Examining the serum metabolome profiles of bighorn sheep captured by the three primary techniques used to capture wild ungulates revealed significant changes in polar metabolite levels between the different animal groups, and trends that persisted throughout the analyses when directly comparing, in a pairwise fashion, specific capture techniques. Results from PLS-DA modeling and analysis of the top 15 metabolites that contribute most (VIP > 1.2) to the separation of the three capture groups revealed that amino acid levels of tryptophan, valine, isoleucine, phenylalanine, and proline were highest in animals captured by dart, with intermediate levels in animals capture using dropnets, and lowest in animals captured using the helicopter method (Fig. 3A). One-way ANOVA analyses identified additional amino acids that displayed similar decreasing level trends from dart to dropnet to helicopter capture (dart > drop net > helicopter) methods, and included arginine, asparagine, aspartate, cysteine, glutamate, and glutamine, glycine, histidine, leucine, lysine, serine, and tyrosine (Fig. 4). These metabolite level changes suggest a shift in amino acid metabolism, and a potentially higher catabolism of these compounds as a function of increasingly more energetically intense and possibly more stressful capture methods such as helicopter capture.
Of these amino acids, aspartate, glycine, and glutamate function as precursors for neurotransmitter synthesis, and may therefore be valuable indicators of the capture techniques’ impacts on animal health and changes to their physiological state. Glutamate is a fundamental component of nitrogen excretion in the urea cycle, and its lower serum levels in animals captured by helicopter support the idea of altered metabolite flow through the urea cycle. In addition to these patterns, decreasing levels of aspartate were observed in samples of dropnet and helicopter captured animals compared to the levels found in the dart-captured animals. The change regarding urea cycle alterations also manifested itself in differential serum urea levels, with fold changes (FC) between the groups decreasing significantly with capture techniques, with a mean FC difference of 1.4 for the dart-captured group, 0.26 for the dropnet-captured group, and − 0.3 for the helicopter-captured animals (Supplementary Table S2). As urea recycling is a prominent feature of ruminant metabolism and urea flux can rapidly change, the urea concentration changes observed between the three capture techniques support an impact on urea cycle intermediates29. While the trend of an overall decrease in urea cycle intermediates parallels a similar trend in amino acid concentrations, the extent to which amino acid metabolism is linked to changes in urea cycle activity is difficult to evaluate due to the nature of nitrogen recycling in the rumen of these ruminants.
Other metabolites found in significantly higher concentrations in the serum samples of dart-captured animals compared to the two other techniques included: formate, glucose, 3-hydroxybutyrate, dimethylamine, carnitine (Fig. 3A). Propionate, which was observed to be higher in the dart and dropnet captured animals than that of helicopter captured animals (Fig. 4) is of interest, as it is the main precursor for glucose synthesis in the liver of ruminants30, and potentially reflect a higher dependence of ruminants on gluconeogenesis due to the almost complete conversion of available dietary carbohydrates to volatile fatty acids in the rumen31. As animal capture via nets increases physical activity as the animals struggle to free themselves from entanglement, generally resulting in longer times animals are under physical restraint, as well as the increased physical exertion and stress as they attempt to flee the pursuing helicopter, the observed decrease in serum propionate levels may reflect increased needs to generate glucose de novo via gluconeogenesis.
This interpretation of the metabolite data is reinforced by the observation of significantly elevated levels of O-acetylcarnitine in the drop net and helicopter net gun animal capture groups compared to the darted animals (Fig. 4). As an important element of the carnitine/acyl-carnitine shuttle and import of fatty acids into the mitochondria for β-oxidation, acyl-carnitine is a major contributor to the flow of acyl groups into the TCA cycle, and a robust indicator of cardiac output and, by extension, TCA cycle activity levels in mammals32. Additional metabolites that displayed distinctly increasing trends based on capture method (dart < dropnet < helicopter), including glycerol, inosine, lactate and pyruvate (Fig. 4). Of these, pyruvate and lactate are particularly relevant to capture techniques, as they represent major components of anaerobic glycolysis. Greater levels of these metabolites in the serum profiles of dropnet and helicopter-captured animals may reflect the greater physical exertion experienced by dropnet and helicopter-captured animals compared to dart-captured animals and the generally longer times animals are under physical restraint. These differences in pyruvate and lactate levels are consistent with our observations that serum glucose levels are lowest in animals captured by helicopter, higher in the dropnet group, and highest in dart-captured animals (Fig. 3A). While we acknowledge that the drugs employed for chemical immobilization have the potential to influence metabolic profiles, we could not discern any notable differences in the metabolomics profiles of darted animals that we could attribute to the drugs. As described, most of the influential metabolites that discriminate the three capture techniques are primarily associated with physical exertion and stress.
