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Salivary cortisol in pigs following adrenocorticotrophic hormone stimulation: Comparison with plasma levels
Br . iel.
1.
(1989) . 145, 362
SALIVARY CORTISOL IN PIGS FOLLOWING ADRENOCORTICOTROPHIC HORMONE STIMULATION : COMPARISON WITH PLASMA LEVELS R . F . PARROT-I', B . H . SIISSON and B . A . BALDWIN A .F.R.C. Institute of Animal Physiology and Genetics Research, Cambridge Research Station, Babraham Hall, Cambridge, CB2 4A T
SUMMARY Four prepubertal pigs were prepared with venous catheters and housed in metabolism cages . Plasma and saliva samples were taken at 15-min intervals over a 105-min period and analysed by radioimmunoassay for total (i .e . free and bound) cortisol content . Adrenocorticotrophic hormone (ACTH) was given i .v . at three different doses (0 . 5, 1 . 0 and 2 . 0 mg) after the second sample and the cortisol responses were compared with pretreatment values and levels observed after saline vehicle administration . Basal levels of salivary cortisol were approximately 10% of those in plasma . ACTH induced significant increases in plasma and salivary cortisol but in no case was a dose/response relationship detected . Plasma cortisol showed a maximum increase of approximately 230°/o whereas salivary cortisol increased only by about 130%, indicating that salivary cortisol is a less sensitive indicator of adrenal activity than plasma cortisol in this species . Estimation of salivary cortisol concentrations may offer practical advantages for the assessment of stress responses in intensively housed pigs .
INTRODUCTION Objective evaluation of current systems of animal husbandry and management are necessary if improvements in the welfare of farm livestock are to be achieved . In the course of such investigations, it is often desirable to measure adrenocortical secretion to determine whether stress responses have been elicited . However, whereas blood samples for hormone analysis can be readily obtained in laboratory situations by using animals surgically prepared with venous catheters, similar procedures cannot be adopted under field conditions . In this respect, the pig presents a particular problem because it is often necessary to use techniques such as vena cava puncture to obtain blood samples and the stress associated with procedures of this type does, of itself, stimulate cortisol release (Nyberg et al., 1988) . There is, therefore, a need to develop a minimally stressful method of sampling pigs if reliable endocrine data are to be obtained . A non-invasive technique of sampling that has proved effective in other species, and may also be of use in the pig, is the collection of saliva . Studies in man have shown that cortisol levels in saliva relate closely to plasma concentrations of the free hormone and are
SALIVARY CORTISOL IN PIGS
30
independent of flow rate (Read el at., 1982 ; Vining et al., 1983 ; Laudat et al., 1988) . In addition, salivary cortisol has been used as an indicator of the stress induced by castration and transportation in calves (Fell & Shutt, 1986 ; Fell, Wells & Shutt, 1986) and Icy varding, transport and confinement in sheep (Fell & Shutt, 1986) . The object of the present study was to examine the feasibility of measuring salivary cortisol concentrations in pigs and to compare the levels with those found in blood samples taken at the same time . The pigs were prepared with venous catheters to minimize the handling effects of blood collection and cortisol levels were measured by radioimmunoassay before and after administration of various doses of adrenocorticotrophic hormone (ACTH) . The doses used were based on the results of an earlier study Rampacek et al., 1984) which showed that 200 i .u . of ACTH produced a large (430)50`(,) increase in plasma cortisol levels in pigs 90 min after intravenous injection . MATERIALS AND METHODS Four prepubertal Large white pigs (3 d 1 9, body weight 30-40 kg) were anaesthetized with closed circuit halothane and prepared with jugular vein catheters using sterile precautions . They were housed in metabolism cages and the catheters were flushed daily with heparinized saline . Sample collection commenced at 11 .15 h and continued at 15-min intervals until 13 .00 h . Directly after the second sample, the animals were injected i .v . with 0 . 