Induction of ovulation in cows with affinity chromatography-purified Human Chorionic Gonadotropin (hCG)

Abstract

Objective of the present study was to purify hCG contained in commercial preparation (Pregnyl® - Organon) by affinity chromatography and to evaluate ovulation rates in cows injected with the purified hormone. A chromatography column containing concanavalin-A sepharose (Con-A) matrix was equilibrated and one hundred thousand international units (UI) of Pregnyl® were applied to the column. Fractions of 3,8µL were collected (4°C) every 5 minutes for 12,5h, to yield a total of 150 fractions. After fraction 76, a column buffer containing 0,3M ±-methylglicoside was added to the column. Protein concentrations were estimated by espectrophotometry (280nm). Two peaks of absorbance were observed, between fractions 14 and 19, and fractions 90 and 100. Fractions 14-19 and 90-150 were sequentially grouped in pairs concentrated by ultrafiltration and analyzed by SDS-PAGE. The first peak of absorbance resulted from proteins contained in the commercial product, eluted from the column and not adsorbed by the Con-A matrix. The second peak represented proteins adsorbed by the column, especially hCG. The fractions containing hCG were grouped in a single sample and protein concentration was measured by the Lowry method. Cows in day 5 of the estral cycle, which had a dominant follicle larger than 8mm, were injected with 0,1, 0,2 or 0,3mg protein of the purificated sample. The ovulation rate 48 hours after treatment was 0%, 50% and 75%, respectively. It was demonstrated that relative concentration of hCG can be increased by affinity chromatography and biological activity of purified hCG was satisfactory preserved.