H. Säkkinen 1, * A. Stien 2 Ø. Holand 3 K. Hove 3 E. Eloranta 4 S. Saarela 1 E. Ropstad 5 1

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1 907 Plasma Urea, Creatinine, and Urea : Creatinine Ratio in Reindeer (Rangifer tarandus tarandus) and in Svalbard Reindeer (Rangifer tarandus platyrhynchus) during Defined Feeding Conditions and in the Field H. Säkkinen 1, * A. Stien 2 Ø. Holand 3 K. Hove 3 E. Eloranta 4 S. Saarela 1 E. Ropstad 5 1 Department of Biology, University of Oulu, Oulu, Finland; 2 Centre for Ecology and Hydrology, Banchory, Kincardineshire, Scotland, United Kingdom; 3 Department of Animal Science, Agricultural University of Norway, Ås, Norway; 4 Department of Physiology, University of Oulu, Oulu, Finland; 5 Department of Reproduction and Forensic Medicine, Norwegian College of Veterinary Medicine, Oslo, Norway Accepted 9/17/01 ABSTRACT Variation in plasma urea and creatinine concentration and plasma urea : creatinine ratio (U : C) were studied in semidomestic free-ranging reindeer (Rangifer tarandus tarandus)on the Norwegian mainland, in wild Svalbard reindeer (Rangifer tarandus platyrhynchus), and in captive reindeer maintained either on a lichen-based diet or a protein-rich concentrate to investigate whether these parameters could be used as indicators of the nutritional status of reindeer. In the mainland animals, plasma creatinine concentration was high in winter and early spring and decreased by two-thirds toward the summer. The overall range in mean plasma creatinine concentration ( SE) was from to mmol/l. Mean plasma urea concentration ( SE) varied from in winter up to mmol/l in summer and autumn. Month of sampling explained 65% and 90% of the variation in plasma urea and creatinine concentrations, respectively, indicating that sea- * Corresponding author; ksakkine@paju.oulu.fi. Physiological and Biochemical Zoology 74(6): by The University of Chicago. All rights reserved /2001/ $03.00 sonality in the diet had the greatest influence on these parameters. Reindeer given lichens as the only feed showed an increase in plasma creatinine and a decrease in plasma urea concentration. Food restriction caused a temporary elevation in urea level but had no significant effect on plasma creatinine concentration. The slight effect of energy intake on urea and creatinine levels was supported by the fact that severe undernutrition in the Svalbard reindeer population had only a small effect on plasma urea and creatinine levels. Protein-rich pellet feed increased plasma urea from around 3 mmol/l to above 10 mmol/ L and reduced creatinine concentrations to less than 100 mmol/ L, suggesting that the protein content of forage is an important determinant of these blood parameters. Mean U : C ratio ( SE) in plasma varied from to Ratios above 20 appeared when protein intake was low and energy intake was restricted or when protein intake was high. Low ratios occurred when protein intake was low but energy intake adequate. Plasma urea and creatinine concentrations and the U : C ratio showed complex dynamics that were affected by both season and the protein and feed intake. We conclude that they appear to be difficult to interpret as single measures of nutritional status of reindeer. Introduction Reindeer are adapted to large seasonal variation in available nutrients in their forage. During summer, the diet consists of green plants and leaves with high nutritive value (Nieminen and Heiskari 1989). In winter, the diet on the Scandinavian mainland is dominated by lichens, which are rich in digestible carbohydrates but low in protein and minerals. In Svalbard, mosses and plants with relatively low digestibility make up the major part of the diet (Staaland 1986). Overgrazing due to increased reindeer populations has become a growing problem in northern parts of Norway (Evans 1996; Johansen et al. 1996). As a result, the availability and quality of winter forage has declined in many herds, and parameters for assessing the nutritional status of reindeer are in demand. To be of practical value, the samples needed to estimate such parameters should be easy to collect under field

