Liver Fatty Acid, Mineral, and Fat-Soluble Nutrients in Wild and Captive Greater Prairie-Chickens

The greater prairie-chicken Tympanuchus cupido pinnatus inhabits tall and mixed grass prairies of the Central Great Plains, whereas the Attwater's prairie-chicken T. c. attwateri is now restricted to two small populations along the Texas Gulf coast. The Attwater's prairie-chicken was federally listed as endangered in 1967, and the U.S. Endangered Species Act provided immediate protection of this grassland obligate after its establishment (Udall 1967; ESA 1973, as amended; USFWS 2021). A captive breeding program currently maintains the Attwater's prairie-chicken. Despite differences in geographic range, the feeding ecologies are similar between the two subspecies and are composed of plant materials (green shoots, buds, and leaves), flowers, seeds, forbs, and insects (Mohler 1952; Korschgen 1962; Cogar 1980; Torres-Poché et al. 2020). Studies using fecal analyses provide dietary details, indicating high diversity and seasonality for both subspecies. Cogar (1980) reported that the Attwater's prairie-chicken diet was composed mainly of herbage throughout the year with minor amounts of seeds and insects. The consumption of seeds and insects was variable, increasing in summer and peaking in autumn. Later, Torres-Poché et al. (2020) used isotopic analyses of fecal samples from Attwater's prairie-chickens to conclude that diets consisted of 61–65% arthropods during summer and autumn and 64% vegetation during winter. Overall, annual diets were 65% animal based and 35% plant based. Rumble et al. (1988) reported that greater prairie-chickens in North Dakota consumed 34 different items over winter, predominantly corn, sunflower, and soybean seeds, along with herbage of mainly agricultural origin. Conversely, Rumble et al. (1988) identified 59 items during spring and summer, primarily dandelion flowers, alfalfa, and sweet clover, with increasing percentages of arthropods (the preferred food of juveniles) later in the season. Although detailed studies of the nutrient content of the diets of both subspecies are lacking, managers commonly use the greater prairie-chicken and domestic chickens as proxy physiologic models for Attwater's prairie-chicken captive breeding programs (Morrow et al. 2019). Diet and tissue nutrient concentrations are a current focus of detailed investigation (Morrow et al. 2019), with a goal of better understanding feeding management and nutritional targets for optimal health and reproduction of the Attwater's prairie-chicken.

Morrow et al. (2019) recently documented reference values for fatty acids, minerals, and fat-soluble micronutrients from wild Attwater's prairie-chicken and greater prairie-chicken eggs as well as egg nutrient concentrations between wild (i.e., free-ranging) and captive Attwater's prairie-chickens. Morrow et al. (2019) showed that all nutrient categories were impacted to some extent by the habitat and availability of dietary items. Samples from wild birds had a higher omega-3:omega-6 (n-3:n-6) fatty acid ratio as well as higher levels of the minerals magnesium, manganese, and zinc and various carotenoid pigments than samples from captive-raised birds. These studies suggest possible impacts on maternal nutrition that could affect embryos and neonatal chicks and support the need for further research to understand optimal nutrition in captive and wild prairie-chicken populations. Our main objectives in the current investigation were to 1) quantify concentrations of fatty acids, macro- and trace minerals, fat-soluble vitamins A and E, and select carotenoids in livers from greater prairie-chickens; 2) compare data to concentrations present in other Galliformes to determine the utility of these indices; and 3) contrast nutrient findings from birds consuming captive or wild diets to determine the potential impacts of diets on these nutrient parameters. We anticipate using the nutrient values obtained to guide improvements to feeding programs and management of the Attwater's prairie-chicken.

