13
J. Zool., Lond. (1995) 235, 99-111 The consequences for deer of ingesting oilseed rape (Brassica nupus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus) A. M. SIBBALD*, G. R. IASON, I. A. BRISTOW, G. C. DAVIDSON Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB9 2QJ, Scotland AND W. H. MACFARLANE SMITH Scottish Crop Research Institute, Invergowrie, Dundee OD2 5DA, Scotland (Accepted I0 December 1993) (With 4 figures in the text) In two experiments, in the growing season March-May, freshly harvested double-low oilseed rape (Brassica napus) plants were fed ad libitum to penned roe deer (Capreolus capreolus) and red deer (Cervus elaphus). In Experiment 1, oilseed rape was fed to five roe and 12 red deer as 60% of their daily dry matter (DM) intake for four weeks, after a two-week dietary change-over period. The rest of the diet comprised cattle-rearing pellets (33%) and freshly cut heather (Calluna vulgaris) plants (7%). In Experiment 2, oilseed rape was fed to seven roe and eight red deer as 100% of the diet for up to six weeks, after a two-week change-over period. In both experiments, Heinz bodies were found in roe deer blood 2-3 weeks after the start of the change-over period, with a fall in mean packed cell volume (PCV) and blood glutathione (GSH) concentration. In Experiment 2, after 5-8 days of feeding on 100% oilseed rape, four roe deer showed signs of inappetance and were changed to a diet of 80% oilseed rape. At the same time, the other three roe deer had very low PCV (< 60% of normal values) and oilseed rape feeding was discontinued for those animals. The red deer showed no symptoms of haemolytic damage nor any change in blood GSH concentration in either experiment, but showed a gradual fall in PCV in Experiment 2. No other ill-effects were observed in any of the animals. Intakes of oilseed rape (gDM/kg0'7S/day) were lower for the roe deer than the red deer in both experiments. The severity of haemolytic anaemia in the roe deer was related to the proportion of oilseed rape in the diet, rather than the amount ingested. It was concluded that the health of roe deer ingesting oilseed rape may be affected if other foods are not available. Contents Page Introduction ................................ 100 Materials and methods ............................ 100 Animals.. ................................ 101 Feeds and feeding. ............................. 101 Measurements and sampling procedures .................... 102 Chemical analyses and haematology ...................... 102 Statistical analysis ............................ 102 Results .................................. 103 Intakes of oilseed rape.. .......................... 103 SMCO and glucosinolates .......................... 104 Blood parameters. ............................. 105 Discussion. ................................. 109 References.. ................................ 111 *Correspondence to: Mrs A. M. Sibbald 99 0 1995 The Zoological Society of London

The consequences for deer of ingesting oilseed rape (Brassica napus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus)

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Page 1: The consequences for deer of ingesting oilseed rape (Brassica napus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus)

J . Zool., Lond. (1995) 235, 99-111

The consequences for deer of ingesting oilseed rape (Brassica nupus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus)

A. M. SIBBALD*, G. R. IASON, I . A. BRISTOW, G. C. DAVIDSON Macaulay Land Use Research Institute, Craigiebuckler, Aberdeen AB9 2QJ, Scotland

AND W. H. MACFARLANE SMITH Scottish Crop Research Institute, Invergowrie, Dundee OD2 5DA, Scotland

(Accepted I0 December 1993)

(With 4 figures in the text)

In two experiments, in the growing season March-May, freshly harvested double-low oilseed rape (Brassica napus) plants were fed ad libitum to penned roe deer (Capreolus capreolus) and red deer (Cervus elaphus). In Experiment 1, oilseed rape was fed to five roe and 12 red deer as 60% of their daily dry matter (DM) intake for four weeks, after a two-week dietary change-over period. The rest of the diet comprised cattle-rearing pellets (33%) and freshly cut heather (Calluna vulgaris) plants (7%). In Experiment 2, oilseed rape was fed to seven roe and eight red deer as 100% of the diet for up to six weeks, after a two-week change-over period.