2 908 H. Säkkinen, A. Stien, Ø. Holand, K. Hove, E. Eloranta, S. Saarela, and E. Ropstad conditions and relatively inexpensive to analyse in adequate numbers for statistical analyses. Blood sampling is easy to perform in semidomesticated herds when the reindeer are gathered for slaughtering and counting and does not require the animals to be sedated. Several blood parameters have been considered for evaluation of the nutritional status of deer. Studies on free-ranging animals have described seasonal patterns in serum urea and creatinine concentrations and in the urea nitrogen : creatinine ratio (Hyvärinen et al. 1975; Nieminen and Timisjärvi 1983; DelGiudice et al. 1992). In captive deer, dietary protein and energy levels have been found to affect several blood parameters (Bjarghov et al. 1976; Seal et al. 1978; Warren et al. 1982; DelGiudice et al. 1987a, 1990; Wolkers et al. 1994). Many of the studies have suggested that serum urea and creatinine concentration and urea nitrogen : creatinine ratio warrant further investigation as nutritional indicators. A decrease in plasma urea concentration has been related to low dietary intake of protein (Bjarghov et al. 1976; Valtonen 1979; Oltner and Wiktorsson 1983) because deer recycle urea when dietary protein intake is low (Robbins et al. 1974; Hove and Jacobsen 1975). An increase in plasma urea concentration during high-protein feeding is well known both in domestic ruminants (Ide et al. 1966; Ropstad et al. 1989; Gonda and Lindberg 1994) and in deer species (Hove and Jacobsen 1975; Bjarghov 1976; Seal et al. 1978). Plasma urea concentration may also increase despite low-protein feeding if energy intake is restricted (Warren et al. 1982). Nutrition related changes in plasma creatinine concentrations of captive deer include increasing levels during fasting (Halse et al. 1976) and during food restriction (Wolkers et al. 1994). In free-ranging white-tailed deer (Odocoileus virginianus), the concentration of serum creatinine has been found to decrease from late winter to summer and to increase subsequently toward autumn (DelGiudice et al. 1992). This seasonal variability has been related to dehydration, changes in muscle mass, excretion of creatinine (DelGiudice et al. 1992; Wolkers et al. 1994), and possibly to changes in production of creatinine (Nieminen and Timisjärvi 1983). As a result of an increase in serum creatinine concentration and a decrease in plasma urea nitrogen concentration, a reduction in serum urea nitrogen : creatinine ratio through autumn has been found in free-ranging white-tailed deer (DelGiudice et al. 1992). Assessing the nutritional status of deer by plasma or serum urea and creatinine concentration and urea : creatinine ratio has received considerable interest. However, the sources of variation within the parameters have gained less attention, and to our knowledge, such studies have not been conducted on reindeer. The aim of this study was to look at sources of variation in plasma urea and creatinine concentrations and in urea : creatinine ratio both in field conditions and in captive reindeer and to evaluate these parameters as tools to describe the nutritional status of reindeer. Material and Methods Free-Ranging Reindeer Seiland Herd. The reindeer of the Seiland herd (Rangifer tarandus tarandus) had their winter pasture around Kautokeino in Finnmark County, Norway (68.6 N, 23 E), from December to May. The lichen reserves in the winter pasture area were small, but no supplementary feed was given to the animals in winter. In May, the herd was moved to the summer pasture on the island of Seiland in Altafjorden (70.3 N, 23.2 E). The pasture on the island was limited in the spring and early summer but improved considerably as summer progressed. Autumn pastures between October and late November, when the herd was moving to the winter pastures, were of moderate quality and consisted of both lichen and some green plants. Svalbard Population. Wild Svalbard reindeer (Rangifer tarandus platyrhynchus) from Nordenskjøldland (78 N, 20 E) were studied on their winter pasture in April 1996 and 1997 and in April May The animals suffered severe starvation during the winter of 1996 due to icing and difficult snow conditions. In 1997 and 1998, the weather and snow conditions were normal, permitting access to the winter forage. Captive Reindeer. Nine adult female reindeer (R. tarandus tarandus) were kept outdoors in m 2 pens, three animals per pen, at the Zoological Gardens of Oulu University, Finland (65 N, 25 E). Group 1 consisted of two pregnant and one nonpregnant females. Group 2 had four pregnant and two nonpregnant females. Three animals in group 2 were given daily oral MgO supplement (H. Säkkinen, E. Eloranta, S Vahtiala, M. Puukka, J. Timisjärvi, S. Saarela, E. Ropstad, unpublished results) that had no significant effect on the parameters studied here ( P 1 0.2). Varying amounts of lichen (Cladonia spp.) or commercial reindeer pellets (Poron Herkku, Raisio Group, Raisio, Finland) were given to animals during four successive feeding periods that lasted altogether 116 d. The feeding regimen is presented schematically