de lípidos para ambos sexos. De este modo, nuestra evaluación brinda una verificación sobre la utilidad de los metabolitos lipídicos del plasma como un indicador de la calidad del hábitat de forrajeo durante la migración. Lay Summary The past several decades have seen a growing body of literature on the utility of plasma-lipid metabolites to assess habitat quality for a multitude of migratory bird species. Research in this area is often focused on 2 key metabolites, β-hydroxybutyrate and triglyceride and their associated relationships with mass change. Triglyceride has a positive relationship with mass change and is often referred to as an indicator of lipid deposition or fattening rates. However, energetic maintenance costs may confound the relationship of triglycerides and lipid deposition. To the best of our knowledge, no studies have attempted to control for potential variation in energy expenditure in conjunction with metabolite sampling during migration. Thus, we believe our paper will be a useful addition to the scientific literature on these metabolites and will be of interest to a broad audience of national and international wildlife managers and avian researchers. INTRODUCTION Wetland loss and degradation have negative consequences for wetland-dependent birds (Czech and Parsons 2002, Ma et al. 2010). Changes in climate, land-use practices, and drainage have contributed to decreases in wetland abundance and quality throughout the upper Midwest and the Prairie Pothole Region (PPR; Dahl 1990, Anteau and Afton 2008a; Anteau et al. 2011, McCauley et al. 2015, McKenna et al. 2017). Wetland degradation can lead to declining food resources (e.g., aquatic vegetation, invertebrates) at important migratory sites, which in turn have direct implications for wetland-obligate species that rely on such habitat resources for nutrient acquisition (Anteau and Afton 2011). Given the myriad of anthropogenic threats to ecological services that wetlands provide, there is a growing need for quick and efficient tools for assessing wetland quality. Avian researchers often employ various measures of body condition as a proxy to quantify fitness of individuals and to make inferences about habitat quality and population health (Brown 1996). The acquisition of energy at migratory stopover areas is paramount for survival and reproduction of many species of migratory birds (Alisauskas and Ankney 1992, McWilliams et al. 2004, Newton 2006, Sedinger and Alisauskas 2014). Many migratory bird species undergo periods of hyperphagia during migration and require high-quality habitat resources at stopover sites to replenish or accumulate lipid reserves (Bairlein 1990, Ramenofsky 1990, Stafford et al. 2014). Plasma-lipid metabolites can be useful for indexing lipid accumulation and catabolism, where free triglycerides (hereafter TRIG) have a positive relationship to mass change and β-hydroxybutyrate (hereafter BOHB) has a negative relationship to mass change (Jenni-Eiermann and Jenni 1994, Williams et al. 1999, Guglielmo et al. 2005, Smith et al. 2007, Anteau and Afton 2008b). Further, plasma-lipid metabolite concentrations provide a quantitative approach to assess spatial and temporal changes in lipid stores and offer utility in assessing foraging habitat quality during migration (Schaub and Jenni 2001, Guglielmo et al. 2002, 2005, Anteau and Afton 2011, Evans-Ogden et al. 2013, Thomas and Swanson 2013). Several researchers have previously used free-living birds to demonstrate that lipid metabolite concentrations vary logically in response to presumed habitat quality (Guglielmo et al. 2005, Seaman et al. 2006, Williams et al. 2007, Lyons et al. 2008). In previous studies, TRIG concentrations have been described as indicators of body mass, mass change, lipid deposition, and fattening rates (Jenni-Eiermann and Jenni 1994, Schwilch and Jenni 2001, Guglielmo et al. 2002, 2005, Owen et al. 2005, Anteau and Afton 2008b, Thomas and Swanson 2013). However, environmental variation in foraging efficiency, maintenance costs, weather events, and disturbance likely influence the allocation of energy income deposited into lipid reserves vs. that which is metabolized outright (Madsen 1995, Biebach 1996, Palm et al. 2013). Because TRIG is present in blood plasma and used during energetically costly activities (Jenni and Jenni-Eiermann 1998, McWilliams et al. 2004), not all TRIG acquired from exogenous sources will be deposited as lipid stores. This is not a trivial concern because during spring migration, environmental conditions or behavioral changes (e.g., pair bonding, maintenance) could change the proportion of free TRIG that gets catabolized or deposited as lipid reserves. Few studies have been able to obtain and track changes in body mass concurrent with plasma metabolite concentrations within a short time period in free-living birds (e.g., 24 hr; Jenni-Eiermann and Jenni 1994, Williams et al. 1999, Anteau and Afton 2008b). Metabolite concentrations can change rapidly (i.e. 10–20 min) in blood plasma of some bird species in response to feeding rates, specifically TRIG and BOHB (Zajac et al. 2006). A primary challenge of this type of study with free-living