The day of ovulation was defined as the day when the largest follicle disappeared between two consecutive days of ultrasonographic evaluation of ovarian structures. To quantify concentrations of progesterone in plasma, blood samples were collected every 12 h from the time of the second treatment of PGF2a until Day 28 or the time of detection of the subsequent behavioral estrus.
Samples of blood for quantitation of progesterone were collected in tubes treated with 30% EdTa (50 ^l for 10ml blood sample) and were centrifuged at 2700 X g for 20 min immediately after collection to minimize degradation of progesterone. Plasma was then harvested and stored at -20°C until assayed for concentrations of progesterone. Samples collected for quantification of LH were allowed to clot at room temperature and then were stored at 4°C for 24 h; they were then centrifuged at 2700 X g for 15 min. Serum was subsequently decanted and stored at -20°C until assayed for concentrations of LH.
Concentrations of progesterone in plasma were analyzed by RIA. Assay procedures included use of a monoclonal antibody to P4-11-BSA (Bios-Pacific, Emeryville, CA), progesterone-11a-hemisuccinate trimethyl ester as radiolabeled tracer, and progesterone (Sigma Chemical Co., St. Louis, MO) as standard.
Intra- and interassay coefficients of variation were 8% and 4%, respectively. Concentrations of LH in serum were analyzed by RIA using an antibody against LH (TEA-RaOLH#35), highly purified ovine LH (LER-1374A) as radiolabeled tracer, and NIH-LH-B7 as standard. Intra- and interassay coefficients of variation were 7% and 11%, respectively.
The experiment was a completely randomized design. Maximum diameter (in millimeters) of the CL was analyzed using the General Linear Models procedure of SAS, including the fixed effects of treatment and day in the model. Function of the CL was analyzed by estimating the total area under the profile of progesterone concentrations in plasma during the estrous cycle. Total progesterone under the curve (arbitrary units) was evaluated using a mathematical equation to determine area and was analyzed with a General Linear Models procedure of SAS considering treatment as the main effect. Comparisons among treatment least-squared means were performed by orthogonal contrasts.