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Remote controlled sampling of cattle and buffalo blood
Camp. Biochem. Physiol. Vol. 89A, No. 2, pp. 231-235, 1988 Printed in Great Britain
0300-9629/88 $3.00 + 0.00 0 1988 Pergamon Journals Ltd
REMOTE CONTROLLED SAMPLING OF CATTLE AND BUFFALO BLOOD J. HATTINGH,* M. F. GANHAO,* F. J. N. KRUGER,* V. DE Vest and G. W. KAY: *Department of General Physiology, School of Dentistry, University of the Witwatersrand, Johannesburg, 2001; TSkukuza, Kruger National Park, 1350 and $Animal and Dairy Science Research Institute, Irene, 1675, South Africa. Telephone: (011) 339-4366 (Received 6 May 1987) Abstract-i. Blood samples were obtained from cattle and buffaloes over extended periods of time by remote controlled methods at 4-hourly intervals. 2. Initial handling of the animals and deliberate exposure to stress at the end of the experimental times resulted in increased plasma concentrations of a number of variables. 3. Between initial handling and applied stress, the concentration of all variables investigated showed little change and no evidence of rhythms. 4. The results obtained reflect true plasma composition of free ranging cattle and buffaloes at rest.
INTRODUCTION
In a recent publication,
the results of a study of plasma constituents at 90-min intervals over a 4%hr period from four unrestrained Nguni cows were reported (Ganhao et al., 1985). The animals were kept in small cubicles in which they could stand or lie down but could not turn. Indwelling jugular catheters were used to sample blood. No circadian, ultradian or diurnal rhythm was found for any of the parameters investigated and basal values over a 48-hr period were established. However, the concentration of certain variables which are usually used as a measure of stress such as ACTH, cortisol, corticosterone and total catecholamines gradually increased after the first 12 hr. This was attributed to the effects of confinement to a small area and the associated limited physical movement. It was suggested that the stress involved could be eliminated by remote controlled blood sampling on free-ranging animals. Remote controlled blood sampling was used in the present study to obtain samples from cattle and wild buffaloes. No circadian or other rhythm was observed in the concentration of the variables investigated in the samples taken at 4-hourly intervals. Applied stress resulted in concentration increases of a number of variables and the results are in agreement with those previously found (Ganhao et d., 1985). No indication was present that the animals experienced discomfort during the experiments and the concentration of the variables examined was a true reflection of the plasma composition at rest. MATERIALS
AND METHODS
Animals and blood sampling
Nguni cows were used at the Animal and Dairy Science Research Institute, Irene and wild buffalo bulls (Syncems cuffer) in the Kruger National Park. In order to place the blood sampling apparatus on the animals, cattle were put into a crush and buffaloes anaesthetized by darting (8-10 mg M99 and 3&50mg Rompun). Blood samples were taken c
aP
89’2A--1
with a syringe immediately before the animals were released (cattle) or the antidote given (buffaloes). The animals were kept in large enclosures in the veld with food and water ad libitum. Remote controlled blood sampling commenced after 2-6 hr. This was repeated every 4 hr thereafter for a variable period of time (hence variable N numbers in Fig. 1). At the end of the experiment a pre-stress sample was taken remotely and the animals were stressed by chasing, noise, the presence of a vehicle, etc., for a period of about 40 min and a final stress sample taken remotely. Cattle were then returned to the crush and buffaloes anaesthetized by remote control and the apparatus removed. The equipment used for blood sampling was attached to a collar, which was fitted around the animal’s neck. The apparatus was connected to the jugular vein with an indwelling catheter (Hattingh, 1985). This was flushed automatically every 15 min with heparinized saline. Undiluted blood samples were taken into a heparinized container as required, the response time of the equipment being about 30 set; animals were virtually unaware that this was being done (except for the sounds of pumps, no other noise is heard) and in many instances remained lying down if they were in that position when sampling commenced. The sample was ejected from the equipment and processed immediately. In the case of buffaloes, the equipment was also used to anaesthetize the animals at the end of the experiment. This was done by priming the machine with the anaesthetic solution before it was placed on the animal and then infusing this when required. Analytical techniques and statistical analyses
Blood samples were immediately centrifuged and the plasma deep frozen. Samples were analysed as soon as possible for a number of variables using standard techniques as described before (Ganhao et al., 1985). Not all analyses were performed for all samples. Profiles on each animal were plotted for each variable with concentration against time. The mean and SD were calculated for cattle and buffaloes for each variable every 4 hr using times as close as possible to the actual sampling times. These results were plotted against time and are reported graphically for those variables which changed in the period after the sampling apparatus was put on and before the applied stress. In addition, overall means and SDS over the last 24 hr of the experiment (excluding samples
231
232
J. HAITINGHet al.
obtained during applied stress) were calculated for all variables. Significance of the differences between means was tested using a Student’s t-test.
