Nutritional effects on serum enzymes and other blood constituents in reindeer calves (Rangifer tarandus tarandus)

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NUTRITIONAL EFFECTS ON SERUM ENZYMES AND OTHER BLOOD CONSTITUENTS IN REINDEER CALVES (RANGIFER 7”RANDUS TARANDUS) R. S. BJARGHOV.P. FJELLHEIM*. K. HOVE?, E. JACOBSEN,

S. SKJENNEBERCAND K. TRY? Norwegian State Reindeer Research. Harstad, Norway (Received 21 Nocrmher

1975)

Four penned reindeer calves (Rang@ tarandus tarandus) were studied from December to April to find differences in blood chemistry reflecting the nutritional condition. Two were fed lichens Abstract-l.

only, while the other two were fed a pelleted reindeer feed (RF-71). 2. Eighteen assays including body weight, hematocrit, organic and inorganic serum constituents were performed to provide a composite picture of the metabolic responses to the difference in quality of available food. 3. The lichen fed animals developed a severe condition of malnutrition with declining body weights and falling levels of hematocrit and serum concentrations of albumin and magnesium. 4. The RF-71 animals showed a slight increase in body weight and no unexpected changes in the blood analyses

INTRODUCTION The reindeer exhibits a seasonal pattern of food intake, activity and growth. (McEwan, 1968a, McEwan & Whitehead, 1969). The winter depression of food intake, growth and weight gain may be at least partly a physiological controlled mechanism independent of quality and quantity of the available food. The snowcovered period is the critical part of the year in the growth cycle of reindeer. During the winter the main part of the food intake is lichens, which contain small amounts of protein. Experiments show that this diet also gives a deficient supply of minerals (Nordfelt et crl., 1961, Jacobsen & Skjenneberg, 1972). Under normal conditions the energy need for maintenance seems to be satisfied, but at the same time lack of protein in the feed is assumed to cause catabolism of muscle tissue. Regarding protein and minerals the winter is not only a maintenance period for the reindeer, but most probably a period of deficit. In the summer the reindeer have to recover nutritionally, and build up stores for the next winter. According to reports from reindeer owners, there often is a great loss of reindeer calves on the winter pastures. Sometimes there also is a need for feeding the whole herd as the winter pastures may be covered by heavy snow or ice. In conjunction with this problem, the Norwegian State Reindeer Research have designed a special brand of pelleted reindeer feed (named RF-71). The present investigation was undertaken to compare the change in body weight and several blood constituents of reindeer fed lichen and RF-71, and to evaluate blood parameters which may give information of the nutritional state of the animal. *Veterinary College of Norway, t Institute for medical biology, Norway.

The measured blood constituents include parameters for eventual reflection of the differences in protein and minerals in the feed.

ANIMALS AND him/s

METHODS

and diets

In late October at an age of 5 months. four male reindeer calves were taken from natural pastures. In a preexperimental period of about I month, two animals (No. 135 & 137) were fed lichens (Cladonia alpestris) at a rate of 1 kg dry matter per day, while the other two (No. 147 & 179) received a pelleted reindeer feed (RF-71) ad lihitum with the exception of the last 10 days when lichens were given ad lihitum. The experimental period lasted from November 20 until April 4. The rations offered in the experimental period and the chemical composition of the feed are given in Table 1 and 2, respectively. The animals in both groups consumed full ration until the middle of March. Then left-overs were noted. Three to four per cent of the offered ration were not eaten by the RF-71 animals while the amount rejected increased in the lichen group to about 70/, and 25$, for animal No. 135 and 137, respectively. The figures for energy intake are calculated from the experiment of Jacobsen & Skjenneberg (1976). The animals were kept in individual pens (3 x 30 m) where food was given once a day (at 8.30 a.m.) in a 2.5 mz sheltered part of the pen. The animals had free access to water or snow. Weighing Every second week before morning feeding weighing to the nearest 100 g was performed with a steelyard connected to a special constructed weighing-bow. The observed weights on the Fig. are averages of weighings on two consecutive days. Blood samples

