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Nutrition-induced changes in blood chemical parameters of pregnant reindeer hinds (Rangifer tarandus tarandus)
Small Ruminant Research 32 (1999) 211±221
Nutrition-induced changes in blood chemical parameters of pregnant reindeer hinds (Rangifer tarandus tarandus) H. SaÈkkinen1,a,*, J. TimisjaÈrvia, E. Elorantaa, U. Heiskarib, M. Nieminenb, M. Puukkac a
Department of Physiology, University of Oulu, Kajaanintie 52 A, 90 220, Oulu, Finland Finnish Game and Fisheries Research Institute, Reindeer Research Station, 99 910, Kaamanen, Finland c Laboratory, Oulu University Hospital, 90 220, Oulu, Finland
b
Accepted 25 September 1998
Abstract Twenty-four pregnant reindeer hinds (Rangifer tarandus tarandus) were allocated to three feeding groups during 110 days experiment from January to May. Group 1 was grazing in a 15 km2 fence, their diet mimicking a good quality winter pasture. Group 2 was fed with lichens. Their energy supply was gradually decreased until it was 30% from the ad libitum amount at the start. Group 3 was fed ad libitum with a pelleted concentrate for reindeer. Lichen fed animals lost 15% of body weight during the experiment. In other groups body weight was either maintained (Group 1) or increased by 18% (Group 3). Serum protein concentration decreased from 77 g/l to 62 g/l in lichen fed animals, accompanied ®rst with a fall in serum urea concentration. When energy supply decreased further, a rise in blood urea occurred. Serum creatinine concentration increased with lichen feeding, and after 2 months it was 61% higher than at the beginning of the experiment. In pellet fed hinds creatinine declined from 190 mmol/l to 143 mmol/l. The birth weight of calves re¯ected the nutritional status of the groups. The average birth weight was 6.3 kg for calves of pellet fed hinds, 5.9 kg in group with mimicked winter diet and 4.0 kg for lichen fed group. Normal winter diet seems to provide enough energy and protein for the maintenance of body weight and for successful calving. Pellet feeding increased body weight, and had a positive effect on birth weights of calves. On poor winter pasture, supplementary feeding can be useful in prevention of malnutrition and in improving of calving success. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Reindeer; Nutrition; Serum proteins; Urea; Serum minerals
1. Introduction Overgrazing of winter pastures due to the increased reindeer population has become an increasing pro*Corresponding author. Tel.: +358-8-553-1240; fax: +358-8553-1061; e-mail: [email protected] 1 Present address: Department of Biology, University of Oulu, P.O. Box 3000, 90 401 Oulu, Finland.
blem in Finland. In many areas the lichen pastures are poor (Kumpula et al., 1997), and supplementary feeding is commonly used, especially in the southern and middle part of the reindeer herding area. The magnitude of supplementary feeding has doubled over the past 10 years, and its annual economical costs equal 25±50% of the slaughtering income at the Finnish reindeer herding area (Kemppainen et al., 1997).
0921-4488/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 9 8 ) 0 0 1 8 4 - 9
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to evaluate whether blood chemical values can be used as an indicator for supplementary feeding.
One third of the supplementary feeding is given as concentrates, usually rich in protein and energy. The natural winter diet of reindeer is dominated by lichens, which are low in protein and fat, but abundant with complex carbohydrates (Nieminen and Heiskari, 1989). Therefore, the physiological effects of supplementary feeding on pregnant females, whose wellbeing is of essential importance for the pro®t of reindeer herding, should be investigated thoroughly. Earlier studies concerning reindeer blood chemistry have been focused on calves and adult non-pregnant animals. Bjarghov et al., 1976 reported, that lichens are de®cient in important nutrients such as minerals and proteins, as indicated by decreased serum mineral and albumin concentrations. Soveri et al., 1992 studied dietary responses of calves kept under natural conditions and on lichen feeding during winter. Lichen fed calves were subjected to slight and moderate weight loss. In addition to a decrease in serum proteins, the concentration of the end products of lipolysis increased, and serum urea level declined very low. Chao et al., 1985 reported that plasma levels of calcium and phosphorus increased with serum alkaline phosphatase and parathyroid hormone in white-tailed deer (Odocoileus virginianus) during pregnancy. The authors suggested this to indicate pregnancy related increase in capacity for calcium absorption and mobilization. Valtonen (1979) studied the renal function of reindeer kept either on high or low protein diet. She found plasma urea concentration to fall on a low protein diet with adequate energy supply, but to increase if combined with insuf®cient intake of energy. Seasonal variation in reindeer serum protein, lipid, glucose and mineral concentrations with highest values in summer, were reported by Nieminen and TimisjaÈrvi (1983). Most of the seasonal variation was suggested to be of dietary origin, determined by annual climatic rhythms. The aim of this study was to compare the effects of pellet feeding, natural winter diet and lichen feeding on blood chemical parameters of pregnant animals and
2. Material and methods 2.1. Animals This experiment was carried out with a permission from the Committee on Animal Experiments of the University of Oulu. Twenty-four pregnant reindeer hinds (Rangifer tarandus tarandus) were used. The animals were corralled at Kaamanen Reindeer Research Station in Northern Finland, 698100 N. The animals were taken from pasture in the middle of January, after which they were randomly allocated to three feeding groups, each consisting of eight animals. The average temperature changed from ÿ118C in January to 48C in the ®rst week of May. The depth of snow cover was 44 cm in January, reached its maximum (81 cm) in April, and was 37 cm in the beginning of May. Group 1 was grazing in a 15 km2 fenced area. The diet of this group was adjusted to mimic a good quality winter pasture. Each animal got 0.2±0.5 kg commercial reindeer pellet (Poron±Herkku, Raisio Group, Raisio, Finland) to cover about one-third of the daily protein and energy supply. Rest of the diet was mainly lichens, hay (Deschampsia ¯exuosa) and blueberry leaves and twigs, that animals were able to ®nd from the fence. Group 2 was fed with lichens (Cladina spp.). Their energy supply was gradually decreased reaching on week 12 30% of the ad libitum amount in the beginning of the study. After this combined pellet and lichen feeding was used. Group 3 was fed ad libitum with commercial reindeer pellet. The energy content of the pellets was calculated using MAFF recommendations (MAFF, 1975, 1984). Digestibility tables for ruminants (Tuori et al., 1995) were used to estimate the digestibility of different compounds of the pellets. Chemical composition and energy content
Table 1 The chemical composition (% DM basis) and the energy value of lichens and the commercial reindeer pellet Poron±Herkku
Lichens Pellets
DM (%)
Energy (MJ ME/kg DM)
Crude protein
Crude fibre
Nitrogen free extracts
Fat
P
Ca
34 86
10.7 9.9
3.4 13.7
41 16
51.0 55.6
2.9 5.0
0.04 0.67
0.1 0.8
H. SaÈkkinen et al. / Small Ruminant Research 32 (1999) 211±221
of lichens and pellets based on the feed analyses are given in Table 1. The pelleted concentrate was composed (%) of 25.5 wheat bran, 25 oat bran, 15 dry
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molasses pulp, 10 wheat, 15 hay (ground), 5 molasses, 2.5 minerals, and 2 vegetable oil. The amount of pellets and lichens eaten by the hinds was measured
Fig. 1. Average weekly energy and dry matter intake (a), protein intake (b) and calcium and inorganic phosphorus intake (c) for groups 2 and 3. Symbols * and * Group 2, & and & Group 3. Solid symbols indicate energy and calcium, open symbols are used for dry matter and phosphorus. Striped bars in (Fig. 1(b)) indicate feeding arrangements for Group 2.