In both experiments, Heinz bodies were found in roe deer blood 2-3 weeks after the start of the change-over period, with a fall in mean packed cell volume (PCV) and blood glutathione (GSH) concentration. In Experiment 2, after 5-8 days of feeding on 100% oilseed rape, four roe deer showed signs of inappetance and were changed to a diet of 80% oilseed rape. At the same time, the other three roe deer had very low PCV (< 60% of normal values) and oilseed rape feeding was discontinued for those animals. The red deer showed no symptoms of haemolytic damage nor any change in blood GSH concentration in either experiment, but showed a gradual fall in PCV in Experiment 2. No other ill-effects were observed in any of the animals.

Intakes of oilseed rape (gDM/kg0'7S/day) were lower for the roe deer than the red deer in both experiments. The severity of haemolytic anaemia in the roe deer was related to the proportion of oilseed rape in the diet, rather than the amount ingested. It was concluded that the health of roe deer ingesting oilseed rape may be affected if other foods are not available.

Contents Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Animals.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Feeds and feeding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Measurements and sampling procedures . . . . . . . . . . . . . . . . . . . . 102 Chemical analyses and haematology . . . . . . . . . . . . . . . . . . . . . . 102 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Intakes of oilseed rape.. . . . . . . . . . . . . . . . . . . . . . . . . . . 103 SMCO and glucosinolates . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Blood parameters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

*Correspondence to: Mrs A. M. Sibbald

99 0 1995 The Zoological Society of London

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100 A. M. SIBBALD ET A L .

Introduction

The past two decades have seen a large increase in oilseed rape (Brassica napus) production in Europe, with the widespread introduction of new ‘double-low’ varieties in the mid 1980s (Gazagnes, 1988). Double-low varieties were selectively bred to contain low levels of erucic acid and glucosinolates in the seeds, in order to improve the performance of animals feeding on the by- products of the oil extraction process. The large-scale introduction of these varieties in Europe coincided with a sudden increase in the reported incidence of oilseed rape poisoning amongst wild roe deer (Capreolus capreolus) (Seifert & Robbelen, 1988). During a particularly severe winter in 1986-7, large numbers of roe deer were found sick or dead in oilseed rape fields in Austria and the cause of their illness was attributed to the ingestion of oilseed rape (Onderscheka et al., 1987).

Ingestion of large quantities of oilseed rape might be expected to affect the health of wild deer, owing to the toxicity of dimethyl-disulphide (DMDS), a rumen breakdown product of S-methyl- cysteine-sulphoxide (SMCO), which has been identified as the ‘kale anaemia factor’ (Smith, 1974). The roe deer poisoned in Austria exhibited a range of symptoms including haemolytic anaemia, together with digestive disorders, disturbance to the central nervous system and haemosiderosis in the kidney, liver and spleen (Onderscheka et al., 1987). Underlying the theory of anti-herbivore plant chemistry (Rhoades, 1979) is the assumption that the effect of plant secondary metabolites in plant herbivore interactions are the result of co-evolutionary processes. It might therefore be expected that the sudden introduction of new genotypes of plants, as in the case of oilseed rape, may have severe effects on herbivores which have neither learnt to avoid, nor had time to adapt to, the ingestion of these compounds. It was suggested that the sudden increase in the incidence of oilseed rape poisoning might have been due to the greater palatability of low-glucosinolate varieties (Tapper, 1989), although such cultivars do not necessarily have low-glucosinolate concentrations in the foliage (Askew, 1990).

Reports of oilseed rape poisoning in wild deer have so far been confined to roe deer, which may be due to the relative infrequency with which other deer species encounter oilseed rape as a food source. However, there may be species differences in metabolism or susceptibility to the toxic compounds. Evidence from sheep, cattle and goats has demonstrated species differences in susceptibility to brassica poisoning (Greenhalgh, Sharman & Aitken, 1969). Preliminary findings with penned red deer (Cervus elaphus) have suggested that they may be able to tolerate oilseed rape in their diet for a number of weeks with no ill effects (Sibbald & Macfarlane Smith, unpubl. data). However, in an experiment with penned roe deer (Fehlberg et al., 1989), oilseed rape ingestion produced symptoms similar to those seen in the wild deer in Austria, leading to death in some animals. In that study, the reactions of individual animals to oilseed rape ingestion were very varied and, although the inclusion of other foods in the diet appeared to reduce the severity of the symptoms, the composition of the diet was not measured.