the buffalo and cattle groups it was clear that no rhythms were present. The overall means and SDS for the last 24 hr of the experiment for the variables investigated are thus reported in Table I, where the N values indicate the number of samples over this period. Figure I shows the profiles for variables which changed with applied stress at the end of the
RESULTS
From the graphs relating to the con~ntration variables
against
time for individual
animals
of
and for
experiments
and/or
which
showed
a change
BUFMKXS l
3321
11
11
1
t
i
1
after
Remote controlled blood sampling
233
t
te----*-
l -
1
i
mmathellnd 4
0
12
t
4:ZHIB
l
I
-
-
-
5
-
=
,
76866533333
Fig. 1. Profiles of the concentration of plasma constituents against time (mean f SD). The numbers below each point are the individual N-values. Mean values which differed significantly from the overall means (P -C0.05) are indicated with an asterisk.
J. HATTINGH el al.
234
Table I. Overall mean values and SDS for the variables investigated for the final 24 hr of the experiment. N values indicate the number of samples over this period. The stress results are the mean values and standard deviations obtained after applied stress Buffaloes _____
Cattle
Control .__._~__~_
.-.
F
SD
N
24 21 26 25 23 24 25
33. I 3.2
I I 7
1.0 17.8 7.1 50
5.1 0.9 2.2 0.5 17.2 I.1 I .4
25 25 25 25 22 21 22 I6
3.5 0.6
N
Haematocrit (%) Glucose (mmol/l) Total lipids (g/l) Ltactate (mmol/l) Cortisol (nmol/l) Free T, (pmol/l) K’ (mmol/l) Total Catecholamines (ng/ml) Norepinephrine (ng/ml) Epinephrine (ng/ml) Dopamine (ngjml) Leu-enkephaline (pg/ml) Met-enkephaline (pg/ml) fi-Endorphine (pg/ml) ol-Endorphine (pg/ml)
~_
Stress
4.1
-
Control
stress
R
SD
N
R
43
2.5
7
31.4
I.1
2
36.5
3.5
5.2
0.6
7
3.6
0.6
2
5.4
0.3
SD
N
x
SD
6.0
2.5
1
2.4
0.3
2
2.8
I.2
1
9.9
3.5
7
1.9
1.2
2
12.1
5.2
I
128.6
44.3
7
38.9
18.1
2
137.5
24.7
2.1 0.6
7
2.1
I.6
0.8 0 I3 0.14 2.51 0.34
7 7
10.6 2.4 7.7 0.5
I I I
1.0 0.17 0.24 4.35 0.59
14.3 2.3 12.1 0.7
2.4 I .o I.1 0.3
1
the equipment was put on. It is clear that, in general, the concentrations of plasma lactate, cortisol, catecholamines, haematocrit, glucose and total lipids all changed during the initial and final periods of the experiments but that they showed little change between. DISCUSSION
The results reported here show that initial handling of the animals when the collar was put on resulted in (significantly) higher mean values for certain variables than what was obtained in the period thereafter. The variables involved are those which are regarded as being indicative of stress, i.e. increases are usually associated with some form of discomfort. Similar increases in the same variables were observed after the animals were deliberately exposed to stress. Between these two procedures the values showed little change and were low. Furthermore, no gradual increase in the values of ACTH, cortisol and catecholamine concentrations was evident as was found previously in cattle confined to cubicles (Ganhao et al., 1985). This also implies that the animals were at rest during the sampling period. It may thus be concluded that representative baseline values for free ranging cattle and buffaloes over a time period were obtained in this study. In addition, it is also clear from Fig. 1. that the concentration of certain variables, e.g. cortisol, haematocrit and glucose may take up to 10 hr to reach baseline values once elevated due to stress. This corresponds to the half-life of certain hormones in blood (Ganong, 1983) and also, in the case of buffaloes, to the known hyperglycaemic and sympatholytic effects of Rompun (Meyer-Jones et ul., 1977). For this reason the overall means reported in Table 1 are only for the last 24 hr of the experiment. Although the N numbers were small in certain cases, especially for buffaloes over a prolonged period of time, the results do permit the conclusion that no rhythms were present in the concentration of the variables investigated when sampling every 4 hr. This agrees with previous observations (Ganhao et al., 1985). However, more frequent sampling, at halfhourly or hourly intervals may well show the exis-