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daily, and the energy, protein and mineral supply was calculated as a weekly group average (Fig. 1a,b,c). Blood samples were drawn from the jugular vein with serum or EDTA- plasma vacutainers (Venoject1, Leuven, Belgium). Blood samples were collected and body weight recorded ®ve times at regular intervals during the experiment. The ®rst sampling and weighing was done when the animals were allocated to feeding groups. The following samples were collected in the beginning of each month until May. Sedatives or medication was not used, the animals were caught and restrained by hand for blood sampling. 2.2. Analyses Serum total proteins were analysed with the biuretmethod (Lowry et al., 1951). The bromcresol green method (Doumas et al., 1971) was used to determine serum albumin concentration. Serum urea was measured enzymatically according to Gutman and Bergmeyer (1974) and creatinine colorimetrically by Jaffe reaction (Fabiny and Ertingshausen, 1971). Cholesterol and triglyserides were measured enzymatically (Allain et al., 1974), blood glucose enzymatically from whole blood after protein precipitation with 0.6 M perchloric acid (Trinder, 1969; Hansen and Freier, 1978) and inorganic phosphate spectrophotometrically in fosfomolybdate form (Daly and Ertingshausen, 1972). Total calcium was analysed with EFEX 5055 -¯ame photometer (Eppendorf, Hamburg, Germany). Serum alkaline phosphatase (SAP) was measured by using kinetic photometric assay according to the recommendations of The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology (1974). All the results are expressed as group means s.d. Statistical analyses were performed using SPSS software programme (SPSS, 1993). Differences between group mean values at different times were assessed by analysis of variance. If the provided F-values indicated signi®cance (P < 0.05), the differences were assessed post hoc by Tukey test. 3. Results Considerable weight changes and differences between the feeding groups were observed during
the experiment (Fig. 2(a)). In lichen fed animals (Group 2) the average body weight loss was 15%. In the two other groups the average body weight either was maintained (Group 1) or increased (Group 3; 18% average body weight increase). At the beginning of the experiment the average serum protein concentration was 73±77 g/l (Fig. 2(b)). During the ®rst two weeks it rose to 83 g/l in Group 3, after which a slight decrease was found. In Groups 1 and 2 serum proteins decreased, being on average 10 and 20% lower in May, respectively. In Group 1 serum albumin concentration was constant throughout the experiment, while it increased by 16% in Group 3 and decreased by 23% in Group 2 (Fig. 2(c)). Groups 1 and 2 had higher serum creatinine levels (197±316 mmol/l) than Group 3 after the ®rst month of the experiment; Group 2 having the highest values throughout (Fig. 3(a)). In Groups 1 and 2 creatinine increased until March, after which it gradually declined to the basal level. In Group 3 creatinine ®rst decreased from 190 mmol/l to 143 mmol/l within 1 month, then gradually increased. Signi®cant differences in serum urea concentration between the feeding groups developed already during the ®rst 2 weeks of the experiment. In the beginning of the experiment serum urea concentration was 12.7± 14.2 mmol/l (Fig. 3(b)). Lichen fed animals (Group 2) had the lowest concentrations, decreasing to 5.2 mmol/l and remaining lower than in the two other groups for the rest of the experiment. SAP activity increased in all three groups towards the end of the experiment (Fig. 3(c)). Group 2 had the lowest SAP activities throughout the experiment. The average increase in SAP activity during the experiment was 94% in Group 1, 30% in Group 2 and 74% in Group 3. After the ®rst sampling the total calcium concentration was lowest in Group 2, in which it decreased gradually from 2.48 to 2.29 mmol/l (Fig. 4(a)). In Groups 1 and 3 total calcium was between 2.52 and 2.36 mmol/l, varying randomly between samplings. Individual subnormal calcium values (<2.2 mmol/l; Hove et al., 1983) were found in Group 2 after 1 month of lichen feeding, and in Group 3 during the ®rst 2 months of the experiment. In the beginning of the experiment inorganic phosphate concentration ranged from 2.6 to 2.8 mmol/l (Fig. 4(b)). Thereafter, it was highest in Group 3 (max
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Fig. 2. Average body weight (a), serum total protein concentration (b) and serum albumin concentration (c) for the feeding groups during the experimental period (days). Symbol ! Group 1, * Group 2, & Group 3. Symbols indicating difference between group mean values: * Difference between groups 1 and 2 at the significance level p < 0.05, ** p < 0.01, *** p < 0.001. # Difference between groups 1 and 3, significance levels as previously. Difference between groups 2 and 3, significance levels as previously.
3.3 mmol/l) and lowest in Group 2 (min 1.6 mmol/l), the values of Group 1 being in the middle (2.2± 2.6 mmol/l). Group 1 had a higher blood glucose (2.9±3.5 mmol/l) than the other feeding groups
(2.5±3.1 mmol/l) throughout the experiment (Fig. 5(a)). At the start, all feeding groups had a mean serum cholesterol of 1.7 mmol/l (Fig. 5(b)). In Group 2 the
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Fig. 3. Average serum creatinine (a), urea (b) and alkaline phosphatase (c) concentration in the feeding groups. Symbols as in (Fig. 2).
changes in cholesterol were seemingly irregular. It decreased ®rst to only 1 mmol/l, rose in April to 2.0 mmol/l, and thereafter decreased again. In Groups 1 and 3 cholesterol varied from 1.3 to 2.0 mmol/l, Group 1 showing an increase after March.
Serum triglycerides showed little variation, from 0.2 to 0.4 mmol/l (Fig. 5(c)). In Groups 1 and 3 a slight increase was observed towards the end of the experiment. Two hinds in Group 2 had an exceptionally high concentration in April (1.81 and
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Fig. 4. Average serum total calcium concentration (a) and inorganic phosphorus concentration (b) in the feeding groups. Symbols as in (Fig. 2).