birds is attaining an adequate sample size of birds recaptured after a short period during which metabolite profiles should reflect mass changes (Guglielmo et al. 2005). For example, Anteau and Afton (2008b) captured, weighed, and marked >3,800 Lesser Scaup (Aythya affinis; hereafter scaup) during 2 spring migration periods and were only able to obtain 22 corresponding metabolite and mass change samples from individuals recaptured within a 24-hr period. Another challenge of studies using free-living specimens is that variation among individuals in feeding rates, diets, energy expenditure (i.e. pairing behaviors, foraging efficiency), and habituation to bait at trap sites may confound the relationship of lipid metabolites to daily mass changes (DMC). Although several evaluations of metabolite changes over time and in response to nutrient acquisition have been conducted using migratory birds held in captivity, none have included waterfowl despite this taxa underpinning conservation planning efforts for wetland and upland habitats throughout North America (North American Waterfowl Management Plan (NAWMP) 2012). Using captive scaup to evaluate TRIG and BOHB relationships with DMC would control for potentially confounding factors such as diet composition and energy expenditures. Moreover, this approach would provide a comparison of energy income and lipid deposition given captive scaup likely have lower energy requirements than free-living scaup (Anteau and Afton 2008b). Such a comparison should provide information regarding the potential utility of TRIG to index lipid deposition rates despite varying environmental conditions that could affect energetic maintenance costs. Additionally, such a comparison could allow for analyses of intrinsic factors (e.g., sex) that may influence plasma-lipid metabolite indices and may clarify an assumption made by Anteau and Afton (2008b) that male and female scaup would exhibit similar metabolite profiles because they consume the same forage items during spring migration. If sex-based differences exist in the relationships of TRIG or BOHB concentrations and mass change, then the index developed based on male-only information may provide biased results in females (Anteau and Afton 2011). Our primary objective was to validate a previously developed plasma metabolite index generated from free-living male Lesser Scaup by controlling for energy expenditure and potential diet effects and testing for differences between sex using captive birds (Anteau and Afton 2008b). We examined the relationship between TRIG, BOHB, and DMC using wild-captured scaup held in short-term captivity and subjected to feeding and fasting treatments (Anteau and Afton 2008b). A metabolite index robust to variation in intrinsic and extrinsic factors would allow a direct evaluation of foraging habitat quality without measuring indirect indicators such as food density, which can be resource-intensive and may not be indicative of nutrient acquisition by foragers. METHODS AND MATERIALS Study Species Scaup are a migratory diving duck species that primarily winter in waters along the Gulf of Mexico, migrates through the Midwest, and breeds from the PPR to the boreal forest of Alaska and Canada (Anteau et al. 2020). Scaup were an ideal study species for this experiment because, as wetland obligates, they derive all their energy from wetland habitat resources (Anteau and Afton 2011). Furthermore, they have exhibited declining population trends and previous investigations have identified female body condition (i.e. low lipid reserves) during spring migration as a likely contributing factor to this decline (Austin et al. 2000, 2006, 2014, Afton and Anderson 2001). Further, a plasma-lipid metabolite index was developed (Anteau and Afton 2008b) and applied to scaup migrating across the landscape during spring migration (Anteau and Afton 2011). Study Area During spring migration 2016 and 2017 (February 20 to March 31), we captured wild scaup at navigation Pool 19 of the Mississippi River (hereafter, Pool 19) near Nauvoo, Illinois. Pool 19 is the largest impounded reservoir on the Mississippi River and extends 70 km from the lock and dam at Keokuk, Iowa northward to lock and dam 18 at Gladstone, Illinois, USA. The lower portion of the pool (i.e. Nauvoo, Illinois southward to Keokuk, Iowa) is wider, has less flow, and is characterized by a substrate that historically supported aquatic vegetation (e.g., Heteranthera dubia, Potamogeton pectinatus, Ceratophyllum demersum, Vallisneria americana, Nelumbo lutea, Sagittaria sp.; Steffeck et al. 1985), but declines in aquatic vegetation communities make benthic invertebrates (e.g., bivalves, gastropods, insects) the primary food of diving ducks on Pool 19 (Thompson 1973, Moore et al. 2010, Hagy et al. 2015). Pool 19 is an important mid-latitude stopover site for scaup, especially during spring migration, and has been a resource for capturing and banding large numbers of scaup since the 1980s (Havera et al. 1992, Anteau and Afton 2009, Arnold et al. 2017). Field Methods We located areas with high numbers of scaup and established bait sites by placing whole kernel corn daily near a post in ~1 m of water until scaup readily fed nearby the site. We deployed modified swim-in/dive-in traps (i.e. funnel traps) baited with corn (Haramis et al. 1982, Anteau and Afton 2008b), monitored traps, and removed birds 2–3 times daily dependent upon trapping success (~0900, 1400, 1800 hr). Following removal from traps, ducks were weighed on a digital balance (±1 g), banded with a U.S. Geological Survey leg band, and transported to outdoor holding pens at Kibbe Field Station in Warsaw, Illinois, USA. To minimize the potential for variation among individuals and unknown food items, we excluded birds from the experiments that had palpable amounts of ingesta in their crop/esophagus at time of capture. Captive Methods We held wild scaup (31 females, 30 males) in captivity for ~2 hr prior to the experiment to further minimize the potential for variation in the amount and type of ingesta present in their guts from wild origins. Our experiment ran for a 24-hr duration, during which time we controlled diet and feeding rates. Following capture, we randomly assigned individuals to 1 of the 2 treatment groups (i.e. fasting or feeding). The goal of the fasting and feeding treatments was to elicit a range of mass loss and gain, respectively, that was similar to that observed from free-living scaup (see Anteau and Afton 2008b). We recognized from pilot work that birds would lose and gain mass at different rates based on individual variation. Scaup selected for fasting treatment were given access to water ad libitum and did not receive food for the duration of stay in captivity. Scaup selected for feeding treatments were provided water ad libitum and manually fed a slurry of food and water with a 60-cc syringe fitted with 30 cm of 4.8-mm diameter, vinyl tubing by inserting the tubing ~3 cm down the esophagus. The slurry was a standardized mixture (gross energy mean = 3,959.42 ± 48.61 cal g–1) of 1.0-part layer crumble (Nutrena Co., Minneapolis, Minnesota, USA), 0.5-part scratch grain (Nutrena Co.), and 0.25-part meal worms (Happy Hen Treats Co., Boerne, Texas, USA). We added water to the food mixture as needed until it flowed through tubing without resistance. We administered food mixture until the esophagus/crop was approximately full as determined by palpation; each feeding bout took ~2 min. Our pilot efforts indicated that feeding to near the point of refusal at 4-hr intervals was required to reasonably ensure that birds in the feeding treatments gained mass during the experiment. To ensure mass gain, we repeated feeding procedures at 4-hr intervals for the duration of the captivity period. Free-living scaup can feed throughout the daily cycle, including nocturnally (Custer et al. 1996, Herring and Collazo 2006). While the volume of food administered was not standardized among individuals, this procedure did standardize the feeding interval and the volume of food relative to the bird’s esophagus, crop, and proventriculus capacity. This procedure simulated foraging bouts to the point of satiation of free-living birds. Prior to entering treatments, we obtained baseline blood samples by extracting 0.5 mL of blood from the tibiotarsal vein anterior the intertarsal joint using a heparinized 1-cc syringe affixed with a 25-ga needle. Approximately 24 hr after recording initial weight and 4-hr post-experimental treatment, we recorded final mass measurements and extracted 0.5 mL of blood as described prior to treatment. We transferred blood to 1.5-mL heparinized micro-centrifuge tubes and centrifuged at 6,000 rpm (2,000× g) for 7 min within 30 min of collection. We extracted plasma using a micropipette, transferred to a sterile vial, and froze within 20 min at –20°C until analysis (Guglielmo et al. 2002, Anteau and Afton 2008b, Janke 2016). Upon completion of experimental procedures, we released scaup near the initial capture site. Animal capture and handling methods used during our study were approved by the Institutional Animal Care and Use Committee at Western Illinois University (#16-06), University of Illinois (#16020), and Illinois Department of Natural Resources (#NH15.5864). The U.S. Geological Survey Bird Banding Laboratory approved markers, blood drawings, and handling methods (#23923). Laboratory Methods We followed established protocols that utilize endpoint assay for measuring total triglycerides (TRIG + glycerol) and glycerol. Free TRIG was calculated by subtracting glycerol from total TRIG (Guglielmo et al. 2002, Anteau 2006, Janke 2016). We measured BOHB by kinetic assay using standard protocols (Guglielmo et al. 2005, Anteau 2006, Janke 2016). Plasma-lipid metabolite assays, both endpoint and kinetic, were completed at Northern Prairie Wildlife Research Center using a microplate spectrophotometer (BioTek, Inc., model EON, Winooski, Vermont, USA). Upon completion of metabolite assays, we censored 2 males and 1 female due to irregular sample quality, thus our analysis included 28 males and 30 females. Data Analysis We used generalized linear models (R Core Team 2017) with a normal distribution and identity link function to evaluate the relationships of sex and plasma-lipid metabolites (i.e. TRIG, BOHB) on mass change of