Oslo, Norway. University of Tromso,

These were taken from the jugular vein immediately before morning feeding every second week, by means of 187

R. S. BJARCIHOVet ~1. Table

I. Weight

of full rations,

Amountfed (dry matter) (g)

GrOUp

fed

Lichen

RF-71 x

energy

content,

Net energy* intake KCal

800

Crude Ca++ protein k/day) (g/day)

25

1.190

1.000

crude protein,

1.820

137

and some minerals

1"lg++

P

(g/dajr) (g/day)

o,e

o,2

a,4

599

2,o

5,6

Net energy for fattening

evacuated @as, tubes (\acutainer).

Care was taken to dislurb the animals as little as possible during the sampling procedure. Serum was separated after a clotting period of Ii 2 hr and sent unfrozen to the laboratory for analysis. Usually the serum samples were analysed within 40 hr. For home measurements (glucose. NEFA, total lipids and GPT) hcparin plasma was separated immediately after sampling and Lcp~ ll-o/cn until anal!sed. EDTA was used ah antlcoagulant lor the blood drawn for hacmatocrit measurements. Hacmatocrit values were read immediately after \ampling.

Alanillc-aminotransferase (ALAT = GPT) was determined according to Reitman & Frankel (1957). The enzyme activities of aspartate- aminotransferase (ASAT = SGOT). and alkaline phosphatase (ALP) in herum wcrc analysed by methods recommended by the Scandinavian Enlyme Committee (1974). The method for determination of total plasma lipids were based on the sulfa phospho-vanillin reaction and the analysis carried out with standard reagents from J. T. Baker C’hemicats NV. Deventer (Netherlands). The plasma NEFA were extracted with heptane and titrated with NaOH according to Dole (1956). Since ruminant blood ma) contain relatively large amounts of \otatile fatty acids. extra washings of the fat soluble heptane phase containing the NEFA were undertaken. C’olourlmctric procedures were used for the detcrmination of inorganic phosphate (Morin & Prox, 1973). cholestcrol (Huang C’Itri.. 1961 1.triglqccridcs (Soloni. 1971) and total proteins (Wcichscthaum. 19461 111se!-urn. Albumin was determined by an indicator method (Doumas ci trl.. 19711 using human serum albumin as a standard. Glucose was measured bq a glucose oxydase reaction (Hochringcr Kit). The serum concentrations of sodium and potassium wcrc determined b) Hame emission photometry, of calcium and magnesium by atomic absorption spectrophotometry a11d of chlorldc by coulomctric titration.

A mean weight loss of 0.6 kg/week were observed in the lichen-fed animals. and a gain of 0.07 kg/week

in the RF-aminals (Fig. la). During the experimental period the lichen-fed animals lost 24$; of their body weight. This is a somewhat higher weight loss than is usually observed during the same period on natural pastures in Norway (Bjarghov rf al., in press). During the last weeks of the experimental period both calves suffered from loss of appetite. McEwan (1966a) has found weight losses amounting to as much as 17”: during the first winter in wild reindeer and caribou. These figures indicate that under-nutrition of the calves in the present experiments was very severe. When calves are fed lichens ad lihirum. weight losses of about 0.35 kg/week are commonly registered (Jacobsen & Skjenneberg, 1972). The reduced intake of food in the last month of the experiment is probably attributable to the development of deficiencies due to the limited content of nutrients other than carbohydrate in the lichens. The observed plasma values in this group are therefore representative for reindeer calves on an extreme lichen diet. Care should be taken, however, to use these data uncritically as reference values for reindeer on natural pastures, where non-lichen vegetation probably contributes significantly to the energy and mineral metabolism (Skjenneberg et d., 1975). The calves fed the pelleted reindeer feed gained about 0.07 kg/week during the experiment. Our weekly weight gains were much lower than those observed by McEwan & Whitehead (1969) during the same period of the year (1.2 kg/week for male reindeer calves). These animals were, however, fed acl lihitum. Blood Alanine-aminotrclnsrus~ (ALAT=GPT) und aspurtale-uminotransferuse (ASAT=SGOT). GPT (Fig. lb) showed values and variations of the same magnitude as found in sheep (Tollersrud et u/., 1971). The values of SGOT activity are more fluctuating (Fig. lc). Great fluctuations in SGOT activities have been found in white-tailed deer (S&al ut a[.. 1972ab). Most probably these fluctuations were caused by the handling of the animals.

