Summary of Study ST003675

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002280. The data can be accessed directly via it's Project DOI: 10.21228/M8X534 This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST003675
Study Title	Polar Metabolite Profiles Distinguish Between Early and Severe Sub-maintenance Nutritional States of Wild Bighorn Sheep
Study Summary	Understanding the metabolic adaptations of wild bighorn sheep (Ovis c. canadensis) to nutritional stress is crucial for their conservation. This study employed 1H nuclear magnetic resonance (NMR) metabolomics to investigate the biochemical responses of these animals to varying sub-maintenance nutritional states. Serum samples from 388 wild bighorn sheep collected between 2014 and 2017 during December through March across Wyoming and Montana were analyzed. Multivariate statistics and machine learning analyses were employed to identify characteristic metabolic patterns and metabolic interactions between early and severe sub-maintenance nutritional states. Significant differences were observed in the levels of 15 of the 49 quantified metabolites, including formate, thymine, glucose, choline, and others, pointing to disruptions in one-carbon, amino acid, and central carbon metabolic pathways. These metabolites may serve as indicators of critical physiological processes such as nutritional intake, immune function, energy metabolism, and protein catabolism, essential for understanding how wild bighorn sheep adapt to nutritional stress. The study has generated valuable insights into molecular networks underlying the metabolic resilience of wild bighorn sheep, highlighting the potential for using specific biochemical markers to evaluate nutritional and energetic states in free-ranging ungulates. These insights may help wildlife man-agers and ecologists compare populations across different times in seasonal cycles, providing information to assess the adequacy of seasonal ranges and support conservation efforts. This research strengthens our understanding of metabolic adaptations to envi-ronmental stressors in wild ruminants, offering a foundation for improving management practices to maintain healthy bighorn sheep populations.
Institute	Montana State University
Department	Chemistry and Biochemistry
Laboratory	PI: Dr. Valerie Copie
Last Name	O'Shea-Stone
First Name	Galen
Address	103 Chemistry and Biochemistry Building
Email	galenoshea@gmail.com
Phone	3035237132
Submit Date	2025-01-15
Num Groups	3
Total Subjects	398
Num Females	398
Analysis Type Detail	NMR
Release Date	2025-02-11
Release Version	1

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Project:

Project ID:	PR002280
Project DOI:	doi: 10.21228/M8X534
Project Title:	Polar Metabolite Profiles Distinguish Between Early and Severe Sub-maintenance Nutritional States of Wild Bighorn Sheep
Project Type:	Untargeted NMR Metabolomics
Project Summary:	Understanding the metabolic adaptations of wild bighorn sheep (Ovis c. canadensis) to nutritional stress is crucial for their conservation. This study employed 1H nuclear magnetic resonance (NMR) metabolomics to investigate the biochemical responses of these animals to varying sub-maintenance nutritional states. Serum samples from 388 wild bighorn sheep collected between 2014 and 2017 during December through March across Wyoming and Montana were analyzed. Multivariate statistics and machine learning analyses were employed to identify characteristic metabolic patterns and metabolic interactions between early and severe sub-maintenance nutritional states. Significant differences were observed in the levels of 15 of the 49 quantified metabolites, including formate, thymine, glucose, choline, and others, pointing to disruptions in one-carbon, amino acid, and central carbon metabolic pathways. These metabolites may serve as indicators of critical physiological processes such as nutritional intake, immune function, energy metabolism, and protein catabolism, essential for understanding how wild bighorn sheep adapt to nutritional stress. The study has generated valuable insights into molecular networks underlying the metabolic resilience of wild bighorn sheep, highlighting the potential for using specific biochemical markers to evaluate nutritional and energetic states in free-ranging ungulates. These insights may help wildlife man-agers and ecologists compare populations across different times in seasonal cycles, providing information to assess the adequacy of seasonal ranges and support conservation efforts. This research strengthens our understanding of metabolic adaptations to envi-ronmental stressors in wild ruminants, offering a foundation for improving management practices to maintain healthy bighorn sheep populations.
Institute:	Montana State University
Department:	Chemistry and Biochemistry
Laboratory:	PI: Dr. Valerie Copie
Last Name:	O'Shea-Stone
First Name:	Galen
Address:	103 Chemistry and Biochemistry Building
Email:	galenoshea@gmail.com
Phone:	3035237132

Subject:

