Adrenal responsiveness to a low-dose ACTH challenge in early and late lactating dairy cows
ABSTRACT To improve the evaluation of the chronic stress conditions, the adrenal responsiveness to low dose ACTH stimulation, in different lactation stages, was checked in 56 multiparous dairy cows from 2 herds (25-350 days in milk). Cows were retrospectively ranked in 3 stages: early (150 DIM) lactation. Herd B (vs. herd A) showed higher basal cortisol and frequency of inflammation. Early stage (vs. others) showed higher basal cortisol, bilirubin, ceruloplasmin and haptoglobin, as well as lower ones of cholesterol and lower rise of plasma cortisol during ACTH challenge (P<0.001). Cortisol peak was also correlated negatively with ceruloplasmin, bilirubin, ROM, and positively with cholesterol, vitamin A and E. Both, basal cortisol and cortisol response to ACTH, are associated to inflammation but in opposite way: basal cortisol positively and cortisol response negatively. This latter results are likely due to lower transcortin synthesis, that could be ensued in early lactating cows suffering inflammation.
Adrenal responsiveness to a low -dose ACTH
challenge in early
and late lactating dairy cow s
Erminio Trevisi, Rosanna Lombardelli,
Andrea Minuti, Giuseppe Bertoni
Istituto di Zootecnica, Università Cattolica del Sacro Cuore, Piacenza, Italy
Corresponding author: Erminio Trevisi. Istituto di Zootecnica, Facoltà di Agraria, Università Cattolica del
Sacro Cuore. Via Emilia Parmense, 84, 29100 Piacenza, Italy – Tel. +39 0523 599278 – Fax: +39 0523
599276 – Email: email@example.com
AbstrAct - To improve the evaluation of the chronic stress conditions, the adrenal responsiveness to
low dose ACTH stimulation, in different lactation stages, was checked in 56 multiparous dairy cows from 2
herds (25-350 days in milk). Cows were retrospectively ranked in 3 stages: early (<75) average (75-150) and
late (>150 DIM) lactation. Herd B (vs. herd A) showed higher basal cortisol and frequency of inflammation.
Early stage (vs. others) showed higher basal cortisol, bilirubin, ceruloplasmin and haptoglobin, as well as
lower ones of cholesterol and lower rise of plasma cortisol during ACTH challenge (P<0.001). Cortisol peak
was also correlated negatively with ceruloplasmin, bilirubin, ROM, and positively with cholesterol, vitamin
A and E. Both, basal cortisol and cortisol response to ACTH, are associated to inflammation but in opposite
way: basal cortisol positively and cortisol response negatively. This latter results are likely due to lower tran-
scortin synthesis, that could be ensued in early lactating cows suffering inflammation.
Key words: Dairy cows, ACTH challenge, Stress assessment, Lactation stage.
Introduction – Several hormones (e.g. ACTH, β endorphins, glucocorticoids, etc.) are involved in the
stress response. The adrenal gland plays a key-role in hormonal reactions to stress, with a marked increase
in glucocorticoids, like cortisol and/or catecholamine; the measurement of plasma cortisol is however more
frequently used to study the stress response (Sapolsky et al., 2000). The proper basal level of cortisol is diffi-
cult to be measured, because affected by several factors; in particular by the time between capture operations
and blood sampling, if higher than 5-10 minutes (from beginning of capture operation to last blood sampling).
Furthermore, there is a wide assumption that chronic stress results in a hyper-reactivity of the adrenal cor-
tex. In fact, the animal’s response to an acute stressor – such as transport (Broom, 1988) or restraint (Trevisi
et al., 2005) – would be greater if the animal had been for long time exposed to other stressors at the farm.
This justifies the use of blood cortisol response to exogenous adrenocorticotrophic hormone administration
(ACTH challenge), as method to evaluate chronic stress condition. Nevertheless, some cautions are needed
regarding the standardization of this method e.g. dosage, distance of time from barn operations, number of
blood samples. In addition, physiological factors, as stage of lactation, could also modify the adrenal activity,
as previously signalled by Shayanfar et al. (1975). Moreover, the suggestion of Ndibualonji et al. (1995) to
reduce ACTH dosage, to have a better physiological response and an irrelevant effect on milk yield, was not
applied. Aim of the study was a better evaluation of chronic stress conditions of dairy cows, by the measure-
ment of adrenal responsiveness to low doses of ACTH in different stages of lactation.
