The mean milk-ejection interval was calculated for the first six milk ejections to predict the time of the seventh milk ejection. One min before the expected seventh milk ejection, the rats were treated with either 5 (JLg relaxin in 0.1 ml saline (n = 8), saline alone (n = 8), or a solution of inactivated relaxin in saline (n = 5). Thirty seconds before the predicted seventh milk ejection, the withdrawal pump was switched on, and blood samples were withdrawn at a rate of approximately one sample every 510 sec. A total of 2 ml was collected from each rat. The timing of the samples was marked on the polygraph record of intramammary pressure. The pump was switched off, and the extension cannula was cut into separate blood samples, which were then transferred to heparinized microtubes and centrifuged. The plasma was stored as described for experiment 1.
Plasma concentrations of oxytocin were measured in unextracted plasma samples using a specific RIA. Rabbit anti-porcine antiserum (85/F2) was prepared by Dr. S. Birkett, Department of Anatomy, University of Bristol, Bristol, UK. The lower limit of detection was 8 pM, and the inter- and intraassay coefficients of variation were 9.2% and 4.6%, respectively, at 20 pM, and 7.1% and 3.5% at 100 pM. Cross-reactivity was 12.5% for mesotocin and 0.03% for all other pituitary and hypothalamic peptides, including oxytocin and vasopressin neurophysins. buy prednisone
In unsuckled rats, mean plasma oxytocin concentrations in relaxin-treated rats were compared with data from saline-treated rats (ANOVA). In the suckled rats, mean oxytocin concentrations were compared between each of the time periods, and a comparison was made between relaxin-treat-ed, saline-treated, and inert-relaxin-treated groups (ANOVA). All data are expressed as mean ± SEM, and significant differences were determined using the Bonferroni’s test at the 5% level.