The aim of the experiments reported here was to determine the susceptibilities of roe and red deer to oilseed rape poisoning. Double-low oilseed rape was fed to captive roe and red deer in order to determine the amount and dietary proportion of oilseed rape likely to have ill-effects in these species, and to allow an assessment of the risk to wild deer in Britain from the widespread cultivation of oilseed rape.

Materials and methods

In Experiment 1, which was carried out between March and May 1991, changes in various blood parameters of roe and red deer were measured when fresh oilseed rape was fed as 60% of the dietary dry

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DEER A N D OILSEED RAPE 101

matter (DM), following a period of feeding on cattle-rearing pellets and fresh heather. Experiment 2 was carried out exactly 1 year later and measured changes associated with feeding oilseed rape as 100% of the diet. Owing to the potential toxicity of oilseed rape, precautions were taken to minimize any damage caused to the animals' health. These precautions included frequent monitoring of blood parameters and general observations of alertness, appetite, etc. Feeding of oilseed rape was stopped for any animal approaching the degree of anaemia reported previously to be life-threatening to roe deer (Fehlberg et al., 1989).

Animals

The number, sex, age and mean live weights of the animals used in the experiments are shown in Table I. In order to acquire animals that would settle in captivity, it was necessary to hand-rear wild-caught roe deer kids. The roe deer kids were removed from their mothers within 2-3 days of birth and artificially reared indoors using a proprietory ewe milk substitute diluted so that the dry matter content was 60% of that recommended for lambs. They were offered fresh leafy browse (mainly birch, Betula sp., beech, Fugus sp. and rowan, Sorbus sp.) from 10 days of age and cattle-rearing pellets (BOCM Silcock Ltd; crude protein 176 g/kgDM) from 8 weeks, and were maintained on a diet of cattle-rearing pellets and freshly cut heather (Calluna vulgaris) plants after weaning. All male roe deer kids were castrated between 2 and 3 months of age. The red deer used in both experiments were obtained from farmed stock and reared naturally at pasture. After weaning they were housed and maintained on a diet of dried grass pellets and hay. Two female and 1 male roe deer and 4 female red deer from Experiment 1 were re-used in Experiment 2.

During both experiments the animals were individually accommodated in adjacent pens (each 1.5 x 2 m), bedded on sawdust and provided with natural lighting. Between experiments the roe deer were group- housed. The red deer were grazed at pasture between May and November.

Feeds and feeding

For the first 6 weeks of each experiment, the roe and red deer were fed a diet of cattle-rearing pellets (75 gDM/kgW0'75/d) and fresh heather (20 gDM/kgW@75/d). In Experiment 1, oilseed rape was then offered ad libitum, while the pellet and heather components of the diet were gradually reduced over a 14-day change- over period until oilseed rape constituted approximately 60% of the total DM intake, with pellets approximately 33% and heather 7%. These proportions were then maintained for each animal by occasional adjustments to the daily pellet ration. In Experiment 2, a similar procedure took place over a 12-day change-over period, until oilseed rape constituted 100% of the diet.

TABLE I Number, sex, age and mean starting live weight ( W ) of roe and red deer used in experiments

Experiment 2 (Age) Experiment 1 (Age)

9 months 21 months 9 months

Sex No. w (kg) No. w (kg) No. w 0%)

2 2

1 2

2 2 76.8

0 4

0 3 0 3

18.5 24.3 20.0 Male 2 Female 3

Male 6 Female 6

Male 3 3

Roe deer

55.0 51.8 Red deer

Control red deer { Female 46.3 48.5

Page 4: The consequences for deer of ingesting oilseed rape (Brassica napus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus)

102 A. M. SIBBALD ET A L .

In both experiments, an autumn-sown double-low variety of oilseed rape (Samourai) was used. Feeding was started at an early stage of plant growth in late March, when the crop was 12 to 15 cm in height, and continued for a total of 42 days in Experiment 1 and 52 days in Experiment 2, by which time the plants were in flower and approximately 70 cm in height. The plants were harvested each morning from 2 adjacent fields (one in each experiment). Whole plants were cut at ground level with a brush-cutter and offered fresh to the animals soon after harvesting. At all times, oilseed rape was fed ad libitum, with a refusal margin of approximately 20%, to ensure that each animal was offered sufficient whole plants to allow for selection of the different parts.