1.18 mmol/l). Other animals in this group had values similar to other feeding groups. The average birth weight of calves was 5.9 kg in Group 1, 4.0 kg in Group 2, and 6.3 kg in Group 3. Two calves in Group 2 with low birth weights (2.4 and 3.6 kg) died soon after birth. The average date of birth, when 50% of the calves in the group were born, was 8th May in Group 3. The calves in Group 1 were born an average six days later and in Group 2 thirteen days later. 4. Discussion Lichen fed animals lost 15% of their body weight during the experiment. Pellet fed hinds gained 18% in body weight, and hinds grazing freely maintained their
body weight. Bjarghov et al., 1976 demonstrated similar effects of pure lichen and pellet feeding on weight of the reindeer calves. Since all the hinds in the present experiment were pregnant, part of the weight gain was attributable to the growing foetus and increase in the weight of amniotic ¯uid and foetal membranes (Roine et al., 1982). Changes in group mean body weights were associated with corresponding changes in serum total protein and albumin concentrations and crude protein intake. Our results are consistent with Bjarghov et al., 1976, where pellet fed calves had a relatively constant concentration of albumin, while a marked decline occurred in lichen fed animals. In line with Preston et al., 1965, blood urea was depended on the amount of protein in the diet. The decrease in serum proteins (Group 2) was ®rst accom-
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Fig. 5. Average serum glucose (a), cholesterol (b) and triglyceride (c) concentration in the feeding groups. Symbols as in (Fig. 2).
panied with a fall in serum urea concentration. However, when the energy supply of Group 2 gradually decreased to 30% of the initial supply, a rise in blood urea occurred (Fig. 3(b)). The elevation was most probably caused by the use of body proteins as an energy source, when not enough energy was provided.
Similar ®ndings are also reported in cattle (Ide et al., 1967), sheep (Leibholtz, 1970), and reindeer (Valtonen, 1979). The creatinine in Group 2 began to increase after 2 weeks of lichen feeding and the rise continued until March, when the group mean was 61% higher than in
H. SaÈkkinen et al. / Small Ruminant Research 32 (1999) 211±221
the beginning of the experiment. An opposite effect was observed in Group 3, where creatinine declined by 24%. Our results differed from those by Nieminen (1980) who found serum creatinine of reindeer hinds to be stable throughout the year, though slightly increased in winter. On the contrary, Halse et al. (1976) showed that creatinine tended to increase during fasting. In black bears (Ursus americanus), the concentration of serum creatinine has been shown to rise concomitantly with a decline in serum urea when food is not available (Nelson et al., 1984). This ®nding is in agreement with our results concerning the effects of lichen feeding. In line with data of Chao et al. (1985), plasma concentrations of alkaline phosphatase were higher with advancing pregnancy. Elevated SAP activities may in part be responsible for Ca absorption and mobilization for the needs of the growing foetus. Magnesium acts as a cofactor for SAP, increasing the activity of apoenzyme (Bosron et al., 1977). Since lichens contain even 20 times less Mg than commercial pellets (Nieminen and Heiskari, 1989), the lowest SAP activity of Group 2 could be attributable to Mg effect. The daily phosphorus intake in Group 2 was only 2.6%, and calcium intake 7.3% of the pellet fed animals (Fig. 1(c)). This was re¯ected as group differences in serum concentrations of phosphorus, but not in calcium. Phosphate concentration decreased by 14% and 28% in Groups 1 and 2 compared to the start. In pellet fed animals (Group 3) inorganic phosphate concentration increased ®rst, but stabilized after 40 days. Again, our results are consistent with Bjarghov et al. (1976). Chao et al. (1985) reported plasma calcium and phosphorus of white-tailed deer to increase during the last trimester of pregnancy. Based on our data on pregnant animals with different mineral supply, it is the feeding rather than pregnancy which seems to in¯uence more plasma mineral levels until calving. In spite of the 70% reduction in energy supply during week 12 of the experiment, Group 2 had a very stable blood glucose level, indicating effective homeostatic control of this parameter. The overall level of blood glucose was similar to results given by Nieminen (1980) for reindeer on the same time of the year. The higher blood glucose of the hinds on mimicked natural diet (Group 1) compared to other groups, could be explained by greater handling stress.
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Blood glucose concentration of reindeer can be increased as a physiological response to excitement (HyvaÈrinen et al., 1976). Group 1 was gathered from a large pen, and blood sampling was more time consuming and stressful for the animals, thus elevating glucose. Since changes in blood glucose level can be very rapid and depend on many factors, it is dif®cult to separate the effects of sampling, nutrition and season from each other. In Group 2 serum cholesterol increased markedly in April, when protein, urea and creatinine levels indicated that animals suffered from protein and energy de®ciency. Seal et al. (1972) and Bjarghov et al. (1976) found no nutritional effects on the plasma triglyceride levels, but the latter reported cholesterol to be higher in animals fed with pellets. Soveri et al. (1992), on the other hand, described a rise in serum triglycerides in reindeer calves fed with lichens. In cattle, Reid et al. (1977) observed a consistent elevation both in serum cholesterol and triglycerides during fasting. In mule deer (Odocoileus hemionus), Torbit et al. (1985) divided starvation process in three phases: increased catabolism of fat and protein reserves (phase I), major mobilization of fat to provide energy and spare body protein (phase II), and reactivation of protein catabolism (phase III). Our ®ndings on lichen fed animals (Group 2) are in agreement with ®rst two phases of this model. Protein catabolism seemed to start ®rst, re¯ected by an increase in serum creatinine and a decrease in serum protein concentration. The second phase occurred around day 80 of the experiment with an increase in serum cholesterol and triglycerides together with a decrease in serum urea and creatinine concentrations. Calves born to Group 2 had 36.5% lower birth weight, and they were born 13 days later compared to Group 3. Higher birth weight can be crucial for the survival of the calves in unpredictable weather conditions in spring. Late born calves have less time to grow and gain weight during the summer, reducing its chances to survive during the ®rst year of life. The reproductive success was clearly reduced in Group 2 because of food deprivation. In turn, supplementary feeding advanced reproduction, as was seen in Group 3. The birth weight and time of calving in Group 1 on mimicked natural diet was in line with earlier records at Kaamanen Reindeer Research Station (Eloranta and Nieminen, 1985).
