Urinary C-peptide as a method for monitoring body mass changes in captive bonobos (Pan paniscus)

In recent years methodological improvements have allowed for more precise estimates of nutrient intake in wild primates. However, estimates of energetic condition have remained relatively imprecise due to the difficulties of estimating digestive efficiency and energy expenditure in these animals. In the absence of a reliable intake-expenditure calculation, a method is needed that directly links changes in energetic condition, such as body mass, to physiological changes that can be detected via markers in body excretions such as urine or feces. One promising marker is C-peptide, a metabolic byproduct of insulin synthesis. Here we present the results of a food restriction experiment carried out in a group of captive bonobos (Pan paniscus). We measured changes in food availability and body mass and determined urinary C-peptide levels with the help of a time-resolved fluoroimmunoassay routinely used for measuring C-peptide in human blood. Urinary C-peptide levels decreased during a period of food restriction and increased again when food availability was continuously increased. During this refeeding phase an increase in body mass was significantly correlated with an increase in urinary C-peptide levels. Our results suggest that urinary C-peptide levels are an accurate indicator of individual energy balance. In conclusion, measuring C-peptide in urine is a promising method to quantify the energetic condition of wild apes.     

Potential applications of urinary C-peptide of insulin for comparative energetics research

The study of comparative energetics offers a valuable way to identify broad ecological principles and assess the functional significance of energetic adaptations during the course of evolution. Yet, the quantification of energetic status for nonhuman primates under natural conditions remains one of the most challenging aspects of comparative energetics research. Here, we report on the development of a noninvasive field method for measuring energetic status in great apes, humans, and possibly other nonhuman primates. Specifically, we have explored measurement of a urinary metabolite of insulin (C-peptide) as a physiological marker of energetic condition in chimpanzees and orangutans. We performed three validation studies and successfully measured C-peptide in urine samples from captive chimpanzees, wild chimpanzees, and wild orangutans. Urinary C-peptide measures gave indications of being a reliable signal of energetic status in both species. For chimpanzees and orangutans in the wild, baseline urinary C-peptide levels were higher during periods of fruit abundance than periods of low fruit availability. Urinary C-peptide levels were also higher for well-fed captive chimpanzees compared with wild chimpanzees. Although sample size was small, top-ranking male chimpanzees showed higher C-peptide levels in the wild than low-ranking males only during the period of fruit abundance. These preliminary results indicate that further development of the urinary C-peptide method could expand opportunities to quantify energetic condition for great apes in the wild and generate new data for comparative research. We highlight specific applications for studying great ape reproduction as well as the nutritional ecology of human foragers.     

Urinary C-peptide tracks seasonal and individual variation in energy balance in wild chimpanzees

C-peptide of insulin presents a promising new tool for behavioral ecologists that allows for regular, non-invasive assessment of energetic condition in wild animals. C-peptide is produced on an equimolar basis with insulin, thus is indicative of the body's response to available glucose and, with repeated measurement, provides a biomarker of energy balance. As yet, few studies have validated the efficacy of C-peptide for monitoring energy balance in wild animals. Here, we assess seasonal and interindividual variation in urinary C-peptide concentrations of East African chimpanzees (Pan troglodytes schweinfurthii). We assayed 519 urine samples from 13 adult male chimpanzees in the Kanyawara community of Kibale National Park, Uganda. C-peptide levels were significantly predicted by the total amount of fruit and the amount of preferred fruit in the diet. However, chimpanzees had very low C-peptide titers during an epidemic of severe respiratory illness, despite highly favorable feeding conditions. Kanyawara males had significantly lower C-peptide levels than males at Ngogo, a nearby chimpanzee community occupying a more productive habitat. Among Kanyawara males, low-ranking males had consistently higher C-peptide levels than dominant males. While counterintuitive, this result supports previous findings of costs associated with dominance in male chimpanzees. Our preliminary investigations demonstrate that C-peptide has wide applications in field research, providing an accessible tool for evaluating seasonal and individual variation in energetic condition, as well as the costs of processes such as immune function and reproduction.     