We analyzed 22 frozen liver samples (1–5 g) from apparently healthy wild-hatched or artificially incubated greater prairie-chickens that died of trauma and sought to include similar representation of sexes in the two groups. We collected 12 samples from wild-hatched, free-ranging birds at a hunter check station in Nebraska on 15 September 2018 from three after-hatch-year and three hatch-year males and three after-hatch-year and three hatch-year females. We determined maturity by molt pattern (Zwickel and Lance 1966; Redfield and Zwickel 1976; Petersen and Braun 1980). We obtained an additional 10 liver samples from greater prairie-chickens (five males and five females) that were artificially incubated, hatched, and raised at the George Miksch Sutton Avian Research Center (Bartlesville, Oklahoma) from 2016 to 2018 as described below. All were younger than 1 year old (hatch-year), with the exception of one female that died in captivity. We fed them a diet of commercial grains (PMI Nutrition International LLC, Brentwood, MO), chopped vegetables, fruit, leafy greens, crickets, and mealworms according to the standard protocol for the Attwater's prairie-chicken captive program.

In preparation for release into the wild, we housed all 10 captive-raised birds in outdoor pens (30 × 100 m) where they could forage on natural vegetation and insects in addition to supplied pelleted diets, whole grains, and frozen vegetables. We sampled four birds immediately following lethal net collisions in these flight pens, including the older female. We sampled six captive-raised birds that died after a soft release that occurred either on 7 or 20 November 2017 on a private ranch southeast of Bassett, Rock County, Nebraska. The soft release included spending 11 or 13 days in acclimation pens (∼6 × 18 m), in which we fed the birds the same type of mixed food as we fed them previously. We made whole grains available on a continuous basis near the release site over the winter. We tracked the released birds (n = 6) with very high frequency transmitters (model A3930 with shortened antenna; Advanced Telemetry Systems, Isanti, MN) for 7–136 days (median = 39 days) before we found their intact carcasses. We excluded the female that was in the wild for the longest time from further analyses because she dispersed ∼16 km from the release site and had presumably transitioned to a completely wild diet, whereas we found the five sampled carcasses within approximately 0.5 km from the release site. When collected, the remaining females weighed 732–772 g (n = 2), and the males weighed 872–917 g (n = 3). We stored harvested livers from all birds at −20°C for up to 14 mo before subsampling for nutrient concentrations.

We sent 1–5 g of frozen tissue via overnight courier on dry ice to Zooquarius Analytical Laboratory (Ithaca, New York) for fatty acid and mineral profiling. Total fatty acids were determined through gas chromatography of methyl esters using the methods of O'Fallon et al. (2007). Macrominerals (calcium, magnesium, phosphorus, potassium, sulfur, and sodium) and trace elements (cobalt, copper, iron, manganese, molybdenum, and zinc) were analyzed using inductively coupled plasma radial spectroscopy following predigestion (Thermo Fisher Scientific Inc., Waltham, MA). Fat-soluble nutrients (vitamins A [as retinol] and E [as α-tocopherol]) and a suite of carotenoids (anhydrolutein, α-carotene, β-carotene, β-cryptoxanthin, lutein, and zeaxanthin) were assayed by high-performance liquid chromatography at Arizona State University (Tempe) using the methodology of McGraw et al. (2006). We report results as percentage of total fatty acids, percent dry weight (dw) for macrominerals, microgram per gram of dw for trace minerals, and microgram per gram of wet weight (ww) for fat-soluble nutrients. Water content (%) was measured by drying at 100°C.

Because of small sample sizes and to avoid having to make assumptions about underlying probability distributions of the data, we used medians as a measure of central tendency. We calculated 95% bias-corrected and accelerated bootstrapped (20,000 replicates) confidence intervals for the medians (boot R package [Canty and Ripley 2021], simpleboot R package [Peng 2019], and R version 4.0.4 [R Core Team 2021]). If a bias-corrected and accelerated confidence interval could not be calculated, we used the percentile method. We first determined if data could be pooled by sex and age, which we considered nuisance variables that were not central to our main objectives, by using a null hypothesis test of no difference between group medians with a critical bootstrapped P value of <0.05. If the null hypothesis was rejected for age, then we used data from only wild and captive hatch-year birds because they made up our largest sample. Similarly, after initial screening of nuisance variables, we compared medians between wild and captive-raised birds using null hypothesis testing of no difference between group medians. For analysis, we split the captive-raised birds between released and nonreleased because of the uncertainty in the proportion of wild and captive diet items represented in the livers of released birds. We used Bonferroni-corrected confidence intervals and P values when making pairwise comparisons between the three groups. For a more complete depiction of differences between wild and released or nonreleased captive-raised birds, we also visually presented the difference in confidence intervals by plotting the kernel density distribution of the bootstrapped medians.