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5. Conclusions Animals on lichen diet lost body weight and had elevated serum urea and creatinine concentrations, indicating catabolism of body proteins. Low serum proteins re¯ected dietary protein de®ciency. The use of body fat caused a rise in serum cholesterol. Feeding with pellets seemed to counteract the signs of malnutrition. Serum protein, albumin and phosphate concentrations increased, indicating adequate dietary protein and mineral intake. Pellet feeding also substantiated an increase in body weight. On good pasture, natural winter diet provided enough energy and proteins for the maintenance of body weight. Serum proteins of hinds on mimicked winter diet decreased, but not to the extent than in animals fed only lichens. Serum inorganic phosphate and creatinine concentrations were in between of lichen and pellet fed animals. Reindeer on good winter pasture seem to accomplish their lichen-based diet with food of better nutritional value in terms of protein and minerals. Supplementary feeding can be used to maintain the normal body condition of reindeer on poor winter pasture, and to improve the productivity of reindeer herding. Acknowledgements This work was supported with a fund by the Finnish Ministry of Agriculture and Forestry. References Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W., Fu, P., 1974. Enzymatic determination of total serum cholesterol. Clin. Chem. 20, 470±475. Bjarghov, R.S., Fjellheim, P., Hove, K., Jacobsen, E., Skjenneberg, S., Try, K., 1976. Nutritional effects on serum enzymes and other blood constituents in reindeer calves (Rangifer tarandus tarandus). Comp. Biochem. Physiol. 55A, 187±193. Bosron, W.F., Anderson, R.A., Falk, M.C., Kennedy, F.S., Vallee, B.L., 1977. Effect of magnesium on the properties of zinc alkaline phosphatase. Biochemistry 16(4), 610±614. Chao, C.C., Brown, R.D., Deftos, L.J., 1985. Metabolism of calcium and phosphorus during pregnancy and lactation in white-tailed deer. Acta Endocrinol. 109, 269±275. The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology, 1974. Recom-
mended methods for the determination of four enzymes in blood. Scand. J. Clin. Lab. Invest. 36, 711±732. Daly, J.A., Ertingshausen, G., 1972. Direct method for determining inorganic phosphate in serum with the `CentrifiChem'. Clin. Chem. 18, 263±265. Doumas, B.T., Watson, W.A., Biggs, H.G., 1971. Albumin standards and the measurements of serum creatinine with bromcresol green. Clin. Chem. Acta 31, 87±96. Eloranta, E., Nieminen, M., 1985. Calving of the experimental herd in Kaamanen during 1970±85. Rangifer, Special Issue No. 1, 115±121. Fabiny, D.L., Ertingshausen, G., 1971. Automated reaction-rate method for determination of serum cholesterol. Clin. Chem. 20, 53. Gutman, I., Bergmeyer, H.U., 1974. Determination of urea with glutamate dehydrogenase as indicator enzyme. In: Bergmyer, H.U. (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, pp. 1794±1798. Halse, K., Skjenneberg, S., Jacobsen, E., Bjarghov, R.S., 1976. Blood magnesium and the renal magnesium threshold in lichen-fed and fasted reindeer. Nord. Vetmed. 28, 529±538. Hansen, J.L., Freier, E.F., 1978. Direct assays of lactate, puryvate, beta-hydroxypuryvate and acetoacetate with centrifugal analyzer. Clin. Chem. 24, 475±479. Hove, K., Horst, R.L., Littledike, E.T., 1983. Effects of 1ahydroxyvitamin D3, 1,25-dihydroxyvitamin D3, 1,24,25-trihydroxyvitamin D3, and 1,25,26-trihydroxyvitamin D3 on mineral metabolism and 1,25-dihydroxyvitamin D concentrations in dairy cows. J. Dairy Sci. 66, 59±66. HyvaÈrinen, H., Helle, T., Nieminen, M., VaÈyrynen, P., VaÈyrynen, R., 1976. Some effects of handling reindeer during gatherings on the composition of their blood. Anim. Prod. 22, 105±114. Ide, Y., Shimbayashi, K., Yonemura, T., 1967. Effect of dietary conditions upon serum and milk urea nitrogen in cows I. Serum and milk urea nitrogen as affected by protein intake. Jpn. J. Vet. Sci. 28, 321±327. Kemppainen, J., Nieminen, M., RekilaÈ, V., 1997. Poronhoidon kuva (In Finnish). Riista- ja kalatalouden tutkimuslaitos, Helsinki. Painorauma, Rauma, 142 pp. Kumpula, J., Colpaert, A., Kumpula, T., Nieminen, M., 1997. The winter pasture resources of the Finnish reindeer management area (In Finnish). Research report 93, Ministry of Agriculture and Forestry, Finnish Game and Fisheries Research Institute, Helsinki, 43 pp. 34 figs. Leibholtz, J., 1970. The effect of starvation and low nitrogen intake on the concentration of free amino-acids in the blood plasma and on the nitrogen metabolism in sheep. Aust. J. Agric. Res. 21, 723±734. Lowry, O.H., Rosenbrough, N.J., Farr, L., Randall, R.J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265. MAFF, 1975. Energy allowances and feeding systems for ruminants. Tech. Bull. 33. Her Majesty's Stationery Office, London, 79 pp. MAFF, 1984. Energy allowances and feeding systems for ruminants. Reference Book 433. Her Majesty's Stationary Office, London, 85 pp.
H. SaÈkkinen et al. / Small Ruminant Research 32 (1999) 211±221 Nelson, R.A., Bech, T.D.I., Steiger, D.L., 1984. Ratio of serum urea to serum creatinine in wild black bears. Science 226, 841±842. Nieminen, M., 1980. Nutritional and seasonal effects on the haematology and blood chemistry in reindeer (Rangifer tarandus tarandus L). Comp. Biochem. Physiol. 66A, 399±413. Nieminen, M., Heiskari, U., 1989. Diets of freely grazing and captive reindeer during summer and winter. Rangifer 9(1), 17± 34. Nieminen, M., TimisjaÈrvi, J., 1983. Blood composition of the reindeer II. Blood chemistry. Rangifer 3(1), 16±32. Preston, R.L., Schrakenberg, D.D., Pfander, W.H., 1965. Protein utilization in ruminants I. Blood urea nitrogen as affected by protein intake. J. Nutr. 86, 281±288. Reid, I.M., Stark, A.J., Isenor, R.N., 1977. Fasting and refeeding in the lactating dairy cow 1. The recovery of milk yield and blood chemistry following a six-day fast. J. Comp. Path. 87, 241±251. Roine, K., Nieminen, M., TimisjaÈrvi, J., 1982. Foetal growth in the reindeer. Acta Vet. Scand. 23, 107±117.
