Energy expenditure and body temperature variations in llamas living in the High Andes of Peru

Some large herbivores exhibit seasonal adjustments in their energy metabolism. Therefore, our aim was to determine if the llama (one of the most extensively kept livestock breeds) exhibits seasonal adjustment of their energy expenditure, body temperature and locomotion, under its natural high altitude Andean habitat. For this purpose, energy expenditure, body temperature and locomotion were measured in seven non-pregnant llama dams for ten months on the Andean High Plateau (4400 m above sea level). Daily energy expenditure was measured as field metabolic rate using the doubly labelled water method at four different measurement times. Additionally, a telemetry system was used to continuously record activity, body temperature (3 min intervals) as well as the position (hourly) of each animal. The results show that llamas adjusted their body temperature and daily energy expenditure according to environmental conditions. Furthermore, llamas under high altitude Andean climatic conditions exhibited a pronounced daily rhythm in body temperature and activity, with low values at sunrise and increasing values towards sunset. Llamas also had remarkably low energy expenditure compared to other herbivores. Thus, despite the domestication process, llamas have not lost the ability to adjust their body temperature and daily energy expenditure under adverse environmental conditions, similar to some wild herbivores.


Results
Climatic conditions. The climatic conditions during the time of our study (13 Nov 2015-15 Sep 2016 were typical for the Andean High plateau with very low T a 's during the night and high T a 's during the day (Fig. 1a). Average daily T a over the entire study period was 4.6 ± 2.7 °C and ranged from −3.7 °C to 10.3 °C. The mean daily minimum T a during our study was −8.1 ± 6.1 °C and ranged from −22.1 °C to 4.6 °C. During the entire study of 308 days, there were 263 days with frost. Mean daily maximum T a was 22.2 ± 3.6 °C and ranged from 9.6 °C to 32.7 °C. The amplitude of daily T a fluctuations, i.e. the difference between daily maximum and minimum T a during the time of the study averaged 30.1 ± 7.3 °C and ranged from 9.5 °C to 45.2 °C. The mean daily relative humidity (RH) was 50.9 ± 17.6%, mean daily maximum RH was 81.0 ± 14.1% (range 41.8-100.0%) and mean daily minimum RH was 17.7 ± 12.4% (range 0.65-61.4%; Fig. 1b). The total precipitation during our study was 424 mm. Precipitation occurred exclusively during the wet season from November to April on 54 of the 308 study days (Fig. 1a). The highest rainfalls occurred on 18 February (31 mm) and 19 January (25 mm). Rainfall on the remaining days ranged between 1 and 18 mm. Natural daylight during our study ranged from 10 to 12 h per day.
Rumen temperature. Over the entire study period, we collected over 760,000 rumen temperature (T r ) measurements at 3 min intervals, ranging from 36.25 °C to 41.17 °C. The average daily T r during our study was 38.46 ± 0.25 °C ( Table 1). The T r followed a diurnal rhythm with the lowest T r usually just after sunrise and the highest T r around late afternoon (Fig. 2). Comparing the minimum T r and maximum T r between months, the lowest recorded minimum T r occurred in September (36.25 °C) and the highest maximum T r in June (40.81 °C). The T r amplitude, i.e. the difference between daily maximum T r and daily minimum T r , was very variable and reached on some days more than 3 °C, increasing from November to September over the entire study. This trend was also evident during the FMR measurements, i.e. the T r amplitude was significantly (P < 0.001) lower in November and March compared to June and September (Table 1, Fig. 2). There was a significant positive relationship between T r and T a over the entire study period (T r , °C = 38.38 + 0.02 * T a , °C; R² = 0.39, F 1,6 = 47.5, P < 0.01, n = 51744, Fig. 3). An example of the adjustment of T r to T a is given in Fig. 4. The figure shows that on days with low T a amplitudes for high Andean conditions as it was the case in March with only 23 °C, T r amplitudes decreased as well even though locomotor activity (LA) was high. Contrarily, on days with large T a fluctuations of more than 37 °C such as in September during the dry season, T r decreased at night much further compared to March. average daily relative humidity (black lines) with daily maxima and minima (grey shaded area) and precipitation (black bars) during the course of the study (308 days) at the study location in the high Andes of Peru (4400 m a.s.l.). Rectangles denote field metabolic rate measurement periods.
www.nature.com/scientificreports www.nature.com/scientificreports/ Field metabolic rate and water turnover. The field metabolic rate (FMR) varied between the four different measurement periods of 15 days each ( March did not differ (P = 0.13), however they did differ (P < 0.001) between the two measurements during the dry season (i.e. June and September). In general, FMR values were higher during the dry than during the wet season ( Table 2). Mixed model analysis revealed that daily distances travelled (P < 0.001, F 1,6 = 36.74, Fig. 5), average T a (P < 0.01, F 1,6 = 17.44), average minimum T a (P < 0.05, F 1,6 = 7.81) and average maximum T a (P < 0.01, F 1,6 = 15.46) had significant effects on FMR.