Nutrient values in deep frozen tissues can remain stable for years in storage; thus, we presume that values reported here are valid and representative (Data S1, Supplemental Material). Although oxidative deterioration of organic compounds (fatty acids and fat-soluble vitamins) in liver tissues may occur after death, we sampled captive or harvested birds within hours after death, and we only sampled released birds that died and that were recovered in cold temperatures. We failed to reject the null hypothesis of no difference between male (n = 6) and female (n = 6) medians for all analyzed nutrients, so we pooled data relative to sex for further analyses. Similarly, few nutrients varied by age in the wild birds; concentrations of two minerals (iron and molybdenum) and the carotenoid anhydrolutein were higher in after-hatch-year (n = 6) than in hatch-year (n = 6) greater prairie-chicken livers, but concentrations of sodium and “other” fatty acid concentrations were lower (Table 1). For these five variables, we only used hatch-year wild birds to compare with captive-raised birds. For captive-raised birds, we rejected the null hypothesis of no difference between median liver nutrient concentrations for five nutrients (behenic acid, sodium, cobalt, zeaxanthin, and water) in the released (n = 5) birds compared with the nonreleased (n = 4) birds (Tables 2–4).

Liver profiles from captive-raised birds that were released showed more differences from wild birds than from nonreleased captive birds (Table 2; Figure 1). Fatty acids that were higher in released birds included saturated fatty acid palmitic (C16:0) and monounsaturated palmitoleic (C16:1n7) and oleic acids (C18:1), contributing to overall higher proportions of both saturated and monounsaturated fatty acids (Figure 2). Conversely, specific acids that were lower in released bird livers than in the livers of wild birds included saturated arachidic acid (C20:0) and polyunsaturated docosahexaenoic acid (C22:6n-3), leading to an overall decrease in total polyunsaturated fatty acids in the released bird livers (Table 2; Figure 2). Of the identified fatty acids that we analyzed, lignoceric acid values were almost twice the level in nonreleased captive-raised birds compared with those levels observed in wild birds, whereas arachidic acid concentrations were approximately half the level found in wild bird livers. Summed proportions of hepatic fatty acid patterns were similar between captive-raised and wild birds, regardless of whether they were released or not, with saturated fats comprising the largest amount (40–45% of total; ∼10% higher in captive-raised birds), followed by polyunsaturated fatty acids (26–35%, ∼25% higher in wild birds). Monounsaturated fatty acids constituted 8–18% of the total (slightly more than 100% higher in captive-raised birds), whereas unidentified fatty acids differed more widely in captive-raised birds (17–34%) and were lower than in wild birds.

While values were similar between captive-raised birds and wild birds for most macrominerals in this study, sulfur was slightly lower in captive-raised greater prairie-chicken livers regardless of release status (Table 3; Figure 3). Sodium was uniquely lower in released bird livers than in either wild or nonreleased bird livers. Trace mineral concentrations, however, exhibited more distinct separations between wild and captive bird samples, with both copper and manganese considerably lower in captive-raised than in wild prairie-chickens. We observed the lowest median levels (Table 3) and highest variability (Figure 3) of these two nutrients in birds that had been released.

Values were similar between captive-raised birds and wild birds for most fat-soluble micronutrients in this study. However, β-carotene was lower in livers from captive-raised greater prairie-chickens (both released and nonreleased) but showed greater variability among wild birds (Table 4; Figure 4). We found lower concentrations of the carotenoid pigment zeaxanthin in livers from nonreleased captive-raised birds than in livers from either wild birds or prairie-chickens that had been released (Table 4).