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Seal, U.S., Verme, L.J., Ozoga, J.J., Erickson, A.W., 1972. Nutritional effects on thyroid activity and blood of white-tailed deer. J. Wildl. Manage. 36, 1041±1052. Soveri, T., Sankari, S., Nieminen, M., 1992. Blood chemistry of reindeer calves (Rangifer tarandus) during the winter season. Comp. Biochem. Physiol. 102A, 191±196. SPSS, 1993. SPSS for Windows, Base System User's Guide, Release 6.0. SPSS, Chicago, IL, USA, 828 pp. Torbit, S.C., Carpenter, L.H., Swift, D.M., Alldredge, A.W., 1985. Differential loss of fat and protein by mule deer during winter. J. Wildl. Manage. 49, 80±85. Trinder, P., 1969. Determination of blood glucose using 4-amino phenazone as oxygen acceptor. J. Clin. Path. 22, 246. Tuori, M., Kaustell, K., Valaja, J., Aimonen, E., Saarisalo, E., Huhtanen, P., 1995. Rehutaulukot ja ruokintasuositukset (In Finnish). Yliopistopaino, Helsinki, 99 pp. Valtonen, M., 1979. Renal responses of reindeer to high and low protein diet and sodium supplement. J. Sci. Agri. Soc. Finl. 51, 381±419.
Nutrition-induced changes in blood chemical parameters of pregnant reindeer hinds (Rangifer tarandus tarandus) H. SaÈkkinen1,a,*, J. TimisjaÈrvia, E. Elorantaa, U. Heiskarib, M. Nieminenb, M. Puukkac a
Department of Physiology, University of Oulu, Kajaanintie 52 A, 90 220, Oulu, Finland Finnish Game and Fisheries Research Institute, Reindeer Research Station, 99 910, Kaamanen, Finland c Laboratory, Oulu University Hospital, 90 220, Oulu, Finland
b
Accepted 25 September 1998
Abstract Twenty-four pregnant reindeer hinds (Rangifer tarandus tarandus) were allocated to three feeding groups during 110 days experiment from January to May. Group 1 was grazing in a 15 km2 fence, their diet mimicking a good quality winter pasture. Group 2 was fed with lichens. Their energy supply was gradually decreased until it was 30% from the ad libitum amount at the start. Group 3 was fed ad libitum with a pelleted concentrate for reindeer. Lichen fed animals lost 15% of body weight during the experiment. In other groups body weight was either maintained (Group 1) or increased by 18% (Group 3). Serum protein concentration decreased from 77 g/l to 62 g/l in lichen fed animals, accompanied ®rst with a fall in serum urea concentration. When energy supply decreased further, a rise in blood urea occurred. Serum creatinine concentration increased with lichen feeding, and after 2 months it was 61% higher than at the beginning of the experiment. In pellet fed hinds creatinine declined from 190 mmol/l to 143 mmol/l. The birth weight of calves re¯ected the nutritional status of the groups. The average birth weight was 6.3 kg for calves of pellet fed hinds, 5.9 kg in group with mimicked winter diet and 4.0 kg for lichen fed group. Normal winter diet seems to provide enough energy and protein for the maintenance of body weight and for successful calving. Pellet feeding increased body weight, and had a positive effect on birth weights of calves. On poor winter pasture, supplementary feeding can be useful in prevention of malnutrition and in improving of calving success. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Reindeer; Nutrition; Serum proteins; Urea; Serum minerals
1. Introduction Overgrazing of winter pastures due to the increased reindeer population has become an increasing pro*Corresponding author. Tel.: +358-8-553-1240; fax: +358-8553-1061; e-mail: [email protected] 1 Present address: Department of Biology, University of Oulu, P.O. Box 3000, 90 401 Oulu, Finland.
blem in Finland. In many areas the lichen pastures are poor (Kumpula et al., 1997), and supplementary feeding is commonly used, especially in the southern and middle part of the reindeer herding area. The magnitude of supplementary feeding has doubled over the past 10 years, and its annual economical costs equal 25±50% of the slaughtering income at the Finnish reindeer herding area (Kemppainen et al., 1997).
0921-4488/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 9 8 ) 0 0 1 8 4 - 9
H. SaÈkkinen et al. / Small Ruminant Research 32 (1999) 211±221
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to evaluate whether blood chemical values can be used as an indicator for supplementary feeding.
One third of the supplementary feeding is given as concentrates, usually rich in protein and energy. The natural winter diet of reindeer is dominated by lichens, which are low in protein and fat, but abundant with complex carbohydrates (Nieminen and Heiskari, 1989). Therefore, the physiological effects of supplementary feeding on pregnant females, whose wellbeing is of essential importance for the pro®t of reindeer herding, should be investigated thoroughly. Earlier studies concerning reindeer blood chemistry have been focused on calves and adult non-pregnant animals. Bjarghov et al., 1976 reported, that lichens are de®cient in important nutrients such as minerals and proteins, as indicated by decreased serum mineral and albumin concentrations. Soveri et al., 1992 studied dietary responses of calves kept under natural conditions and on lichen feeding during winter. Lichen fed calves were subjected to slight and moderate weight loss. In addition to a decrease in serum proteins, the concentration of the end products of lipolysis increased, and serum urea level declined very low. Chao et al., 1985 reported that plasma levels of calcium and phosphorus increased with serum alkaline phosphatase and parathyroid hormone in white-tailed deer (Odocoileus virginianus) during pregnancy. The authors suggested this to indicate pregnancy related increase in capacity for calcium absorption and mobilization. Valtonen (1979) studied the renal function of reindeer kept either on high or low protein diet. She found plasma urea concentration to fall on a low protein diet with adequate energy supply, but to increase if combined with insuf®cient intake of energy. Seasonal variation in reindeer serum protein, lipid, glucose and mineral concentrations with highest values in summer, were reported by Nieminen and TimisjaÈrvi (1983). Most of the seasonal variation was suggested to be of dietary origin, determined by annual climatic rhythms. The aim of this study was to compare the effects of pellet feeding, natural winter diet and lichen feeding on blood chemical parameters of pregnant animals and
2. Material and methods 2.1. Animals This experiment was carried out with a permission from the Committee on Animal Experiments of the University of Oulu. Twenty-four pregnant reindeer hinds (Rangifer tarandus tarandus) were used. The animals were corralled at Kaamanen Reindeer Research Station in Northern Finland, 698100 N. The animals were taken from pasture in the middle of January, after which they were randomly allocated to three feeding groups, each consisting of eight animals. The average temperature changed from ÿ118C in January to 48C in the ®rst week of May. The depth of snow cover was 44 cm in January, reached its maximum (81 cm) in April, and was 37 cm in the beginning of May. Group 1 was grazing in a 15 km2 fenced area. The diet of this group was adjusted to mimic a good quality winter pasture. Each animal got 0.2±0.5 kg commercial reindeer pellet (Poron±Herkku, Raisio Group, Raisio, Finland) to cover about one-third of the daily protein and energy supply. Rest of the diet was mainly lichens, hay (Deschampsia ¯exuosa) and blueberry leaves and twigs, that animals were able to ®nd from the fence. Group 2 was fed with lichens (Cladina spp.). Their energy supply was gradually decreased reaching on week 12 30% of the ad libitum amount in the beginning of the study. After this combined pellet and lichen feeding was used. Group 3 was fed ad libitum with commercial reindeer pellet. The energy content of the pellets was calculated using MAFF recommendations (MAFF, 1975, 1984). Digestibility tables for ruminants (Tuori et al., 1995) were used to estimate the digestibility of different compounds of the pellets. Chemical composition and energy content
Table 1 The chemical composition (% DM basis) and the energy value of lichens and the commercial reindeer pellet Poron±Herkku
Lichens Pellets
DM (%)
Energy (MJ ME/kg DM)
Crude protein
Crude fibre
Nitrogen free extracts
Fat
P
Ca
34 86
10.7 9.9
3.4 13.7
41 16
51.0 55.6
2.9 5.0
0.04 0.67
0.1 0.8
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of lichens and pellets based on the feed analyses are given in Table 1. The pelleted concentrate was composed (%) of 25.5 wheat bran, 25 oat bran, 15 dry
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molasses pulp, 10 wheat, 15 hay (ground), 5 molasses, 2.5 minerals, and 2 vegetable oil. The amount of pellets and lichens eaten by the hinds was measured
Fig. 1. Average weekly energy and dry matter intake (a), protein intake (b) and calcium and inorganic phosphorus intake (c) for groups 2 and 3. Symbols * and * Group 2, & and & Group 3. Solid symbols indicate energy and calcium, open symbols are used for dry matter and phosphorus. Striped bars in (Fig. 1(b)) indicate feeding arrangements for Group 2.