Material and methods – The research was carried out in two herds of North-East Italy, and concerned 56
multiparous dairy cows after 25 days in milk (DIM), 26 from herd A and 30 from herd B. In the herd A cows
were kept in a tie-stall equipped with auto feeder and received the forages twice a day; in the herd B, cows
were kept in loose-housing stalls and received a total mixed ration once a day. Twenty five out of 56 animals (7
Ital.J.anIm.S cI. vol. 8 (S uppl. 2), 661-663, 2009661
from herd A and 18 from herd B) were treated in 2 stages: early (<75 DIM) and average-late (>75 DIM) lacta-
tion. The others were treated only one time during the lactation (25-35 0DIM). Almost 3 hours after morning
meal and far from any other worker operations, cows were captured, using any care to avoid fright, immedia-
tely bled from jugular vein and i.v. treated with 20 µg (=2IU) of a synthetic analogue of ACTH (ACTH1-24, Sy-
nacthen® – Novartis Pharma AG - Stein, CH). Blood samples were also taken 30 and 60 minutes after ACTH
injection, leaving cows restrained in the rack. All the blood samples were collected in vacuum Li-heparin tubes,
and immediately stored in iced water. After the blood sampling, the body condition score was evaluated and
the presence of injury or clinical diseases, the parity, calving data and milk yield of the last two milkings were
recorded. In the blood samples, packed cell volume (PCV) was determined and, after centrifugation (3500g for
16min. at 6°C), plasma cortisol was measured by RIA method (Coat-A-Count; DPC, Los Angeles, CA, USA).
Only the samples taken before challenge were also analysed for metabolic profile according to Bertoni et al.
(2008). Reactive oxygen metabolites (ROM), vitamin A and E were also analyzed, but only in herd B.
The integrated response of cortisol over 60 min were evaluated as area under the curve. For the statistical
evaluation, data were subjected to ANOVA using GLM procedure (SAS Inst. Inc., Cary, NC, release 9.1) inclu-
ding in the model herd and stage of lactation (<75, 75-150, >150 DIM) as main factors. Cows treated in two
stages of lactation were also subjected to ANOVA using the REPEATED statement in the MIXED procedure
of SAS. Finally, Pearson correlations were calculated considering overall data and within stage of lactation.
results and conclusions – The herd A, vs.
herd B, showed higher milk yield (38.6 vs. 35.5; kg/d
P<0.001). As to the blood, some parameter differen-
ces were already observed before ACTH challenge:
lower levels of cortisol (3.6 vs. 5.3; ng/ml P<0.05) and
ceruloplasmin (2.6 vs. 3.4; µmol/l P<0.01), but higher
levels of PCV (0.31 vs. 0.29 l/l; P<0.01), glucose (4.0
vs. 3.8 mmol/l; P<0.01), cholesterol (6.7 vs. 4.5 mmol/l;
P<0.01) and albumin (37.8 vs. 36.1 g/l; P<0.05). These
differences cannot be easily justified; in fact, diet and
management were adequate in both herds. Neverthe-
less, from some of the above mentioned parameters
it can be suggested a higher frequency of inflamma-
tory conditions in herd B; according to Bertoni et al.
(2008), these latter could be explained by the higher
ceruloplasmin levels and the lower cholesterol and al-
bumin levels in this herd. Contrariwise no differences
between two herds were showed in cortisol response
after ACTH challenge. The peak of cortisol level was
observed 30 minutes after treatment in both herds,
and the average value of plasma cortisol at 30 and
60 minutes after treatment was 47.9 and 36.7 ng/ml
respectively. Afterwards, cows were retrospectively
ranked in 3 groups according to their DIM: early (<75
DIM), average (75-150 DIM) and late (>150 DIM)
lactation. Early, in comparison to average and late
groups, showed higher levels of basal cortisol (P<0.05
vs. late group only, Figure 1.a). On the contrary, early
group showed a lower rise of plasma cortisol during ACTH challenge (P<0.001) respect to the other groups
(figure 1.a). The integrated response of cortisol over 60 minutes measured as area under the curve was conse-
quently lower (P<0.001) in cows in early stage of lactation 1747 vs. 2229 and 2322 ng/ml x 60 minutes for ear-
Figure 1. Plasma cortisol level (ng/ml)
during ACTH challenge in all
treated cows (1.a), ranked in
3 stages of lactation (<75; 75-
150; >150 DIM), or in subjects
(1.b) where challenge has been
repeated on the same cows
in two phases (<75 and >150
DIM). a,b= P< 0.05; A,B= P< 0.01.
minutes from ACTH challenge
< 75 DIM
> 150 DIM
minutes from ACTH challenge
< 75 DIM
> 150 DIM
Ital.J.anIm.S cI. vol. 8 (S uppl. 2), 661-663, 2009662
proc. 18th nat. congr. aSpa, palermo, Italy
ly, average and late groups respectively.
Similar results were observed conside-
ring only the cows treated in 2 stages of
lactation: <75 and >75 DIM (figure 1.b).