5, 1 . 0 or 2 . 0 mg (i .e . 50, 100 or 200 Lu .) of the synthetic ACTH tetracosactrin ('Synacthen', Ciba Ltd, Horsham, W . Sussex) or physiological saline as a control . Since Synacthen is supplied in I ml aliquots containing 250°g, the 2 . 0 mg dose was given in an 8 ml volume and, therefore, an equivalent volume of saline was administered as the control injection . Blood was collected in 10 ml heparinized sample tubes (Monovette', Sarstedt Ltd, Beaumont Leys, Leics .), centrifuged, and the resultant plasma stored at -30ûC pending analysis . Saliva samples were collected immediately after blood withdrawal by allowing the pig to chew on three cotton buds (Johnson &,Johnson Ltd, Slough, Berks .) until they were thoroughly moistened (about 30 s) . The cotton buds were centrifuged for 5 min to remove the saliva, which was then stored at -30 ûC . Concentrations of cortisol in plasma and saliva were measured by radioimmunoassay using a previously described method (Heap, Silver & Walters, 1981) . Since cortisol in saliva reflects the free plasma fraction (Vining & McGinley, 1987) it should, theoretically, not be necessary to extract saliva with an organic solvent to dissociate cortisol from binding proteins . However, because pig saliva is frequently contaminated with particulate matter and since a proportion of salivary cortisol may be protein-bound (14 Chu & Ekins, 1988), it was considered advisable to subject the saliva to the same dichloromethane extraction procedure as the plasma . The results, therefore, represent total cortisol content (i .e . free and bound fractions) of both saliva and plasma . In addition, because salivary cortisol values are usually less than 10%, of those in plasma (Read el al., 1982) the plasma was diluted 1 in 5 so that results from both types of sample fell within similar regions of the standard curve . RESULTS Plasma and salivary cortisol concentrations before and after treatment with ACTH are
364
BRITISH VETERINARY JOURNAL, 145, 4
after saline injection' were significantly (P<0 . 05 ; two-tailed paired t-test) greater than mean pretreatment values, indicating an effect of handling . Salivary cortisol concentrations, however, were not similarly influenced by handling . Plasma cortisol showed a maximum increase of approximately 230% (15-50 pmol/1) after AC'T'H administration whereas salivary cortisol only increased by about 130°/0 (1 . 3-3 nmol/1) . Analysis of variance indicated that there were no differences between the cortisol responses to three dose levels of ACTH for either plasma or saliva . Therefore, the data from tests with the three dose levels were combined in a further analysis which showed that plasma levels of cortisol were significantly greater than both pretreatment and saline control values commencing 30 min after injection (range, P> 0 . 005-0 . 001 ; two-tailed paired t-test) . However, salivary cortisol concentrations differed from pretreatment and saline control levels starting 45 min after injection (range, P> 0 . 03-0 . 001 ; two-tailed paired t-test) . PLASMA CORTISOL (nmol/I)
70SALINE
0 .5mg ACTH
1 .0mg ACTH
2 .0mg ACTH
-I-
r -
6o50403020-
I
I
I
I
100SALIVARY
5-
CORTISOL (nmol/I)
4TT
3-
I
1
I
I
II I
2-
T
I
I
0 - 15 0 15 30 45 60 75 90 -15 0 15 30 45 60 75 90 -15 0 15 3045 60 75 90 -15 0 15 30 45 60 75 90 TIME(min)
Cortisol concentrations (c±SEN!) in plasma and saliva of prepubertal pigs sampled at 15min intervals before and after administration of various doses of ACTH or saline vehicle . Significant differences are described in the text . Fig . 1 .
DISCUSSION
In the present study, it was found to be relatively straightforward to collect saliva samples from pigs in sufficient quantities to permit measurement of cortisol content by radioimmunoassay . As in other species, the total (i .e . free and bound) amount of cortisol present in plasma was greater than that in saliva and there was a positive relationship between the cortisol content of the two compartments, as judged by the response to ACTH . However, salivary cortisol appeared to be a less sensitive indicator of pituitary/ adrenocortical activity than plasma cortisol .
