title: Coumestrol and its metabolite in mares' plasma after ingestion of phytoestrogen-rich plants: potent endocrine disruptors inducing infertility.
abstract: Phytoestrogens exist in plants that are present in forages fed to horses. They may compete with 17-β estradiol and influence the estrous cycle. Therefore, the objective was to determine whether coumestrol from clover-mixed pastures is present in mare's plasma after their ingestion (experiment I), and when this phytoestrogen was present in mare's plasma after ingestion (experiment II). The effect of a long-term ingestion of phytoestrogens on estrous cycle disruption was assessed (experiment III; clinical case). Experiment I was carried out in nonpregnant anestrous and cyclic Lusitano mares (n = 14) kept on clover and grass-mixed pastures, and supplemented with concentrate and hay or cereal straw. Blood and feedstuff were obtained from November to March. In experiment II, stabled cyclic Lusitano mares (n = 6) were fed for 14 days with increasing amounts of alfalfa pellets (250 g to 1 kg/day). Sequential blood samples were obtained for 8 hours after feed intake on Day 0 (control) and on Days 13 and 14 (1 kg/day alfalfa pellets). Experiment III mares were fed with a mixture of alfalfa and clover haylage for 5 months (group 1; n = 4) or for 9 months (group 2; n = 12). Estrous cycle was determined on the basis of plasma estradiol (E2), progesterone (P4), and ultrasound (experiment III). Concentrations of phytoestrogen coumestrol and its metabolite methoxycoumestrol were determined by high-performance liquid chromatography coupled with mass spectrometry. Phytoestrogens decreased in pasture from November until March (P < 0.01) (experiment I), but were always detected in mares' plasma. In experiment II, plasma-conjugated forms of coumestrol and methoxycoumestrol were higher on Days 13 and 14 than in control (P < 0.05). The highest concentrations of conjugated form of coumestrol were at 1.5 and 4 hours (P < 0.001), whereas its free forms peaked at 1 and at 3.5 hours after ingestion (P < 0.05). Methoxycoumestrol-conjugated form concentration was the highest at 1.5 and 5 hours (P < 0.001), whereas its free form peaked at 1 hour (P < 0.05) and at 1.5 hours (P < 0.001). Long-term intake of coumestrol caused lack of ovulation, uterine edema, and uterine fluid accumulation (experiment III). Coumestrol and methoxycoumestrol in both forms were higher in group 2 (while still ingesting haylage) than in group 1, after haylage withdrawal (P < 0.001). These data show that in the mare, coumestrol and its metabolite increase in blood after ingestion of estrogenic plants and can influence reproduction in mares as potent endocrine disruptors.
authors.affiliation: CIISA, Faculty of Veterinary Medicine, Technical University of Lisbon, Lisboa, Portugal. gmlfdias@fmv.utl.pt
authors.forename: G
authors.initials: G
authors.lastname: Ferreira-Dias
authors.forename: M
authors.initials: M
authors.lastname: Botelho
authors.forename: A
authors.initials: A
authors.lastname: Zagrajczuk
authors.forename: M R
authors.initials: MR
authors.lastname: Rebordão
authors.forename: A M
authors.initials: AM
authors.lastname: Galvão
authors.forename: P Pinto
authors.initials: PP
authors.lastname: Bravo
authors.forename: K
authors.initials: K
authors.lastname: Piotrowska-Tomala
authors.forename: A Z
authors.initials: AZ
authors.lastname: Szóstek
authors.forename: W
authors.initials: W
authors.lastname: Wiczkowski
authors.forename: M
authors.initials: M
authors.lastname: Piskula
authors.forename: M J
authors.initials: MJ
authors.lastname: Fradinho
authors.forename: D J
authors.initials: DJ
authors.lastname: Skarzynski
mesh_terms: D000821:Animal Feed / Q000032:analysis / Q000633:toxicity*; D000818:Animals; D003375:Coumestrol / Q000097:blood* / Q000633:toxicity*; D004435:Eating / Q000502:physiology; D052244:Endocrine Disruptors / Q000633:toxicity*; D030762:Estrous Cycle / Q000187:drug effects; D005260:Female; D006736:Horses / Q000097:blood; D007246:Infertility / Q000139:chemically induced* / Q000662:veterinary; D010060:Ovulation / Q000187:drug effects; D048789:Phytoestrogens / Q000008:administration & dosage / Q000097:blood / Q000633:toxicity*; D010944:Plants / Q000737:chemistry / Q000633:toxicity
chemical_list: D052244:Endocrine Disruptors; D048789:Phytoestrogens; D003375:Coumestrol
keywords: Alfalfa; Clover-mixed pastures; Coumestrol; Hyperestrogenicity; Mare; Phytoestrogens