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daily, and the energy, protein and mineral supply was calculated as a weekly group average (Fig. 1a,b,c). Blood samples were drawn from the jugular vein with serum or EDTA- plasma vacutainers (Venoject1, Leuven, Belgium). Blood samples were collected and body weight recorded ®ve times at regular intervals during the experiment. The ®rst sampling and weighing was done when the animals were allocated to feeding groups. The following samples were collected in the beginning of each month until May. Sedatives or medication was not used, the animals were caught and restrained by hand for blood sampling. 2.2. Analyses Serum total proteins were analysed with the biuretmethod (Lowry et al., 1951). The bromcresol green method (Doumas et al., 1971) was used to determine serum albumin concentration. Serum urea was measured enzymatically according to Gutman and Bergmeyer (1974) and creatinine colorimetrically by Jaffe reaction (Fabiny and Ertingshausen, 1971). Cholesterol and triglyserides were measured enzymatically (Allain et al., 1974), blood glucose enzymatically from whole blood after protein precipitation with 0.6 M perchloric acid (Trinder, 1969; Hansen and Freier, 1978) and inorganic phosphate spectrophotometrically in fosfomolybdate form (Daly and Ertingshausen, 1972). Total calcium was analysed with EFEX 5055 -¯ame photometer (Eppendorf, Hamburg, Germany). Serum alkaline phosphatase (SAP) was measured by using kinetic photometric assay according to the recommendations of The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology (1974). All the results are expressed as group means s.d. Statistical analyses were performed using SPSS software programme (SPSS, 1993). Differences between group mean values at different times were assessed by analysis of variance. If the provided F-values indicated signi®cance (P < 0.05), the differences were assessed post hoc by Tukey test. 3. Results Considerable weight changes and differences between the feeding groups were observed during
the experiment (Fig. 2(a)). In lichen fed animals (Group 2) the average body weight loss was 15%. In the two other groups the average body weight either was maintained (Group 1) or increased (Group 3; 18% average body weight increase). At the beginning of the experiment the average serum protein concentration was 73±77 g/l (Fig. 2(b)). During the ®rst two weeks it rose to 83 g/l in Group 3, after which a slight decrease was found. In Groups 1 and 2 serum proteins decreased, being on average 10 and 20% lower in May, respectively. In Group 1 serum albumin concentration was constant throughout the experiment, while it increased by 16% in Group 3 and decreased by 23% in Group 2 (Fig. 2(c)). Groups 1 and 2 had higher serum creatinine levels (197±316 mmol/l) than Group 3 after the ®rst month of the experiment; Group 2 having the highest values throughout (Fig. 3(a)). In Groups 1 and 2 creatinine increased until March, after which it gradually declined to the basal level. In Group 3 creatinine ®rst decreased from 190 mmol/l to 143 mmol/l within 1 month, then gradually increased. Signi®cant differences in serum urea concentration between the feeding groups developed already during the ®rst 2 weeks of the experiment. In the beginning of the experiment serum urea concentration was 12.7± 14.2 mmol/l (Fig. 3(b)). Lichen fed animals (Group 2) had the lowest concentrations, decreasing to 5.2 mmol/l and remaining lower than in the two other groups for the rest of the experiment. SAP activity increased in all three groups towards the end of the experiment (Fig. 3(c)). Group 2 had the lowest SAP activities throughout the experiment. The average increase in SAP activity during the experiment was 94% in Group 1, 30% in Group 2 and 74% in Group 3. After the ®rst sampling the total calcium concentration was lowest in Group 2, in which it decreased gradually from 2.48 to 2.29 mmol/l (Fig. 4(a)). In Groups 1 and 3 total calcium was between 2.52 and 2.36 mmol/l, varying randomly between samplings. Individual subnormal calcium values (<2.2 mmol/l; Hove et al., 1983) were found in Group 2 after 1 month of lichen feeding, and in Group 3 during the ®rst 2 months of the experiment. In the beginning of the experiment inorganic phosphate concentration ranged from 2.6 to 2.8 mmol/l (Fig. 4(b)). Thereafter, it was highest in Group 3 (max
H. SaÈkkinen et al. / Small Ruminant Research 32 (1999) 211±221
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Fig. 2. Average body weight (a), serum total protein concentration (b) and serum albumin concentration (c) for the feeding groups during the experimental period (days). Symbol ! Group 1, * Group 2, & Group 3. Symbols indicating difference between group mean values: * Difference between groups 1 and 2 at the significance level p < 0.05, ** p < 0.01, *** p < 0.001. # Difference between groups 1 and 3, significance levels as previously. Difference between groups 2 and 3, significance levels as previously.
3.3 mmol/l) and lowest in Group 2 (min 1.6 mmol/l), the values of Group 1 being in the middle (2.2± 2.6 mmol/l). Group 1 had a higher blood glucose (2.9±3.5 mmol/l) than the other feeding groups
(2.5±3.1 mmol/l) throughout the experiment (Fig. 5(a)). At the start, all feeding groups had a mean serum cholesterol of 1.7 mmol/l (Fig. 5(b)). In Group 2 the
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Fig. 3. Average serum creatinine (a), urea (b) and alkaline phosphatase (c) concentration in the feeding groups. Symbols as in (Fig. 2).
changes in cholesterol were seemingly irregular. It decreased ®rst to only 1 mmol/l, rose in April to 2.0 mmol/l, and thereafter decreased again. In Groups 1 and 3 cholesterol varied from 1.3 to 2.0 mmol/l, Group 1 showing an increase after March.