Total body water of individual animals ranged from 56 to 71% of body mass. Average total body water was significantly lower in November (61.4 ± 5.35%) compared to March (66.8 ± 4.0%), but did not differ between all other measurement months (Table 2). Similarly, total water intake (TWI) in September was 3.75 ± 0.51 L d −1 , significantly lower compared to all other measurement months, while TWI did not differ between November, March and June.  www.nature.com/scientificreports www.nature.com/scientificreports/ Locomotor activity and distances covered. In our study, animals were herded to (07.00 h) and from (17.00 h) the grazing grounds every day approximately at the same time, thus LA followed a strong diurnal pattern over the entire study period, similar to T a . An example of that pattern for LA as well as for T a and T r is depicted in Fig. 4. During the FMR measurements average daily LA was significantly higher (P < 0.001) during March (29.71 ± 1.80%) and June (28.07 ± 1.38%), compared to November (25.05 ± 1.59%) and September (23.05 ± 2.75%). The same trend was evident when dividing the data into night (i.e. when animals stayed in the corral) and day (i.e. when animals were out grazing; Table 2).
Average daily distances traveled by the animals varied over the study period. Over the entire study the mean daily distance travelled was 4.67 ± 1.41 km and ranged from 1.03 km to 12.06 km. During the FMR measurements the daily distances travelled in June were significantly (P < 0.001) higher compared to all other FMR measurement months, but no differences (P > 0.05) were detected between November, March and September (Table 2).  www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Our study is the first measuring FMR using the doubly labelled water method in the llama in its natural habitat of the Peruvian high Andes. Furthermore, we combined FMR data with data from a telemetry system measuring T r , activity and distances traveled by GPS. These are the first continuously recorded long-term T r and activity measurements for a camelid in the high Andes. Our data show that llamas spend substantially more energy when traveling long distances. However, compared with other ruminants and herbivores llamas have a lower FMR. Furthermore, considering the harsh climate of the Andes, llamas seem to adjust their T b according to T a to save energy.
Our present data on FMR in llamas kept in their natural habitat of the Andes are similar to results reported recently for llamas measured in a temperate lowland environment 20 , which ranged from 17.48 to 25.87 MJ d −1 . However, considering the much larger daily T a fluctuations in the high Andes (Fig. 1), the present FMR values suggest that llamas adjusted their FMR according to T a . Several studies have reported reductions in FMR in domestic and wild ungulates during adverse environmental conditions [10][11][12]21 . Our results from llamas in the Andes support these findings. The range of T a in which T b is regulated by sensible heat loss and thus does not require additional energy for thermoregulation is called the thermal neutral zone (TNZ). Although the TNZ of the llama has not been measured, results from guanacos, which is the wild ancestor of the llama, suggest that the TNZ lies somewhere in the range of −15.5 to 20 °C 22,23 , i.e. −15.5 °C being the lower critical temperature and 20 °C the upper critical temperature outside which the animal needs additional energy to regulate T b . Assuming a similar TNZ for the llama, animals in our study were outside their TNZ for some portions of the day during all FMR measurement periods when average T a increased above 20 °C (Table 2). Thus, the increased FMR measured in June and September can be partially explained by the increased average T a amplitudes as evidenced by correlations between the FMR and T a variables. However, it needs to be emphasised that these are average T a variables over FMR measurement periods of 15 days each. On some individual days during the FMR measurements T a ranged between −19 °C and 28 °C and thus were even further outside the suggested TNZ. The FMR measured in June (26.22 ± 1.48 MJ d −1 ) was nearly 100% higher than that in November (13.15 ± 1.77 MJ d −1 ). This can partly be explained by the longer distances the animals travelled in June compared to all other measurement periods (Table 2, Fig. 5). However, FMR was significantly affected by T a and thus animals seemed to have increased their energy expenditure not only due to the longer distances traveled but also due to differences in T a .
The course of daily T a in our study was typical for the High Andean climate with very low T a at night and moderate to high T a during the day (Figs 1 and 2). Thus daily T a amplitudes reached 45 °C on some days. With increasing T a amplitudes, T r amplitudes increased as well, similar to results found in a previous study on llamas kept in a temperate environment 20 . However, the daily T a and T r fluctuations in the previous study were much smaller compared to the present results. Although a comparison between both locations has to be treated with caution (due to random effects etc.), the data show that T r and T a amplitudes were correlated in both studies (Fig. 6). The results from the high Andes, however, suggest a higher flexibility in regulating T r according to T a in llamas kept at these altitudes (~4400 m a.s.l.).