1
tence of rhythms in these animals which do not sleep for long periods of time (Ruckebusch, 1972). The overall mean values reported here for the variables investigated over 24 hr compare well with those found earlier in cattle confined to cublicles (Ganhao et al., 1985) and to those obtained from buffaloes shot in the brain (Hattingh et al., 1984a). Certain differences are however evident, e.g. catecholamine and cortisol concentrations in buffaloes and lactate and cortisol concentrations in cattle. The present results differ significantly from those obtained from anaesthetized animals in the case of certain variables (Hattingh et al., 1984b). It is suggested that the results obtained by remote controlled blood sampling are a truer reflection of the plasma composition of the animals studied because confinement to cubicles with the consequent limited physical movement is unnatural and a shot in the brain may for instance lead to mass sympathetic discharge and other effects (Hattingh et al., 1984a). Anaesthetization itself also causes stress. The present results thus reflect the plasma composition of free-ranging animals and may be used in an initial approach to quantify the stress response (Hattingh, 1986). Remote controlled blood sampling has been used by other workers. Bubenik and Bubenik (1979) reported on a remote controlled self contained blood sampler which could be used in animals such as horses, cows, white-tailed deer and red deer. Falke et al. (1985) used a pressure controlled remote sampling device in seals, and Birke (1986) mentioned that the technique is used to study the way in which horses cope with strenuous exercise. The device used by Farrel er al. (1970) was pre-programmed and could not be controlled remotely. The results of the present study, the first quantitative baseline observations on free ranging animals taken over a period of time, indicate the value of this technique. It is now possible to study the effects of different stressors on wild animals without the interference of handling, anaesthetization and other influences.
Acknowledgements-The financial support of the FRD, National Parks Board and University of the Witwatersrand
Remote controlled blood sampling is gratefully acknowledged. Technical help was rendered by Mr J. A. N. Grobbelaar and others. REFERENCES
Birke L. (1986) Equine athletes: blood, sweat and biochemistry. New Scientist 110, 48-52. Bubenik G. A. and Bubenik A. B. (1979) Remote-controlled self-contained blood sampler. C.A.L.A.S./A.C.T.A.L. Proc. 5 l-53.
Falke J. K., Hill R. D., Quist J., Schneider R. C., Guppy M., Liggins G. C., Hachachka P. W., Elliot R. E. and Zap01 W. M. (1985) Seal lungs collapse during free diving: evidence from arterial nitrogen tensions. Science 229, 556558. Farrel D. J., Corbett J. L. and Long R. A. (1970) Automatic sampling of blood and ruminal fluid of grazing sheep. Res. Vet. Sci. 11, 217-220. Ganhao M. F., Hattingh J., Kay G. W., Cornelius S. T. and Grobbelaar J. A. N. (1985) Plasma constituents of Nguni cows over forty-eight hours. J.S.A. Vet. Ass. 56, 177-180.
235
Ganong W. F. (1983) Review of Medical Physiology, 12th edn. Lange, Los Altos, California. Hattingh J., Wright P. G., de Vos V., McNairn I. S., Ganhao M. F., Silove M., Wolverson G. and Cornelius S. T. (1984a) Blood composition in culled elephants and buffaloes. J.S.A. Vet. Ass. 55, 157-164. Hattingh J., Wright P. G., de Vos V., Levine L., Ganhao M. F., McNairn 1. S., Russel A., Knox C., Cornelius S. T. and Bar-Noy J. (1984b) Effect of etorphine and succinyldichohne on blood composition in elephant and buffalo. S.A.J. Zool. 19, 286290. Hattingh J. (1985) Remote controlled blood sampling. S.A.J.
Sci. 81, 644.
Hattingh J. (1986) Physiological measurement S.A.J.
of stress.
Sci. 82, 612-614.
Meyer-Jones L., Bosch W. H. and McDonald L. E. (1977) Veterinary Pharmacology and Therapeutics, 4th edn, pp. 363-367. Iowa State University Press, Iowa. Ruckebusch Y. (1972) The relevance of drowsiness in the circadian cycle of farm animals. Anim. Behaa. 20, 637-643.
