Brains were sectioned by microtome at 40 m in the coronal plane and every third section was Nissl stained

Brains were sectioned by microtome at 40 m in the coronal plane and every third section was Nissl stained. either implanted with T (T-treated; N=8) or received empty implants (Controls; N=8) for 21 days. After capture in Papallacta, birds were acclimated to the housing 5,15-Diacetyl-3-benzoyllathyrol facility for 14 to 25 days (depending on capture date) prior to implantation (implantation occurred on experiment Day 0). Three days prior to implantation (Day -3), body mass (Pesola spring scale to nearest 0.1 g), wing chord length (wing rule to nearest mm), CP length, and fat score (scale 0C3 based on visual inspection of the furcular and abdominal regions), were measured in all birds and a ~250 l blood sample was collected (see below). Also on Day -3, birds were separated into T-treated or Control groups balanced by body size and condition. On Day 0, T-treated birds received two 10mm T-filled subcutaneous silastic implants (1.47mm inner diameter; Dow Corning) along the left flank and Control birds received two empty (control) silastic implants of the same size (see below for surgical details). Similar sized implants were previously used in this species to 5,15-Diacetyl-3-benzoyllathyrol elevate plasma T to high, physiologically relevant, breeding levels [Moore et al., 2004b]. On Day 21 5,15-Diacetyl-3-benzoyllathyrol post-implantation, body mass, CP length, and fat score were measured and a second blood sample was collected and stored. Birds were given a terminal intramuscular injection of 7.5 mg sodium pentobarbital and transcardially perfused with 0.9% heparinized saline (150 IU/10 ml) followed by 10% neutral buffered formalin. Brains and testes were removed within five minutes of perfusion. Brains were post-fixed in 10% formalin, and were stored under refrigeration until delivery to the University of Washington (Seattle, WA). Post-perfusion, the gonads were removed and testis diameter and lengths were measured (to 0.01mm using digital calipers) and volume was calculated using the formula for ellipsoid cylinders (4/3*(0.5 width)2*(0.5 length)). Experiment 1 (capture to tissue collection) occurred during the non-breeding period in the subjects source population (Papallacta). Experiment 2 To determine whether T induced growth of the song nuclei occurred through androgen and/or estrogen receptors, birds were treated with androgen receptor antagonist flutamide 5,15-Diacetyl-3-benzoyllathyrol (FLU), and/or ATD, which inhibits the aromatase enzyme necessary for conversion of androgens to estrogens. After capture in Pintag, birds were acclimated to the housing facility for 65C75 days prior to implantation. Three days prior to implantation (Day -3) a blood sample was collected for all birds and wing chord length, body mass, and fat scores were used to separate birds into four groups (n=6 per group: T treated (T-treated), T with FLU (T+FLU), T with FLU and ATD (T+FLU+ATD), and Controls. Due to limited facilities and birds, we did not include a T with ATD group. On Day 0, T-treated birds received two 10 mm T-filled and four empty silastic implants (one T-filled and two blanks along each flank). T+FLU birds received two 10 mm T-filled, two 10 mm FLU-filled, and two empty silastic implants (one of each type along each flank). T+FLU+ATD received two 10 mm T-filled, two 10 mm FLU-filled, and two 10 mm ATD-filled silastic implants (one of each type along each flank). Controls received six empty 10 mm silastic implants (three along each flank). Surgical sites were checked on Days 2, 7, and 14. On Day 27, body mass, CP length, and fat score were measured, a second blood sample was collect, the birds were perfused, and their brains were stored as previously described. The testes were measured as above and were also weighed using a digital scale. Three birds were removed Cav3.1 from the study, one T+FLU and two T+FLU+ATD, due to a lack of healing at the site of implantation and their repeated loss of implants. The capture of subjects for Experiment 2 and acclimation to the facilities occurred during the nonbreeding season in the source population (Pintag) but hormone treatment occurred during the early breeding season. Behavioral observations During both experiments individual behavior was monitored on three separate days (Exp 1: days 4, 11, 18; Exp 2: days 6, 16, 25) starting within 30 minutes of dawn. Observations were conducted from behind a blind positioned five meters either east or west of the aviary. Each of the two positions allowed for half of the birds in the aviary to be observed at one time (equal numbers of each treatment). When observations on one side were completed, observations on the other side were started 15 minutes after moving between the blinds to allow the birds to recover from the disturbance. Individuals on each side of the aviary were observed for singing rate for 24 minutes (Experiment 1) or 36 minutes (Experiment 2) totaling 48 and 132 minutes of observation time.

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