In our study T r decreased during the night and increased during the day. These daily T r fluctuations were higher during the dry season (May-September) than during the wet season (November-April) and similar to the T a fluctuations (Figs 2 and 4), suggesting that animals followed a shallow daily hypometabolism. Reducing the metabolic rate to save energy has been known for a long time to be employed by many small mammals weighing less than 10 kg (for review see Heldmaier et al. 3 ; Ruf and Geiser 5 , Geiser 24 ) but not for larger animals with the exception of bears and badgers. But there is increasing evidence, that also larger mammals such as red deer 10 , ibex 11 and horses can reduce their metabolic rate to save energy. The average daily T r fluctuations we report www.nature.com/scientificreports www.nature.com/scientificreports/ here were lowest in November (1.44 °C) and highest in September (1.61 °C, Table 2). These values are in the range of previously reported T b amplitudes for zebras (1.7 °C) 25 alpacas (1.5 °C) 26 , angora goats (1.4 °C) 27 , blesbok (1.4 °C) 26 , impalas (1.1 °C) 28 and pronghorn (1.0 °C) 29 . However, these values and our results are means of several animals over a number of days. The highest individual T r amplitudes in our study over a period of ten months ranged from 2.50-3.44 °C (Table 1). Even higher amplitudes of 4-7 °C have been found for the Arabian oryx 8 , springbok 30 and camel 31 . The daily T r fluctuations observed in our study were larger than the circadian variations of llamas under temperate conditions (37.5-38.6 °C) 32 and suggest that the animals used heterothermy, possibly to reduce energy expenditure. Furthermore, these daily T r fluctuations followed the daily photoperiod and daily T a cycle over the entire study period as evidenced by the correlation between T r and T a (Fig. 3). Similar results have been also found for ibex 11 , red deer 10 and horses 9,33-35 . Because animals were herded every morning at around the same time to the pastures, activity increased sharply at that time and thus possibly resulted in an increase of T r . In earlier studies on herbivores, T b or T r fluctuations decreased with decreasing average T a 10,11 . In the present study, however, daily T r fluctuations increased with decreasing average T a and higher T a amplitudes (Tables 1 and  2, Figs 2 and 4). The increased T r amplitudes could be explained by a decrease in pasture quality during the dry season. Thus, energy needs might have been compromised, which could have led to increased heterothermy by lowering the minimum T r and thus increasing the T r amplitude 26 . However, our body mass and body condition score data do not support this suggestion (Table 2). Therefore, it is more likely that animals lowered their T r at night to increase the capacity to store heat during the day and thus reducing energetic costs as has been shown in a number of herbivores such as the eland 36 , Arabian oryx 37 , giraffe 38 , Arabian sand gazelle 39 , Thompson's gazelle, Grant's gazelle 40 and the Asian elephant 41 .
In an earlier study 20 47 ). Based on these data, a phylogenetic corrected regression equation was derived (FMR, MJ d −1 = 1.23 BM 0.63±0.12 ) from which a predicted FMR of 28.9 MJ d −1 for the llama could be computed. The predicted FMR was about 10% and 30% higher than the actual measured FMR in that study in summer and winter, respectively. In the present study, we derived a separate phylogenetic corrected regression equation (Fig. 7) 48 , camelids in general and the llama in particular seem to have exceptionally low energy expenditures compared to other herbivores, which might be an adjustment to the harsh Andean climatic conditions and low food supply at high altitudes. An even lower FMR has been reported for the giant panda 49 . Contrarily, predicted FMR values from phylogenetic corrected regression equations for alpacas did not deviate much from actual values (Fig. 7). The relative higher FMR in alpacas compared to llamas might be due to their additional metabolic requirements for Figure 7. Relationship between field metabolic rate (FMR, measured using the doubly labelled water method) and body mass in herbivores. Data are from seven herbivores (black dots) published elsewhere (see text for details) and from the llama of the present study (black circles) at four different measurement periods under high Andean climatic conditions (4400 m a.s.l.). The regression line was derived using the phylogenetic least square approach, excluding the data from the llama. However, the maximum likelihood (ML) of lambda was 0 and thus the regression line represents an ordinary least square regression.
www.nature.com/scientificreports www.nature.com/scientificreports/ fine fibre production. In this context studies on high altitude adaptation of oxygen transport properties of blood and circulation could give further insight into the energy metabolism in camelids. Among other features, a high blood oxygen affinity assures a sufficient blood saturation. Interestingly, many of the special blood and circulation properties found in South American camelids are also described for camels 18,19 . However, camels do not live in high altitudes, but Old and New world camelids share their capability to survive in arid climates.