on the upper part of Figure 3. Lichens were given ad lib. for 6 d to both groups beginning February 4 (period 1). Next, group 1 continued to receive lichens ad lib. while lichens were given at 80% of ad lib. to group 2 for 51 d (period 2). Group 2 was again given lichens ad lib. beginning on day 58, and group 1 continued with lichens ad lib. (period 3). Then, group 2 continued to receive lichens ad lib. while group 1 was gradually switched from lichens ad lib. to the commercial pelleted reindeer feed beginning on day 77 (period 4). Feed samples of lichens and the pelleted concentrates were taken daily, frozen, and combined for chemical analyses that were performed at Soil Analysis Service Viljavuuspalvelu Oy, Mikkeli, Finland. The chemical composition of lichens and the pelleted concentrates (per kg dry mass) was 2.7% and 15.1% crude protein, 43.6% and 17.1% crude fibre, 2.5% and 4.4%

3 Plasma Urea, Creatinine, and U : C Ratio of Reindeer 909 crude fat, 50.3% and 55.5% N-free extract, 0.12 and 2.60 g Na, 1.0 and 11.0 g K, 0.51 and g Ca, and 0.18 and 3.40 g Mg, respectively. The metabolizable energy (ME) contents of the feeds were 10.7 MJ/kg dry mass for lichens and 9.9 MJ/kg dry mass for the pelleted concentrates. The pelleted concentrates contained 25.5% wheat bran, 25.0% oat bran, 15.0% dry molasses pulp, 15.0% ground hay, 5.0% wheat, 5.0% molasses, 2.5% minerals, and 2.0% vegetable oil. The animals had free access to water throughout the study. The ad lib. feeding was regulated so that there was still some edible material left in the orts. Daily measurements of the given feeds and orts for both groups, together with the estimates of dry mass content of the given feeds and orts, were used to calculate the daily dry mass intakes of the groups. The dry matter content of the given feeds and orts was determined by drying samples of 100 g at 95 C to a constant weight. The ME content of lichens was calculated according to Tuori et al. (1995) based on the chemical composition and digestibility estimates for lichens (Jacobsen and Skjenneberg 1977), whereas the ME content of the pellets was based on manufacturer information. The daily dry mass intake, ME content of the feeds, and the chemical analyses were used to calculate the intakes of ME and crude protein within each feeding period (Table 1). The experimental protocol was approved by the Committee on Animal Experiments of the University of Oulu. Blood Sampling and Weighing of the Animals Jugular venous blood was collected with heparinized vacutainers from adult female reindeer. Animals were restrained by hand without using sedatives or medication. Plasma was separated by centrifugation within 2 8 h and stored at 20 C until analysed. The Seiland animals were blood sampled in March ( n p 137), May ( n p 89), June ( n p 10), October ( n p 100), and November ( n p 99) 1997 and in March ( n p 108), May ( n p 63), July ( n p 34), and October ( n p 102) In Svalbard, blood sampling took place from April 14 to April 30, 1996 ( n p 74); from April 15 to April 26, 1997 ( n p 81); and from April 17 to May 9, 1998 ( n p 144). The captive animals were sampled three times a week before feeding at 9 a.m. Weighing was done when blood was sampled, except for the first 3 mo of 1997 in the Seiland herd. Captive animals were weighed once a week. Seiland animals and captive reindeer were weighed with electronic livestock weighing scales (Farmer Tronics, Give, Denmark and Evofarm, Teknoscale, Vantaa, Finland). Svalbard reindeer were weighed with a spring scale (Salter Industries, West Bromwich, United Kingdom). All scales had an accuracy of 0.1 kg. Chemical Analyses Plasma urea concentration was measured with Technicon Axon autoanalyzer (Miles, Tarrytown, N.Y.), clinical method SM4-2150E94 based on a method by Tiffany et al. (1972). Plasma creatinine concentration was also analysed with the autoanalyzer, clinical method SM4-2141E94 using enzymatic Jaffe reaction automated by Chasson et al. (1961) and further improved by Rossignol et al. (1984). The chemical analyses were performed at the Central Laboratory, Norwegian School of Veterinary Medicine. Table 1: The average intake of metabolizable energy (ME) and crude protein (CP) and the average body mass change (DM) of the captive reindeer Period ME (MJ/d/animal) CP (g/d/animal) DM Pregnant (kg/d/animal) DM Nonpregnant (kg/d/animal) P1 (February 4 9): Group Group P2 (February 10 April 1): Group Group P3 (April 2 20): Group Group P4: Group 1: April 21 May 8 a May 9 29 a Group 2: April 21 May a Commercial reindeer pellet Poron Herkku (Raisio Group, Finland) and some lichens were given to group 1.