Subject ID:	SU003807
Subject Type:	Mammal
Subject Species:	Ovis canadensis canadensis
Taxonomy ID:	112262

Factors:

Subject type: Mammal; Subject species: Ovis canadensis canadensis (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Sheep type	Nurtitional State
SA401802	1372	Blood serum	Mtn	Early-submaint
SA401803	1629	Blood serum	Mtn	Early-submaint
SA401804	1630	Blood serum	Mtn	Early-submaint
SA401805	1358	Blood serum	Mtn	Early-submaint
SA401806	1346	Blood serum	Mtn	Early-submaint
SA401807	1347	Blood serum	Mtn	Early-submaint
SA401808	1359	Blood serum	Mtn	Early-submaint
SA401809	1348	Blood serum	Mtn	Early-submaint
SA401810	1360	Blood serum	Mtn	Early-submaint
SA401811	1361	Blood serum	Mtn	Early-submaint
SA401812	1627	Blood serum	Mtn	Early-submaint
SA401813	1349	Blood serum	Mtn	Early-submaint
SA401814	1362	Blood serum	Mtn	Early-submaint
SA401815	1363	Blood serum	Mtn	Early-submaint
SA401816	1364	Blood serum	Mtn	Early-submaint
SA401817	1350	Blood serum	Mtn	Early-submaint
SA401818	1351	Blood serum	Mtn	Early-submaint
SA401819	1352	Blood serum	Mtn	Early-submaint
SA401820	1365	Blood serum	Mtn	Early-submaint
SA401821	1373	Blood serum	Mtn	Early-submaint
SA401822	1628	Blood serum	Mtn	Early-submaint
SA401823	1626	Blood serum	Mtn	Early-submaint
SA401824	1366	Blood serum	Mtn	Early-submaint
SA401825	1614	Blood serum	Mtn	Early-submaint
SA401826	1743	Blood serum	Mtn	Early-submaint
SA401827	1729	Blood serum	Mtn	Early-submaint
SA401828	1727	Blood serum	Mtn	Early-submaint
SA401829	1738	Blood serum	Mtn	Early-submaint
SA401830	1733	Blood serum	Mtn	Early-submaint
SA401831	1611	Blood serum	Mtn	Early-submaint
SA401832	1612	Blood serum	Mtn	Early-submaint
SA401833	1613	Blood serum	Mtn	Early-submaint
SA401834	1615	Blood serum	Mtn	Early-submaint
SA401835	1625	Blood serum	Mtn	Early-submaint
SA401836	1616	Blood serum	Mtn	Early-submaint
SA401837	1617	Blood serum	Mtn	Early-submaint
SA401838	1618	Blood serum	Mtn	Early-submaint
SA401839	1619	Blood serum	Mtn	Early-submaint
SA401840	1620	Blood serum	Mtn	Early-submaint
SA401841	1621	Blood serum	Mtn	Early-submaint
SA401842	1622	Blood serum	Mtn	Early-submaint
SA401843	1623	Blood serum	Mtn	Early-submaint
SA401844	1624	Blood serum	Mtn	Early-submaint
SA401845	1353	Blood serum	Mtn	Early-submaint
SA401846	1367	Blood serum	Mtn	Early-submaint
SA401847	1725	Blood serum	Mtn	Early-submaint
SA401848	1675	Blood serum	Mtn	Early-submaint
SA401849	1667	Blood serum	Mtn	Early-submaint
SA401850	1668	Blood serum	Mtn	Early-submaint
SA401851	1669	Blood serum	Mtn	Early-submaint
SA401852	1670	Blood serum	Mtn	Early-submaint
SA401853	1671	Blood serum	Mtn	Early-submaint
SA401854	1672	Blood serum	Mtn	Early-submaint
SA401855	1673	Blood serum	Mtn	Early-submaint
SA401856	1674	Blood serum	Mtn	Early-submaint
SA401857	1676	Blood serum	Mtn	Early-submaint
SA401858	1664	Blood serum	Mtn	Early-submaint
SA401859	1677	Blood serum	Mtn	Early-submaint
SA401860	1567	Blood serum	Mtn	Early-submaint
SA401861	1568	Blood serum	Mtn	Early-submaint
SA401862	1569	Blood serum	Mtn	Early-submaint
SA401863	1570	Blood serum	Mtn	Early-submaint
SA401864	1574	Blood serum	Mtn	Early-submaint
SA401865	1575	Blood serum	Mtn	Early-submaint
SA401866	1579	Blood serum	Mtn	Early-submaint
SA401867	1580	Blood serum	Mtn	Early-submaint
SA401868	1666	Blood serum	Mtn	Early-submaint
SA401869	1663	Blood serum	Mtn	Early-submaint
SA401870	1354	Blood serum	Mtn	Early-submaint
SA401871	1651	Blood serum	Mtn	Early-submaint
SA401872	1355	Blood serum	Mtn	Early-submaint
SA401873	1356	Blood serum	Mtn	Early-submaint
SA401874	1357	Blood serum	Mtn	Early-submaint
SA401875	1374	Blood serum	Mtn	Early-submaint
SA401876	1368	Blood serum	Mtn	Early-submaint
SA401877	1369	Blood serum	Mtn	Early-submaint
SA401878	1370	Blood serum	Mtn	Early-submaint
SA401879	1375	Blood serum	Mtn	Early-submaint
SA401880	1371	Blood serum	Mtn	Early-submaint
SA401881	1652	Blood serum	Mtn	Early-submaint
SA401882	1662	Blood serum	Mtn	Early-submaint
SA401883	1653	Blood serum	Mtn	Early-submaint
SA401884	1654	Blood serum	Mtn	Early-submaint
SA401885	1655	Blood serum	Mtn	Early-submaint
SA401886	1656	Blood serum	Mtn	Early-submaint
SA401887	1657	Blood serum	Mtn	Early-submaint
SA401888	1658	Blood serum	Mtn	Early-submaint
SA401889	1659	Blood serum	Mtn	Early-submaint
SA401890	1660	Blood serum	Mtn	Early-submaint
SA401891	1661	Blood serum	Mtn	Early-submaint
SA401892	1726	Blood serum	Mtn	Early-submaint
SA401893	1739	Blood serum	Mtn	Early-submaint
SA401894	1714	Blood serum	Mtn	Early-submaint
SA401895	1736	Blood serum	Mtn	Early-submaint
SA401896	1735	Blood serum	Mtn	Early-submaint
SA401897	1741	Blood serum	Mtn	Early-submaint
SA401898	1716	Blood serum	Mtn	Early-submaint
SA401899	1731	Blood serum	Mtn	Early-submaint
SA401900	1730	Blood serum	Mtn	Early-submaint
SA401901	1715	Blood serum	Mtn	Early-submaint