SALIVARY CORTISOL IN PIGS
365
Salivary cortisol is considered to relate directly to the free component of the plasma, although salivary levels are somewhat reduced by metabolism to cortisone in man (Vining & McGinley, 1987) and probably also in the pig (W .D . Booth & S .H . Lambie, personal communication) . In spite of this, there is a virtually linear relationship between total salivary and plasma cortisol in man under normal conditions and following adrenal suppression with dexamethasone (Read et al., 1982) . However, stimulation with ACTH to produce cortisol levels outside the normal physiological range results in a greater increase in salivary than plasma cortisol (Read et al., 1982 ; Laudat et al., 1988) . The suggested explanation for this effect is that the cortisol binding globulin (CBG) rapidly becomes saturated as plasma levels rise, resulting in a greater increase in the free plasma fraction, which is then reflected in the saliva (Read et al., 1982 ; Vining et al., 1983) . Enhanced sensitivity of salivary cortisol has also been demonstrated in sheep ; in a preliminary study, the lowest dose of ACTH used in the present experiment (0 . 5 mg) produced a 410% increase in plasma cortisol after 45 min and a 561% increase in salivary cortisol (Parrott & Misson, unpublished findings) . However, the results of the present study indicate that a different relationship exists in the growing pig . Not only were the increases in cortisol in both plasma and saliva much smaller, but the salivary response was delayed and reduced in comparison with the plasma . The reason for this difference is unknown although factors such as age, sex and genotype have been shown to influence the CBG content of porcine plasma (Nyberg et al., 1988) and may have a bearing on the situation . In the present experiment, there were no differences in the cortisol responses of plasma or saliva to the three doses of ACTH administered . However, in a previous report using larger (185-day-old) pigs, the highest dose of ACTH used here (2 . 0 mg) produced an increase in cortisol more than twice that seen in this study (Rampacek et al., 1984) . Moreover, considering the fact that 185-day-old pigs would weigh much more than those in the present experiment, the amount of ACTH given per kg would also have been much less . This seems to suggest that the pigs used in the present study were relatively insensitive to the effects of exogenous ACTH . This may be related to the fact that another report showed that prepubertal boars aged between 100 and 160 days, the range used in the present experiment, had significantly (P< 0 . 01) lower basal cortisol levels than either younger or older animals (Allrich et al., 1982) . Since the pigs in this experiment failed to show a differential response to the various doses of ACTH, or a particularly large response to the highest dose, the results raise the possibility that the low basal cortisol levels of pigs in the 100-160 day age range may be due to adrenal insensitivity, rather than reduced activity at the hypothalamic or pituitary level . Although the present data are derived from a small sample of pigs, the results indicate that salivary cortisol may be a useful indicator of plasma cortisol in this species . The disadvantage of having a measure of adrenal activation that appears to be less sensitive than that of plasma cortisol may be outweighed by the possibility of collecting samples for analysis without causing pain and distress, or the need for restraint . This investigation was carried out using regularly handled caged animals where sample collection was relatively easy . Clearly, therefore, some degree of artifice may be necessary to apply the procedure under normal husbandry conditions . Nevertheless, the technique may he useful for the assessment of welfare in specific situations, such as that of the tethered sow .
366
BRITISH VETERINARY JOURNAL, 145, 4
ACKNOWLEDGEMENTS The authors are grateful to Mr . D . Brown of the A .F .R .C . statistics group for carrvin,g out the analysis .
REFERENCES ALLRICH, R. D ., CHRIS I'ENSOX, R . K ., FORD, J . J . & ZIMMIER\LAN, D . R . (1982) . ,Journal of Animal Science 55, 1139 . CHU, F . W . & EKIVS, R . P . (1988) . Journal of Endocrinology 11, Supplement, Abstract 69 . FELL, L . R . & SHU'l'r, D . A . (1986) . Proceedings of the Australian Society for Animal Production 16, 203 . FELL, L . R ., WELLS, R . & SHUrr, D. A . (1986) . Australian Veterinary Yournal63, 16 . HEAP, R . B ., SILVER, A .& W .ALTERS, D . E . (1981) . Quarterly Journal of Experimental Physiology 66, 129 . LAUDrcr, M . H ., CERDAS, S ., FOURNIER, C ., GUHIAx, D ., GUILHAUNIE, B . & Lu ION, J . P . (1988) . journal of Clinical Endocrinology and Metabolism 66, 343 . NYBERC, L ., LUNDSTRONI, K ., EDtoRS-LiLJA, I . & RL'yDCRE., M . (1988) . Journal of Animal Science 66, 1201 . RANIPACEK, G . B ., KRAELIVG, R . R ., FoNDA, E . S . & BARB, C . B . (1984) . Journal of Animal 401 . READ, G. F ., RIAD-FAHMY, D ., WALKER, R . F . & GRIFFITHS, K . (ed) (1982) . Immunoassays of
Science 58,
Steroids in Saliva : Proceedings of Ninth Tenovus Workshop . Cardiff: Alpha-Omega Publishing Ltd . VINING, R . F . MCGmu .V, R . A . (1987) . Journal of Steroid Biochemistry 27, 81 . VINING, R . F ., MCGNLEY, R . A ., MAKSI~j ris, J . J . & YHo, K . (1983) . Annals of Clinical Biochemistry 20, 329 .