The TWI calculated during the FMR measurement periods did not differ between November, March and June but was significantly lower in September, i.e. at the end of the dry season (Table 2). Interestingly T r amplitudes were highest at the end of the dry season in September and FMR decreased during the dry season from June to September ( Table 2), suggesting that animals not only conserved energy but also water towards the end of the dry season. This is in agreement with previous studies suggesting that animals, especially camelid species with a pronounced low metabolism living in resource poor environments have an adaptive advantage because not only less energy resources are required but also less water is lost during respiration 48 .
In conclusion, our study provides evidence that llamas kept at the Andean High Plateau have an exceptionally low energy expenditure compared to other ruminants. Furthermore, llamas seem to adjust their T b according to T a which must involve some trade-offs that allow them to save energy instead of keeping their T b constant. Understanding these trade-offs may provide further insights into the adaptations of animals allowing them to survive in extreme environments such as the high Andes.

Methods
Animals and study site. Procedures performed in our study were in accordance with the Peruvian animal ethics regulations and approved by the Peruvian National Ministry for Health (SENASA 2016-0009809). The study was conducted for 308 days from November 2015 to September 2016 at the research station Toccra (Centro de Desarrollo Alpaquero de Toccra) of the non-governmental organisation DESCOSUR (Centro de Estudios y Promoción del Desarollo del Sur, Arequipa, Peru) at an altitude of 4400 m a.s.l., approx. 80 km to the North of the city of Arequipa in the Andes of Southern Peru (15°44′21″S, 71°26′33″W). The study area is characterised by a semi-arid climate with an average annual rainfall of 400-500 mm and T a ranging from as low as −25 °C at night to as high as 30 °C during day time. The average year is divided into a wet season (November-April) when nearly all of the annual rainfall occurs and a dry season (May-October).
Study animals originated from a large female llama herd of 210 animals kept under a traditional Andean herding system, i.e. animals were led to pasture in the morning shortly after sunrise at approx. 07:00 h and were herded back into a corral before sunset at approx. 17:00 h where they stayed throughout the night partly to protect them from their only predator, the nocturnal puma (Puma concolor). During the day animals roamed freely on the pasture of the High Andean plateau consisting mainly of the ecosystems pajonal (dry with tall bunch grasses) and bofedal (wet with grasses and herbs). The bofedales are formed by impenetrable stone and clay layers upon which melting water accumulates. No additional feeding was practiced and water was available throughout the year by natural surface water. For the present study a total of seven non-pregnant adult llama dams with an average age of 5.7 ± 1.5 years and a mean body mass of 125.4 ± 15.2 kg were randomly chosen and kept together with the rest of the herd.

Measurements.
Climate. The T a (resolution: 0.0625 °C) and RH (resolution: 0.04%) were recorded continuously throughout the study with miniature data loggers at 30 min intervals at approx. 1.5 m above the ground (i-Button, DS1923#F5, Maxim Integrated Products, Sunnyvale, CA, USA). Precipitation data were obtained from a nearby weather station at approx. 10 km distance to the farm (15° 58′43″S, 71° 12′48″W).
Telemetry and body condition score. We equipped seven animals with a telemetry system (GPS Plus-3 Store on Board collar, Vectronic Aerospace GmbH, Berlin, Germany) described in detail elsewhere 50 . In brief, the telemetry system consists of two units, a ruminal unit (22 × 80 mm, 100 g) and a collar unit (450 g). The ruminal unit was administered perorally after animals were immobilized with an anaesthetic drug (Xylacin, Rompun ® ; Bayer HealthCare, Leverkusen, Germany, 4 mg/100 kg body mass). The ruminal unit measured T r every 3 min, which was transmitted via short-distance UHF link to a data logging system located in the collar unit 50 . Furthermore, LA was also recorded every 3 min with two different activity sensors and expressed in % of the maximum value recorded. All data were recorded for 308 days and stored in the collar unit and downloaded via a laptop. Additionally the position of each animal was recorded every 60 min using a GPS device located on the back of the collar (GPS Plus-3 Store on Board collar, Vectronic Aerospace GmbH, Berlin, Germany). The body condition score, a palpable and visual assessment of the degree of fatness of individual animals was recorded during the four FMR measurement times according to a point system (scale: 0 = emaciated to 5 = obese) described in detail elsewhere 51 .  52,53 . At the beginning and at the end of the FMR measurements, body mass was recorded for each llama using a mobile scale (Weighing System MP 800, resolution: 0.1 kg, Patura KG, Laudenbach, Germany) and a blood sample of 5 ml was drawn from the Vena jugularis of every animal to estimate the background isotopic enrichment of 2 H and 18 O in the body fluids (method D; Speakman and Racey 54 ). After taking the background sample, each llama was injected intravenously with approximately 0.16 g of DLW per kg body mass, (65% 18 O and 35% 2 H). The individual dose of each llama was determined prior to the injection according to its body mass. The actual dose given was gravimetrically measured by weighing the syringe before and after administration to the nearest 0.01 g (Digital Scale LS200, G&G GmbH, Neuss, Germany). The llamas were then held in a corral with no access to food or water for an 8-h www.nature.com/scientificreports www.nature.com/scientificreports/ equilibration period, after which a further 5 ml blood sample was taken. After dosing, additional blood samples were taken at 7 and 15 days to estimate the isotope elimination rates.