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Collection:

Collection ID:	CO003800
Collection Summary:	A total of 388 serum samples were obtained from wild bighorn sheep populations in Wyoming (7 locations) and Montana (4 locations) via helicopter netgun capture techniques as described below: Animal captures occurred from December through March during the winters of 2014–15, 2015–16, and 2016–17, when all animals were on senescent native forages resulting in sub-maintenance diets. The majority of these animals, 385, were captured using net guns fired from a helicopter. This method required close pursuit by the helicopter, normally for 2–5 min, until a small net was deployed from a shoulder mounted gun that entangled the animal. A handler was place on the ground to physically restrain the captured animal via a blindfold and hobbles. Animals captured in remote wilderness were processed and released at the capture site, generally within 15–35 min of capture. In other situations with good ground access, the blindfolded and hobbled animals were placed in transport bags and slung under the helicopter on a long cable to a central processing site where they were processed, with blood samples normally collected between 20 and 60 min after capture. Large nets suspended over baited sites (dropnet) were dropped on 104 animals. Once the net was dropped, a large crew of handlers physically restrained the animals with blindfolds and hobbles, extracted each animal from the net, and carried it to a central processing area within 100 m of the dropnet site. Because the large nets captured 10–30 animals each, captured animals were queued for processing with blood samples generally drawn from 20 to 90 min following deployment of the nets. Ground-based delivery of immobilizing drugs, i.e. a cocktail of butorphanol, azaperone, and medetomidine; via dart rifles was used to capture 73 animals12. Animals were approached to within 5–15 m for effective dart delivery. Once stuck by the dart, the animals normally ran 5–20 m, and resumed pre-darting behaviors with their social group until the drugs began to take effect, causing the darted animal to bed down until sedated, normally 20–35 min following drug delivery. Sedated animals were blindfolded and hobbled, sampled, and drug antagonists administered, with processing time normally requiring 10–20 min. For all capture techniques, a blood sample was drawn from the jugular vein of each animal and immediately placed under refrigeration until serum was harvested 2–6 h after capture. Serum was frozen at − 20 °C for transport to research facilities where all samples were stored at − 80 °C until further processed. The majority of the samples originated from unique animals, but small numbers of marked animals were repeatedly captured and sampled in consecutive years in three of the Wyoming herds.
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR003816
Treatment Summary:	Due to seasonal changes, these wild animals were sustained on senescent native forages, resulting in sub-maintenance diets from late summer/early fall until the onset of the growing season the following spring. Animals captured in December months (n = 170) were classified as “early submaintenance” (Early-SM), animals captured in January (n = 35) were classified as “moderate submaintenance” (Mod-SM), and animals captured from February through March (n=183), which subsisted approximately 6 months on senescent forages, were classified as “severe submaintenance” (Se-vere-SM).

