. Albihn 1991; Bage et al., 1997 . Marginally elevated, so-called suprabasal, plasma
˚
Ž
y1 y1
. progesterone concentrations 0.5–1.0 nmol l
vs. - 0.5 nmol l as normality were
measured during oestrus and could either be a consequence of an incomplete pre-ovula- tory luteolysis or be caused by an increased progesterone release from the adrenal
glands. In different ways, increased levels of progesterone are known to delay the onset of oestrus: inhibition of the pre-ovulatory LH-surge by negative feedback on the
Ž .
hypothalamus Stoebel and Moberg, 1982; Duchens et al., 1994, 1995b , inhibition of Ž
endometrial oxytocin receptor formation McCracken et al., 1984; Lamming and Mann, .
Ž 1995 and subsequent release of uterine PGF
Porter and Behrman, 1971; Beard et al.,
2 a
. 1994 . Prolonged exposure to progesterone leads to a variety of uterine responses all of
Ž which may be due to an inhibitory effect on the synthesis of receptors for oxytocin for
. reviews, see Silvia et al., 1991; McCracken et al., 1999 .
The present study was designed to measure plasma progesterone and cortisol response to ACTH-treatment in ovariectomised RBH, thereby investigating the relation-
ship between adrenocortical function and reproductive physiology and, eventually, to evaluate the potential influence of stress as a component of the repeat breeding
syndrome.
2. Materials and methods
2.1. Animals Ten clinically healthy heifers of the Swedish Red and White breed were studied: five
Ž .
Ž repeat breeders RBH aged 2.5–4 years, with a mean weight of 633 kg range: 560–700
. Ž
. Ž
kg and five virgin heifers VH aged 2–2.5 years, with a mean weight of 401 kg range: .
340–500 kg . An RBH was hereby defined as a heifer that had failed to conceive after three or more inseminations in spite of normal, regular oestrous cycles and no palpatory
pathological findings in the genital tract. The VH group consisted of normal, sexually mature heifers never mated or inseminated prior to the experiment. All animals were
purchased from farms declared free from bovine virus diarrhea virus and bovine leucosis virus. Prior to the experiment, they were clinically and gynaecologically monitored for
at least three consecutive sexual cycles. Mean plasma progesterone concentration around ovulation, determined from six blood samples collected 24 h before and after ovulation,
y1
Ž .
y1
was 0.7 nmol l in RBH i.e. suprabasal concentration and 0.3 nmol l
in VH. The heifers were kept tethered in tie stalls and fed hay and water ad libitum. They
had become accustomed to handling several months before the actual trial started. During the 2 days of the experimental period, efforts were made to feed and manage the
heifers according to normal routines in order to minimise adrenocortical activity in response to experimental procedures or disturbed routines. One single person, familiar to
the animals, carried out all sampling.
2.2. Experimental procedure Prior to the trial, the heifers were bilaterally ovariectomised by lateral laparotomy
Ž .
Duchens et al., 1996 . The experiment was initiated when circulating hormones of
ovarian origin, represented by plasma progesterone, had reached a constant basal level
y1
Ž .
- 0.2 nmol l
in all animals a minimum of 7 days were required . Blood was Ž
w
collected via an indwelling jugular vein catheter central venous catheter set, Cook .
Veterinary Products, Australia . The catheter was inserted under local anaesthesia 1 day prior to the sampling period, or alternatively without local anaesthesia at least 2 h before
Ž the first sample on day 1 to avoid stress effects from the catheterisation Alam and
. Dobson, 1986 . A silicone tubing connected to the catheter was stitched to the heifer’s
neck to reduce stress at sampling or treatment injection. The heifers served as their own controls during a 2-day experiment. As a pretreat-
Ž .
Ž ment control s day 1 , 5 ml saline natrium chloride 9 mgrml, Kabi Pharmacia,
. Ž
. Sweden was injected. On the day of treatment s day 2 , a synthetic analogue of
Ž
w
. ACTH Synachten , Ciba, Switzerland was administered. In order to obtain a physio-
Ž .
w
logical adrenocortical response, a suitable dose of ACTH, 60 mg 48 IU Synachten Ž
made up to a volume of 5 ml in saline solution, was selected Alam and Dobson, 1986; .
van der Kolk and Breukink, 1991; Hein and Allrich, 1992; Verkerk et al., 1994 . Blood samples were collected every 30 min during a period of 6 h. Directly after the third
blood sample, NaCl or ACTH was injected i.v. via the catheter. Sampling always started at 0800 h to avoid effects from circadian and ultradian rhythms in plasma cortisol
Ž .
concentrations Lefcourt et al., 1993 . Immediately after collection, the samples were centrifuged at 3000 = g for 10 min and the plasma was separated and stored at y208C
to await hormone assay. During the experiment, the behaviour and reactions of the animals were noted in order to explain unexpected elevations of plasma cortisol.
2.3. Hormone determinations Blood plasma concentrations of progesterone were determined by a modified lumi-
Ž nescence immunoassay already validated for bovine plasma Forsberg et al., 1993;
. Duchens et al., 1995a . The intra-assay coefficient of variation was 7.04 and the
corresponding inter-assay variation was 10.6. The detection limit of the assay was 0.1 nmol l
y1
. Cortisol was measured by a Coat-A-Count, solid phase radioimmunoassay Ž
.
y1
Diagnostic Products, Los Angeles CA, USA . The detection limit was 5.5 nmol l .
According to the manufacturer, the antiserum shows low cross-reactivity with proges- Ž
. terone 0.15 . The intra-assay variation was between 2.2 and 6.3. The inter-assay
coefficient of variation was between 3.8 and 5.2. 2.4. Statistical analyses
The heifers served as their own controls, thereby reducing the influence of individual variation. Individual baseline levels of plasma progesterone and cortisol were calculated
Ž .
from the day 1 saline treatment analytical results plus the first three pretreatment samples of day 2. Mean hormone concentration SD was calculated for each animal.
Values within the limits of the mean SD were selected and subjected to a repeated calculation of mean SD with an additional exclusion of values beyond the range.
Altogether, this procedure was repeated three times to obtain a smoothed baseline, which would serve as a cut-off point for significant rises in individual hormone
concentrations. Baseline values are presented with range. Total production of proges- terone and cortisol after ACTH administration was calculated from the area under each
ŽŽ .
. respective curve according to the formula: R s Ý T q T
r2 300 min, where i s 0,
i iq30
30 . . . 300 min. The duration of significantly increased plasma hormone concentrations was determined in relation to individual baseline levels. Comparison of the mean group
data concerning total hormone production and duration of significantly increased plasma Ž
w
hormone concentrations was done by a Student’s t-test using Minitab Minitab Release
. 10.2, Minitab, PA, USA .
In accordance with the lower detection limit of the assays, cortisol concentrations were fixed at 5.5 nmol l
y1
and progesterone concentrations at 0.1 nmol l
y1
. The concentrations of cortisol and progesterone in the baseline samples were transformed to
Ž .
logarithms in base 10 and differences between groups tested by a repeated measures analysis of variance for effect of group, time and group-time interaction using the
Ž MIXED procedure of the statistical analysis systems package SAS Institute, Cary, NC,
. USA, 1996 . Least-square means were obtained for combination of effects and were
compared using Student’s t-test. Correlations between cortisol and progesterone concentrations were calculated for
baseline samples and after treatment, respectively. Results are presented as mean SEM. Differences P - 0.05 were considered signifi-