A control group of red deer were fed a diet of dried grass pellets (850 gDM/h/d) and hay (ad libitum) throughout each experiment.

Measurements and sampling procedures

Weights of fresh oilseed rape, pellets and heather offered to and refused by individual animals were measured daily. The DM contents of samples of material offered and refused were determined daily for oilseed rape, and weekly for pellets and heather, by oven-drying for 24 h at 85 "C.

Twice weekly, duplicate 500 g samples of whole oilseed rape plants were taken at random from the day's harvest. These samples were separated into leaf, petiole, stem and flower bud (or flower if present), weighed and stored at -20 "C. In Experiment 1 only, weekly samples of oilseed rape plants refused by each animal were also separated and weighed, in order to estimate individual intakes of the different plant parts.

At weekly intervals throughout both experiments, lOml blood samples were taken by jugular venepunc- ture into evacuated heparinized tubes. During key periods of oilseed rape feeding (weeks 4 and 5 of Experiment 1 and weeks 1-4 of Experiment 2), blood samples were taken twice weekly. The control red deer were blood sampled every fortnight in Experiment 1, and every week in Experiment 2.

Chemical analyses and haematology

The SMCO content in samples of plant material was determined by the method of Griffiths & Macfarlane Smith (1989) and glucosinolates by the method of Macfarlane Smith & Griffiths (1988).

Heinz body count (HBC) was determined on whole blood after staining with Brilliant Cresyl Blue, using the method described by Duncan & Milne (1993). Packed cell volume (PCV) was determined on whole blood, using a Hawksley micro-haematocrit system. A 2 ml sub-sample of whole blood was retained for glutathione (GSH) analysis, which was carried out after storage overnight at 4"C, using the method of Beutler, Duran & Kelly (1963). The rest of the blood was centrifuged at 2500rpm for 10min and the supernatant plasma stored at -20 "C. Plasma concentrations of urea, creatinine and gamma glutamyl transpepsidase (GGTP) were subsequently determined by continuous flow analysis. Urea was measured using the Berthelot reaction (Wilcox et al., 1966), creatinine using the Jaffe reaction (Butler, 1975), and GGTP using the method of Fuke et al. (1976). Plasma alkaline phosphatase activity was measured using a commercial diagnostic kit (Sigma procedure no. DG 1245, Sigma, Poole, Dorset).

Statistical analysis

The effects of oilseed rape feeding, deer species and time were examined by analysis of variance using the GENSTAT 5 package (Lawes Agricultural Trust, 1987). Intakes of oilseed rape and the estimated intakes of SMCO or glucosinolates by the 2 deer species were compared within specific time periods, using the mean daily intake for each animal. Intakes of oilseed rape in the 2 different experiments were similarly compared within deer species. The effects of time on SMCO and glucosinolate concentrations in the plants were analysed using variation within weeks as the error term. Effects of oilseed rape feeding and time on PCVs in the red deer and the controls were compared using the variation between animals as the error term. The effects of oilseed rape feeding on other blood parameters were analysed within species of deer, using variation between individual animals and between sampling dates to provide the error term.

Page 5: The consequences for deer of ingesting oilseed rape (Brassica napus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus)

DEER AND OILSEED RAPE

Results

(a) 75-

60 - h

2 n m.

0 45- ?

0) Y

UJ

a, Y

. v

30- .- a a m Iz:

15-

0

103

..: : .. , .'.. '. . . . . . . . . . . . . . .... ... . . . . . . . . . . . . . . . . . . _ . . . . . .... _. _.

I I I I I I 0 10 20 30 40 50 60

Intakes of oilseed rape

In Experiment 1, after the dietary proportion of oilseed rape was established at the end of week 2, daily intakes of oilseed rape remained fairly constant (Fig. la). The mean intakes of oilseed

Days from start of rape feeding

. . I . . . . . (b) 75- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

0 I I 1 I I 1 0 10 20 30 40 50 60

Days from start of rape feeding FIG. 1. Mean intakes of rape dry matter by (a) roe deer (-) and red deer (. .... .) in Experiment 1 and (b) Group A

roe deer (-) and red deer (. . . . . .) in Experiment 2.