Table 2. Diets: Chemical composition

Er"s m.*tser

1,1cr.en

,*

iii'-;l

91'A

1

I

orgaxc matter

Ash

98,6

134

94,4

5.6

Ether extract

:ntde fiber

N-frse extract

Ca++

Mg'+

3,1

197

32,6

a,2

0,lO

0,03

O,O5

13,7

7,2

11,o

62,5

0,59

0,20

0,56

Crude protein

P

Nutritional

effects on serum enzymes

Fig. 1. Plotted curves of body weight, alanine-aminotransferase (ALAT = GPT), aspartate-aminotransferase (ASAT = SCOT), and alkaline phosphatase. Closed triangles and circles for animal No. 179 and 147, respectively-both fed RF-71. Open triangles and circles for animal No. 135 and 137. resnectively-both fed lichen.

Alkaline phosphatase (ALP). The calves fed RF-71 had higher ALP activities than calves fed lichen. The ALP activities of the RF-71 animals increased throughout the experiment. High ALP activities in serum of juvenile mammals are generally considered to reflect bone growth. This would mean in the present experiment, that the calves fed RF-71 should have higher bone growth than calves fed lichen. According to empirical knowledge, good nutrition results in early growing antlers. The steeper elevation in the ALP activity in Feb-Mar for the calves fed RF-71 is perhaps correlated to the growing antlers. Total lipid and cholesteroL Higher serum levels of total lipids and cholesterol were observed in the RF-71 group than in the lichen group (Fig. 2a & b). These differences most probably reflect the differences in lipid content and nutritional value of the two diets (Tables 1 & 2). For white-tailed deer, however, Seal et al. (1972b) found higher serum cholesterol level in a group on moderate diet (69 mg/lOO ml) compared with a group on high diet (61 mg/lOOml). Triglycerides. The apparent lack of nutritional effects on the plasma triglycerid levels (Fig. 2c) are in good agreement with the results of Seal et al. (1972b) in white-tailed deer. Non-rsterijied ,fatty acids (NEFA). Plasma concen-

trations of NEFA (Fig. 2d) did not show a clear relation to the diets. Elevated plasma NEFA levels are a common and well documented finding in various species (also ruminants) during periods of negative energy balance and fat mobilization. (Reid & Hinks, 1962; Kronfeld, 1965; Holmes & Lambourne, 1970; Pehrson, 1971). In the present investigation no systematic trend in plasma NEFA levels attributable to dietary differences could be detected. The animals losing weight had NEFA levels lower or comparable with the two other animals during most of the experiment. Pre-feeding NEFA levels between 0.5 and 1.5 m-equiv/l suggests a certain amount of lipolysis (Reid & Hinks, 1960; Kronfeld, 1965). The general tendency to increased NEFA levels as the winter progresses fits well in with the concomitant tendency to decreases in pre-feeding glucose levels. One of the lichen-fed animals shows a very different pattern (Fig. 2d bottom) which is difficult to explain. TotaL protein. The RF-71 animals had plasma protein levels in the same range as observed in first year for caribou and reindeer (6-8 g/100 ml, McEwan & Whitehead, 1969) while the lichen animals were in the lower range or below these values (Fig. 3a). The values for the lichen animals are in agreement with findings of Ullrey et al. (1967) in protein deficient white-tailed deer. Values observed in wild yearling

R. S.

190

73

5

40

.-

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.-G..._,

---s--d-_,

,p

t-c20 t

tb)

Fig.