Analysis of the serum profiles of animals captured using the immobilizing dart method compared to those of animals captured using helicopter net gun capture, revealed persistence of several of the metabolite level trends that were observed when evaluating metabolome differences between all three techniques (Figs. 4, 5). PLS-DA analysis indicated significantly elevated levels of glycerol, lactate, and inosine in the helicopter capture group compared to the dart capture group (Fig. 3C). Lower levels of inosine in darted animals paralleled trends in elevated lactate levels for the helicopter capture group, potentially representing a robust indicator of the metabolic impact of the two different capture techniques, as the serum concentration of inosine was almost 8 times greater in the helicopter capture group compared to dart (Fig. 5, Supplementary Table S1). A similar trend was noticeable for glycerol, as its serum levels were over two orders of magnitude higher in helicopter versus dart-captured animals (Supplementary Table S2). These metabolite level changes suggest an increase in fatty acid catabolism in the helicopter captured animals, due to increased energy (ATP) needs resulting from the increased exertion as these animals attempt to evade capture. Changes in the levels of these three metabolites (glycerol, lactate, inosine) highlight the impact of the dart versus helicopter capture techniques on the serum metabolite profiles of animals captured by these two very different approaches.
The pairwise analysis of the polar metabolite profiles of dart versus helicopter-captured groups also highlighted specific changes in the serum concentrations of amino acids, including tryptophan, lysine, and cysteine, which serves as a source of precursors for TCA cycle activity, via production of pyruvate, which was increased in the serum profiles of animals captured by helicopter (Fig. 5). In contrast, serum levels of asparagine, aspartate, valine, and proline were significantly lower in the helicopter-captured animals (Fig. 5). These amino acids are vitally important for diverse central carbon energy metabolic processes, and are used to generate additional intermediates such as fumarate, succinyl-CoA, and α-ketoglutarate. Changes in these amino acid levels may thus reflect significant changes in central carbon metabolism and energy-generating processes in dart versus helicopter captured animals, and the significant impact of these capture techniques on the physiology of wild bighorn sheep. Other metabolites found in lower concentration in the serum samples of helicopter-captured animals included formate, dimethylamine, and urea (Fig. 5). Changes in the levels of these metabolites reflected changes in urea metabolism which mimicked what was observed when comparing all capture techniques (Figs. 4, 5), and provide additional evidence for the impact of capture technique on nitrogen metabolism and the urea cycle.
In animals captured using the dropnet method compared to the immobilizing dart technique, similar serum metabolite patterns to those identified in animals captured by helicopter versus dart were observed. PLS-DA analysis indicated lower concentrations of serum glycerol, lactate, and inosine in the dart versus dropnet-captured animals (Fig. 3B), very similar to what was observed when comparing the dart versus helicopter groups (Supplementary Fig. S4). Interestingly, ketoleucine (2-oxoisocaproate) levels were higher in the sera of animals captured by dropnet compared to helicopter in which it was the lowest (Fig. 4). This apparent change in ketoleucine concentrations suggests that capture may induce a change in branch chain amino catabolism which is further supported by observations of decreasing levels of valine, leucine and isoleucine from dart to dropnet to helicopter capture techniques (Fig. 4)33. Similar to what was observed when comparing the serum profiles of dart versus helicopter-captured animals, the levels of several key amino acids involved in energy production were altered, all lower in the serum samples of dropnet-captured animals compared to dart-captured animals, and included lysine, arginine, cysteine, glutamate, phenylalanine, serine, and tryptophan (Supplementary Fig. S4). The extent of these changes were within the same orders of magnitude as to what was observed in the dart versus helicopter capture. These metabolite patterns suggest comparable shifts in central carbon energy metabolism when animals were captured by the dart compared to the helicopter or dropnet method, the latter yielding very similar metabolite profiles to those observed for animals captured by helicopter, albeit in a seemingly less dramatic fashion as reflected in fewer specific metabolite level changes being significant (18 vs 29 when comparing dropnet and helicopter to the dart-captured group, respectively). A key metabolite discriminating dart from dropnet capture techniques involved choline, which was significantly lower in concentration in the dropnet-captured animals (FC = − 0.4) compared to the dart-captured group (FC = 0.6) (Fig. 4 and Supplementary Table S2). This trend in choline level was also noticeable when all three capture techniques were analyzed together, with dropnet and helicopter serum samples exhibiting lower choline levels compared to dart, similar to what was seen when the dart and helicopter groups were compared (Fig. 4 and Supplementary Table S2). The importance of choline level changes was also highlighted in the PLS-DA loading vectors importance values (Supplementary Fig. S3), with choline being one of the driving factors that separated the three capture groups, specifically contributing to the separation of the dropnet group from the two other capture groups (Supplementary Fig. S3). These data suggest that dropnet and helicopter capture techniques have a greater impact on key metabolic pathways associated with choline metabolism including the Kennedy pathway, which accounts for ~ 95% of choline utilization to generate phosphatidylcholine and phosphatidylethanolamine34. Other potentially impacted processes included intermediates of the one-carbon metabolism cycle, of which choline and betaine are main contributors35. Additional evidence supporting changes in one-carbon cycle involved the decrease in betaine levels when all three capture techniques were compared, with dart having highest level of betaine, followed by dropnet, and then helicopter (Fig. 4). Overall, the trends in metabolite level changes observed when comparing dart versus helicopter capture groups persisted in the dart versus dropnet-capture comparisons, with a few exceptions as presented above.