Reproductive Energetics of Female Mantled Howlers (<Emphasis Type="Italic">Alouatta palliata</Emphasis>)

The energetic costs of reproduction have an important influence on the life histories of female primates. At present, however, the interplay of female reproductive state, food availability, and strategies aimed at maintaining energy balance has been described for only a few species, limiting our ability to understand intra- and interspecific variation in female life histories. We assessed how female mantled howlers (Alouatta palliata) are affected by reproductive seasonality, and whether they alter their behavior to cope with the energetic demands of reproduction. From August 2013 to July 2015 we measured the reproductive state, behavior (1100 h of focal animal observations), and energetic condition (312 urine samples collected for C-peptide analysis) of 7 adult females, and assessed food availability (weekly phenological sampling of 397 food trees). Female behavior did not vary with reproductive state or reproductive seasonality. There were, however, differences in how females responded to variation in food availability according to reproductive state. Cycling and gestating females spent more time feeding than lactating females, and cycling females less time resting than females in other reproductive states, when food was more available. C-peptide concentrations were unaffected by either individual or overall variation in reproductive state, except for cycling females, whose concentrations increased during periods of high food availability. The energetic condition of female mantled howlers is broadly maintained over different stages of reproduction, but is sensitive to variation in food availability.     

Orangutan Energetics and the Influence of Fruit Availability in the Nonmasting Peat-swamp Forest of Sabangau,Indonesian Borneo

Data on energy intake and the effects of fluctuations in fruit availability on energy intake for African apes, and orangutans in mast-fruiting habitats, indicate that orangutans may face greater energetic challenges than do their African counterparts. Comparable data on orangutans in nonmasting forests, which experience lower fluctuations in fruit availability, have been lacking, however, complicating interpretations. We conducted a 46-mo study of orangutan energetics in the nonmasting Sabangau peat-swamp forest, Indonesian Borneo. Sabangau orangutans experienced periods of negative energy balance apparently even longer than in mast-fruiting habitats, as indicated by comparisons of observed energy intake with theoretical requirements and analysis of urinary ketones. Daily energy intake was positively related to fruit availability in flanged males, but not in adult females or unflanged males. This may represent different foraging strategies between age-sex classes and suggests that fruit availability is not always an accurate indicator of ape energy intake/balance. Urinary ketone levels were not generally related to fruit availability, daily energy intake, day range, or party size. This is probably due to low energy intake, and consequently high ketone production, throughout much of the study period. Comparisons with published results on African apes support the hypothesis that orangutans are unique among hominoids in regularly experiencing prolonged periods of negative energy balance. This has important effects on orangutan behavior and socioecology, and has likely been a key factor driving the evolutionary divergence of orangutans and African apes.     

A Noninvasive Method for Estimating Nitrogen Balance in Free-Ranging Primates

The vital role of body protein as an energy reserve has received little focus in studies of wild primates. Owing to the relatively low protein content of fruit, some frugivorous primates could face a protein deficit if body protein is catabolized for energy during periods of low fruit availability. Such an imbalance can be detected if fatty acids, amino acids, and nitrogen (N) catabolites are reincorporated or recycled back to tissues. Here we describe a method to quantify protein recycling by measuring standardized urea concentration and N isotope signatures from urine samples collected from wild Bornean orangutans (Pongo pygmaeus wurmbii). Our overall goal was to explore if concentrations of urea and ??¹⁵N values could be used as indicators of the amount of protein consumed and the degree of protein recycling, respectively, in wild, free-ranging primates. We examine the relationships between urea concentration, ??¹⁵N values, protein intake, and fruit availability. Urea concentration increased with fruit availability, reflecting a slight increase in protein consumption when fruit was abundant. However, we found no relationship between ??¹⁵N values and fruit availability, suggesting that orangutans avert a negative protein balance during periods of low fruit availability. These noninvasive methods complement recent advances in primate energy balance research and will contribute to our understanding of adaptations of primates during periods of fruit shortage.     