2. Plotted

curves

of total lipids, cholesterol,

triglycerides, and non esterified Symbols are as in Fig. 1.

male white-tailed deer in generally good condition were also in the same range as the present lichen-fed animals (Seal & Erickson, 1969). For dairy cows. (Payne rt ~11.. 1970) and penned pregnant white-tailed deer. (Seal Pf ul.. 1972b) it is shown that there were small or no significant differences in total serum protein between groups on high and moderate diets. This experiment, however, indicates that a considerable difference in the protein intake can be reflected in the concentration of total serum protein. ~l~u~~n. The animals fed RF-71 show a relative constant concentration of albumin, the values Iying between 4.5 and 5 g/l00 ml (Fig. 3b). The two calves fed lichen show a lower and markedly declining albumin concentration during the experiment period. In dairy cows Payne et cd. (1970) found that albumin varied considerably and tended to be especially high in cows on pasture. ~ypoalbuminaemia occurred in herds. with low protein intakes. Herds grazing highly fertilized pastures tended to have high levels of albumin. The albumin values from the present experiment give good support to this observation. However. no significant effect of dietary protein on blood albumin concentration was found for whitetailed deer (Ullrey rr al., 1967) and Colombian blacktailed deer (Bandy ct al.. 1957). The lower concentration and declining trend in albumin and total protein for the lichen fed animals most probably reflect that the diet is deficient in proteins.

fatty xids (NEFA).

L’rru. Serum urea concentrations were about twice as high in the RF-71 group as in the other (Fig. 30). The levels observed in the present experiment were higher than those observed earlier in reindeer on comparable diets (Hove & Jacobsen. 1975). This experiment indicates that serum urea nitrogen in reindeer calves is related to protein intake. The assumption is in agrreement with previous reports dealing with other ruminant species (Preston ut trl.. 1965; McEwan 1968b; Payne et d.. 1970; Seal L’Itl/.. 1972: and Tore11 pt al., 1974). However, as Preston et al. (1965) pointed out, the relationship can be modified by several other factors, of which not all are yet defined. In the last 2-3 weeks an increasing plasma urea level was observed for the lichen fed animals. This occurred during the period of decreasing appetite, and most probably reflects an increased catabolism of body protein at this time (Leibholz, 1970). Glncosr. Plasma glucose levels (Fig. 3d) were in the same range as observed both in penned and free ranging reindeer (Luick et al., 1973). The absence of hypoglycamic plasma indicates that the intake of glycogenic precursors are sufficient to maintain plasma glucose within the normal range on a pure lichen diet. ~~~~ut#~rjt.Changes in the hematocrit pattern during the experimental period (Fig. 3e) resemble that of albumin, total lipid. magnesium, and body weight. A positive correlation between hematocrit and level of nutrition is well documented in various ruminant species (Rosen & Bischoff, 1952: Payne CT trl.. 1970:

Nutritional effects on serum enzymes

I91

Fig. 3. Plotted curves of total protein, albumin, urea, glucose and hematocrit. Symbols are as in Fig. 1.

and Holmes & Lambourne, 1970). Among other parameters McEwan & Whitehead (1969) observed hematocrit during the first year in caribou and reindeer. In the period from November to April the hematocrit values showed a decreasing tendency. These results resemble the present. McEwan & Whitehead (1969) suggested that the lowering of hematocrit could possibly be ascribed to protein or iron deficiency or both. lnorgan~~ phosphorus. As seen in Fig. 4a there is a clear difference in the serum concentration of inorganic phosphate between the groups fed RF-71 and lichen. Most probably this is caused by the dietary difference. It has been demonstrated that dietary conditions affect phosphorus levels in the blood of sheep (Becker & Smith, 1950; Long et al., 19653, dairy cow (Payne er nl., 1970) and white-tailed deer (Teeri et al., 1958; Seal et al., 1972b). The inorganic phosphorus for reindeer calves is within the range stated for other ruminants (Long et al., 1965; McEwan, 1968b; Payne et al., 1970).