4 910 H. Säkkinen, A. Stien, Ø. Holand, K. Hove, E. Eloranta, S. Saarela, and E. Ropstad Pregnancy Status The pregnancy of captive animals was confirmed before the onset of the experiment by transrectal ultrasound (Scanner 100, Pie Medical, Maastricht, Netherlands) with a 5-MHz linear probe placed in the rectum with a plastic extension. Statistical Analyses We analysed body mass, plasma creatinine, plasma urea, and plasma urea : creatinine ratios using generalised linear models (McCullagh and Nelder 1989). Plasma creatinine concentration of the Seiland herd was modeled assuming a quasi-poisson error distribution because the variance increased with increasing mean levels. Overdispersion with respect to a Poisson distribution was corrected by scaling the deviance and variance of estimates by the ratio of the residual deviance to the residual degrees of freedom (McCullagh and Nelder 1989). In the other analyses of data from the Seiland herd and Svalbard reindeer population, models were fitted assuming a normal error distribution. In all models, the effect of predictor variables on the response variable were assumed to be linear by using the identity link function. For the captive animals the data consisted of replicated measurements from the same animals, and the focus of the analysis was whether individuals showed the same pattern in the response variables. To control for between-individual variation in the average level of the response variables, we fitted linear mixed models with the identity of the reindeer as a random effect. Time since onset of the experiment, pregnancy, and group were fitted as fixed effects. The slope parameters for other continuous variables such as body mass were fitted as random coefficients with reindeer identity as the grouping variable. This was done to allow the estimation of the across-individual average slope and to test whether this differed from zero (Davidian and Giltinan 1995). Pearson s correlation coefficient was used for simple correlations. Figure 1. The mean values ( SE, error bars partially under the symbols) for body mass (a), plasma creatinine concentration (b), plasma urea concentration (c), and plasma U : C ratio (d ) in the Seiland herd and in the Svalbard reindeer population. Results Seiland Herd In the Seiland herd, the mean body mass varied significantly among months ( F4, 490 p 6.80, P! 0.001), with a 10% loss over the winter months (Fig. 1a). The mean body mass was 4.2 kg higher in October 1998 than in October 1997 ( F1, 490 p 11.1, P! 0.001). The mean plasma creatinine concentration also varied among months ( F5, 733 p 378, P! 0.001) and between years ( F1, 733 p 204, P! 0.001; Fig. 1b). The range of mean creatinine concentrations was from to mmol/l. It was highest in the winter months and early spring and decreased by two-thirds toward the summer, being lowest in June July. Between-month variation explained 65% of the variation in creatinine. Small effects of variation between years (7%) and the year-month interaction (3%) were also seen. The relationship between body mass and plasma creatinine concentration was in general positive but explained only 9% of the variation in creatinine levels. Plasma urea concentration was low during the winter and high in summer and autumn (Fig. 1c). It varied significantly among months ( F5, 747 p 843, P! 0.001) and between years ( F1, 747 p 131, P! 0.001), with a significant month-year interaction ( F2, 747 p 566, P! 0.001). The overall range in the mean plasma urea concentration was from to mmol/l. Body mass explained only 0.8% of the variation in urea concentration, whereas the month of sampling explained 89.5%. Mean plasma U : C ratio ranged from to

5 Plasma Urea, Creatinine, and U : C Ratio of Reindeer The ratio was highest in June October, lowest in November March (Fig. 1d), and showed considerable variation between months of sampling ( F5, 732 p 954, P! 0.001) and also between years ( F3, 732 p 300, P! 0.001). The plasma U : C ratio was mainly determined by the urea concentration, which varied by factor of 7, while plasma creatinine concentration varied by only a factor of 3. Svalbard Population In the Svalbard population, mean body mass was more than 20% lower in April 1996 than in April 1997 and in April May 1998 ( F2, 273 p 193, P! 0.001; Fig. 1a). The mean plasma creatinine level was slightly lower than, but similar to, the levels observed in the Seiland herd in May (Fig. 1b). The variation between years was significant ( F2, 281 p 11.1, P! 0.001) but explained only 7% of the variation in the creatinine concentrations. Plasma creatinine levels showed a decrease over the sampling