While diet impacts health and immune function, growth, and reproductive performance, there is limited information on nutritional profiles and optimal nutrient balance for most species. Therefore, we generally rely on information extrapolated from other species fed under controlled conditions that might or might not duplicate the diverse ingredients or habitat variability encountered naturally. For the Attwater's prairie-chicken, nutritional requirements and physiology of domestic poultry species and data obtained from the more common greater prairie-chicken are used to refine managed feeding protocols for captive breeding and recovery programs. The notable differences in tissue nutrient concentrations that we identified between wild and captive-raised prairie-chickens may reflect ingredient profiles in the diets consumed by these birds and form a foundation for targeted changes.

Poultry studies show that fatty acids in some tissues such as muscle and adipose correlate directly with dietary fatty acid profiles, while liver responses seem to be dependent on the class of fatty acid considered, with n-3 polyunsaturated and saturated fatty acids showing the greatest correlations (Kanakri et al. 2018). General patterns in the proportions of hepatic fatty acids reported for domestic chickens (45.6% saturated, 23.6% monounsaturated, and 30.8% polyunsaturated; Majewska et al. 2016) were more similar to overall values for captive-raised than wild birds in our study, which is not surprising considering that primary commercial diets that we used were based on domestic poultry nutrient recommendations and ingredients. However, some interesting differences are apparent in that the released birds differed significantly in all three categories from values obtained in wild birds (Table 2). Wild birds in particular showed higher levels of polyunsaturated fatty acids (linoleic [C18:2n6], linolenic [C18:3n3], and docosahexaenoic and eicosapentaenoic [C20:3n3] acids) and lower levels of monounsaturated fatty acids (oleic and palmitoleic acids) than are typically seen in poultry studies. It is likely that the consumption of a higher percentage of green plant materials by wild birds (Rumble et al. 1988), with accompanying higher levels of n-3 linolenic acid (Rajaram 2014), underlies the observed higher values of polyunsaturated fatty acids and variability seen in wild birds. Additionally, consumption of insects (even seasonally) by wild greater prairie-chickens may contribute to the change in fatty acid categories observed between wild and captive birds; insect oil inclusion decreases liver monounsaturated fatty acids and increases liver polyunsaturated fatty acids in domestic poultry chicks (Benzertiha et al. 2019).

Health benefits associated with the consumption of n-3 fatty acids led to numerous livestock feeding trials with supplemental n-3, resulting in increased deposition in tissues (i.e., eggs, milk, meat, and organs) over days to weeks, with changes to tissue fatty acid profiles reported within 4 weeks after consumption of these products (Kanakri et al. 2018). In a study of prairie-chicken egg composition, Morrow et al. (2019) found that a change in lipids added to manufactured pellets fed to captive birds resulted in an n-3:n-6 ratio shift (from 1:7 to 1:4) following replacement of soy oil with flax oil but did not achieve the 1:1 ratio noted in egg tissues of wild birds. Liver tissues from wild greater prairie-chickens in our study contained numerically higher levels of total n-3 fatty acids (linolenic, docosahexaenoic, and eicosapentaenoic acids) than liver tissues from captive-fed birds. Our calculated n-3:n-6 ratios of 1:15, 1:14, and 1:25 for wild, nonreleased captive-raised, and captive-raised released birds, respectively, are similar to the value of 1:21 reported by Majewska et al. (2016) for poultry fed commercially formulated diets. Grains in commercial feeds typically contain a lower n-3:n-6 ratio than green leafy plants (Simopoulos 2002). This likely contributes to differences in these fatty acid fractions, particularly for birds released late in the growing season and offered mature grains. The similarity of the fatty acid fractions in the nonreleased birds and the wild birds is probably due to dietary ingredients, primarily linolenic acid from flax oil and marine ingredients in the pelleted captive diet and green plant fractions in the wild diet. Pellets fed to captive greater prairie-chickens contained both algae and fish meal as components (PMI Nutrition International LLC, Brentwood, MO), which is reflected in the higher levels of docosahexaenoic acid in the livers of nonreleased captive-raised birds. Dilution of that dietary component after release and adaptation to a wild diet could explain the lower level of docosahexaenoic acid in livers from released captive-raised birds. More detailed nutrient analyses of individual dietary components on a seasonal basis would clarify speculation. Dietary sources of other significantly different fatty acids (arachidic, behenic, and lignoceric acids) among groups are unknown at this time.