Page 6: The consequences for deer of ingesting oilseed rape (Brassica napus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus)

104 A. M. SIBBALD ET A L .

rape by the two species during the two-week change-over period were not different when expressed on a metabolic live weight basis, but during weeks 3-6 the mean intake was lower for the roe than the red deer (45.3 vs. 54.5 gDM/kgW@75/day, P < 0.05).

In Experiment 2, after 5-8 days of ingesting 100% oilseed rape, pellets and heather were re-introduced to the diet on an individual basis for four of the roe deer (thereafter referred to as Group A), since their intakes were declining rapidly. Individual animals had shown a drop in oilseed rape intake of between 50% and 75% over two days. Oilseed rape intakes by these animals then recovered (Fig. lb) and the dietary proportion was held at approximately 80% thereafter, with pellets adjusted to contribute approximately 13% and heather 7% of the total DM intake. At the same time, oilseed rape feeding was stopped completely for the other three roe deer (thereafter referred to as Group B), since their packed cell volumes (PCV) fell below 60% of normal levels (see Materials and methods). There was no difference in the mean oilseed rape intakes of Groups A and B during the change-over period. The red deer remained on the diet of 100% oilseed rape throughout Experiment 2, with intakes consistently higher than those of the roe deer in Group A (67.2 vs. 48.5 gDM/kgW0'75/day, P < 0.01 for weeks 4-7).

SMCO and glucosinolates

Concentrations of SMCO and total glucosinolates measured in the leaves and stem, and

TABLE I1 Weekly mean concentrations of SMCO and total glucosinolates, measured in leaf and stem and calculated for the whole

oilseed rape plant

Experiment 1

SMCO (g/kgDM)

Week

1 2 3 4 5 6

S.E.

Leaf

3.40 2.65 2.10 1.45 1.70 2.65

0.479

Stem

5.90 4.90 3.25 2.15 1.95 2.50

0.730

Whole plant 4.34 3.47 2.62 1.88 1.96 2.51

Glucosinolates (mmol/kgDM)

Leaf Stem Whole plant

3.15 3.98 3.48 3.05 4.02 3.50 1.01 1.72 1.42 0.19 0.78 0.59 0.08 0.43 0.40 0.14 0.23 0.35

0.643 0.781

Experiment 2

Week

1 2 3 4 5 6 /

S.E.

Leaf

1.85 1.98 1.55 1.20 1.50 0.90 1.23

0.556

SMCO (g/kgDM)

Stem

2.82 2.58 1.90 1.40 2.33 1.62 2.55

0.461

Whole plant 1.95 2.32 1.61 1.35 1.87 1.43 2.08

Glucosinolates (mmol/kgDM)

Leaf Stem Whole plant

6.49 12.94 8.17 4.14 13.68 6.95 3.93 11.73 7.18 2.39 8.43 5.13 3.28 6.22 4.78 1.67 4.65 3.51 1.03 2.72 2.08

0.868 1.414

Page 7: The consequences for deer of ingesting oilseed rape (Brassica napus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus)

DEER A N D OILSEED RAPE 105

TABLE 111 Mean intakes of SMCO and total glucosinolates in Experiment I , calculated from estimated intakes of the different plant

parts

Intake of SMCO (g/kgW) Intake of total glucosinolate (mmol/kgW)

Week Roe deer Red deer S.E. Roe deer Red deer S.E.

1 2 3 4 5 6

32.4 13.3 4.53 55.1 52.2 5.90 52.8 49.8 4.18 36.3 34.4 2.70 45.4 39.2 2.73 53.1 51.0 2.07

25.9 10.3 3.53 55.6 51.1 5.76 28.3 26.6 2.20 11.9 11.3 0.85 9.1 7.9 0.54 7.3 9.3 0.30

calculated for the whole oilseed rape plant, are shown in Table 11. There was a significant decrease over time in the total glucosinolate concentration of leaf and stem in both experiments (P < 0.01). Although there was no consistent trend over time in SMCO concentrations, there were significant variations from week to week in the leaf (P < 0.05) and stem (P < 0.01) in Experiment 1.