随机限食和重喂食小鼠能量收支和生长发育的可塑性

为阐明能量收支和生长发育的可塑性对动物适应食物资源变化的作用和意义,将断乳后的雄性KM小鼠40只随机限食4周,再重喂食4周。采用封闭式流体压力呼吸计测定基础代谢率(BMR)。限食使摄食量显著增加,BMR和活动行为降低,胴体和生殖腺重量显著降低。重喂食后上述指标均恢复到对照组水平,表现出显著的可塑性变化。结果表明,KM小鼠能通过摄食量、BMR、活动行为和身体组成的可塑性调节以适应难以预测的食物资源变化。     

随机饥饿和重喂食对小鼠能量代谢和行为的影响

为阐明能量代谢和行为的可塑性对动物适应食物资源变化的意义,将成年雄性KM 小鼠随机饥饿驯化4
周,再重喂食驯化4 周。采用食物平衡法测定摄食量、封闭式流体压力呼吸计测定基础代谢率(BMR) 和非颤
抖性产热(NST)、观察法测定行为。随机饥饿使摄食量、消化道重量显著增加,BMR 和NST 显著降低。与对照
组相比,饥饿组休息行为显著增加,活动显著降低。重喂食后,上述指标均恢复到对照组水平,表现出显著的
可塑性。研究结果表明,动物适应难以预测的食物资源短缺的主要策略包括:增加自由取食期间的摄食量;降
低BMR、NST 和活动行为,从而保存身体贮存的能量。能量代谢和活动行为在较短的时间尺度内表现出显著的
可塑性对小鼠适应不可预测的食物资源短缺的应激环境具有重要意义。     

Digestive parameters and water turnover of the leopard tortoise

Leopard tortoises (Stigmochelys pardalis) experience wide fluctuations in environmental conditions and unpredictable availability of food and water within the Nama-Karoo biome. It was hypothesised that tortoises fed two diets differing in preformed water and fibre content would have differing food intake, gut transit rate, assimilation efficiency, faecal and urinary water loss, and urine concentrations. It was predicted that tortoises fed these contrasting diets would attempt to maintain energy and water balance by altering their digestive parameters. Leopard tortoises fed lucerne (Medicago sativa) had a low food intake coupled with long gut transit times, which resulted in the lowest amount of faecal energy and faecal water lost. Tortoises fed tomatoes (Solanum lycopersicum) had higher food intake and faster gut transit times, but more energy and water was lost in the faeces. However, daily energy assimilated and assimilation efficiency were comparable between tortoises fed the two diets. Urine osmolality was significantly different between tortoises on the two diets. Results indicate that leopard tortoises can adjust parameters such as transit rate, food intake, water loss and urine osmolality to maintain body mass, water and energy balance in response to a high fibre, low water content and a low fibre, high water content diet. This study suggests that this digestive flexibility allows leopard tortoises in the wild to take advantage of unpredictable food and water resources.     

Urinary C-peptide levels in male bonobos (Pan paniscus) are related to party size and rank but not to mate competition

Within- and between-species variation in male mating strategies has been attributed to a multitude of factors including male competitive ability and the distribution of fertile females across space and time. Differences in energy balance across and within males allow for the identification of some of the trade-offs associated with certain social and mating strategies. Bonobos live in groups with a high degree of fission–fusion dynamics, there is co-dominance between the sexes and a linear dominance hierarchy among males. Males compete over access to females, breeding is aseasonal, and females exhibit sexual swellings over extended time periods. In this study we use urinary C-peptide (UCP) levels in male bonobos (Pan paniscus) obtained from 260 urine samples from a wild bonobo community, to quantify male energy balance during mate competition and levels of gregariousness in the species. Although high ranking males are more aggressive, spend more time in proximity to maximally tumescent females, and have higher mating frequencies, we found no indication that mate guarding or mate competition affected male energy balance. Our results showed a positive correlation between monthly mean UCP levels and mean party size. When traveling in large parties, high ranking males had higher UCP levels than those of the low ranking males. These results support the hypothesis that patterns of fission–fusion dynamics in bonobos are either linked to energy availability in the environment or to the energetic costs of foraging. The finding of a rank-bias in UCP levels in larger parties could also reflect an increase in contest competition among males over access to food.     