Poultry studies show that changes to tissue storage of minerals, as a result of dietary changes, are seen in 7–21 days to as long as 5–8 weeks. Hence, our 3- to 6-week study should be sufficient to reflect changes in status due to different diets. With the exception of sulfur, for which we found no comparative data, macromineral concentrations overlapped widely among greater prairie-chicken groups, and values were within ranges reported for the livers of domestic poultry, such as chickens, turkeys, ducks, geese, and quail (Puls 1994a; Cieślik et al. 2011; Majewska et al. 2016; Duman et al. 2019; USDA 2021) as well as wild Canada geese Branta canadensis (Belinsky and Kuhnlein 2000) and willow ptarmigan Lagopus lagopus (Pulliainen and Tunkkari 1986). Therefore, we consider our findings to be reasonable guideline reference values for the species. Similarly, serum biochemistry values and hematology parameters reported for captive-raised Attwater's prairie-chickens were within the expected ranges for a variety of avian species (West and Haines 2002). Tissue sodium concentrations are a poor indicator of sodium intake (Puls 1994a); hence, the distinctly lower sodium values seen in released bird livers may be spurious. Generally, grains and forage plants are deficient in sodium. Diets consumed by the released birds (presumably composed of agricultural seeds, mature foliage, or crop residues available at the time of release) may lack sodium as opposed to their previous supplemented pellets or the mixed diets (including insects) of wild birds. Similarly, the higher sulfur values in wild greater prairie-chickens than we observed in captive-raised groups may reflect dietary ingredient differences but require future investigation.

Some essential trace minerals that we measured in this study appear to differ between wild and captive-raised birds and differ from values considered normal in domestic poultry, as might be expected due to their selective breeding for rapid growth and meat quality. In particular, hepatic copper levels of captive-raised birds were below published ranges of ∼17–36 mg/kg dw (Puls 1994a) and 15–20 mg/kg dw (Majewska et al. 2016) for granivores. Hepatic copper levels for all the birds in our study were at the lower ends of published ranges for more herbivorous avian species, including 13–15 mg/kg dw in grouse and ∼15–100 mg/kg dw in grazing Anseriformes (Pullianen and Tunkkari 1986; Puls 1994a; Cieślik et al. 2011). While manganese levels in all groups of greater prairie-chickens were also within the expected range of 5–15 mg/kg in granivorous birds (Puls 1994a; Majewska et al. 2016), Pullianen and Tunkkari (1986) reported much higher and seasonally variable manganese values of 25–100 mg/kg for wild willow ptarmigan. Iron and molybdenum levels in both captive and wild greater prairie-chickens (∼1,650–1,900 and 8–11 mg/kg, respectively) were higher than expected values of 200–1,100 mg/kg of iron and 1.5–3 mg/kg of molybdenum in domestic poultry (Puls 1994a; Majewska et al. 2016; Duman et al. 2019). We do not know the significance of these differences at this time, but they could have health implications for prairie-chickens. High iron can lead to oxidative damage, while copper and manganese are critical cofactors of antioxidant enzyme systems. High molybdenum also interferes with copper uptake. Morrow et al. (2019) noted differences in egg tissue copper, manganese, and zinc levels between wild and captive prairie-chicken populations, with wild bird levels higher than those observed in captive-raised birds. Additionally, copper deficiency in poultry and other species changes liver and adipose tissue fatty acid profiles, resulting in lower saturated fats and higher proportions of polyunsaturated fats, similar to the hepatic fatty acid pattern differences that we observed between captive-raised and wild birds from our study samples.