Serum triglycerides showed little variation, from 0.2 to 0.4 mmol/l (Fig. 5(c)). In Groups 1 and 3 a slight increase was observed towards the end of the experiment. Two hinds in Group 2 had an exceptionally high concentration in April (1.81 and
H. SaÈkkinen et al. / Small Ruminant Research 32 (1999) 211±221
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Fig. 4. Average serum total calcium concentration (a) and inorganic phosphorus concentration (b) in the feeding groups. Symbols as in (Fig. 2).
1.18 mmol/l). Other animals in this group had values similar to other feeding groups. The average birth weight of calves was 5.9 kg in Group 1, 4.0 kg in Group 2, and 6.3 kg in Group 3. Two calves in Group 2 with low birth weights (2.4 and 3.6 kg) died soon after birth. The average date of birth, when 50% of the calves in the group were born, was 8th May in Group 3. The calves in Group 1 were born an average six days later and in Group 2 thirteen days later. 4. Discussion Lichen fed animals lost 15% of their body weight during the experiment. Pellet fed hinds gained 18% in body weight, and hinds grazing freely maintained their
body weight. Bjarghov et al., 1976 demonstrated similar effects of pure lichen and pellet feeding on weight of the reindeer calves. Since all the hinds in the present experiment were pregnant, part of the weight gain was attributable to the growing foetus and increase in the weight of amniotic ¯uid and foetal membranes (Roine et al., 1982). Changes in group mean body weights were associated with corresponding changes in serum total protein and albumin concentrations and crude protein intake. Our results are consistent with Bjarghov et al., 1976, where pellet fed calves had a relatively constant concentration of albumin, while a marked decline occurred in lichen fed animals. In line with Preston et al., 1965, blood urea was depended on the amount of protein in the diet. The decrease in serum proteins (Group 2) was ®rst accom-
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Fig. 5. Average serum glucose (a), cholesterol (b) and triglyceride (c) concentration in the feeding groups. Symbols as in (Fig. 2).
panied with a fall in serum urea concentration. However, when the energy supply of Group 2 gradually decreased to 30% of the initial supply, a rise in blood urea occurred (Fig. 3(b)). The elevation was most probably caused by the use of body proteins as an energy source, when not enough energy was provided.
Similar ®ndings are also reported in cattle (Ide et al., 1967), sheep (Leibholtz, 1970), and reindeer (Valtonen, 1979). The creatinine in Group 2 began to increase after 2 weeks of lichen feeding and the rise continued until March, when the group mean was 61% higher than in
H. SaÈkkinen et al. / Small Ruminant Research 32 (1999) 211±221
the beginning of the experiment. An opposite effect was observed in Group 3, where creatinine declined by 24%. Our results differed from those by Nieminen (1980) who found serum creatinine of reindeer hinds to be stable throughout the year, though slightly increased in winter. On the contrary, Halse et al. (1976) showed that creatinine tended to increase during fasting. In black bears (Ursus americanus), the concentration of serum creatinine has been shown to rise concomitantly with a decline in serum urea when food is not available (Nelson et al., 1984). This ®nding is in agreement with our results concerning the effects of lichen feeding. In line with data of Chao et al. (1985), plasma concentrations of alkaline phosphatase were higher with advancing pregnancy. Elevated SAP activities may in part be responsible for Ca absorption and mobilization for the needs of the growing foetus. Magnesium acts as a cofactor for SAP, increasing the activity of apoenzyme (Bosron et al., 1977). Since lichens contain even 20 times less Mg than commercial pellets (Nieminen and Heiskari, 1989), the lowest SAP activity of Group 2 could be attributable to Mg effect. The daily phosphorus intake in Group 2 was only 2.6%, and calcium intake 7.3% of the pellet fed animals (Fig. 1(c)). This was re¯ected as group differences in serum concentrations of phosphorus, but not in calcium. Phosphate concentration decreased by 14% and 28% in Groups 1 and 2 compared to the start. In pellet fed animals (Group 3) inorganic phosphate concentration increased ®rst, but stabilized after 40 days. Again, our results are consistent with Bjarghov et al. (1976). Chao et al. (1985) reported plasma calcium and phosphorus of white-tailed deer to increase during the last trimester of pregnancy. Based on our data on pregnant animals with different mineral supply, it is the feeding rather than pregnancy which seems to in¯uence more plasma mineral levels until calving. In spite of the 70% reduction in energy supply during week 12 of the experiment, Group 2 had a very stable blood glucose level, indicating effective homeostatic control of this parameter. The overall level of blood glucose was similar to results given by Nieminen (1980) for reindeer on the same time of the year. The higher blood glucose of the hinds on mimicked natural diet (Group 1) compared to other groups, could be explained by greater handling stress.
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Blood glucose concentration of reindeer can be increased as a physiological response to excitement (HyvaÈrinen et al., 1976). Group 1 was gathered from a large pen, and blood sampling was more time consuming and stressful for the animals, thus elevating glucose. Since changes in blood glucose level can be very rapid and depend on many factors, it is dif®cult to separate the effects of sampling, nutrition and season from each other. In Group 2 serum cholesterol increased markedly in April, when protein, urea and creatinine levels indicated that animals suffered from protein and energy de®ciency. Seal et al. (1972) and Bjarghov et al. (1976) found no nutritional effects on the plasma triglyceride levels, but the latter reported cholesterol to be higher in animals fed with pellets. Soveri et al. (1992), on the other hand, described a rise in serum triglycerides in reindeer calves fed with lichens. In cattle, Reid et al. (1977) observed a consistent elevation both in serum cholesterol and triglycerides during fasting. In mule deer (Odocoileus hemionus), Torbit et al. (1985) divided starvation process in three phases: increased catabolism of fat and protein reserves (phase I), major mobilization of fat to provide energy and spare body protein (phase II), and reactivation of protein catabolism (phase III). Our ®ndings on lichen fed animals (Group 2) are in agreement with ®rst two phases of this model. Protein catabolism seemed to start ®rst, re¯ected by an increase in serum creatinine and a decrease in serum protein concentration. The second phase occurred around day 80 of the experiment with an increase in serum cholesterol and triglycerides together with a decrease in serum urea and creatinine concentrations. Calves born to Group 2 had 36.5% lower birth weight, and they were born 13 days later compared to Group 3. Higher birth weight can be crucial for the survival of the calves in unpredictable weather conditions in spring. Late born calves have less time to grow and gain weight during the summer, reducing its chances to survive during the ®rst year of life. The reproductive success was clearly reduced in Group 2 because of food deprivation. In turn, supplementary feeding advanced reproduction, as was seen in Group 3. The birth weight and time of calving in Group 1 on mimicked natural diet was in line with earlier records at Kaamanen Reindeer Research Station (Eloranta and Nieminen, 1985).
