Episodic Gonadotropin Secretion: MATERIALS AND METHODS
All animal rearing, handling, cannulation, and euthanasia procedures were approved by the University of Arkansas Institutional Animal Care and Use Committee. One-day-old broiler breeder chicks were used in two experiments. Birds were banded, vaccinated, and reared on a 23L:1D photoperiod with ad libitum food and water intake in floor pens. At 8 wk of age, males were placed on a restricted diet to maintain a weight gain of approximately 0.75% of their initial body weight per week. At the same time, photoperiod was reduced to 8L:16D as recommended by the breeder. At 20 wk of age, males were placed in a 14L:10D photoperiod that was gradually raised to 16L:8D by 28 wk of age. After 28 wk of age, 14 birds were transported to a surgery and animal isolation suite and confined in individual cages (0.6 X 0.5 X 0.5 m). Birds were provided with the same photoperiod and feed as described before and were checked at least twice each day for signs of stress or discomfort. At caging, birds were fitted with a canvas saddle ‘‘jacket’’ (~22 X 15 cm) similar to those previously described.
One week after caging, birds were cannulated via the jugular vein using a procedure less invasive than that previously described for chickens and similar to that described for use in turkeys. Cannulas, swivels, gauze, and needles used during the procedure were autoclaved before use. Silicone tubes were treated with TDMAC-heparin (Polysciences, Inc., Warrington, PA) to reduce thrombus formation and, therefore, assist in the maintenance of patent cannulas for the duration of the experiment. Sodium citrate (5 mg/ml) and gentamicin sulfate (0.5 mg/ml; Phoenix Pharmaceutical, Inc., St. Joseph, MO) were added to sterile physiological saline (saline-citrate) to prevent clotting and bacterial contamination of cannulas during blood sampling and reinfusion of blood cells.
Birds were restrained by wrapping the legs and wings with a medical-grade adhesive tape. After laying the bird on the surgical table, the neck was supported by placing a 7-cm plastic cylinder below the opposite side of the intended site of puncture. The lower third of the right side of the neck was locally anesthetized with 1 ml of 2% lidocaine hydrochloride and washed with 75% ethanol. Next, a 17-gauge needle with a 14-gauge catheter was inserted in the jugular vein, the needle was removed, and medical silicone tubing (length, 1.4 m; inner diameter, 0.64 mm; outer diameter, 1.19 mm; Helix Medical, Inc., Carpinteria, CA) was pushed 15 cm toward the heart. A 3-ml disposable syringe containing saline-citrate was connected to the opposite end of the tubing with a 18-gauge blunt needle and was used to flush the cannula before insertion in the jugular vein and to check blood flow after insertion. Next, the needle was removed, and a small sleeve (length, 1 cm) of silicone tubing (inner diameter, 0.77 mm; outer diameter, 1.65 mm) that was previously fitted with a 4-0 suture by going in and out on one side of the tube (0.5 cm apart) was placed around the wall of the cannula. The sleeve that served to anchor the cannula was sutured to the skin 0.5 cm cephalad from the point where the cannula emerged from the skin. A second sleeve was placed approximately 5 cm from the first and sutured to the skin after a half-loop of the cannula was formed between the two anchor points. The cannula was then threaded to the back saddle through latex tubing (inner diameter, 47 mm; outer diameter, 64 mm; Fisher Scientific International, Pittsburgh, PA) that was attached to the saddle and sutured to the skin close to the second sleeve. The cannula exited the saddle through a hole and was then threaded through an autoclavable button-tether-swivel unit (Instech Labs, Plymouth Meeting, PA). Birds were returned to their cages, and the spring tether (length, 60 cm) and the single-channel swivel (Instech 375 series) were mounted with a clamp on the top of the cage. The cannula was attached to the bottom of the swivel and looped around and down through the top of the tether. One end of a medical silicon tubing (length, 6 m; inner diameter, 0.77 mm; outer diameter, 1.65 mm) was connected to the other extreme of the swivel, and the other end was affixed to a syringe pump (KDS220; Fisher). The pump, which was located in a remote place out of sight of the birds, was calibrated to deliver a continuous infusion of 0.3 ml/h of the saline-citrate solution, except when blood samples were obtained (see below). This procedure allowed frequent blood sampling in unrestrained birds with free access to feed and water.