period from mid-april to mid-may (Fig. 2; F1, 280 p 34.6, P! 0.001). There was no significant difference between the years in this decrease ( F2, 278 p 1.41), even though the decrease was significant only within 1996 (estimated slope p 2.01, SE p 0.61) and 1998 (estimated slope p 1.12, SE p 0.22) and not within 1997 (estimated slope p 0.52, SE p 0.70). The de- crease in plasma creatinine concentration from mid-april to mid-may was not due to a decrease in body mass (F1, 279 p 0.02, P p 0.88). The mean plasma urea concentration was highest in 1996 ( mmol/l) and lowest in 1998 ( mmol/l; Fig. 1c). The variation between years was significant (F2, 270 p 16.0, P! 0.001) but explained only 11% of the variation in urea concentrations. Body mass did not affect plasma urea concentrations ( F1, 281 p 1.25, P p 0.26). Both plasma creatinine and urea concentrations had consistent levels between the years, and so the plasma U : C ratio was quite stable, ranging from 30 to 34 (Fig. 1d). Nevertheless, plasma U : C ratio was significantly lower in 1998 than in 1996 and 1997 ( F p 8.35, P! 0.01). 2, 281 Captive Reindeer All captive reindeer lost weight when lichens were given as the only feed (Fig. 3a). The range of relative body mass losses varied from 4% to 25%. The reduction in body mass was less in pregnant females than in nonpregnant females in periods 2 and 3 ( F2, 248 p 36.7, P! 0.001), but the overall weight loss was similar between treatment groups. The loss rates were highest in period 1 and decreased with time in periods 2 and 3 (Table 1). In period 4, the reindeer in group 1 showed a marked increase Figure 2. Plasma creatinine concentration in the Svalbard reindeer population over the sampling periods from mid-april to mid-may

6 Figure 3. The mean values ( SE) for body mass (a), plasma creatinine concentration (b), plasma urea concentration (c), and plasma U : C ratio (d ) of the captive reindeer. Feeding periods (P1 P4) as defined in Table 1.

7 Plasma Urea, Creatinine, and U : C Ratio of Reindeer 913 in body mass when given protein-rich pellets, while the body mass of animals on a lichen diet (group 2) continued to decline. The pregnant animals increased their weight just before calving and lost weight when the calf was born. The mean plasma creatinine concentration increased up to 250 mmol/l during lichen feeding in period 1 and the first half of period 2 (Fig. 3b). The mean plasma creatinine concentration stayed high until period 4. In period 4, it decreased to less than 100 mmol/l in animals fed the protein-rich pellet diet but decreased also in group 2, which was still on the lichen diet ( F1, 90 p 7.12, P! 0.01). This decrease in group 2 was unrelated to body mass change ( F1, 90 p 0.74, P p 0.39) and occurred at the same time of year as the decrease observed in 1996 and 1998 in the Svalbard population. The mean plasma urea concentration decreased from 5 8 mmol/l to around 2 mmol/l after the onset of lichen feeding in period 1 (Fig. 3c). At the start of period 2, mean urea concentration increased and became higher in group 2 than in group 1 (difference mmol/l). In period 3, urea dropped quickly in group 2 to the level of group 1. When the protein-rich pellets were given to group 1 in period 4, urea increased quickly to 10 mmol/l, while it remained low in group 2 under lichen feeding. Body mass change did not significantly affect plasma urea concentration ( F1, 247 p 1.19, P p 0.28). The pattern of plasma U : C ratio followed plasma urea concentration closely (Fig. 3d). Low ratios were found in periods 2 and 3 for group 1 and in periods 3 and 4 for group 2. The reduction in U : C ratio was seen in both feeding groups, as their body weights declined when fed lichens. After the start of pellet feeding, the U : C ratio of group 1 increased, and 15- fold U : C ratios compared with ratios during period 2 were achieved by the end of the experiment. Discussion Our study demonstrates large seasonal changes in plasma creatinine concentrations of free-ranging reindeer, with high levels in winter and early spring and a threefold decrease toward the summer. Earlier studies have reported the occurrence of these seasonal changes in deer, but their extent and regularity have not received much previous attention (Nieminen 1980; Nieminen and Timisjärvi 1983; DelGiudice et al. 1992). We suggest that the major factors influencing plasma creatinine concentration in reindeer are changes in their protein and feed intake and that the low protein intake of the captive reindeer feeding on lichen likely increased plasma creatinine concentration by decreasing the glomerular filtration rate and excretion of