All blood samples were drawn into EDTA blood tubes. Whole blood samples were transported to the city of Arequipa and were pipetted into 1 ml glass vials and stored at −20 °C until determination of 18 O and 2 H enrichment. Samples were sent from Peru to Europe by airmail. Blood samples were vacuum distilled 55 , and water from the resulting distillate was analysed using a Liquid Isotope Water Analyser (Los Gatos Research, USA) at the University of Aberdeen, Aberdeen, Scotland, UK. Samples were run alongside five lab standards for each isotope and IAEA International standards (SMOW, GISP and SLAP) to correct for daily machine variations and delta values were converted to ppm. Isotope enrichments were converted to values of CO 2 production using a two pool model as recommended for this size of animal 56 . We chose the assumption of a fixed evaporation of 25% of the water flux, since this has been shown to minimize error in a range of applications 57,58 . Specifically carbon dioxide production rate (r CO2 ) per day in mols was calculated using equation A6 from Schoeller et al. 59 . The daily amount of energy expended measured as FMR was calculated from carbon dioxide production by assuming a respiration quotient of 0.85. Total body water (mols) was calculated using the intercept method 53 from the dilution spaces of both oxygen and hydrogen under the assumption that the hydrogen space overestimates total body water by 4% and the oxygen-18 space overestimates it by 1% 59 . The TWI (l/day) that consists of drinking water, preformed water ingested in food and metabolic water was estimated as the product of the deuterium space and the deuterium turnover rate 60 .
Statistical Analysis. The measurements of T r had declines that could be attributed to the ingestions of water and cold food. These data points were removed by visually checking the raw data. In this cleaned data set, T r values ranged from 36.25 to 41.17 °C. In total 2156 individual days were available for data analysis of LA and T r . For each animal, hourly and daily means were calculated using R 3.5.0 61 .
To compare T r (Table 1) and various physiological and behavioural variables ( Table 2) during the time of FMR measurements a mixed model was used with animal as a random factor to adjust for repeated measurements and month (i.e. FMR measurement periods) as a fixed factor using the MIXED procedure in SAS version 9.4 (SAS, Inst. Inc., Cary, NC). An integrated post-hoc test (Tukey) was used to detect differences between means with a 5% significance level. Data are expressed as LS-Means or means ± sd where appropriate. To adjust for repeated measurements in all other analysis we included animal ID as a random factor in a mixed model using the MIXED procedure in SAS. Thus, slopes and intercepts in Figs 3, 5 and 6 are adjusted for repeated measurements. Additionally we included body mass as a covariate and month as a fixed factor in a mixed model analysis to test whether various variables had an effect on FMR. Daily distances between continuous GPS locations for each animal were calculated with the program package 'geosphere' 62 in R version 3.5.0 61 .
To test for the generality of the relation between body mass and FMR in herbivores, published data and our results were assessed using the PGLS approach in order to account for the potential lack of independence between species, because of their shared evolutionary history. The statistical procedure has been described in detail elsewhere [63][64][65][66][67] . In brief, the phylogeny was derived from a published mammalian supertree which includes 4510 species with updated branch lengths derived from dated estimates of divergence times 68 . The supertree for mammals was pruned to include only the species of concern, i.e. herbivores (n = 8), using the ' Analysis in phylogenetics and evolution' package (APE 69 ) and the ' Analysis of evolutionary diversification' package (GEIGER 70 ) in R. The method of PGLS was implemented for the trait data using the 'Comparative analyses of phylogenetics and evolution' package (CAPER 71 ) in R using Pagel's branch length transformations (lambda, λ) 72 .

Data Availability
The data analysed during the current study are available from the corresponding author on reasonable request.