Validation of a Novel Collection Device for Non-Invasive Urine Sampling from Free-Ranging Animals

Recent advances in non-invasively collected samples have opened up new and exciting opportunities for wildlife research. Different types of samples, however, involve different limitations and certain physiological markers (e.g., C-peptide, oxytocin) can only be reliably measured from urine. Common collection methods for urine to date work best for arboreal animals and large volumes of urine. Sufficient recovery of urine is thus still difficult for wildlife biologists, particularly for terrestrial and small bodied animals. We tested three collection devices (two commercially available saliva swabs, Salivette synthetic and cotton, and cotton First aid swabs) against a control to permit the collection of small volumes of urine from the ground. We collected urine samples from captive and wild macaques, and humans, measured volume recovery, and analyzed concentrates of selected physiological markers (creatinine, C-peptide, and neopterin). The Salivette synthetic device was superior to the two alternative devices. Concentrations of creatinine, absolute C-peptide, C-peptide per creatinine, absolute neopterin, and neopterin per creatinine measured in samples collected with this device did not differ significantly from the control and were also strongly correlated to it. Fluid recovery was also best for this device. The least suitable device is the First aid collection device; we found that while absolute C-peptide and C-peptide per creatinine concentrations did not differ significantly from the control, creatinine concentrations were significantly lower than the control. In addition, these concentrations were either not or weakly correlated to the control. The Salivette cotton device provided intermediate results, although these concentrations were strongly correlated to the control. Salivette synthetic swabs seem to be useful devices for the collection of small amounts of urine from the ground destined for the assessment of physiological parameters. They thus provide new opportunities for field studies to incorporate physiological markers, particularly on smaller bodied and terrestrial animals and where urine collection is difficult.     

Urinary C-peptide measurement as a marker of nutritional status in macaques

Studies of the nutritional status of wild animals are important in a wide range of research areas such as ecology, behavioural ecology and reproductive biology. However, they have so far been strongly limited by the indirect nature of the available non-invasive tools for the measurement of individual energetic status. The measurement of urinary C-peptide (UCP), which in humans and great apes shows a close link to individual nutritional status, may be a more direct, non-invasive tool for such studies in other primates as well and possibly even in non-primate mammals. Here, we test the suitability of UCPs as markers of nutritional status in non-hominid primates, investigating relationships between UCPs and body-mass-index (BMI), skinfold fatness, and plasma C-peptide levels in captive and free-ranging macaques. We also conducted a food reduction experiment, with daily monitoring of body weight and UCP levels. UCP levels showed significant positive correlations with BMI and skinfold fatness in both captive and free-ranging animals and with plasma C-peptide levels in captive ones. In the feeding experiment, UCP levels were positively correlated with changes in body mass and were significantly lower during food reduction than during re-feeding and the pre-experimental control condition. We conclude that UCPs may be used as reliable biomarkers of body condition and nutritional status in studies of free-ranging catarrhines. Our results open exciting opportunities for energetic studies on free-ranging primates and possibly also other mammals.     

Radioimmunoassay of estrone conjugates from urine dried on filter paper

Hormonal analysis of urine from free-ranging primates has been limited due to the difficulty of preserving samples under field conditions. Drying urine on filter paper is an alternative for field preservation. This study describes a new laboratory method for eluting steroids from filter paper with methanol, along with a series of experiments used to develop and validate this method. The overall elution recovery of estrone sulfate (ES) from filter paper was 86.4%. Estrone conjugate (E1C) levels in humans and captive orangutans were analyzed by radioimmunoassay (RIA). Values from samples dried on filter paper were significantly correlated with values from matched frozen samples, with elution efficiencies ranging between 97.1% and 102.4%. Creatinine (Cr) measurements from frozen urine compared to urine dried on filter paper were also significantly correlated (r=0.96) with an elution efficiency of 101.7%. After the samples were stored for 2 years, the absolute values of E1C and Cr were significantly lower but were still significantly correlated with frozen urine values. These data demonstrate the effectiveness of filter paper as a medium for preserving urinary steroid samples, and the efficiency of methanol as a solvent for eluting E1C and Cr. This method thus provides a viable alternative to the traditional procedure of freezing urine for field studies, where freezers are not available.     

Dietary and Energetic Responses of Pongo abelii to Fruit Availability Fluctuations

Like many primates, orangutans face fluctuations in fruit availability. We show that in Ketambe (Sumatra), fruit availability fluctuations have very little influence on orangutan diet. Most importantly, the percentages of fruits including figs, fruits excluding figs, and figs in their diet do not differ significantly between periods of varying fruit availability. In addition, there is no trace of ketones in orangutan urine samples during periods of varying fruit availability. Unlike 1 orangutan population in Borneo, orangutans in Sumatra experience no prolonged negative energy budget because of fluctuations in fruit availability. Dietary data from other Sumatran and Bornean orangutan populations indicate that the pattern might be representative for a more general difference between Sumatra and Borneo, and we discuss how this might have affected orangutan reproduction and life history for the 2 species.     