Experiment 1. Blood samples (1 ml) from four birds were collected at 10-min intervals for 4 h commencing on the day of cannulation (Day 0) and for 12 h on each of Days 1 and 2. On Day 0, sampling started 2-5 h after cannulation, and samples throughout the experiment were obtained during the photophase of the photoperiod. Samples were immediately centrifuged (8000 X g for 3 min). The plasma portions were stored at —20°C until LH, FSH, and testosterone concentrations were determined, and the red blood cells were reconstituted in saline-citrate. Between samples, the contents of the cannula were flushed, and an additional 1 ml of saline-citrate was returned to the bird. Every third sample (every 30 min), each bird received his own reconstituted red blood cells through the cannula. In addition, a single blood sample was obtained via a brachial wing vein immediately before cannulation and within 2 min after handling the birds. After cannulation, hourly blood samples were obtained from the cannula for 8 h on Day 0 and for 12 h on each of Days 1 and 2. Plasma was stored at —20°C until concentrations of corticosterone were determined.
At the end of Day 2, birds were killed, and the left and right testis were recovered, inspected for gross anatomy, and weighed.
Experiment 2. Blood samples (1 ml) from 10 birds were collected at 10-min intervals for 8 h commencing 1 day after cannulation. Samples throughout the experiment were obtained during the photophase of the photoperiod. Samples were immediately centrifuged (8000 X g for 3 min). The plasma portions were stored at —20°C until LH and FSH were determined, and the red blood cells were reconstituted in saline-citrate. Between samples, the content of the cannula was flushed, and an additional 1 ml of saline-citrate was returned to the bird. Every third sample (every 30 min), each bird received his own reconstituted red blood cells through the cannula.
The LH concentrations were measured by RIA using reagents provided by the USDA-ARS Animal Hormone Program. Briefly, sodium phosphate buffer (NaPO4; 20 |xl, 0.25 M, pH 7.4), 125I (500 |xCi in 2 |xl of NaPO4, 0.25 M), and chloramine-T (580 ng in 10 |xl NaPO4, 0.25 M) were added to a vial that contained 5 |xg of chicken LH (USDA-cLH-I-3). After 5 min of incubation, the reaction mixture was transferred to a Sephadex G-25 column (Bio-Rad, Hercules, CA) that was used to separate [125I]LH form free 125I.
Antiserum against LH (USDA-AcLH-5) was diluted 1:20 000 in PBS-BSA (0.1%) containing 1:400 normal rabbit serum (Sigma Chemical, St. Louis, MO). Two hundred microliters of the dilution was added to culture tubes (12 X 75 mm) containing standards (USDA-cLH-K-3; 0.5-6.4 ng) in 500 |xl of PBS-BSA or unknown (100 |xl of plasma plus 400 |xl of PBS-BSA). Iodinated LH (100 |xl; 30000 cpm in PBS-BSA) was added to each tube, and tubes were incubated for 48 h at 4°C. Then, 200 |xl of sheep anti-rabbit gamma globulin (1:40 dilution) were added. After incubation for 16 h at 4°C, 3.0 ml of PBS (4°C) were added to each tube. Tubes were centrifuged for 30 min (1900 X g), after which the supernatant was aspirated and the radioactivity in the pellet was quantified with a gamma counter.
Addition of 5 and 10 ng of LH to 1 ml of serum resulted in 108% and 114% recovery (n = 4). When different volumes of serum (50, 100, and 150 |xl) were assayed, concentrations were parallel to the LH standard curve. Inter- and intraassay coefficients of variation (CVs) were 8.2% and 7.2%, respectively (n = 4).
Concentrations of FSH in plasma (150 |xl) were quantified in duplicate by RIA. The USDA-cFSH-K-1 was used to prepare standards (0.25-8.0 ng) in 300 |xl of PBS-BSA (0.1%). The inter- and intraassay CVs were 2.4% and 9.7%, respectively (n = 4).
