Hereford-cross beef heifers (n=30), 15 to 20 months of age and weighing between 235 and 405 kg (average 336 kg), were chosen from a herd of 51 heifers maintained in outdoor pens at the University of Saskatchewan Goodale Research Farm (52° North and 106° West). Heifers were fed alfalfa/grass hay and concentrate to gain approximately 1.3 Kg per day and had water ad libitum during the experimental period from October to December. Heifers were initially examined by transrectal ultrasonography (MyLab5 VET, Canadian Veterinary Imaging, Georgetown, Ontario Canada) to detect the presence of a CL (i.e., confirm post-pubertal status; . Animal procedures were performed in accordance with the Canadian Council on Animal Care and were approved by University of Saskatchewan Protocol Review Committee.
Treatments and examinations
Heifers in which a CL was detected were given two luteolytic doses of PGF (12 h apart) and monitored by ultrasonography for ovulation. Five to eight days later, the two largest ovarian follicles were ablated by transvaginal ultrasound-guided follicular aspiration to synchronize follicular wave emergence which was expected to occur 1 to 1.5 days later [19, 20]. Prostaglandin (500 μg cloprostenol, Estrumate, Schering-Plough Animal Health, Pointe-Claire, QC, Canada) was given intramuscularly at 60 and 72 h after follicular ablation to induce regression of the CL and shift from a non-ovulatory to an ovulatory follicular wave . At the time of follicular wave emergence (Day 0; i.e., 1.5 days after follicle ablation), heifers were assigned randomly to two groups and given an intravaginal device containing 1 g of letrozole (letrozole group, n=15) or a placebo (letrozole-free) intravaginal device (control group, n=15). Devices were inserted on Day 1 and were kept in place until Day 7 or until ovulation was detected, which ever occurred first.
Intravaginal devices were prepared using a Cue-Mate spine (Bioniche Animal Health, Bellville, ON, Canada) assembled with two blank (progesterone-free) silicone pods that were coated with a gel-based vehicle containing letrozole or vehicle only (control). The vehicle contained the following (all ingredients % w/w): letrozole 10%, gelatin 20% (Gelatin type B, Fisher Scientific, Pittsburgh, PA, USA), polymer 65% (prepared by mixing distilled water 68%, Poloxamer 188 12% and Poloxamer 407 20%, both from Spectrum Chemical, New Brunswick, NJ, USA) and distilled water 5%.
The observations from ultrasound examinations were recorded on a sketch sheet in which each ovary and its structures (CL and follicles ≥ 4 mm in diameter) were represented by size and location . Ovulation was defined as the disappearance of any follicle ≥8 mm between two consecutive daily examinations and was confirmed by the subsequent development of a CL . Follicular wave emergence was defined as occurring 1.5 days after follicular ablation . The dominant follicle of a wave was defined as the largest antral follicle of that wave .
Collection of blood samples
Blood samples were collected by coccygeal venipuncture into 10 mL heparinized vacuum tubes (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA). Samples were collected daily from pre-treatment follicular wave emergence (Day 0) to 12 days after the subsequent ovulation. In a subset of letrozole-treated animals (n=4), frequent blood samples were collected using an in-dwelling jugular catheter, as previously described , at the time of catheter placement and 0, 10, 20, 30 min, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h after treatment for measurement of plasma letrozole concentration. Blood samples were centrifuged at 1500 × g for 20 min and plasma was separated and stored in plastic tubes at -20°C.
Plasma LH concentrations were determined in duplicate using a double-antibody radioimmunoassay (NIDDK-bLH4) [25, 26]. The minimum and maximum values along the standard curve were 0.06 and 8 ng/mL, respectively. All samples were analyzed in a single assay; the intra-assay coefficient of variation was 11.4% for low reference samples (mean, 0.9 ng/mL) and 12.2% for high reference samples (mean, 2.1 ng/mL).
Plasma FSH concentrations were determined in duplicate using a double-antibody radioimmunoassay using NIDDK-anti-oFSH-1 primary antibody and expressed as USDA bovine FSH-Il units [25, 26]. The minimum and maximum values along the standard curve were 0.12 and 16 ng/mL, respectively. All samples were analyzed in a single assay; the intra-assay coefficients of variation were 7.9 and 6.5%, for low (mean, 2.4 ng/mL) and high reference samples (mean 4.9 ng/mL), respectively.