Bornean orangutans on the brink of protein bankruptcy

Protein is a limiting resource that is essential to the growth, maintenance and reproduction of tropical frugivores, yet few studies have examined how wild animals maintain protein balance. During chronic periods of fruit scarcity, Bornean orangutans (Pongo pygmaeus) often catabolize their own fat reserves despite unusually low metabolic requirements. Such energy deficits suggest a marginal existence, and raise the possibility that orangutans also endure periods of negative protein balance. To test this hypothesis, we conducted the first study of protein cycling in a wild primate. Our five year analysis of urinary metabolites revealed evidence of protein recycling when fruit was scarce. During these periods, orangutans consumed more leaves and bark, proteinaceous but tough foods that yielded a mean daily intake of 1.4 g protein kg(-1) metabolic mass. Such an amount is inadequate for humans and one-tenth the intake of mountain gorillas, but sufficient to avert, perhaps narrowly, a severe protein deficit. Our findings highlight the functional and adaptive value of traits that maximize protein assimilation during periods of ecological exigency.     

Intra-and interpopulational differences in orangutan (Pongo pygmaeus) activity and diet: implications for the invention of tool use

Tool manufacture and use have been described for wild orangutans (Pongo pygmaeus), with appreciable interpopulational differences in tool complexes. The ecological factors that contribute to these differences require investigation. Significant interpopulational differences in diet suggest that ecological factors contribute to variation in tool-based insect foraging. Using 4 years of behavioral data from the Suaq Balimbing Research Station (Sumatra, Indonesia), we tested predictions of two ecological hypotheses for the invention of tool use for insect foraging. We found limited evidence for inter- and intrasexual differences, as well as temporal variation, in activity budget and diet. However, differences did not correspond to variation in either rate of tool use or specialization on tool-based insectivory. Compared to other populations, orangutans at Suaq Balimbing ate significantly more insects. Low temporal variation in insectivory and an abundance of social insects at Suaq Balimbing suggest that insects formed a staple in the diet rather than a fallback food. Our findings do not support the hypothesis that tool use is a response to the low availability of primary food sources. Rather, greater opportunities for invention likely contributed to insect-extraction tool use at Suaq Balimbing.     

Changes in Orangutan Caloric Intake,Energy Balance,and Ketones in Response to Fluctuating Fruit Availability

I used novel quantitative measures to assess the impact of fruit seasonality on changes in diet and energy balance during 4918 hours of observation on <60 orangutans in Gunung Palung National Park, West Kalimantan, Indonesia, from September 1994 through September 1995. During this period a mast fruiting occurred, resulting in large fluctuations in fruit availability, measured by monitoring of 558 orangutan fruit trees. Orangutan diet varied tremendously in accordance with these fluctuations. During the month of highest fruit production, 100% of the orangutan diet was composed of fruit. In contrast, during the fruit-poor period as little as 21% of the diet was fruit, while 37% was bark. Nutritional analyses of 78 of the foods most commonly eaten during this study show that mast foods were significantly higher in caloric content than were nonmast foods, which translates into substantial changes in caloric intake. During the month of highest fruit consumption (January), males consumed an estimated 8422 kcal/day and females consumed 7404 kcal/day. During the month of lowest fruit consumption (May), males consumed 3824 kcal/day and females consumed 1793 kcal/day. These differences were significantly different between the 2 months for both males and females. In May, males ate significantly more kcal/day than females did. Time spent feeding did not differ between the two periods for either males or females. I assessed the impact of these changes in diet and caloric intake on orangutan physiological functioning by measuring ketones in 257 urine samples collected from adult males and females. Ketones—products of fat metabolism—were present in urine only during the fruit-poor period. These data suggest that orangutans are highly efficient at storing fat during fruit-rich periods and utilizing the reserves during times of fruit shortfall. This response may have important implications for orangutan survivorship, reproduction, and behavior.     

