Effect of adrenal stimulation on bovine plasma steroids

Brunner, Donaldson & Hansel (1969) showed that ACTH suppressed corpus luteum formation when administered during Days 2 to 8 of the bovine oestrous cycle. We found that adrenal suppression by betamethasone treatment from Day 10 increased the length of the bovine oestrous cycle: plasma concentrations of oestradiol and androstenedione were unaltered but testosterone levels were reduced by 13 % (Kanchev, Dobson, Ward & Fitzpatrick, 1976). The present experiment was undertaken to investigate the effect on plasma steroids of adrenal stimulation achieved by a synthetic adrenocortico- trophin (ACTH). Five Friesian heifers, 36 months old, were given an intramuscular injection of 2 mg Synacthen Depot (Ciba) equivalent to 200 i.u. ACTH. They were bled by jugular venepuncture before and 2, 6 and 24 h after injection. Plasma was stored at —15°C until analysed. Progesterone and testosterone were measured by the methods described in detail by Kanchev et al (1976). Cortisol was determined in 0-2 ml samples to which 1 ml ethanol was added. After centrifugation the supernatant was decanted and analysed. The antibody AB2 (raised against cortisol-3-CMO-BSA) was donated by Dr P. H. Rowe and had low cross-reactions with other steroids known to occur in bovine plasma (Cook, Rowe & Dean, 1973). The antibody (01 ml at 1:1000) was incubated overnight with 0-1 ml (1,2- 3H)cortisol (sp. act. 55 Ci/mmol : Radiochemical Centre, Amersham) and the bound and free fractions were separated with 1 ml 2-5% charcoal. The average (± S.E.M.) recovery of (3H)cortisol added to plasma samples as internal standard in 8 assays was 80-6 ± 4-6%. The water blank was <0-5 ng/ml. When known amounts of cortisol were added, the recovery was 102 ± 17 %. The intra- and inter-assay coefficients of variation were 13-3% and 9-9% respectively. Chromatography is not obligatory with this method, as it is with those involving competitive protein binding, isotope dilution or spectro- photometry, and because cortisol is the principal secretory product of the bovine adrenal cortex (Venkataseshu & Estergreen, 1970) this radioimmunoassay should give an accurate assessment of adrenal function.