0300-9629/88 $3.00 + 0.00 0 1988 Pergamon Journals Ltd
REMOTE CONTROLLED SAMPLING OF CATTLE AND BUFFALO BLOOD J. HATTINGH,* M. F. GANHAO,* F. J. N. KRUGER,* V. DE Vest and G. W. KAY: *Department of General Physiology, School of Dentistry, University of the Witwatersrand, Johannesburg, 2001; TSkukuza, Kruger National Park, 1350 and $Animal and Dairy Science Research Institute, Irene, 1675, South Africa. Telephone: (011) 339-4366 (Received 6 May 1987) Abstract-i. Blood samples were obtained from cattle and buffaloes over extended periods of time by remote controlled methods at 4-hourly intervals. 2. Initial handling of the animals and deliberate exposure to stress at the end of the experimental times resulted in increased plasma concentrations of a number of variables. 3. Between initial handling and applied stress, the concentration of all variables investigated showed little change and no evidence of rhythms. 4. The results obtained reflect true plasma composition of free ranging cattle and buffaloes at rest.
INTRODUCTION
In a recent publication,
the results of a study of plasma constituents at 90-min intervals over a 4%hr period from four unrestrained Nguni cows were reported (Ganhao et al., 1985). The animals were kept in small cubicles in which they could stand or lie down but could not turn. Indwelling jugular catheters were used to sample blood. No circadian, ultradian or diurnal rhythm was found for any of the parameters investigated and basal values over a 48-hr period were established. However, the concentration of certain variables which are usually used as a measure of stress such as ACTH, cortisol, corticosterone and total catecholamines gradually increased after the first 12 hr. This was attributed to the effects of confinement to a small area and the associated limited physical movement. It was suggested that the stress involved could be eliminated by remote controlled blood sampling on free-ranging animals. Remote controlled blood sampling was used in the present study to obtain samples from cattle and wild buffaloes. No circadian or other rhythm was observed in the concentration of the variables investigated in the samples taken at 4-hourly intervals. Applied stress resulted in concentration increases of a number of variables and the results are in agreement with those previously found (Ganhao et d., 1985). No indication was present that the animals experienced discomfort during the experiments and the concentration of the variables examined was a true reflection of the plasma composition at rest. MATERIALS
AND METHODS
Animals and blood sampling
Nguni cows were used at the Animal and Dairy Science Research Institute, Irene and wild buffalo bulls (Syncems cuffer) in the Kruger National Park. In order to place the blood sampling apparatus on the animals, cattle were put into a crush and buffaloes anaesthetized by darting (8-10 mg M99 and 3&50mg Rompun). Blood samples were taken c
aP
89’2A--1
with a syringe immediately before the animals were released (cattle) or the antidote given (buffaloes). The animals were kept in large enclosures in the veld with food and water ad libitum. Remote controlled blood sampling commenced after 2-6 hr. This was repeated every 4 hr thereafter for a variable period of time (hence variable N numbers in Fig. 1). At the end of the experiment a pre-stress sample was taken remotely and the animals were stressed by chasing, noise, the presence of a vehicle, etc., for a period of about 40 min and a final stress sample taken remotely. Cattle were then returned to the crush and buffaloes anaesthetized by remote control and the apparatus removed. The equipment used for blood sampling was attached to a collar, which was fitted around the animal’s neck. The apparatus was connected to the jugular vein with an indwelling catheter (Hattingh, 1985). This was flushed automatically every 15 min with heparinized saline. Undiluted blood samples were taken into a heparinized container as required, the response time of the equipment being about 30 set; animals were virtually unaware that this was being done (except for the sounds of pumps, no other noise is heard) and in many instances remained lying down if they were in that position when sampling commenced. The sample was ejected from the equipment and processed immediately. In the case of buffaloes, the equipment was also used to anaesthetize the animals at the end of the experiment. This was done by priming the machine with the anaesthetic solution before it was placed on the animal and then infusing this when required. Analytical techniques and statistical analyses
Blood samples were immediately centrifuged and the plasma deep frozen. Samples were analysed as soon as possible for a number of variables using standard techniques as described before (Ganhao et al., 1985). Not all analyses were performed for all samples. Profiles on each animal were plotted for each variable with concentration against time. The mean and SD were calculated for cattle and buffaloes for each variable every 4 hr using times as close as possible to the actual sampling times. These results were plotted against time and are reported graphically for those variables which changed in the period after the sampling apparatus was put on and before the applied stress. In addition, overall means and SDS over the last 24 hr of the experiment (excluding samples
231
232
J. HAITINGHet al.
obtained during applied stress) were calculated for all variables. Significance of the differences between means was tested using a Student’s t-test.