(Accepted for publication 4 October 19881
1.
(1989) . 145, 362
SALIVARY CORTISOL IN PIGS FOLLOWING ADRENOCORTICOTROPHIC HORMONE STIMULATION : COMPARISON WITH PLASMA LEVELS R . F . PARROT-I', B . H . SIISSON and B . A . BALDWIN A .F.R.C. Institute of Animal Physiology and Genetics Research, Cambridge Research Station, Babraham Hall, Cambridge, CB2 4A T
SUMMARY Four prepubertal pigs were prepared with venous catheters and housed in metabolism cages . Plasma and saliva samples were taken at 15-min intervals over a 105-min period and analysed by radioimmunoassay for total (i .e . free and bound) cortisol content . Adrenocorticotrophic hormone (ACTH) was given i .v . at three different doses (0 . 5, 1 . 0 and 2 . 0 mg) after the second sample and the cortisol responses were compared with pretreatment values and levels observed after saline vehicle administration . Basal levels of salivary cortisol were approximately 10% of those in plasma . ACTH induced significant increases in plasma and salivary cortisol but in no case was a dose/response relationship detected . Plasma cortisol showed a maximum increase of approximately 230°/o whereas salivary cortisol increased only by about 130%, indicating that salivary cortisol is a less sensitive indicator of adrenal activity than plasma cortisol in this species . Estimation of salivary cortisol concentrations may offer practical advantages for the assessment of stress responses in intensively housed pigs .
INTRODUCTION Objective evaluation of current systems of animal husbandry and management are necessary if improvements in the welfare of farm livestock are to be achieved . In the course of such investigations, it is often desirable to measure adrenocortical secretion to determine whether stress responses have been elicited . However, whereas blood samples for hormone analysis can be readily obtained in laboratory situations by using animals surgically prepared with venous catheters, similar procedures cannot be adopted under field conditions . In this respect, the pig presents a particular problem because it is often necessary to use techniques such as vena cava puncture to obtain blood samples and the stress associated with procedures of this type does, of itself, stimulate cortisol release (Nyberg et al., 1988) . There is, therefore, a need to develop a minimally stressful method of sampling pigs if reliable endocrine data are to be obtained . A non-invasive technique of sampling that has proved effective in other species, and may also be of use in the pig, is the collection of saliva . Studies in man have shown that cortisol levels in saliva relate closely to plasma concentrations of the free hormone and are
SALIVARY CORTISOL IN PIGS
30
independent of flow rate (Read el at., 1982 ; Vining et al., 1983 ; Laudat et al., 1988) . In addition, salivary cortisol has been used as an indicator of the stress induced by castration and transportation in calves (Fell & Shutt, 1986 ; Fell, Wells & Shutt, 1986) and Icy varding, transport and confinement in sheep (Fell & Shutt, 1986) . The object of the present study was to examine the feasibility of measuring salivary cortisol concentrations in pigs and to compare the levels with those found in blood samples taken at the same time . The pigs were prepared with venous catheters to minimize the handling effects of blood collection and cortisol levels were measured by radioimmunoassay before and after administration of various doses of adrenocorticotrophic hormone (ACTH) . The doses used were based on the results of an earlier study Rampacek et al., 1984) which showed that 200 i .u . of ACTH produced a large (430)50`(,) increase in plasma cortisol levels in pigs 90 min after intravenous injection . MATERIALS AND METHODS Four prepubertal Large white pigs (3 d 1 9, body weight 30-40 kg) were anaesthetized with closed circuit halothane and prepared with jugular vein catheters using sterile precautions . They were housed in metabolism cages and the catheters were flushed daily with heparinized saline . Sample collection commenced at 11 .15 h and continued at 15-min intervals until 13 .00 h . Directly after the second sample, the animals were injected i .v . with 0 . 