scaup. We examined residuals and determined that the relationship between BOHB and DMC was curvilinear; hence, we natural-log-transformed BOHB (Jenni-Eiermann et al. 2002, Anteau and Afton 2008b). We used backward elimination procedures (α = 0.05) to evaluate main effects and interaction terms for BOHBln, TRIG, and sex. We considered the potential for carryover effects given birds’ recent time spent in the wild and evaluated initial metabolite values as covariates. Baseline values for TRIG and BOHBln prior to entering treatments did not improve model fit (P > 0.426). We used a dataset of BOHBln and TRIG values from female scaup (n = 43) lethally collected at Pool 19 in spring 2004 and 2005 (see Anteau and Afton 2011) to compare our predictive model with that of Anteau and Afton (2008b). We compared both model-predicted values of mass change (g) and Z-standardized model-predicted values of mass change from each index to make comparisons of model correspondence (Janke 2016). We examined summary statistics for mass change predictions and standardized mass change predictions and conducted linear regressions between both indices. We considered perfect correspondence between indices to have a slope = 1, intercept = 0, and r2 = 1. RESULTS Female DMC ranged from –65 to 61 g; 92% of females in the feeding treatment gained mass and 100% of females in the fasting treatment lost mass. DMC for males ranged from –91 to 21 g; 25% of males in the feeding treatment gained mass and 100% of males in the fasting treatment lost mass. Concentrations of TRIG ranged from 0.32 to 1.97 in female scaup and 0.44 to 2.14 (mmol L–1) in male scaup (Table 1). Concentrations of BOHB ranged from 0.19 to 1.61 and 0.21 to 1.40 (mmol L–1) for female and male scaup, respectively (Table 1). Table 1. Sample size (n), minimum, maximum, mean values, and upper (UCI) and lower (LCI) 95% CIs for TRIG and BOHB concentrations and DMC (grams) for captive Lesser Scaup in the fasting (Fast) and feeding (Feed) treatments. . Captive female . . . Captive male . . . . CF alla . Fast . Feed . CM allb . Fast . Feed . n 30 17 13 28 15 13 TRIG Minimum 0.32 0.32 0.73 0.44 0.44 0.50 Maximum 1.97 1.01 1.97 2.14 1.65 2.14 Mean 0.94 0.6 1.39 0.91 0.66 1.19 95% LCI 0.76 0.50 1.15 0.72 0.50 0.90 95% UCI 1.13 0.71 1.63 1.09 0.82 1.48 BOHB Minimum 0.19 0.46 0.19 0.21 0.49 0.21 Maximum 1.61 1.61 0.40 1.40 1.40 0.57 Mean 0.58 0.78 0.31 0.64 0.89 0.34 95% LCI 0.46 0.64 0.27 0.50 0.73 0.29 95% UCI 0.69 0.92 0.34 0.77 1.05 0.40 DMC Minimum –65.00 –65.00 –28.00 –90.00 –90.00 –27.00 Maximum 61.00 –20.00 61.00 21.00 –33.00 21.00 Mean –12.97 –45.29 22.28 –34.64 –55.53 –10.54 95% LCI –27.70 –50.94 17.20 –44.80 –64.73 –18.36 95% UCI 1.73 –39.65 41.42 –24.40 –46.34 –2.72 . Captive female . . . Captive male . . . . CF alla . Fast . Feed . CM allb . Fast . Feed . n 30 17 13 28 15 13 TRIG Minimum 0.32 0.32 0.73 0.44 0.44 0.50 Maximum 1.97 1.01 1.97 2.14 1.65 2.14 Mean 0.94 0.6 1.39 0.91 0.66 1.19 95% LCI 0.76 0.50 1.15 0.72 0.50 0.90 95% UCI 1.13 0.71 1.63 1.09 0.82 1.48 BOHB Minimum 0.19 0.46 0.19 0.21 0.49 0.21 Maximum 1.61 1.61 0.40 1.40 1.40 0.57 Mean 0.58 0.78 0.31 0.64 0.89 0.34 95% LCI 0.46 0.64 0.27 0.50 0.73 0.29 95% UCI 0.69 0.92 0.34 0.77 1.05 0.40 DMC Minimum –65.00 –65.00 –28.00 –90.00 –90.00 –27.00 Maximum 61.00 –20.00 61.00 21.00 –33.00 21.00 Mean –12.97 –45.29 22.28 –34.64 –55.53 –10.54 95% LCI –27.70 –50.94 17.20 –44.80 –64.73 –18.36 95% UCI 1.73 –39.65 41.42 –24.40 –46.34 –2.72 aCombined sample of captive females (CF) from both treatments. bCombined sample of captive males (CM) from both treatments. Open in new tab Table 1. Sample size (n), minimum, maximum, mean values, and upper (UCI) and lower (LCI) 95% CIs for TRIG and BOHB concentrations and DMC (grams) for captive Lesser Scaup in the fasting (Fast) and feeding (Feed) treatments. . Captive female . . . Captive male . . . . CF alla . Fast . Feed . CM allb . Fast . Feed . n 30 17 13 28 15 13 TRIG Minimum 0.32 0.32 0.73 0.44 0.44 0.50 Maximum 1.97 1.01 1.97 2.14 1.65 2.14 Mean 0.94 0.6 1.39 0.91 0.66 1.19 95% LCI 0.76 0.50 1.15 0.72 0.50 0.90 95% UCI 1.13 0.71 1.63 1.09 0.82 1.48 BOHB Minimum 0.19 0.46 0.19 0.21 0.49 0.21 Maximum 1.61 1.61 0.40 1.40 1.40 0.57 Mean 0.58 0.78 0.31 0.64 0.89 0.34 95% LCI 0.46 0.64 0.27 0.50 0.73 0.29 95% UCI 0.69 0.92 0.34 0.77 1.05 0.40 DMC Minimum –65.00 –65.00 –28.00 –90.00 –90.00 –27.00 Maximum 61.00 –20.00 61.00 21.00 –33.00 21.00 Mean –12.97 –45.29 22.28 –34.64 –55.53 –10.54 95% LCI –27.70 –50.94 17.20 –44.80 –64.73 –18.36 95% UCI 1.73 –39.65 41.42 –24.40 –46.34 –2.72 . Captive female . . . Captive male . . . . CF alla . Fast . Feed . CM allb . Fast . Feed . n 30 17 13 28 15 13 TRIG Minimum 0.32 0.32 0.73 0.44 0.44 0.50 Maximum 1.97 1.01 1.97 2.14 1.65 2.14 Mean 0.94 0.6 1.39 0.91 0.66 1.19 95% LCI 0.76 0.50 1.15 0.72 0.50 0.90 95% UCI 1.13 0.71 1.63 1.09 0.82 1.48 BOHB Minimum 0.19 0.46 0.19 0.21 0.49 0.21 Maximum 1.61 1.61 0.40 1.40 1.40 0.57 Mean 0.58 0.78 0.31 0.64 0.89 0.34 95% LCI 0.46 0.64 0.27 0.50 0.73 0.29 95% UCI 0.69 0.92 0.34 0.77 1.05 0.40 DMC Minimum –65.00 –65.00 –28.00 –90.00 –90.00 –27.00 Maximum 61.00 –20.00 61.00 21.00 –33.00 21.00 Mean –12.97 –45.29 22.28 –34.64 –55.53 –10.54 