Estimated mean intakes of SMCO and total glucosinolates by the two deer species in Experiment 1 were different in the first week of oilseed rape feeding (P < 0.05; Table 111) but not thereafter. Intakes of total glucosinolates decreased significantly between weeks 2 and 6.

Blood parameters

Heinz bodies were not seen before oilseed rape feeding, but appeared in all the roe deer 2-3 weeks after the start of the change-over period (Fig. 2a and b). In both experiments, HBCs peaked within a week of reaching the target proportion of oilseed rape in the diet. The mean peak values (f S.E.) were 36.3% (33.6) in Experiment 1,45.5% (46.3) for Group A and 50.2% (f6.0) for Group B in Experiment 2. Heinz bodies were not seen in significant numbers in the red deer or the controls at any time.

Roe deer PCVs fell from a mean of 52.8% (fl.36), measured over the six weeks before oilseed rape feeding in Experiment 1, to 429% (fl.28) after 15 days with oilseed rape as 60% of the diet (Fig. 3a). In Experiment 2, roe deer PCVs fell from a weekly mean value of 549% (fl.02) to 40.3% (fl-33) in Group A, and to 284% (f2.60) in Group B, seven days after the change to 100% oilseed rape in the diet (Fig. 3b). Red deer PCVs did not fall relative to those of the control group when oilseed rape was 60% of the diet. However, in Experiment 2 there was a significant decline in PCVs in the red deer ingesting oilseed rape, from a mean of 51.8% (fl.15) before oilseed rape feeding to 40.1% (fl.71) after 30 days on 100% oilseed rape (P < 0.001).

GSH concentrations in the roe deer fell from a mean value of 73.4 (f5.59)mg/lOOml erythrocytes, measured over the six weeks before oilseed rape feeding in Experiment 1, to 55.8 (f4.67) mg/100 ml erythrocytes after three weeks with oilseed rape as 60% of the diet (Fig. 4a). In Experiment 2 (Fig. 4b), GSH concentrations for Group A roe deer fell from a six-week mean value of 76.9 (414.26) to 35.9 (f7.78)mg/lOOml erythrocytes after three weeks with oilseed rape as 80% of the diet. In Group B roe deer, GSH concentrations fell to a minimum value of 16.6 (f0.87) mg/100ml erythrocytes two weeks after the change to 100% oilseed rape, although these

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106

(b’ 50-

40 -

h

8 E 30- v

3 0 0 x -0 x 20- N c Q) I .-

10 -

A. M. SIBBALD ET A L .

\

L - - - - - - - \

Days from start of rape feeding

I

I I I 20 40 60

1 80

Days from start of rape feeding

FIG. 2. Heinz body counts for (a) roe deer in Experiment 1 (-) and (b) Group A roe deer (-) and Group B roe deer (- -) in Experiment 2.

animals had remained on 100% oilseed rape for no more than eight days after the change-over. In all cases, GSH concentrations in the roe deer did not begin to rise again until oilseed rape feeding was stopped. Neither the red deer ingesting oilseed rape nor the red deer controls showed any significant changes in GSH concentrations in either experiment.

Page 9: The consequences for deer of ingesting oilseed rape (Brassica napus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus)

DEER AND OILSEED RAPE

(a) 60-

50 - h

v 8 Q,

- k 9 40- - - a, 0 U Q, Y 0

2 30-

20 4 0

107

- 277 ...... ... .... - .........: - - - . . . . .

I 1 I I I 1 -20 0 20 40 60 80

I I I I I 1 4 0 -20 0 20 40 60 80

Days from start of rape feeding

FIG. 3. Packed cell volumes (PCV) of (a) roe deer (-), red deer (- - - - -) and control deer (- -) in Experiment 1 and (b) Group A roe deer (-), Group B roe deer (- -), red deer (- - - - -) and control deer (- -) in Experiment 2.