Concentrations of corticosterone were quantified by RIA similar to that previously described with the following modifications. Duplicate 200-|xl aliquots of assay buffer (PBS, 0.01 M, pH 7.0, with 0.1% gelatin) containing standards (0, 31.25, 62.5, 125, 250, 500, 1000, 2000, and 4000 pg/ml) or unknown plasma samples were extracted in borosilicate glass tubes (12 X 75 mm) with 2 ml of ethyl acetate by mixing at room temperature for 30 min. At 5 min after mixing, duplicate aliquots (1 ml) of the solvent layer were decanted in borosilicate glass tubes (12 X 75 mm), and the solvent was evaporated under nitrogen gas. Standards and samples were reconstituted in 400 |xl of assay buffer. Corticosterone antiserum was diluted 1:16 000 in assay buffer, and 100 |xl were added to each tube. Radioactively labeled [125I]corticosterone (ICN Pharmaceuticals, Inc., Costa Mesa, CA) was diluted in assay buffer, and 100 |xl (20 000 cpm) were added to each tube. After incubation for 24 h at 4°C, 200 |xl of sheep anti-rabbit gamma globulin (1:40 dilution) were added, followed by 500 |xl of 6% polyethylene glycol to each tube, and then incubated at 4°C for 60 min. Tubes were centrifuged for 30 min (1900 X g), the supernatant aspirated, and the radioactivity in the pellet quantified with a gamma counter. Addition of 10 pg of corticosterone to 1 ml of plasma resulted in 109% recovery (n = 4). When different volumes of plasma (50, 100, and 150 |xl) were assayed, concentrations were parallel to the corticosterone standard curve. Similarly, when different volumes of the supernatant (500 and 1000 ml) were assayed, concentrations were parallel to the corticosterone standard curve. Inter- and intraassay CVs were 13.8% and 7.7%, respectively (n = 4).
Testosterone was quantified using a solid-phase RIA (ICN testosterone kit; ICN Pharmaceuticals). The addition of 10 ng of testosterone to 1 ml of plasma resulted in 109% recovery (n = 8). When different concentrations of plasma were assayed, concentrations were parallel to the standard curve. Inter- and intraassay CVs were 1.7 and 4.9%, respectively.
Experiment 1. Analysis of variance and orthogonal contrasts were used to determine differences in LH, FSH, testosterone, and corticosterone between Day 0 and Day 1 and between Day 0 and Day 2.
The number of LH, FSH, and testosterone pulses was analyzed by using Pulsar (software modified for the PC by Gitzen and Ramirez, Urbana, IL). Considering the frequency at which samples were collected, we set G as 99 to exclude one sample pulse that, by our definition of pulses, cannot occur. Other G values were G = 3.6, G = 2.9, G = 2.5, and G = 2.1. Additionally, time-series analysis and the mean ± 1 SD were used to evaluate LH, FSH, and testosterone pulse frequency. Pulse amplitude was defined as the difference between the greatest value during the pulse and the nadir within 30 min before the pulse. Analysis of variance with split-plot units was performed to evaluate the number of pulses associated with each method on Day 1 and Day 2. The effect of method was in the main plot, and bird(method) was used to test significant method effects. Day of bleeding and the interaction of day with method were in the subplot, and the residual error was used to test significant effects of day and the interaction.
The coincidence between LH and FSH and between LH and testosterone pulses was determined by categorical data analysis. Endogenous FSH and testosterone pulses were considered to be associated with endogenous LH pulses (sensitivity) when the FSH or testosterone pulse occurred within the LH pulse length, as determined by Pulsar. The percentage of FSH and testosterone pulses that were not associated with LH pulses was called the false-positive rate, and the percentage of LH pulses that were not associated with FSH and testosterone pulses was called the false-negative rate.
Experiment 2. The number of LH and FSH pulses was analyzed by using Pulsar. The same G parameters described in experiment 1 were used. Pulse amplitude for LH and FSH was defined as the difference between the greatest value during the pulse and the nadir within 30 min before the pulse. The coincidence between LH and FSH pulses was determined by categorical data analysis. Endogenous FSH pulses were considered to be associated with endogenous LH pulses (sensitivity) when the FSH pulse occurred within the LH pulse length, as determined by Pulsar. The percentage of FSH pulses that were not associated with LH pulses was called the false-positive rate, and the percentage of LH pulses that were not associated with FSH pulses was called the false-negative rate.