Magnesium. Serum magnesium levels were identical and within the normal range of other ruminant species when the experiment started. (Dairy cows: Payne et al., 1970; white-tailed deer: Seal et al., 1972b). During the experiment serum magnesium in the lichen-fed animals declined to values well within the range where dairy cows are highly susceptible to hypomagnesaemic teatanies (Fig. 4b). No such symptoms were observed, however. The ma~esium requirements of dairy cows have been estimated to about 50@750ppm of the diet. The magnesium content of the present diet was only about 300 ppm. Observations of low plasma magnesium levels in lichen-fed and undiseased calves on natural winter pastures clearly indicate that magnesium deficiencies may be a problem of considerable ~rn~r~n~e in reindeer husbandry (Bjarghov et al., in prep.). Calcium. Serum calcium levels show small effect of this experiment’s dietary conditions (Fig. 4~). This is in agreement with observations on dairy herd (Payne et al., 1970) and white-tailed deer (Teeri et al., 1958;

192

R. S.

Fig. 4. Plotted

curves of inorganic

phosphorus,

BJARGHOV

uf (I/.

magnesium.

calcium,

potassium,

sodium

and chloride.

Symbols are as in Fig. 1.

Seal clt trl.. 1972b). The serum calcium concentration is similar to values reported for caribou (McEwan, 1968b), sheep (Long et cd., 1965) white-tailed deer (Teeri rt u/., 1958; Seal et al., 1972b) and dairy herds (Payne ~‘1trl.. 1970) all falling into the range between 9 and 12mg/lOOml. The serum calcium concentration declines slightly in the lichen fed animals. Since the ALP activity does not show any increase, the fall in the calcium concentration is probably not caused by a calcium and/or vitamin D deficiency. Most probably the changes in calcium concentration is a reflection of the fall in the albumin concentration. Potassium. It seems to be a nutritional related effect on the serum potassium concentration of the animals in this experiment (Fig. 4d). Higher potassium levels were also found in the sera of dairy herds grazing highly fertilized pasture (Payne et ctl., 1970). For animals on both diets the elevation of serum potassium at the end of this experiment is perhaps reflecting an elevation in muscle protein catabolism. The potassium values determined in this experiment are similar to concentrations reported for caribou (M&wan. 1968b), but lower than values reported for sheep (Long et trl., 1965). Sodium tmrl chloride. The sodium level appeared to fluctuate somewhat more than the other inorganic

electrolytes studied. (From 136 to 154 m-equivll. Fig. 4e). The same fluctuation is observed in both groups. The fluctuations agree with values reported for sheep (Long rt al., 1965). Plasma sodium for caribou is reported by McEwan (1968b) to be 147 m-equiv,‘l. Chloride seems to follow changing in sodium (Fig. 4e). CONCLUSIONS

The measurements which have been performed in this study, clearly demonstrate that immediately after the start of a pure lichen feeding, the body weight, hematocrit and serum magnesium levels start to fall. Somewhat later there occurs a similar fall in the serum albumin level. These findings strongly indicate that a pure lichen diet is deficient in important nutrients. Body weight, hematocrit, albumin, and magnesium are good indicators for the nutritional state of reindeer. With this big difference in diets, however, analyses of total protein, urea, total lipids, cholesterol, and inorganic phosphorous will give information in assessing the nutritional state of the reindeer. The feeding of reindeer with RF-71 seems to counteract all signs of a deficiency state and also substantiates an increase in body weight,