Insulin Levels,Physical Activity,and Urinary Catecholamine Excretion of Obese and Non-Obese Rhesus Monkeys

The hypothesis that spontaneous obesity in rhesus monkeys is associated with abnormalities in energy expenditure was tested. Obese (n=7) and non-obese (n=5) monkeys were described in terms of body size and composition, food intake, and physical activity. Additionally, the relationships among fasting and stimulated insulin levels in serum, C-peptide levels in serum and urine, and urinary catecholamines were examined. Obese animals had primarily abdominal deposition of excess body fat, as indicated by markedly elevated abdominal circumferences and skin-fold thicknesses. Food intake did not differ between groups. Physical activity was much lower in the obese group. Obese monkeys had markedly higher serum insulin and C-peptide levels in the fasted state and in response to an intravenous glucose challenge. Urinary excretion of C-peptide and catecholamines was measured during successive 2-day periods of ad libitum feeding, food deprivation, and refeeding in order to examine potential differences between groups in sympathoadrenal activity and their relationship to insulin secretion. C-peptide excretion was greater for obese and decreased for both groups during food deprivation. Urinary dopamine (DA), norepinephrine (NE), and epinephrine (E) levels were significantly greater for obese animals in all conditions. DA excretion was lowest during deprivation and E excretion was lowest during refeeding, whereas NE excretion was relatively unaffected by feeding condition. The overall patterns of C-peptide and catecholamine excretion were qualitatively similar for both groups, and there were no reliable differences between obese and non-obese in their responses to the feeding manipulation. The results suggest that hyperinsulinemia associated with obesity in rhesus monkeys is linked to increased catecholamine secretion and a resistance to cate-cholaminergic action.     

The influence of ambient temperature and the energy and protein content of food on nitrogenous excretion in the Egyptian fruit bat (Rousettus aegyptiacus)

The diets of frugivorous and nectarivorous vertebrates contain much water and generally have high energy but low protein contents. Therefore, we tested the prediction that to save energy under conditions of high energy demands and high water intake, frugivorous Egyptian fruit bats (Rousettus aegyptiacus) will increase both the absolute quantity and the proportion of ammonia in their urine. We also examined whether such changes occur when protein intake is low and water intake is high. We did three feeding trials. In trials 1 and 2, bats were fed one of four liquid diets containing constant soy protein concentrations but varying in sucrose concentration and were kept at ambient temperatures (T(a)) of 30 degrees Celsius and 12 degrees Celsius, respectively. In trial 3, bats were kept at Ta=12 degrees Celsius and fed one of four liquid diets with equal sucrose concentrations but varying protein concentrations. In trial 1, food intake at a sucrose concentration of 256 mmol/kg H(2)O was initially high but decreased to a constant rate with further increases in sucrose concentration, while in trial 2, food intake decreased exponentially with increasing sucrose concentration. As predicted, at 12 degrees Celsius with varying sucrose concentration, both the absolute quantity and the fraction of ammonia in the bats' urine increased significantly with food intake (P<0.02), while the absolute quantity of urea and the fraction of urea nitrogen excreted decreased significantly with food intake (P<0.03). Varying sucrose concentration had no significant effect on nitrogen excretion at Ta=30 degrees Celsius. Varying protein concentration had no significant effect on nitrogen excretion at Ta=12 degrees Celsius. We suggest that Egyptian fruit bats can increase ammonia excretion in response to increased energetic demands, and we calculate that they can save energy equal to approximately 2% of their daily metabolic rate by doing so.     

Influence of sexual maturity on feeding, growth and energy stores of wild Atlantic salmon parr

Maturing Salmo salar parr had significantly lower short-term energy stores (indicated by hepato-somatic index) in August, and significantly lower energy stores and growth rate (indicated by RNA : DNA) in September than immature parr captured from the same stream sites on the same date. There were no significant differences in gut fullness or protein concentrations, suggesting that up to early September the main energetic consequences of maturation were a reduction in allocations to growth and short-term energy storage, but not a mobilization of long-term stored energy in the form of proteins. These are the first observations of relative food intake and energy storage for maturing parr under natural conditions, and also the first to assess growth effects on wild fish before completion of the maturation process.