The low cobalt in released versus nonreleased captive-raised birds may indicate a marginal vitamin B12 status that requires 2–5 mo to deplete (Puls 1994a). Vitamin B12, containing cobalt at its core, is provided by supplemented commercial pellets and through either microbial production or consumption of animal-based foodstuffs, but vitamin B12 can be lacking in solely plant-based diets. Trace mineral status from the current captive diets appears suboptimal, particularly copper, manganese, and possibly long-term storage of cobalt; interactions among trace minerals warrant further investigation.

Dietary impacts of fat-soluble vitamins and carotenoid pigments should be reflected in tissues within days to weeks of consumption (McGraw et al. 2006). In this study, vitamin E (α-tocopherol) values were at the high end of the range of 4–40 μg/g ww reported for a variety of domestic poultry species, including ducks, turkeys, and chickens (Puls 1994b) as well as quail (Sahin et al. 2002), and should be considered an appropriate guideline for the species. No histologic evidence of vitamin E imbalances (such as exudative tissues, cardiomyopathy, and muscular dystrophies) exists for captive greater prairie-chickens over years of managed care. The vitamin E concentrations of about 200 IU/kg dw in manufactured diets were developed over several years of modification and appear to result in comparable tissue levels displayed in wild birds. While appropriate for greater prairie-chickens, these values may or may not be appropriate for Attwater's prairie-chickens due to species-specific variability reported elsewhere (Morrow et al. 2019).

Vitamin A nutrition is not as clearly defined and is impacted by multiple factors. Hepatic vitamin A values (measured as retinol) reported in the literature vary widely by species, with concentrations ranging from about 2 μg/g ww in quail to 60–300 μg/g ww in chickens (Puls 1994b) compared with the prairie-chicken concentrations of approximately <1–8 μg/g that we report here. In addition, vitamin A values in captive studies are highly dependent on the dietary provisioning of preformed vitamin A at various concentrations (Puls 1994b). Wild greater prairie-chickens, consuming only plant-based materials or insects containing low or undetectable vitamin A concentrations (Oonincx and Finke 2020), need to convert carotenoid precursors into active retinol to meet vitamin requirements. The variability of median retinol levels in wild liver samples (Figure 4) may be reflective of variable carotenoid concentrations of native food. Those differences have not yet been quantified nor have utilization pathways been determined for this species. Few comparative liver carotenoid concentrations are reported for domestic poultry; however, chicken hepatic β-carotene and lutein levels of 0.4 and 6–9 μg/g ww, respectively (Puls 1994b), are considerably lower than greater prairie-chicken values that we report here.

Lutein, zeaxanthin, β-cryptoxanthin, and β-carotene represent major carotenoids identified in a variety of seeds eaten by birds (McGraw et al. 2002) and plant-based diets eaten by other species. These carotenoids are absorbed in the intestine and transported throughout the body, and some are converted by tissue enzymes to other metabolites that result in nutrient or pigment activity. Additionally, carotenoids are important for optimal immune function, reproductive success, and embryonic development (Nabi et al. 2020). Birds and fish poorly utilize carotenes and preferentially accumulate polar hydroxy- and ketocarotenoids. The higher levels of all carotenoids that we observed in this study reflect dietary sources for wild greater prairie-chicken and likely contribute to health status and ornament pigmentation. Wild eggs from Attwater's and greater prairie-chickens displayed higher levels of total carotenoids than captive-fed birds, including β-carotene, zeaxanthin, and anhydrolutein (Morrow et al. 2019). The significantly lower β-carotene concentrations in both captive-raised groups than observed in wild birds likely reflects considerably reduced levels of green forage in the captive diets or a seasonal lack of green plants in the released birds. Dierenfeld and Morrow (2021) analyzed native plants eaten by wild Attwater's prairie-chickens in Texas and measured greater than tenfold higher β-carotene levels in native plants than in manufactured pelleted feed and lutein concentrations approximately eightfold higher in manufactured pellets than in wild food plants (n = 9 species). The substantially higher zeaxanthin concentrations in the wild as well as captive-raised released greater prairie-chickens suggests that corn comprises a significant portion of diets for these two groups during the sampling periods because zeaxanthin is a primary carotenoid pigment found in corn. Lastly, the anhydrolutein pigment difference in age groups of wild birds suggests a possible bioaccumulation of a carotenoid with bioconversion in the liver (McGraw et al. 2002), presumably derived from dietary lutein or through β-cryptoxanthin as an intermediary. Anhydrolutein may have both vitamin and ornamentation pigment coloration activity. The importance of carotenoid and vitamin A nutrition to prairie-chicken health and reproduction deserves further investigation. Contrasting retinol and carotenoid profiles in both liver (this study) and egg yolk (Morrow et al. 2019) tissues between wild and captive-raised birds suggests that absorption, storage, and metabolism of these nutrients provided by various native foods may provide useful guidelines for the preparation of captive diets.