95% LCI –27.70 –50.94 17.20 –44.80 –64.73 –18.36 95% UCI 1.73 –39.65 41.42 –24.40 –46.34 –2.72 aCombined sample of captive females (CF) from both treatments. bCombined sample of captive males (CM) from both treatments. Open in new tab Our final model for predicting mass change with metabolite concentrations included BOHBln, TRIG, and the interaction of sex and TRIG (R2 = 0.68, F3,54 = 37.79, P < 0.001; Figure 1). Triglycerides were positively associated with mass change (t57 = 5.45, P ≤ 0.001), but the relationship differed by sex (t = –3.95, P ≤ 0.001). BOHBln was negatively associated with mass change (t57 = –4.18, P ≤ 0.001) but did not differ by sex (t57 = –0.01, P = 0.99). We found no support for a main effect of sex (t57 = 0.58, P = 0.57). Therefore, we derived the following predictive equations for DMC in scaup: Figure 1. Open in new tabDownload slide Linear relationships of TRIG and natural-log transformed BOHBln to DMC of (A) male and (B) female Lesser Scaup held in short-term captivity during spring migration 2016 and 2017. Solid circles depict TRIG concentration, and open circles depict BOHBln concentration. Figure 1. Open in new tabDownload slide Linear relationships of TRIG and natural-log transformed BOHBln to DMC of (A) male and (B) female Lesser Scaup held in short-term captivity during spring migration 2016 and 2017. Solid circles depict TRIG concentration, and open circles depict BOHBln concentration. Female DMC=−69.82+41.36(TRIG)−26.73(BOHBln) Male DMC=−69.82+19.93(TRIG)−26.73 (BOHBln) Correspondence of non-standardized predictions for free-living male and captive female indices was positive (r2 = 0.79), but estimates suggested a directional bias between the 2 estimation methods (female slope = 0.65, 95% confidence interval (CI) = 0.54 to 0.75; intercept = 21.83, 95% CI = –14.10 to –29.6; Figure 2). Standardized predicted values of DMC also corresponded positively between models (r2 = 0.79), but there was no directional bias between the 2 estimation methods (female slope = 0.89, 95% CI = 0.75 to 1.03; intercept = 0; 95% CI = –0.14 to 0.14; Figure 3). Figure 2. Open in new tabDownload slide Boxplots represent the minimum, first quartile, median, third quartile, and maximum values of predicted mass change from scaup collected at Pool 19 of our the Mississippi River during springs 2004–2005 (data from Anteau and Afton 2011) and calculated from Anteau and Afton’s (2008b) free-living male (FLM) model and captive female (CF) model. Figure 2. Open in new tabDownload slide Boxplots represent the minimum, first quartile, median, third quartile, and maximum values of predicted mass change from scaup collected at Pool 19 of our the Mississippi River during springs 2004–2005 (data from Anteau and Afton 2011) and calculated from Anteau and Afton’s (2008b) free-living male (FLM) model and captive female (CF) model. Figure 3. Open in new tabDownload slide Boxplots represent the minimum, first quartile, median, third quartile, and maximum values of Z-standardized, predicted mass change from scaup collected at Pool 19 of the Mississippi River during springs 2004–2005 (data from Anteau and Afton 2011) and calculated from Anteau and Afton’s (2008b) free-living male (FLM) and our captive female (CF) regression models. Figure 3. Open in new tabDownload slide Boxplots represent the minimum, first quartile, median, third quartile, and maximum values of Z-standardized, predicted mass change from scaup collected at Pool 19 of the Mississippi River during springs 2004–2005 (data from Anteau and Afton 2011) and calculated from Anteau and Afton’s (2008b) free-living male (FLM) and our captive female (CF) regression models. DISCUSSION We observed a similar relationship between DMC and TRIG and BOHB concentrations with previous studies of migratory birds, indicating that plasma metabolites can be valuable indicators of energy income and, presumably by extension, foraging habitat quality (Jenni-Eiermann and Jenni 1994, Williams et al. 1999, Guglielmo et al. 2002, 2005, Smith et al. 2007, Anteau and Afton 2008b, 2011). Rate of mass change estimates for BOHBln (26.73) were similar to those reported by Anteau and Afton (28.65; 2008b) and did not differ by sex. Correspondingly, research with Western Sandpipers (Calidris mauri) showed that concentrations of BOHB did not differ by age or sex (Guglielmo et al. 2002). This result suggests that BOHB concentration is a reliable indicator of negative mass change because it responds to changes in energetic costs and food intake correspondingly with rates of lipolysis regardless of age and sex (Janke 2016). In contrast to previous findings, our results indicated that plasma TRIG concentrations provided an index of energy income rather than lipid deposition, per se, particularly for male scaup. In landscape-level applications of plasma metabolite indices, our findings suggest limitations where habitat and life history events are not static. Within a study site and species, raw estimates of lipid dynamics may be useful for interpreting relative habitat quality, but different metabolite concentrations across studies and species require standardization to be useful (Janke 2016). Although the