Plasma concentrations of estradiol were determined using a commercial radioimmunoassay kit (Double Antibody Estradiol; Diagnostic Products Corp., Los Angeles, CA, USA). The procedure was carried out at the Department of Animal Health and Biomedical Sciences, University of Wisconsin–Madison, as previously described [27, 28], with the following modifications: Standards (0.78–100 pg/mL) were prepared in steroid-free (charcoal-treated) bovine plasma. The standards (250 μL in duplicate) and plasma samples (500 μL in duplicate) were extracted with 3 mL of diethyl ether, frozen in a dry-ice/methanol bath, decanted into assay tubes, and dried overnight under a fume hood. The dried samples and standards were re-suspended with 100 μL of assay buffer (0.1% gelatin in PBS). The intra- and inter-assay coefficients of variation were 10.5 and 10.6% for high reference samples (mean 11.1 pg/mL), and 14.8 and 12.3% for low reference samples (mean 2.6 pg/mL), respectively. The sensitivity of the assay was 0.1 pg/mL.
Plasma progesterone concentrations were determined in duplicate using a commercial solid-phase radioimmunoassay kit (Coat-A-Count; Diagnostic Products Corporation, Los Angeles, CA, USA). The range of the standard curve was 0.1 to 40.0 ng/mL. All samples were analyzed in a single assay; the intra-assay coefficients of variation were 9.7% and 5.8 % for low- (mean, 1.7 ng/mL) and high-reference samples (mean, 18.7 ng/mL), respectively.
Plasma letrozole concentrations were quantified using liquid chromatography tandem mass spectrometry (LCMS/MS), as described . Briefly, letrozole was extracted from 250 mL plasma with 250 mL of 0.1M ammonium acetate followed by the addition of 5 mL methyl t-butyl ether (MTBE) and vortexed for 15 s. The organic layer was removed and transferred to a fresh 15 mL plastic tube and dried by gentle nitrogen gas flow. The dried extract was reconstituted in 1 mL of 100% ethanol. Separation was accomplished by HPLC (Agilent 1200; Agilent, Santa Clara, CA, USA) fitted with an analytical column (50 × 2.1 mm, 3 mm particle size; Betasil C18; Thermo Scientific, Waltham, MA, USA) operated at 35°C. Mass spectra were collected using a tandem mass spectrometer (SCIEX 3000; Applied Bioscience, Foster City, CA, USA) fitted with an electrospray ionisation source, operated in the negative ionisation mode. Quantification was performed using Analyst 1.4.1 software provided by SCIEX (Applied Bioscience). The minimum and maximum values along the standard curve were 0.25 and 500 ng/mL, respectively. The limit of quantification used in this method was 0.25 ng/mL and the mean recovery was 70%.
The following pharmacokinetic parameters were determined: C
(maximum observed plasma concentration of letrozole), t
(time to reach C
), t1/2 (terminal elimination half-life), AUC (area under the plasma letrozole concentration-time curve from zero to infinity calculated as AUC
). The concentration of letrozole in plasma as a function of time (C–t) data for each heifer was analyzed by non-compartmental techniques using a computer modeling program (WinNonLin Standard Edition Version 2.1, Pharsight Corporation, Mountain View, CA, USA). Peak letrozole concentration in plasma (Cmax) and time to peak letrozole concentration (tmax) were determined using observed values. The apparent terminal rate constant (λ) was determined by linear regression of the last 6–8 points on the terminal phase of the logarithmic plasma concentration vs time curve. The area under the C–t curve until the final plasma sample (AUClast) was determined using the linear trapezoidal rule. The total area under the curve extrapolated to infinity (AUC0-∞) was calculated by adding the Clast obs/λ + AUClast. The terminal half-life (T1/2λ) was calculated as ln2 ⁄λ. The mean residence time (MRT) was calculated as the area under the moment curve extrapolated to infinity (AUMC0-∞) ⁄AUC0-∞. Systemic clearance (ClS) was determined using the dose divided by AUC0-inf. The apparent volume of distribution (Vλ/f) was calculated by clearance divided by λ. Absolute bioavailability was calculated by comparing letrozole AUClast obtained using intravaginal devices to the AUClast obtained after a single iv injection of letrozole (unpublished data), corrected by dose (Bioavailability = (AUClast intravag/Doseintravag)/(AUClast iv/Doseiv) × 100).
Statistical analyses were done using the Statistical Analysis System software package (SAS Learning Edition 9.1, 2006; SAS Institute Inc., Cary, NC, USA). Time-series data (hormone concentrations, follicle and CL diameter profiles) were analyzed by repeated measures using the PROC MIXED procedure. The main effects were treatment (letrozole and control), time, and their interactions (noted in figures as treatment, day and treat*day, respectively). Single-point measurements (dominant follicle diameter at device placement, maximum diameter of extant dominant follicle, intervals from ablation to wave emergence, and from device placement to ovulation) were analyzed by t-tests. Individual time point comparisons between treatment groups were performed using least significant difference test. Significance was defined as P ≤ 0.05.