In both experiments, plasma urea concentrations were higher during oilseed rape feeding than during the six weeks before, in roe deer (12-7 vs. 9.8 and 11-9 vs. 7-0mM/1 in Expts 1 and 2; P < 0.001) and red deer (9.4 vs. 7-6 and 12-6 vs. 8-4 in Expts 1 and 2; P < 0.001), but there were no changes in the control animals. There were no increases in mean plasma

Page 10: The consequences for deer of ingesting oilseed rape (Brassica napus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus)

108

80- a,

0

c

2 $ 60-

E

7 40-

I cn

- 0 0

v E"

C!.l 20-

0

,.v /'f--7 - -

-..* , _: -. .-. . -, . .. .. -._- . .i ........ .... _ - ...... .- . - .- 0 :'

I I I I I 1

(b) 100

c ...... /

/' . . x

0

/ \ .. 2 ....

s ...... .......

1 /-- :j??:\ _ . . - .; ' \'. \-w ;.. .../. < ....

\ ', /

" I I I I I I I -40 -20 0 20 40 60 80

Days from start of rape feeding FIG. 4. Plasma glutathione (GSH) concentrations of (a) roe deer (-), red deer (- - - - -) and control deer (- -) in

Experiment 1 and (b) Group A roe deer (-), Group B roe deer (- -), red deer (- - - - -) and control deer (- -) in Experiment 2.

creatinine or GGTP concentrations in either experiment. Plasma alkaline phosphatase was higher during oilseed rape feeding than during the six weeks before, in red deer ingesting oilseed rape (93.1 vs. 67.1 iu/l, P < 0.001) and control red deer (76.8 vs. 42.3 iu/l, P < 0.05) in Experiment 2.

Page 11: The consequences for deer of ingesting oilseed rape (Brassica napus): feeding experiments with roe deer (Capreolus capreolus) and red deer (Cervus elaphus)

DEER A N D OILSEED RAPE

Discussion

109

Total intakes of DM by both deer species declined as the proportion of oilseed rape in the diet increased. This is consistent with the observation that intakes of brassicas by ruminants are generally lower than those of other herbages of similar digestibility (Burnett, 1988). It might have been expected that intakes of total DM by the different deer species, when offered the same proportion of oilseed rape in the diet, would have been similar per kg W0'75 (Kleiber, 1961), but intakes by the roe deer were lower. Estimated intakes of SMCO and glucosinolates are expressed per kg W, since the scaling factor of 0.75 is not necessarily appropriate to the action of toxic compounds within the body. On this basis, intakes of SMCO and glucosinolates were similar for both species in Experiment 1 .

In every case, ingestion of oilseed rape by the roe deer led to classic symptoms of haemolytic anaemia within two weeks. Heinz bodies are stainable granules of denatured haemoglobin found on the outer walls of damaged erythrocytes before they are lost from the blood, so that the percentage of cells affected (HBC) will be related to the subsequent fall in PCV and give an indication of the extent of the anaemia. The difference in severity of the anaemia between the two experiments was associated with differences in the proportion of oilseed rape in the diet, rather than the amount ingested, although in each experiment the response varied between individual animals. There were no signs of anaemia in the red deer with 60% oilseed rape, while ingestion of 100% oilseed rape caused only a gradual fall in PCV, with no sign of the Heinz body formation that is characteristic of haemolytic damage, nor any signs of impairment of the erythrocyte anti- oxidant system. At present, we have no explanation for the slow fall in PCVs seen in the red deer, since haematological screening showed no other differences between the animals ingesting oilseed rape and the controls.

The difference in response to oilseed rape feeding between the two species of deer represents a real difference in susceptibility, since intakes of the plant toxins by the two species in these experiments were similar. Although individual selection of various plant parts was not measured in Experiment 2, there were no differences in Experiment 1 between the calculated mean intakes of SMCO resulting from differences in selectivity by the roe and red deer. It is possible that the roe and red deer metabolize SMCO differently and that circulating levels of DMDS were higher in the roe deer, although these were not measured. Alternatively, it is possible that the erythrocytes themselves are more susceptible to oxidative damage by DMDS and a detailed study of the erythrocyte anti-oxidant systems of the two deer species may help to explain the difference in susceptibility to oilseed rape poisoning.

