Tion period samples had been frozen at (M tl and Palme.Europe PMC Funders Author Manuscripts Europe PMC Funders Author ManuscriptsHorm Behav. Author manuscript; accessible in PMC February .Stocker et al.PageBetween experiments there was a minimum of a twoweek period so that you can give the birds time for you to restabilize relationships within the group that were potentially disturbed because of the separation. Due to the fact hormone levels differ with age of the person and season (Breuner St e et al,we tested the initial group,of which all ravens hatched in ,among December and Might plus the second one,where all but two men and women hatched in ,among December and May well . All procedures had been authorized by the Austrian Federal Ministry of Science,Research and Economy (approval quantity: BMWFW.WFIIb). Deciding upon one of the most appropriate enzyme immunoassay ACTH challenge To date the enzyme immunoassay made use of to measure corticosterone metabolites (CM) in ravens had been validated only biologically (St e et al. To add a physiological validation and to test the possible suitability of a different,much more recently developed assay we performed an ACTH challenge on four ravens in September . Two knowledgeable researchers caught PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22065305 two males and two females out of their dwelling aviary. Subsequently,a veterinarian conducted a health check on all birds just before administering the ACTH (Synacthen: gml,Novartis) injection within the right brachial vein. Each person . ml of synthetic ACTH. Instantly following,the ravens had been released in malefemale pairs in two separate compartments of their house aviary and all droppings have been collected for the following four hours. Droppings were also collected two hours prior to capturing to measure the basal corticosterone metabolite concentration on that day. All samples were analyzed with two diverse enzyme immunoassays: (A) an oxoetiocholanolone assay (working with oxoetiocholanolone as typical and ML240 web antibodies raised in rabbits against androstaneol,dioneCMO: bovine serum albumin; sensitivity: ng CMg dropping,detailed assay description see M tl et al,which turned out to be one of the most proper assay for ravens following the biological validation (St e et al. This assay will not only crossreact with CO steroids but also with CO metabolites that possess a oloxo structure. The second assay (B) was a cortisone assay (using pregnene,diol,,trione as typical and antibodies raised in rabbits against pregnene,diol,,trioneHS: bovine serum albumin; sensitivity: ng CMg dropping,detailed assay description in St e et al,that is appropriate to measure CM excretion in many bird species e.g. northern wheatears,Oenanthe oenanthe (Eikenaar et al,chicken,Gallus gallus domesticus,and quail,Coturnix japonica (Rettenbacher et al. St e et al. An additional oxoetiocholanolone EIA (detailed assay description see Palme and M tl,was initially also taken into consideration,but currently the immunogram showed that this assay will not be suitable for measuring CM in raven droppings. Despite the fact that assay A at the same time as B could detect a CM peak (Figit seems that this peak is related for the short,but stressful occasion of catching as an alternative to to the ACTH treatment itself,which within the bird treated last happened min after catching. This time delay was brought on by the thorough health-related examination of every of your four birds before the injection. The time lag in between the get started of catching as well as the appearance from the CM peak ranges from to min with an . . fold CM boost in assay A,and from to min making use of assay B,Europe PMC Funders Author M.