direction of the relationship between TRIG and mass change is consistent with Anteau and Afton (2008b), our slope estimates for TRIG (females: 41.36, males: 19.93) were greater than previously reported for free-living male scaup (11.82; Anteau and Afton 2008b). Captive females had a similar range of mass change as previously reported for free-living male scaup (Anteau and Afton 2008b; Table 1). In contrast, captive females had lower concentrations of TRIG than did free-living males (Tables 1 and 2). This result suggests that TRIG values may be affected by energetic maintenance costs, life history events, and initial refueling rates at sites during migration. Such effects could impart a directional bias if estimates of TRIG are not standardized (Janke 2016). Table 2. Sample size (n), minimum (Min.), maximum (Max.), mean values, and upper (UCI) and lower (LCI) 95% CIs for TRIG and BOHB concentrations and DMC (grams) for male free-living Lesser Scaup (Anteau and Afton 2008b). . Free-living Male . . . . FLM alla . MLOb . MGOc . N 22 12 10 Min. TRIG 0.39 0.39 0.81 Max. TRIG 3.77 1.71 3.77 Mean TRIG 1.56 1.02 2.20 95% LCI 1.16 0.79 1.56 95% UCI 1.95 1.26 2.84 Min. BOHB 0.13 0.24 0.13 Max. BOHB 2.06 2.06 0.36 Mean BOHB 0.45 0.64 0.23 95% LCI 0.36 0.31 0.18 95% UCI 0.54 0.96 0.28 Min. DMC –68.00 –68.00 2.00 Max. DMC 61.00 –1.00 61.00 Mean DMC –5.91 –27.25 19.7 95% LCI –19.47 –39.59 5.65 95% UCI 7.65 –14.91 33.75 . Free-living Male . . . . FLM alla . MLOb . MGOc . N 22 12 10 Min. TRIG 0.39 0.39 0.81 Max. TRIG 3.77 1.71 3.77 Mean TRIG 1.56 1.02 2.20 95% LCI 1.16 0.79 1.56 95% UCI 1.95 1.26 2.84 Min. BOHB 0.13 0.24 0.13 Max. BOHB 2.06 2.06 0.36 Mean BOHB 0.45 0.64 0.23 95% LCI 0.36 0.31 0.18 95% UCI 0.54 0.96 0.28 Min. DMC –68.00 –68.00 2.00 Max. DMC 61.00 –1.00 61.00 Mean DMC –5.91 –27.25 19.7 95% LCI –19.47 –39.59 5.65 95% UCI 7.65 –14.91 33.75 aTotal sample of free-living male (FLM) scaup (Anteau and Afton 2008b). bCategorized by mass loss observed (MLO) in free-living specimens. cCategorized by mass gain observed (MGO) in free-living specimens. Open in new tab Table 2. Sample size (n), minimum (Min.), maximum (Max.), mean values, and upper (UCI) and lower (LCI) 95% CIs for TRIG and BOHB concentrations and DMC (grams) for male free-living Lesser Scaup (Anteau and Afton 2008b). . Free-living Male . . . . FLM alla . MLOb . MGOc . N 22 12 10 Min. TRIG 0.39 0.39 0.81 Max. TRIG 3.77 1.71 3.77 Mean TRIG 1.56 1.02 2.20 95% LCI 1.16 0.79 1.56 95% UCI 1.95 1.26 2.84 Min. BOHB 0.13 0.24 0.13 Max. BOHB 2.06 2.06 0.36 Mean BOHB 0.45 0.64 0.23 95% LCI 0.36 0.31 0.18 95% UCI 0.54 0.96 0.28 Min. DMC –68.00 –68.00 2.00 Max. DMC 61.00 –1.00 61.00 Mean DMC –5.91 –27.25 19.7 95% LCI –19.47 –39.59 5.65 95% UCI 7.65 –14.91 33.75 . Free-living Male . . . . FLM alla . MLOb . MGOc . N 22 12 10 Min. TRIG 0.39 0.39 0.81 Max. TRIG 3.77 1.71 3.77 Mean TRIG 1.56 1.02 2.20 95% LCI 1.16 0.79 1.56 95% UCI 1.95 1.26 2.84 Min. BOHB 0.13 0.24 0.13 Max. BOHB 2.06 2.06 0.36 Mean BOHB 0.45 0.64 0.23 95% LCI 0.36 0.31 0.18 95% UCI 0.54 0.96 0.28 Min. DMC –68.00 –68.00 2.00 Max. DMC 61.00 –1.00 61.00 Mean DMC –5.91 –27.25 19.7 95% LCI –19.47 –39.59 5.65 95% UCI 7.65 –14.91 33.75 aTotal sample of free-living male (FLM) scaup (Anteau and Afton 2008b). bCategorized by mass loss observed (MLO) in free-living specimens. cCategorized by mass gain observed (MGO) in free-living specimens. Open in new tab Our comparison of predictions for DMC between our captive scaup model and the model from free-living scaup published by Anteau and Afton (2008b) suggests that there was a directional bias between the 2 studies. Specifically, TRIG estimates for captive female scaup were greater than those of free-living males. Therefore, predictions made about mass change for free-living male scaup would be inflated when using a captive female-derived model. Possible explanations for this difference in directionality include variation in energetic maintenance costs, diets, stress levels, and other physiological effects between captive and free-living scaup. For example, diets of birds held in captivity may have been nutritionally different than wild birds (i.e. high energy and digestibility), and diet composition has potential to influence metabolite concentrations independent of mass changes (Seaman et al. 2005, Smith et al. 2007). Further, it is possible that male scaup in captivity had different rates of absorption than females given the same diet, and that could have potential to impact plasma TRIG values. However, birds analyzed by Anteau and Afton (2008b) were captured in traps baited with corn and may have had similar dietary effects, especially on TRIG concentrations of birds that gained mass (Seaman et al. 2005). Although we attempted to minimize stress in captive scaup by limiting handling time due to potential effects of stress on lipid metabolite concentrations (Jenni and Jenni-Eiermann 1996, Jenni-Eiermann and Jenni 1997, Dietz et al. 2009), wild birds held in captivity likely experienced greater short-term stress responses than free-living birds analyzed by Anteau and Afton (2008b) and it has been suggested that stress associated with capture may influence metabolite