Nutritional

effects on serum enzymes

193

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40, 350-356. BJAKGHO~ R. S.. JACOI~SLN E. & SKJ~.NNI~IRG S. (1976) Nutritional effect on weight gain and winter survival of reindeer calves (Rm~qifi~ turmdus rarund~s) (In press). BJARGHO~ R. S., JACOBSEN E. & SKJENNEBERC S. (1976) Effect of spring emigration on some blood constituents of reindeer (Runyjfk tmrmlu.s fccrmdus) in different physical condition. (In prep.). DOLE V. P. (1956) A relation between non esterified fatty acids in plasma and the metabolism of glucose. J. c/in. Inrvsr. 35, 150 156. DOUMAS B. T., WATWN W. A. & BIGGS H. G. (1971) Albumin standards and the measurement of serum albumin with Bromcresol green. Cliniu chim. Acts. 31, 87-96. Hove: K. & JACOBSEN E. (1975) Renal excretion of urea in reindeer. Effect of nutrition. Acta oef. scared. 16, 513-519. HOLMES J. H. G. & LAMBOURNE L. J. (1970) The relation between plasma free fatty acid concentration and the digestihlc’energy intake of cattle. Rrs. wt. Sci. 11, 27-36. HL i\\c, T. C’.. CHIN C. P.. WI:~LI K V. & RAFTI.RY A. (1961) A stable reagent for the Licbermann-Burchard reaction. .4llU/>,f. Cht~rrl. 33. 1405m 1407. JA~OHSEN E. & SKJENNEBERG S. (1972) Effects of supplemental feeding with protein and minerals to reindeer calves on a lichen diet. Report No. 5. Norwegian State Reindeer Research, Harstad. JACOBSEN E. & SKJENNERERG S. (1976) Fordsyelighet av lav og tilskuddsfbr til rein. Forskning og forsok i landbruket. 26(2), 287-305. KRONFELD D. S. (1965) Plasma non-esterified fatty acid concentration in the dairy cow: responses to nutritional and hormonal stimuli, and significance of ketosis. Vet. Rec. 77 (2), 30-35. LE~BHOLZ J. (1970) The effect of starvation and low nitrogen intakes on the concentration of free amino acids in the blood plasma and on the nitrogen metabolism in sheep. Aust. J. uqric. Rex 23, 723-734. LONG C. H., ULLREY D. E.. MILLER E. R., VINCENT B. H. & ZUTANT C. L. (1965) Sheep hematology from birth to maturity. III. Serum calcium, phosph&us, magneslum. sodium and potassium. J. Anim. Sci. 24, 145-150. Lurch J. R., PERSON S. J., CAMERON R. D. & WHITE R. G. (1973) Seasonal variations in glucose metabolism of reindeer (Runyij& tclrundus L.) estimated with (U-14C) glucose and (3-3H) glucose. Br. J. Nuts. 29, 245~259. M~E~AK E. H. (1968a) Growth and development of the barren-ground caribou. Il. Postnatal growth rates. Can. .1. Loo/. 46, 1023%1029. MC.EWAN E. H. (1968b) Hematological studies of barren ground caribou. Catl. J. Zool. 46, 1031&1036. M~EWAN E. H. & WHITEHEAD P. E. (1969) Changes in the blood constituents of reindeer and caribou occurring hith age. Ctrrr. J. Zoo/. 47. 557-562.

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Acknowledyrmenr-This The Agricultural Research

work was partly supported Council of Norway.

by

REFERENCES

W. D.. WOOD A. J. & COWAN I. Met. (1957) The effect of age and the plane of nutrition on the blood chemistry of the Columbian black-tailed deer. (0tlocoileu.s Hw~ionu,s Coltrmhianus). Carl. J. Zool. 35, 283-289. BE~KFR D. E & SMITH S. E. (1950) A chemical and morstudy of normal sheep blood. Cot&l I/ef. phological