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5. Conclusions Animals on lichen diet lost body weight and had elevated serum urea and creatinine concentrations, indicating catabolism of body proteins. Low serum proteins re¯ected dietary protein de®ciency. The use of body fat caused a rise in serum cholesterol. Feeding with pellets seemed to counteract the signs of malnutrition. Serum protein, albumin and phosphate concentrations increased, indicating adequate dietary protein and mineral intake. Pellet feeding also substantiated an increase in body weight. On good pasture, natural winter diet provided enough energy and proteins for the maintenance of body weight. Serum proteins of hinds on mimicked winter diet decreased, but not to the extent than in animals fed only lichens. Serum inorganic phosphate and creatinine concentrations were in between of lichen and pellet fed animals. Reindeer on good winter pasture seem to accomplish their lichen-based diet with food of better nutritional value in terms of protein and minerals. Supplementary feeding can be used to maintain the normal body condition of reindeer on poor winter pasture, and to improve the productivity of reindeer herding. Acknowledgements This work was supported with a fund by the Finnish Ministry of Agriculture and Forestry. References Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W., Fu, P., 1974. Enzymatic determination of total serum cholesterol. Clin. Chem. 20, 470±475. Bjarghov, R.S., Fjellheim, P., Hove, K., Jacobsen, E., Skjenneberg, S., Try, K., 1976. Nutritional effects on serum enzymes and other blood constituents in reindeer calves (Rangifer tarandus tarandus). Comp. Biochem. Physiol. 55A, 187±193. Bosron, W.F., Anderson, R.A., Falk, M.C., Kennedy, F.S., Vallee, B.L., 1977. Effect of magnesium on the properties of zinc alkaline phosphatase. Biochemistry 16(4), 610±614. Chao, C.C., Brown, R.D., Deftos, L.J., 1985. Metabolism of calcium and phosphorus during pregnancy and lactation in white-tailed deer. Acta Endocrinol. 109, 269±275. The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology, 1974. Recom-
mended methods for the determination of four enzymes in blood. Scand. J. Clin. Lab. Invest. 36, 711±732. Daly, J.A., Ertingshausen, G., 1972. Direct method for determining inorganic phosphate in serum with the `CentrifiChem'. Clin. Chem. 18, 263±265. Doumas, B.T., Watson, W.A., Biggs, H.G., 1971. Albumin standards and the measurements of serum creatinine with bromcresol green. Clin. Chem. Acta 31, 87±96. Eloranta, E., Nieminen, M., 1985. Calving of the experimental herd in Kaamanen during 1970±85. Rangifer, Special Issue No. 1, 115±121. Fabiny, D.L., Ertingshausen, G., 1971. Automated reaction-rate method for determination of serum cholesterol. Clin. Chem. 20, 53. Gutman, I., Bergmeyer, H.U., 1974. Determination of urea with glutamate dehydrogenase as indicator enzyme. In: Bergmyer, H.U. (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, pp. 1794±1798. Halse, K., Skjenneberg, S., Jacobsen, E., Bjarghov, R.S., 1976. Blood magnesium and the renal magnesium threshold in lichen-fed and fasted reindeer. Nord. Vetmed. 28, 529±538. Hansen, J.L., Freier, E.F., 1978. Direct assays of lactate, puryvate, beta-hydroxypuryvate and acetoacetate with centrifugal analyzer. Clin. Chem. 24, 475±479. Hove, K., Horst, R.L., Littledike, E.T., 1983. Effects of 1ahydroxyvitamin D3, 1,25-dihydroxyvitamin D3, 1,24,25-trihydroxyvitamin D3, and 1,25,26-trihydroxyvitamin D3 on mineral metabolism and 1,25-dihydroxyvitamin D concentrations in dairy cows. J. Dairy Sci. 66, 59±66. HyvaÈrinen, H., Helle, T., Nieminen, M., VaÈyrynen, P., VaÈyrynen, R., 1976. Some effects of handling reindeer during gatherings on the composition of their blood. Anim. Prod. 22, 105±114. Ide, Y., Shimbayashi, K., Yonemura, T., 1967. Effect of dietary conditions upon serum and milk urea nitrogen in cows I. Serum and milk urea nitrogen as affected by protein intake. Jpn. J. Vet. Sci. 28, 321±327. Kemppainen, J., Nieminen, M., RekilaÈ, V., 1997. Poronhoidon kuva (In Finnish). Riista- ja kalatalouden tutkimuslaitos, Helsinki. Painorauma, Rauma, 142 pp. Kumpula, J., Colpaert, A., Kumpula, T., Nieminen, M., 1997. The winter pasture resources of the Finnish reindeer management area (In Finnish). Research report 93, Ministry of Agriculture and Forestry, Finnish Game and Fisheries Research Institute, Helsinki, 43 pp. 34 figs. Leibholtz, J., 1970. The effect of starvation and low nitrogen intake on the concentration of free amino-acids in the blood plasma and on the nitrogen metabolism in sheep. Aust. J. Agric. Res. 21, 723±734. Lowry, O.H., Rosenbrough, N.J., Farr, L., Randall, R.J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265. MAFF, 1975. Energy allowances and feeding systems for ruminants. Tech. Bull. 33. Her Majesty's Stationery Office, London, 79 pp. MAFF, 1984. Energy allowances and feeding systems for ruminants. Reference Book 433. Her Majesty's Stationary Office, London, 85 pp.
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Seal, U.S., Verme, L.J., Ozoga, J.J., Erickson, A.W., 1972. Nutritional effects on thyroid activity and blood of white-tailed deer. J. Wildl. Manage. 36, 1041±1052. Soveri, T., Sankari, S., Nieminen, M., 1992. Blood chemistry of reindeer calves (Rangifer tarandus) during the winter season. Comp. Biochem. Physiol. 102A, 191±196. SPSS, 1993. SPSS for Windows, Base System User's Guide, Release 6.0. SPSS, Chicago, IL, USA, 828 pp. Torbit, S.C., Carpenter, L.H., Swift, D.M., Alldredge, A.W., 1985. Differential loss of fat and protein by mule deer during winter. J. Wildl. Manage. 49, 80±85. Trinder, P., 1969. Determination of blood glucose using 4-amino phenazone as oxygen acceptor. J. Clin. Path. 22, 246. Tuori, M., Kaustell, K., Valaja, J., Aimonen, E., Saarisalo, E., Huhtanen, P., 1995. Rehutaulukot ja ruokintasuositukset (In Finnish). Yliopistopaino, Helsinki, 99 pp. Valtonen, M., 1979. Renal responses of reindeer to high and low protein diet and sodium supplement. J. Sci. Agri. Soc. Finl. 51, 381±419.