concentrations (Guglielmo et al. 2002). However, Schwilch and Jenni (2001) reported no difference in TRIG or BOHB concentrations for Eurasian Reed Warblers (Acrocephalus scirpaceus) that had been handled when compared with newly arrived individuals and TRIG levels did not change in response to prolonged and repeated stress in European Starlings (Sturnus vulgaris; Cyr et al. 2007). The impact of stress was not clearly evident on metabolite concentrations, but it may have affected the dietary energy absorption rates in the group that was fed, particularly males. Stress levels likely differed across studies and should be evaluated further as a potential mechanism affecting these metabolite indices. Despite potential differences between species and studies, standardizing predictions from metabolite indices removed the directional bias among the estimation methods. Thus, the directionality and general relationship of lipid metabolites with DMC appears consistent, and that there is utility with these indices (Janke 2016). Similarly, standardized model predictions of mass change derived from scaup and Garden Warblers (Sylvia borin) were similar (Janke 2016). Accordingly, it appears that standardized predictions of DMC are generally robust to variation in intrinsic and extrinsic factors (e.g., energetic maintenance costs, stress, etc.), but we urge that researchers be cognizant that the relationship between TRIG concentration and lipid deposition likely varies by these extrinsic factors. At first glance, our finding of sex-based differences in the relationship of TRIG and DMC for spring-migrating scaup calls into question the assumption made by Anteau and Afton (2008b) that TRIG would correspond to mass gain similarly between sexes. Dombrowski et al. (2003) noted that female Northern Pintail (Anas acuta) exhibited higher concentrations of TRIG in blood serum compared to males at the same stopover sites during spring. Guglielmo et al. (2002) documented substantial differences in the relationship between TRIG and body mass among male and female Western Sandpipers. Among scaup in our feeding treatment, 92% of females gained mass, but only 25% of males gained mass, even though both sexes were given the same high-carbohydrate diet and fed in excess of their presumed daily energy needs. Despite males losing mass in the feeding trial, their TRIG concentrations were similar to those of females in the feeding trial (Table 1). The lack of mass gain was not likely attributable to males being at or above optimal mass at the start of the experiment; initial mass of males that lost mass averaged 732 g while those that gained mass averaged 788 g. Moreover, calorimetry of the diet we used indicated that it exceeded estimated daily energy requirements for scaup such that both sexes should have been energetically satiated (Miller and McA Eadie 2006, Gross et al. 2020). There are 2 non-competing mechanisms we can propose to explain the sex-based difference in the relationship between mass change and TRIG. First, differing energy demands between sexes results in morphological and physiological differences during spring that resulted in males depositing far less lipids than females, despite lipid income in excess of their daily energetic requirements (see Heitmeyer 1988, Loesch et al. 1992). Based on the optimal body mass hypothesis, there are mobility disadvantages for increasing lipid reserves beyond what is needed (Lima 1986, Rogers 1987). Accordingly, males may not increase body lipids during spring migration due to relatively lower energetic demands of males relative to females during migration and pre-breeding (Anteau and Afton 2004). Additionally, if females have greater energetic demands than males, then females may have invested more in gut morphology than males to handle increased throughput, making females better morphologically equipped to take advantage of the feeding treatment. Second, behavioral differences between sexes resulted in differing levels of activity and (or) stress that increased maintenance costs and (or) decreased dietary nutrient absorption of males. Male scaup are driven to form pair bonds during spring migration, particularly because of the male-biased sex ratio and breeding chronology of the species (Weller 1965, Afton 1985, Afton and Anderson 2001). Perhaps, males being able to see but not interact with the females caused an increase in activity and stress of males which increased maintenance costs and (or) dietary absorption rates resulting in lower lipid deposition rates. Canvasbacks (Aythya valisineria) are a similar species that is considered to rely more on stored energy during breeding relative to Lesser Scaup. This suggests that sex-based differences in the need for mass gain on spring stopovers should be greater for Canvasbacks given that female Canvasbacks likely carry greater lipid reserves than female scaup during migration. However, a captive experiment on Canvasbacks, with the same procedures as used here, did not find a sex-based difference in the relationship of mass change and TRIG (Bouton 2018). It remains an open question whether the sex-based difference we observed in the relationship of mass