5, 1 . 0 or 2 . 0 mg (i .e . 50, 100 or 200 Lu .) of the synthetic ACTH tetracosactrin ('Synacthen', Ciba Ltd, Horsham, W . Sussex) or physiological saline as a control . Since Synacthen is supplied in I ml aliquots containing 250°g, the 2 . 0 mg dose was given in an 8 ml volume and, therefore, an equivalent volume of saline was administered as the control injection . Blood was collected in 10 ml heparinized sample tubes (Monovette', Sarstedt Ltd, Beaumont Leys, Leics .), centrifuged, and the resultant plasma stored at -30ûC pending analysis . Saliva samples were collected immediately after blood withdrawal by allowing the pig to chew on three cotton buds (Johnson &,Johnson Ltd, Slough, Berks .) until they were thoroughly moistened (about 30 s) . The cotton buds were centrifuged for 5 min to remove the saliva, which was then stored at -30 ûC . Concentrations of cortisol in plasma and saliva were measured by radioimmunoassay using a previously described method (Heap, Silver & Walters, 1981) . Since cortisol in saliva reflects the free plasma fraction (Vining & McGinley, 1987) it should, theoretically, not be necessary to extract saliva with an organic solvent to dissociate cortisol from binding proteins . However, because pig saliva is frequently contaminated with particulate matter and since a proportion of salivary cortisol may be protein-bound (14 Chu & Ekins, 1988), it was considered advisable to subject the saliva to the same dichloromethane extraction procedure as the plasma . The results, therefore, represent total cortisol content (i .e . free and bound fractions) of both saliva and plasma . In addition, because salivary cortisol values are usually less than 10%, of those in plasma (Read el al., 1982) the plasma was diluted 1 in 5 so that results from both types of sample fell within similar regions of the standard curve . RESULTS Plasma and salivary cortisol concentrations before and after treatment with ACTH are
364
BRITISH VETERINARY JOURNAL, 145, 4
after saline injection' were significantly (P<0 . 05 ; two-tailed paired t-test) greater than mean pretreatment values, indicating an effect of handling . Salivary cortisol concentrations, however, were not similarly influenced by handling . Plasma cortisol showed a maximum increase of approximately 230% (15-50 pmol/1) after AC'T'H administration whereas salivary cortisol only increased by about 130°/0 (1 . 3-3 nmol/1) . Analysis of variance indicated that there were no differences between the cortisol responses to three dose levels of ACTH for either plasma or saliva . Therefore, the data from tests with the three dose levels were combined in a further analysis which showed that plasma levels of cortisol were significantly greater than both pretreatment and saline control values commencing 30 min after injection (range, P> 0 . 005-0 . 001 ; two-tailed paired t-test) . However, salivary cortisol concentrations differed from pretreatment and saline control levels starting 45 min after injection (range, P> 0 . 03-0 . 001 ; two-tailed paired t-test) . PLASMA CORTISOL (nmol/I)
70SALINE
0 .5mg ACTH
1 .0mg ACTH
2 .0mg ACTH
-I-
r -
6o50403020-
I
I
I
I
100SALIVARY
5-
CORTISOL (nmol/I)
4TT
3-
I
1
I
I
II I
2-
T
I
I
0 - 15 0 15 30 45 60 75 90 -15 0 15 30 45 60 75 90 -15 0 15 3045 60 75 90 -15 0 15 30 45 60 75 90 TIME(min)
Cortisol concentrations (c±SEN!) in plasma and saliva of prepubertal pigs sampled at 15min intervals before and after administration of various doses of ACTH or saline vehicle . Significant differences are described in the text . Fig . 1 .
DISCUSSION
In the present study, it was found to be relatively straightforward to collect saliva samples from pigs in sufficient quantities to permit measurement of cortisol content by radioimmunoassay . As in other species, the total (i .e . free and bound) amount of cortisol present in plasma was greater than that in saliva and there was a positive relationship between the cortisol content of the two compartments, as judged by the response to ACTH . However, salivary cortisol appeared to be a less sensitive indicator of pituitary/ adrenocortical activity than plasma cortisol .