Treatment ID:

TR003816

Treatment Summary:

Due to seasonal changes, these wild animals were sustained on senescent native forages, resulting in sub-maintenance diets from late summer/early fall until the onset of the growing season the following spring. Animals captured in December months (n = 170) were classified as “early submaintenance” (Early-SM), animals captured in January (n = 35) were classified as “moderate submaintenance” (Mod-SM), and animals captured from February through March (n=183), which subsisted approximately 6 months on senescent forages, were classified as “severe submaintenance” (Se-vere-SM).

Sample Preparation:

Sampleprep ID:	SP003814
Sampleprep Summary:	Serum samples were prepared for small molecule polar metabolite extraction and 1H NMR metabolomics as follows: Samples were thawed at room temperature following storage at − 80 °C with reagents kept at − 20 °C until used. A 1:3 500 μL serum: 1500 μL acetone solution was added to 2 mL plastic, flat-cap conical vials13. The resulting solution was mixed thoroughly by inverting the sample tubes 10 times, and incubated at − 20 °C for 60 min to allow for protein precipitation, followed by sample centrifugation at 13,000×g for 30 min at room temperature. Clarified supernatants containing the polar metabolite mixtures were subsequently transferred to new 2.0 mL tubes and dried overnight using a Speedvac vacuum centrifuge with no heat, and stored at − 80 °C until further use. For NMR, dried metabolite extracts were resuspended in 600 μL of NMR buffer consisting of 25 mM of NaH2PO4/Na2HPO4, 0.4 mM of imidazole, 0.25 mM of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) in 90% H2O/10% D2O, pH 7.0.

Sampleprep ID:

SP003814

Sampleprep Summary:

Serum samples were prepared for small molecule polar metabolite extraction and 1H NMR metabolomics as follows: Samples were thawed at room temperature following storage at − 80 °C with reagents kept at − 20 °C until used. A 1:3 500 μL serum: 1500 μL acetone solution was added to 2 mL plastic, flat-cap conical vials13. The resulting solution was mixed thoroughly by inverting the sample tubes 10 times, and incubated at − 20 °C for 60 min to allow for protein precipitation, followed by sample centrifugation at 13,000×g for 30 min at room temperature. Clarified supernatants containing the polar metabolite mixtures were subsequently transferred to new 2.0 mL tubes and dried overnight using a Speedvac vacuum centrifuge with no heat, and stored at − 80 °C until further use. For NMR, dried metabolite extracts were resuspended in 600 μL of NMR buffer consisting of 25 mM of NaH2PO4/Na2HPO4, 0.4 mM of imidazole, 0.25 mM of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) in 90% H2O/10% D2O, pH 7.0.

Analysis:

Analysis ID:	AN006036
Analysis Type:	NMR
Num Factors:	5
Num Metabolites:	47
Units:	Micromolar

NMR:

NMR ID:	NM000304
Analysis ID:	AN006036
Instrument Name:	Bruker 600 MHz Avance III
Instrument Type:	FT-NMR
NMR Experiment Type:	1D-1H
NMR Comments:	Bruker 600 MHz Avance III NMR Spectrometer (actively shielded) with 600 MHz TCI (H-C/N-D05Z) LT (liquid helium cooled) Helium CryoProbeTM. Available working temperature range: -40oC+80oC. Three channels: 1H, X1=13C, X2=15N, lock. Auto-tune/Auto-shim. SampleJetTM automated sample loading system.
Spectrometer Frequency:	600 MHz