change and TRIG would translate to free-living scaup. This difference would not apply to free-living scaup if it was caused more exclusively by our second mechanism (sex-based differences in captive activity and stress), which we suspect is more likely. Regardless of the mechanism, this result also provides evidence that TRIG is more indicative of energy income than deposition. As plasma-lipid metabolites can change rapidly in migrating birds in response to foraging bouts (Zajac et al. 2006), we attempted to minimize potential sources of variation in metabolite responses by standardizing diet and maintenance costs of scaup. By restricting movements, manually feeding birds to the point of satiation, and restricting energy income through fasting, we controlled for a presumably large amount of variation in energy income and expenditure occurring in free-living populations. Thus, fasting birds should have catabolized lipid reserves and produced BOHB resulting from lipolysis (Ramenofsky 1990, Boismenu et al. 1992), whereas force-fed birds should have deposited lipids as indicated by higher TRIG levels produced from energy income. Despite these controls and a substantially larger sample size, our model fit (R2 = 0.68) was slightly less than for free-living populations (R2 = 0.75) reported previously by Anteau and Afton 2008b. There was considerable individual variation in mass change within each treatment (feed and fasted), especially for males. We suspect that underlying physiological differences between male and female scaup during spring, potentially in combination with an interacting effect of stress, is the best explanation for these results. In spite of these differences, our results indicate that using lipid metabolites to broadly indicate mass change and/or food acquisition in Lesser Scaup to predict foraging habitat quality is robust to the issues of forage timing, abundance, and diet composition as questioned by Anteau and Afton (2008b). However, the considerable individual variation in mass change response within our treatments, even in females, suggests the need for robust sample sizes for studies using this technique to index stopover site quality. CONCLUSIONS Our results provide information on the utility of TRIG as a measure of energy income and BOHB as a reliable predictor of negative mass change in birds. When predictions of mass change are standardized, it appears that metabolite indices are robust to variations in energy expenditure and should be useful indicators of daily lipid dynamics. Consequently, our findings should be useful for interpretation of TRIG and BOHB concentrations in landscape-level inferences about habitat quality and refueling performance for migratory birds (Seaman et al. 2006, Lyons et al. 2008, Anteau and Afton 2011). We suggest that future research explore the potential mechanism(s) for sex-based differences in the relationship of TRIG and mass change, evaluate temporal variation in TRIG concentrations to better understand rates of change, and test effects of diet on metabolite concentrations for scaup. ACKNOWLEDGMENTS We thank R. Smith (Illinois Department of Natural Resources); C. Hine, A. Yetter, and others (Illinois Natural History Survey); A. Afton (Louisiana State University); R. Klaver (Iowa State University); and J. Lamer and S. Jenkins (Western Illinois University) for contributions to the study design, in-kind contributions, and logistical support for this project. We thank A. Janke for providing support with metabolite assays and helpful comments on earlier drafts of our manuscript. We thank A. Bouton, K. Farinosi, S. Lynch, J. Norman, M. Ryckman, and numerous volunteers for field assistance. We extend a sincere thank you to R. Leonard for his hospitality and care for field crews during scaup capture and banding operations. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Data used in this paper are owned, held, and managed by Western Illinois University. The findings and conclusions in this article are those of the author(s) and the U.S. Geological Survey and do not necessarily represent the views of the U.S. Fish and Wildlife Service. Funding statement: We thank the Mississippi Flyway Council and Department of Biological Sciences and Kibbe Field Station at Western Illinois University for funding. We thank Illinois Natural History Survey and Northern Prairie Wildlife Research Center for in-kind support. Ethics statement: All methods were in compliance with the Western Illinois University Institutional Animal Care and Use Committee (Protocol # 16-06), U.S. Geological Survey Bird Banding Laboratory (Permit # 23923), and Illinois Department of Natural Resources (Permit #NH15.5864). Author contributions: E.J.S., H.M.H., M.J.A., and C.N.J. designed the study; E.J.S. collected the data; E.J.S., H.M.H., M.J.A., and C.N.J. analyzed the data; and all authors wrote or substantially edited the paper. Data depository: Analyses reported in this article can be reproduced using the data provided by Smith et al. (2021). LITERATURE CITED Afton , A. D . ( 1985 ). 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OrnithologyOxford University Press

Published: May 8, 2021

References