the buffalo and cattle groups it was clear that no rhythms were present. The overall means and SDS for the last 24 hr of the experiment for the variables investigated are thus reported in Table I, where the N values indicate the number of samples over this period. Figure I shows the profiles for variables which changed with applied stress at the end of the
RESULTS
From the graphs relating to the con~ntration variables
against
time for individual
animals
of
and for
experiments
and/or
which
showed
a change
BUFMKXS l
3321
11
11
1
t
i
1
after
Remote controlled blood sampling
233
t
te----*-
l -
1
i
mmathellnd 4
0
12
t
4:ZHIB
l
I
-
-
-
5
-
=
,
76866533333
Fig. 1. Profiles of the concentration of plasma constituents against time (mean f SD). The numbers below each point are the individual N-values. Mean values which differed significantly from the overall means (P -C0.05) are indicated with an asterisk.
J. HATTINGH el al.
234
Table I. Overall mean values and SDS for the variables investigated for the final 24 hr of the experiment. N values indicate the number of samples over this period. The stress results are the mean values and standard deviations obtained after applied stress Buffaloes _____
Cattle
Control .__._~__~_
.-.
F
SD
N
24 21 26 25 23 24 25
33. I 3.2
I I 7
1.0 17.8 7.1 50
5.1 0.9 2.2 0.5 17.2 I.1 I .4
25 25 25 25 22 21 22 I6
3.5 0.6
N
Haematocrit (%) Glucose (mmol/l) Total lipids (g/l) Ltactate (mmol/l) Cortisol (nmol/l) Free T, (pmol/l) K’ (mmol/l) Total Catecholamines (ng/ml) Norepinephrine (ng/ml) Epinephrine (ng/ml) Dopamine (ngjml) Leu-enkephaline (pg/ml) Met-enkephaline (pg/ml) fi-Endorphine (pg/ml) ol-Endorphine (pg/ml)
~_
Stress
4.1
-
Control
stress
R
SD
N
R
43
2.5
7
31.4
I.1
2
36.5
3.5
5.2
0.6
7
3.6
0.6
2
5.4
0.3
SD
N
x
SD
6.0
2.5
1
2.4
0.3
2
2.8
I.2
1
9.9
3.5
7
1.9
1.2
2
12.1
5.2
I
128.6
44.3
7
38.9
18.1
2
137.5
24.7
2.1 0.6
7
2.1
I.6
0.8 0 I3 0.14 2.51 0.34
7 7
10.6 2.4 7.7 0.5
I I I
1.0 0.17 0.24 4.35 0.59
14.3 2.3 12.1 0.7
2.4 I .o I.1 0.3
1
the equipment was put on. It is clear that, in general, the concentrations of plasma lactate, cortisol, catecholamines, haematocrit, glucose and total lipids all changed during the initial and final periods of the experiments but that they showed little change between. DISCUSSION
The results reported here show that initial handling of the animals when the collar was put on resulted in (significantly) higher mean values for certain variables than what was obtained in the period thereafter. The variables involved are those which are regarded as being indicative of stress, i.e. increases are usually associated with some form of discomfort. Similar increases in the same variables were observed after the animals were deliberately exposed to stress. Between these two procedures the values showed little change and were low. Furthermore, no gradual increase in the values of ACTH, cortisol and catecholamine concentrations was evident as was found previously in cattle confined to cubicles (Ganhao et al., 1985). This also implies that the animals were at rest during the sampling period. It may thus be concluded that representative baseline values for free ranging cattle and buffaloes over a time period were obtained in this study. In addition, it is also clear from Fig. 1. that the concentration of certain variables, e.g. cortisol, haematocrit and glucose may take up to 10 hr to reach baseline values once elevated due to stress. This corresponds to the half-life of certain hormones in blood (Ganong, 1983) and also, in the case of buffaloes, to the known hyperglycaemic and sympatholytic effects of Rompun (Meyer-Jones et ul., 1977). For this reason the overall means reported in Table 1 are only for the last 24 hr of the experiment. Although the N numbers were small in certain cases, especially for buffaloes over a prolonged period of time, the results do permit the conclusion that no rhythms were present in the concentration of the variables investigated when sampling every 4 hr. This agrees with previous observations (Ganhao et al., 1985). However, more frequent sampling, at halfhourly or hourly intervals may well show the exis-