None of the other symptoms previously associated with oilseed rape poisoning (Onderscheka et al., 1987; Fehlberg et al., 1989) was observed in any of the animals. However, some of the symptoms of severe oilseed rape poisoning could be the result of a more prolonged anaemia, which was not allowed to occur in these experiments. The time course of the recovery of normal PCV in the roe deer in these experiments was independent of the size of the reduction in PCV, and occurred irrespective of whether the animals continued to ingest oilseed rape. This is because the natural replacement of damaged red blood cells and greater resistance of the younger cells to oxidative damage (Penny, David & Wright, 1964) will temporarily reverse changes in HBC and PCV and can give rise to a cyclical pattern of HBC and PCV if brassica diets are fed for a considerably longer period (Greenhalgh, Sharman & Aitken, 1970). Continued feeding of oilseed rape beyond the six or seven weeks would almost certainly have produced a recurrence of the

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110 A. M . SIBBALD ET A L

symptoms. The lower GSH concentrations in the blood of the roe deer produced by oilseed rape feeding in these experiments, probably due to the action of glucosinolate breakdown products (Duncan & Milne, 1993), might then have contributed to the development of progressively severe anaemia. Low red cell GSH concentrations have been implicated in susceptibility to anaemia (Tucker & Kilgour, 1973), although there is evidence of compensatory adaptations in other parts of the blood antioxidant defence mechanisms of sheep with a genetic GSH deficiency (McPhail, Morrice & Duthie, 1993).

The increase in plasma urea concentrations seen in all the animals ingesting oilseed rape was consistent with metabolic changes found during oilseed rape feeding (Fehlberg et al., 1989) rather than evidence of disturbed kidney function. Although elevated alkaline phosphatase concentra- tions may be associated with liver damage, plasma levels in the red deer stayed within the normal range for most species (Kerr, 1989) and a similar elevation was observed in the control deer which had not ingested oilseed rape.

Another possible reason for the absence of other symptoms of oilseed rape poisoning in the roe deer in these experiments is the fact that they were well nourished and kept indoors. It is likely that an anaemic response, which appeared to have little effect on these animals, might have serious consequences for wild deer under more adverse conditions. The outbreak of rape poisoning in Austria was associated with particularly harsh weather, which would have both reduced the availability of other food sources and increased the energy costs of foraging. The roe deer which succumbed to poisoning were reported to be in poor body condition, with little or no fat reserves (Onderscheka et al., 1987), which could have reduced their tolerance to the toxicity of the plants (Bidlack, Brown & Mohan, 1986) as well as to the effects of the resulting anaemia.

Since the severity of the anaemic response in the roe deer appeared to be related to the proportion of rape in the diet, rather than the quantity of plant toxins ingested, it is possible that the presence of other food material in some way altered the metabolism of these toxins, thus alleviating their effects. This apparently protective effect may be specific to certain food types. The risk to roe deer in Britain from ingesting oilseed rape can only be assessed from the results of this particular study in conjunction with a knowledge of the likely inclusion of oilseed rape in the diet of wild deer. Approximately 380,000 hectares of land in Britain are currently used for oilseed rape production. In a recent field study, it was shown that, in an area with a relatively large proportion of cultivated land planted with oilseed rape, less than 20% of radio locations of tagged roe deer were in oilseed rape crops (Reynolds & Tapper, 1990). The proportion of time spent feeding on rape will undoubtedly vary with the availability of other food sources and the proximity of oilseed rape crops to sheltered woodland, but it seems unlikely that roe deer in Britain will ingest oilseed rape as more than 60% of their diet under normal circumstances. The results of these feeding experiments suggest that the risk of poisoning with 60% rape in the diet is small, at least when the rest of the diet is nutritionally adequate. However, the rapid fall in PCVs seen in some of the roe deer when only rape was fed, together with the developing inappetance shown by the others, suggests that some roe deer may be at risk if other foods are in short supply.

This research was undertaken with funding under the MAFF Open Contract Scheme which is gratefully acknowledged. We wish to thank the Forestry Commission Wildlife and Conservation Research Branch and their District Managers in Scotland for advice and help in the provision of suitable roe deer kids, and R. W. Mayes and J. A. Milne for helpful comments on the manuscript.

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DEER AND OILSEED RAPE 111

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