Phytoestrogen coumestrol and its metabolite in mares' plasma after clover mixed pasture and alfalfa pellets ingestion

Abstract

Phytoestrogens are estrogens present in forages fed to horses. Phenolic rings in phytoestrogens enable their binding to cells estrogen receptors and may compete with 17-β-estradiol and influence the estrous cycle. The aim of the study was to determine whether phytoestrogens from clover mixed pasture and alfalfa are present in mare's plasma after their ingestion. In experiment I, Lusitano mares (n=14) kept on mixed pastures with clover and grasses were bled monthly, from November to March. Pasture was collected, lyophilized and frozen. In Experiment II, stabled mares (n=6) were fed with increasing amounts of alfalfa pellets (from 250 g to 1 kg/day), added to their regular feed, for 14 days. They were bled before alfalfa inclusion (control), and on days 13 and 14 at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7 and 8 hours after feed intake. Coumestrol and its metabolite 4′-metoxycoumestrol concentrations were determined in plasma and feeds by HPLC. Data were analyzed by one-way ANOVA. Phytoestrogens were present in pasture and alfalfa samples, being 4′-metoxycoumestrol in the highest concentration. They decreased in pasture from November until March (P<0.01). However, they were always identified in mares' plasma, regardless of feeding system. In Experiment II, both active and conjugated forms of coumestrol and 4′-metoxycoumestrol in plasma were higher on days 13 and 14 than in control samples (P<0.05). Coumestrol (free form) rose and reached a peak at 3.5 h after ingestion (P<0.05), while its conjugated form peaked at 1.5 and 4 h (P<0.001). The free form of its metabolite peaked at 1.5 h and the conjugated form at 0.5, 1.5 and 5 h (P<0.001). These findings show that, in the mare, coumestrol and its metabolite increase in blood after estrogenic plants ingestion. Further studies should be carried out to evaluate their influence on reproduction in mares.