creatinine. The previous assumption is supported by the different anatomy of the reindeer kidney compared to the kidney of domesticated ruminants. Reindeer kidney has a thick cortex in relation to the medulla and therefore has a limited capacity to concentrate urine (Valtonen and Eriksson 1977). To excrete protein metabolites, reindeer on high-protein diets increase their water intake, urine production, and glomerular filtration rate compared with reindeer on low-protein diets (Valtonen 1979). Excretion of creatinine to urine also has a tendency to increase with higher protein intake in white-tailed deer (DelGiudice et al. 1995). This is supported by the marked drop in plasma creatinine of our captive reindeer subsequently fed high-protein concentrates (group 1). Furthermore, sampling month explained 65% of the variation in plasma creatinine of the Seiland herd, and the lowest creatinine levels occurred in summer, when the protein content of their diet is highest (Nieminen and Heiskari 1989) and body mass is increasing. Glomerular filtration rate may also increase with increasing NaCl intake in ruminants (Potter 1961; Weeth and Lesperance 1965). Ingested amounts of NaCl up to 10 g daily did not have this effect in reindeer (Valtonen 1979), which indicated that change in salt intake did not strongly contribute to the decrease in plasma creatinine concentration of our pellet-fed animals. Amount of muscle mass has been considered an important factor on production rate and concentration of serum creatinine (Rodwell 2000), but in our study, body mass explained only 9% of the variability in plasma creatinine of the Seiland herd. The plasma creatinine concentration has been suggested to increase when protein derived from muscle is used as an energy source at times of nutritional deprivation or after muscle damage, in both cases by increasing the net production of creatinine (Nieminen and Timisjärvi 1983; Jurado and Mattix 1998). However, our results indicate that insufficient energy intake affects plasma creatinine concentration only to a minor extent since no difference in plasma creatinine level was seen between the restricted and ad lib. fed captive reindeer in period 2. This was also evident in the Svalbard population, in which only a minor effect on plasma creatinine occurred despite over 20% reduction in mean body mass. Our analysis suggests that not all of the seasonal variation in plasma creatinine concentrations is due to changes in food quality or in body weight. The mean plasma creatinine concentration decreased in captive reindeer (group 2, period 4) without any change in the feeding regimen and irrespective of body mass change. Correspondingly, in the Svalbard population, creatinine showed a significant decrease over the sampling period from mid-april to mid-may. One possible interpretation of our findings is that the function of reindeer kidney shows innate seasonality that is not directly related to dietary and/or body mass changes but follows the expected seasonality in food quality and availability. Free-ranging white-tailed deer also show a seasonal pattern in their serum creatinine concentration (DelGiudice et al. 1992). The authors suggested that an increase in creatinine concentration between summer and winter could in part be explained by dehydration associated with nutritional deprivation. In the present study, the captive animals had free access to water. Dehydration was evaluated by measuring packed-cell

8 914 H. Säkkinen, A. Stien, Ø. Holand, K. Hove, E. Eloranta, S. Saarela, and E. Ropstad volume and plasma protein concentration, which are known to increase with dehydration in domestic animals (Kaneko 1989) and in deer (DelGiudice et al. 1987a). Since the concentration of neither parameter became elevated during the study (H. Säkkinen, A. Stien, Ø. Holand, K. Hove, E. Eloranta, S. Saarela, E. Ropstad, unpublished results), dehydration was an unlikely cause of increasing plasma creatinine concentrations in the captive animals. Consistent with earlier studies (Nieminen and Timisjärvi 1983; DelGiudice et al. 1992), plasma urea concentrations of the free-ranging animals showed significant seasonal variation, with high concentrations in summer and autumn and low concentrations in winter. Based on our results from the captive animals, changes in dietary protein intake seem to have the greatest influence on these seasonal differences, whereas energy intake plays a less significant role. In the captive reindeer, mean plasma urea concentration decreased to around 2 mmol/l after lichens had been fed ad lib. for 60 d (crude protein intake g/d). In reindeer with a similar