1
tence of rhythms in these animals which do not sleep for long periods of time (Ruckebusch, 1972). The overall mean values reported here for the variables investigated over 24 hr compare well with those found earlier in cattle confined to cublicles (Ganhao et al., 1985) and to those obtained from buffaloes shot in the brain (Hattingh et al., 1984a). Certain differences are however evident, e.g. catecholamine and cortisol concentrations in buffaloes and lactate and cortisol concentrations in cattle. The present results differ significantly from those obtained from anaesthetized animals in the case of certain variables (Hattingh et al., 1984b). It is suggested that the results obtained by remote controlled blood sampling are a truer reflection of the plasma composition of the animals studied because confinement to cubicles with the consequent limited physical movement is unnatural and a shot in the brain may for instance lead to mass sympathetic discharge and other effects (Hattingh et al., 1984a). Anaesthetization itself also causes stress. The present results thus reflect the plasma composition of free-ranging animals and may be used in an initial approach to quantify the stress response (Hattingh, 1986). Remote controlled blood sampling has been used by other workers. Bubenik and Bubenik (1979) reported on a remote controlled self contained blood sampler which could be used in animals such as horses, cows, white-tailed deer and red deer. Falke et al. (1985) used a pressure controlled remote sampling device in seals, and Birke (1986) mentioned that the technique is used to study the way in which horses cope with strenuous exercise. The device used by Farrel er al. (1970) was pre-programmed and could not be controlled remotely. The results of the present study, the first quantitative baseline observations on free ranging animals taken over a period of time, indicate the value of this technique. It is now possible to study the effects of different stressors on wild animals without the interference of handling, anaesthetization and other influences.
Acknowledgements-The financial support of the FRD, National Parks Board and University of the Witwatersrand
Remote controlled blood sampling is gratefully acknowledged. Technical help was rendered by Mr J. A. N. Grobbelaar and others. REFERENCES
Birke L. (1986) Equine athletes: blood, sweat and biochemistry. New Scientist 110, 48-52. Bubenik G. A. and Bubenik A. B. (1979) Remote-controlled self-contained blood sampler. C.A.L.A.S./A.C.T.A.L. Proc. 5 l-53.
Falke J. K., Hill R. D., Quist J., Schneider R. C., Guppy M., Liggins G. C., Hachachka P. W., Elliot R. E. and Zap01 W. M. (1985) Seal lungs collapse during free diving: evidence from arterial nitrogen tensions. Science 229, 556558. Farrel D. J., Corbett J. L. and Long R. A. (1970) Automatic sampling of blood and ruminal fluid of grazing sheep. Res. Vet. Sci. 11, 217-220. Ganhao M. F., Hattingh J., Kay G. W., Cornelius S. T. and Grobbelaar J. A. N. (1985) Plasma constituents of Nguni cows over forty-eight hours. J.S.A. Vet. Ass. 56, 177-180.
235
Ganong W. F. (1983) Review of Medical Physiology, 12th edn. Lange, Los Altos, California. Hattingh J., Wright P. G., de Vos V., McNairn I. S., Ganhao M. F., Silove M., Wolverson G. and Cornelius S. T. (1984a) Blood composition in culled elephants and buffaloes. J.S.A. Vet. Ass. 55, 157-164. Hattingh J., Wright P. G., de Vos V., Levine L., Ganhao M. F., McNairn 1. S., Russel A., Knox C., Cornelius S. T. and Bar-Noy J. (1984b) Effect of etorphine and succinyldichohne on blood composition in elephant and buffalo. S.A.J. Zool. 19, 286290. Hattingh J. (1985) Remote controlled blood sampling. S.A.J.
Sci. 81, 644.
Hattingh J. (1986) Physiological measurement S.A.J.
of stress.
Sci. 82, 612-614.
Meyer-Jones L., Bosch W. H. and McDonald L. E. (1977) Veterinary Pharmacology and Therapeutics, 4th edn, pp. 363-367. Iowa State University Press, Iowa. Ruckebusch Y. (1972) The relevance of drowsiness in the circadian cycle of farm animals. Anim. Behaa. 20, 637-643.