Besides some obvious milk yield diffe-
rences (39; 34; 35 kg/d in early, average
and late groups respectively), the cows
in early stage of lactation showed lower
levels of cholesterol and higher levels of
total bilirubin, ceruloplasmin and hap-
toglobin (Table 1) suggesting again a
relationship with inflammation. In the
attempt to evaluate the factors respon-
sible of the different cortisol response
between early and average-late lacta-
tion, correlations with cortisol (basal
and raise after challenge) were estimated. The basal cortisol level appeared negatively correlated with DIM
(r=-0.41, P<0.001), PCV (r=-0.25, P<0.05), cholesterol (r=-0.30, P<0.01), GGT (r=-0.26, P<0.05), and positively
correlated with ceruloplasmin (r=0.27, P<0.01) and total bilirubin (r=0.31 P<0.001). Significant correlationsceruloplasmin (r=0.27, P<0.01) and total bilirubin (r=0.31 P<0.001). Significant correlations (r=0.27, P<0.01) and total bilirubin (r=0.31 P<0.001). Significant correlations
– but with reverse sign - were also observed between cortisol response after ACTH challenge and checked pa-
rameters. In particular, the peak cortisol correlations were negative with ceruloplasmin (r=-0.24, P<0.05), total
bilirubin (r=-0.35, P<0.001), ROM (r=-0.32, P<0.05) and positive with DIM (r=0.48, P<0.001), glucose (r=0.24,
P<0.05), PCV (r=0.25, P<0.05), cholesterol (r=0.35, P<0.001), vitamin A (r=38, P<0.01) and E (r=0.52, P<0.001).
Therefore, it seems that both, basal cortisol level and cortisol response to ACTH, are correlated to inflamma-
tory phenomena, but not the same way. Higher basal cortisol in inflamed cows could mean a higher adrenal Higher basal cortisol in inflamed cows could mean a higher adrenal
reactivity, then a chronic stress condition, as suggested by Sapolskysuggested by Sapolsky et al. (2000). On the contrary, the cortisol
response to ACTH challenge is lower during inflammation (e.g. negatively correlated with parameters which
are risen by these phenomena and positively with the others: cholesterol, vitamin A and E), suggesting that
inflamed cows would be in better welfare conditions (Broom, 1988). Because it is obvious that severe inflam-
mation means on the contrary a welfare depression, the more likely hypothesis of this discrepancy could be: a)
the lower doses of ACTH we have utilized are still too high and cortisol level reaches always the maximum; b)
the maximum level of cortisol is also influenced by circulating transcortin which in turn is reduced – as other
liver synthesized proteins (Greenspan(GreenspanGreenspan et al., 1986) – in case of inflammation.To conclude the proper evalua-) – in case of inflammation. To conclude the proper evalua-
tion of adrenal response needs an ACTH dose which causes a cortisol peak not affected by transcortin levels.
The research was partially supported by Associazione Italiana Allevatori.
references – bertoni, G., Trevisi, E., Han, X., Bionaz, M., 2008. Effects of inflammatory conditions on
liver activity in puerperium period and consequences for performance in dairy cows. J. Dairy Sci. 91:3300-3310.
broom, D.M., 1988. The scientific assessment of animal welfare. Appl. Anim. Behav. Sci. 20:5-19. Greenspan,
F.S., Forsham, P.H., 1986. In “Basic & Clinical Endocrinology” LMP, Los Altos, California. ndibualonji, B.B.,
Dehareng, D., Van Eenaeme, C., Godeau J.M. 1995. Response of milk yield, plasma cortisol, amino acids, urea
and glucose to a single low-dose administration of adrenocorticotrophic hormone in lactating cows. Vet. Res.
26:32-42. sapolsky, R.M., Romero, L.M., Munck, A.U., 2000. How do glucocorticoids influence stress responses?
Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev. 21:55-89. shayanfar,
F., Head, H.H., Wilcox, C.J., Thatcher, W.W., 1975. Adrenal responsiveness in lactating holstein cows. J. Dairy
Sci. 58:870-8. trevisi, E., Lombardelli, L., Bionaz, M., Bertoni, G., 2005. Plasma cortisol level in relationship to
welfare conditions in dairy cows. 56th Annual Meeting EAAP, Uppsala, Sweden, 5-8 June, 11:106.
phic hormone in lactating cows. Vet. Res.Vet. Res.
Table 1. Differences in blood plasma obtained before
ACTH challenge in cows ranked in 3 stages
of lactation: early (< 75DIM) average (75-
150DIM) and late (>150DIM).
A,B= P< 0.01.
a,b= P< 0.05;
Stage of lactation
Ital.J.anIm.S cI. vol. 8 (S uppl. 2), 661-663, 2009663
proc. 18th nat. congr. aSpa, palermo, Italy