Clinical changes in ovariectomized ewes exposed to phytoestrogens and 17 beta-estradiol implants.

Eight Swedish Finewool Landrace ewes, ovariectomized 5 months earlier and kept on nonestrogenic hay, were each fed 3.5 kg red clover silage, corresponding to 6.1 g phytoestrogens (of which 3.5 g was formononetin) per day, for 14 days in November (short days). In January (short days), two groups (3 each) of these ewes received one or two 17 beta-estradiol sc implants. In May (long days), one of two new groups (4 each) of these ewes was reexposed to phytoestrogens for another 14 days while the other served as a control. Physical examination of ewes for changes in reproductive organs was carried out two or three times per week during each feeding/treatment, and continued until observed changes disappeared. Clinically significant changes occurred in the reproductive organs of ewes fed red clover. Vulva color changed from pale to pink and red, and there were enlargements of the vulva, uterus, and udder. In addition, teat length and circumference increased, and secretion of milky fluid began. These changes were similar, but more pronounced during treatment with 17 beta-estradiol, particularly teat circumference. The changes in vulva were more dramatic in May than in November and resembled those observed in ewes treated with estradiol. Our data show that a daily intake of 3.5 g formononetin for 14 days caused the increase of teat size and changes in the color of the vulva and in uterus weight in ovariectomized ewes.

Effects of oestrogenic silage on some clinical and endocrinological parameters in ovariectomized heifers.

The influence of phytoestrogens was studied in 3 ovariectomized Swedish Friesian heifers fed 20 kg of 100% red clover silage per heifer/day for 14 days. Behaviour, reproductive organs and pituitary response to exogenous gonadotropin-releasing hormone (GnRH) injections were monitored. Clinical effects like oedema and mucous discharge in the vulva, presence of milky fluid in the mammae and increases in teat size and the cross-sectional distance of the uterus were observed in heifers fed red clover silage. Fluid accumulation in the uterus, visualized by means of ultrasonography, had still not disappeared 30 days after the red clover silage had been completely withdrawn. Red clover silage appeared to reduce the magnitude and duration of the pituitary response to GnRH injections.

Liquid chromatographic determination of the estrogens daidzein, formononetin, coumestrol, and equol in bovine blood plasma and urine.

A liquid chromatographic (LC) method is described for the determination of the plant estrogens diadzein, formononetin, and coumestrol and the estrogenically active metabolite equol in bovine blood plasma and urine. The blood and urine samples are incubated overnight with and without beta-glucuronidase/sulfatase for analysis of both free and conjugated forms of estrogens. Samples are applied to Extrelut columns, extracted with ethyl acetate, and evaporated to dryness. Residues from urine samples are dissolved in methanol, diluted with water, acidified with HCl, and purified by injection through a Sep-Pak C18 cartridge. This eluate is used for LC analysis. Residues from blood samples are dissolved in benzene-petroleum ether (1 + 1), extracted with ammonium hydroxide, acidified with glacial acetic acid, and extracted with ethyl acetate. The ethyl acetate extract is evaporated, dissolved in 80% methanol, injected onto a LC reverse-phase column, and separated in a linear gradient system between 40 and 80% methanol in phosphate buffer. Quantitation is performed by means of UV and fluorescence responses. The method was sensitive enough to determine 0.4 ng/mL of daidzein and formononetin and 0.1 and 13 ng/mL of coumestrol and equol, respectively, in blood, and 130, 80, and 7 ng/mL of daidzein, formononetin, and coumestrol, respectively, and 4 micrograms/mL of equol in urine. The applicability of the method was checked by the determination of total and free plant estrogens in blood samples from a dairy cow fed a normal diet.