The polar metabolite profiles obtained from serum samples of animals captured by dropnet and helicopter were more similar to each other than those of dart-captured animals. Nevertheless, PLS-DA analysis indicated 15 metabolites that contributed to the separation of dropnet versus helicopter capture groups (Fig. 2D), which were all lower in concentration in the serum samples of helicopter-captured animals except for lactate (Fig. 3D). Metabolites whose levels were higher in the dropnet capture group compared to the helicopter group included formate, 3-methyl-2-oxovalerate, glucose, tryptophan, valine, isoleucine, 2-oxoisocaproate, proprionate, phenylalanine 3-hydroxybutyrate, carnitine (Fig. 3D, VIP > 1.3). These changes were consistent with the trends observed when the serum profiles of dart-captured animals were compared to those of the helicopter and the dropnet-captured groups (Fig. 4). Some serum amino acid levels were significantly lower in the helicopter capture group compared to dropnet, but the magnitude of these differences was less pronounced than the one observed when the dart-capture group was compared to the helicopter or to the dropnet capture groups. The differentiating trends between helicopter and dropnet were reflected in the PLS-DA models, although the validation metrics (Q2 ~ 0.6, R2 ~ 0.7 and CER < 0.08) were marginal, suggesting that the PLS-DA model may be slightly overfit for this pairwise capture group comparison (Supplementary Fig. S2). Univariate and volcano plot analysis indicated that level changes of only three metabolites, including formate, propionate, and 3-methyl-2-oxovalerate contributed to the separation of the dropnet from the helicopter capture groups (Supplementary Fig. S5).
In conclusion, we have found that different animal capture techniques result in distinct and broad serum metabolic changes in wild bighorn sheep. Serum metabolite profile differences were most significant when the dart-captured animals were compared to the other animal groups captured by dropnet or helicopter methods. Metabolite level changes were less pronounced when the serum metabolite profiles of dropnet-captured animals were compared to those of the helicopter-captured group.
The differences in metabolic profiles documented in this study were attributed primarily to differences in physical activity and stress caused by the different capture methods. Both dropnet and helicopter capture rely on nets with consequential physical struggles as the animals attempt to escape entanglement. Both also involve significant time between capture and animal restraint with blindfolds and hobbles aimed at reducing physical activity, but no doubt causing stress, continued muscle exertion, and elevated heart, respiration, and metabolic rates. While there existed considerably variability in the time animals were manipulated when subjected to each capture technique, in general, animals captured with dropnets and helicopters were physically retrained for long periods of time. In contrast, cautiously approaching animals on the ground and delivering immobilization drugs via a dart rifle appear to result in minimal physical exertion and, while darted animals were also blindfolded and hobbled for all handling, processing and sampling, physical exertion and stress appear to be minimal due to sedation. This study has thus demonstrated that the three animal capture techniques examined here, which are the primary techniques employed to capture most wild ruminants, have wide ranging impacts on the metabolism of bighorn sheep, as reflected in significant and broad ranging changes in serum polar metabolite profiles. Most notable appears to be a significant shift in central carbon energy metabolism due to the nature of the type of capture technique employed.
The field of metabolomics has considerable potential to enhance the assessment of the health and physiological state of wild animals, and to guide efforts aimed at improving their conservation and management. Of particular interest for wild ruminants is the development of quantitative analytical tools to accurately characterize their body reserves, nutritional status, and disease state, which are the primary limiting factors influencing wild animal populations.
Controlled experimental studies with captive animals will provide the most rigorous approach to developing metabolomics-based tools, but ethical constraints limit experimental protocols involving disease processes, and preclude experimental protocols that mimic the type of severe and prolonged nutritional deprivation routinely experienced by wild ruminants inhabiting seasonal environments. Thus, complementary observational studies of wild animals will be needed to realize the full potential of metabolomics for wildlife conservation and management. Our findings suggest that when designing such studies that require the capture of wild animals, it may be prudent to employ a single capture technique, if possible, to reduce confounding factors that may alter serum metabolome profiles. The more dramatic changes that were observed in the polar serum metabolite profiles of animals captured using the dropnet and helicopter techniques suggest that administration of tranquilizers as soon as animals are restrained may be warranted to mitigate short-term physiological impacts36,37.
Source: Ecology - nature.com