crude-protein intake, almost 93% of the filtered urea was recycled, with an associated decline in plasma urea concentration to 0.85 mmol/l (Hove and Jacobsen 1975). This suggests that the captive animals were recycling urea to save endogenous protein. Consistent with earlier findings from domestic ruminants (Ide et al. 1966; Gonda and Lindberg 1994) and reindeer (Hove and Jacobsen 1975; Bjarghov et al. 1976), feeding with high-protein concentrates raised plasma urea concentrations quickly to above 10 mmol/l, which can be attributed to increased dietary intake of nitrogen and a consequent decrease in urea recycling (Robbins et al. 1974; Valtonen 1979) when urea is increasingly excreted into the urine (Thornton 1970). The effects of dietary protein intake on plasma urea concentrations were confounded by the effects of energy intake. Plasma urea concentration was about 3.82 mmol/l higher in reindeer for which food intake was restricted by 20% during period 2 than in the animals fed ad lib. Our results are in agreement with Warren et al. (1982), who reported a similar increment in white-tailed deer on low-energy forage. The elevation of urea despite low protein content of the diet may be the result of a decrease in urea recycling when energy intake is insufficient (Valtonen 1979) or to increased production of urea due to muscle catabolism. Food-restricted red deer had elevated plasma urea nitrogen levels associated with a lower protein : DNA ratio of muscle tissue (Wolkers et al. 1994). The captive reindeer given lichens as the only feed were in declining nutritional condition, with up to 25% body mass losses. However, body mass loss did not have a significant effect on their plasma urea level. Together with the findings that plasma urea concentration was affected by both protein and energy intake and that body mass explained only 0.8% of the variability in plasma urea concentrations of the Seiland herd, plasma urea does not appear to be an easily interpretable indicator of nutritional condition. The pattern of U : C ratio was mainly determined by plasma urea concentration since the magnitude of change in plasma urea was much larger than in creatinine. As a consequence, the plasma U : C ratio of free-ranging mainland reindeer was highest in summer and autumn and decreased during winter. In the captive reindeer, ratios above 20 appeared when low protein intake was combined with low energy intake and declining body mass (group 2, period 2) or when both protein and energy intake were high and body weight increasing (group 1, period 4). A reduction in U : C ratio was seen in both feeding groups when low protein intake was combined with adequate energy intake (period 3) accompanied by a slowdown of the mass loss rate in both groups. This kind of reduction has been interpreted to reflect a shift in metabolism to conserve endogenous protein (Ramsay et al. 1991) and has been reported in urea nitrogen : creatinine ratio of both white-tailed deer (DelGiudice et al. 1987b, 1992) and red deer (Wolkers et al. 1994) during nutritional restriction. However, the variability in plasma U : C ratios in relation to protein and feed intake and irrespective of body mass changes indicate that plasma U : C ratio is also difficult to interpret as an index of nutritional condition. Conclusions We have found that plasma urea and creatinine concentrations and the plasma U : C ratio of free-ranging reindeer show seasonal changes that could be predicted as an index of nutritional status. However, we have also demonstrated that these parameters show complex dynamics that are affected by both season and the protein and energy intake of the reindeer. The plasma concentrations of urea and creatinine and plasma U : C ratios therefore appear difficult to use as single measures of nutritional status of reindeer, especially during nutritional restriction, but provide physiologically interesting information about the seasonal adaptation of this northern species. Acknowledgments The field studies were accomplished with the support from the Reindriftens fagråd, Norway; the Norwegian Research Council, Norway; and Natural Environment Research Council, Great Britain. The experimental part of the study was supported by the Finnish Ministry of Agriculture and Forestry. We also thank the Central Laboratory, Norwegian School of Veterinary Medicine, for excellent analytical help and the Zoological Gardens of Oulu University for animal care during the feeding experiment. We would also like to acknowledge Dr. Jouni Timisjärvi and Dr. Steve Albon for their comments on the manuscript.

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