Despite an attempt to simulate natural feeding habits of captive prairie-chickens by housing them in large, vegetated pens containing a variety of plants and invertebrate prey species, some nutritional differences with potential health consequences appear when comparing their liver analyses with those from wild prairie-chickens. Further implications for different nutrients are reflected in birds transitioned for release programs. Assuming that the wild birds represent target nutrient levels, nutrient concentrations in diets supplied to captive-raised birds may need to be modified to achieve those levels. Particularly, trace minerals copper and manganese and the β-carotene (relative to other carotenoids) content of captive diets should be targeted based on data from this comparison. Other positive nutritional strategies to consider are making supplemental feeds with oil seeds that have elevated polyunsaturated fatty acid profiles, adding ingredients such as green plant materials with higher carotenoid levels, and ensuring that sources of salts mineralized with trace elements (at least copper, manganese, and cobalt) are readily available at release sites. Recognizing and understanding nutrient status and limitations will guide more nutritionally optimal dietary protocols. Even timing releases over periods of higher resource diversity (including insects) to better duplicate nutrient diversity may be a practical option. Finally, the adequacy of using these greater prairie-chicken values as a reference when formulating the captive diets for Attwater's prairie-chickens needs to be evaluated as data become available.

Please note: The Journal of Fish and Wildlife Management is not responsible for the content or functionality of any supplemental material. Queries should be directed to the corresponding author for the article.

Data S1. Hepatic fatty acid, mineral, and fat-soluble micronutrient concentrations in wild and captive-raised greater prairie-chickens Tympanuchus cupido collected from 2017 to 2018 in Oklahoma and Nebraska.

Available: https://doi.org/10.3996/JFWM-22-003.S1 (16 KB XLSX)

Reference S1. Rumble MA, Newell JA, Toepfer JE. 1988. Diets of greater prairie chickens on the Sheyenne National Grasslands. Pages 49–54 in Bjugstad AJ, technical coordinator. Prairie chickens on the Sheyenne National Grasslands. Fort Collins, Colorado: U.S. Department of Agriculture. General Technical Report RM-159.

Available: https://doi.org/10.3996/JFWM-22-003.S2 (83 KB PDF) and https://www.fs.usda.gov/research/treesearch/22788 (August 2022)

Reference S2.[USFWS] U.S. Fish and Wildlife Service. 2021. Attwater's greater prairie-chicken (Tympanuchus cupido attwateri) 5-year review: summary and evaluation. Eagle Lake and Houston: Attwater Prairie Chicken National Wildlife Refuge and Texas Coastal Ecological Services.

Available: https://doi.org/10.3996/JFWM-22-003.S3 (1.286 MB PDF) and https://ecos.fws.gov/docs/tess/species_nonpublish/995.pdf (August 2022)

We thank the late Dr. John Toepfer, Brandon Gibson, Rich Lackaff, William Vodehnal, and Nebraska hunters for assistance in obtaining samples in Nebraska; Nebraska Game and Parks Commission and U.S. Fish and Wildlife Service for permits and logistic support; and funding from the Mohamed bin Zayed Species Conservation Fund and the National Fish and Wildlife Foundation. We also thank the Associate Editor, two anonymous reviewers, and John Hoolihan for their helpful critique that greatly improved this manuscript.

Any use of trade, product, website, or firm names in this publication is for descriptive purposes only and does not imply endorsement by the U.S. Government.