SALIVARY CORTISOL IN PIGS
365
Salivary cortisol is considered to relate directly to the free component of the plasma, although salivary levels are somewhat reduced by metabolism to cortisone in man (Vining & McGinley, 1987) and probably also in the pig (W .D . Booth & S .H . Lambie, personal communication) . In spite of this, there is a virtually linear relationship between total salivary and plasma cortisol in man under normal conditions and following adrenal suppression with dexamethasone (Read et al., 1982) . However, stimulation with ACTH to produce cortisol levels outside the normal physiological range results in a greater increase in salivary than plasma cortisol (Read et al., 1982 ; Laudat et al., 1988) . The suggested explanation for this effect is that the cortisol binding globulin (CBG) rapidly becomes saturated as plasma levels rise, resulting in a greater increase in the free plasma fraction, which is then reflected in the saliva (Read et al., 1982 ; Vining et al., 1983) . Enhanced sensitivity of salivary cortisol has also been demonstrated in sheep ; in a preliminary study, the lowest dose of ACTH used in the present experiment (0 . 5 mg) produced a 410% increase in plasma cortisol after 45 min and a 561% increase in salivary cortisol (Parrott & Misson, unpublished findings) . However, the results of the present study indicate that a different relationship exists in the growing pig . Not only were the increases in cortisol in both plasma and saliva much smaller, but the salivary response was delayed and reduced in comparison with the plasma . The reason for this difference is unknown although factors such as age, sex and genotype have been shown to influence the CBG content of porcine plasma (Nyberg et al., 1988) and may have a bearing on the situation . In the present experiment, there were no differences in the cortisol responses of plasma or saliva to the three doses of ACTH administered . However, in a previous report using larger (185-day-old) pigs, the highest dose of ACTH used here (2 . 0 mg) produced an increase in cortisol more than twice that seen in this study (Rampacek et al., 1984) . Moreover, considering the fact that 185-day-old pigs would weigh much more than those in the present experiment, the amount of ACTH given per kg would also have been much less . This seems to suggest that the pigs used in the present study were relatively insensitive to the effects of exogenous ACTH . This may be related to the fact that another report showed that prepubertal boars aged between 100 and 160 days, the range used in the present experiment, had significantly (P< 0 . 01) lower basal cortisol levels than either younger or older animals (Allrich et al., 1982) . Since the pigs in this experiment failed to show a differential response to the various doses of ACTH, or a particularly large response to the highest dose, the results raise the possibility that the low basal cortisol levels of pigs in the 100-160 day age range may be due to adrenal insensitivity, rather than reduced activity at the hypothalamic or pituitary level . Although the present data are derived from a small sample of pigs, the results indicate that salivary cortisol may be a useful indicator of plasma cortisol in this species . The disadvantage of having a measure of adrenal activation that appears to be less sensitive than that of plasma cortisol may be outweighed by the possibility of collecting samples for analysis without causing pain and distress, or the need for restraint . This investigation was carried out using regularly handled caged animals where sample collection was relatively easy . Clearly, therefore, some degree of artifice may be necessary to apply the procedure under normal husbandry conditions . Nevertheless, the technique may he useful for the assessment of welfare in specific situations, such as that of the tethered sow .
366
BRITISH VETERINARY JOURNAL, 145, 4
ACKNOWLEDGEMENTS The authors are grateful to Mr . D . Brown of the A .F .R .C . statistics group for carrvin,g out the analysis .
REFERENCES ALLRICH, R. D ., CHRIS I'ENSOX, R . K ., FORD, J . J . & ZIMMIER\LAN, D . R . (1982) . ,Journal of Animal Science 55, 1139 . CHU, F . W . & EKIVS, R . P . (1988) . Journal of Endocrinology 11, Supplement, Abstract 69 . FELL, L . R . & SHU'l'r, D . A . (1986) . Proceedings of the Australian Society for Animal Production 16, 203 . FELL, L . R ., WELLS, R . & SHUrr, D. A . (1986) . Australian Veterinary Yournal63, 16 . HEAP, R . B ., SILVER, A .& W .ALTERS, D . E . (1981) . Quarterly Journal of Experimental Physiology 66, 129 . LAUDrcr, M . H ., CERDAS, S ., FOURNIER, C ., GUHIAx, D ., GUILHAUNIE, B . & Lu ION, J . P . (1988) . journal of Clinical Endocrinology and Metabolism 66, 343 . NYBERC, L ., LUNDSTRONI, K ., EDtoRS-LiLJA, I . & RL'yDCRE., M . (1988) . Journal of Animal Science 66, 1201 . RANIPACEK, G . B ., KRAELIVG, R . R ., FoNDA, E . S . & BARB, C . B . (1984) . Journal of Animal 401 . READ, G. F ., RIAD-FAHMY, D ., WALKER, R . F . & GRIFFITHS, K . (ed) (1982) . Immunoassays of
Science 58,
Steroids in Saliva : Proceedings of Ninth Tenovus Workshop . Cardiff: Alpha-Omega Publishing Ltd . VINING, R . F . MCGmu .V, R . A . (1987) . Journal of Steroid Biochemistry 27, 81 . VINING, R . F ., MCGNLEY, R . A ., MAKSI~j ris, J . J . & YHo, K . (1983) . Annals of Clinical Biochemistry 20, 329 .
(Accepted for publication 4 October 19881