Free and conjugated isoflavones in the plasma of sheep following ingestion of oestrogenic clover

1967 ◽  
Vol 18 (4) ◽  
pp. 647 ◽  
Author(s):  
DA Shutt ◽  
A Axelsen ◽  
HR Lindner
Keyword(s):  
Plasma Level ◽  
Total Concentration ◽  
Red Clover ◽  
Subterranean Clover ◽  
Inhibitory Effect ◽  
Water Soluble ◽  
Biochanin A ◽  
Uterine Weight ◽  
Sheep Grazing

The plasma of sheep grazing various species of oestrogenic clover contained water-soluble conjugates of daidzein, formononetin, genistein, and biochanin A, chiefly in the form of glucuronides, in concentrations up to 40 µg/100 ml. In sheep pen-fed on red clover or subterranean clover (cv. Dinninup and Yarloop) the total concentration of these four isoflavones 3–5 hr after feeding ranged from 150 to 720 µg/100 ml plasma, of which 1–2% only was present in "free" (i.e. unconjugated) form; by 24 hr the plasma level had fallen to < 5 µg/100 ml plasma. Preconditioning the pen-fed sheep to oestrogenic clover, or adding lucerne to the diet, had no significant effect on the concentration of "free" or conjugated isoflavones in the plasma and had no inhibitory effect on the uterine weight response. Comparison of the composition of circulating isoflavones to that of the clover ingested indicated partial demethylation of formononetin to daidzein and of biochanin A to genistein, and suggested that genistein and biochanin A were degraded more rapidly than formononetin or daidzein in the sheep. Equol, a metabolite previously isolated from the urine of sheep treated with formononetin, was present in the plasma at a concentration of 50–170 µg/100 ml 24 hr after feeding.

Study of the fate of phyto-oestrogens in the sheep by determination of isoflavones and coumestrol in the plasma and adipose tissue

1967 ◽  
Vol 18 (2) ◽  
pp. 305 ◽  
Author(s):  
HR Lindner
Keyword(s):  
Adipose Tissue ◽  
Plasma Levels ◽  
Growth Response ◽  
Red Clover ◽  
Subterranean Clover ◽  
Biochanin A ◽  
Blood Levels ◽  
Sheep Grazing ◽  
Fat Depots ◽  
Uterine Growth

A method is presented for the specific determination in plasma or adipose tissue of sheep of the phyto-oestrogens genistein (G), biochanin A (BA), formononetin (F), daidzein (D), pratensein, and coumestrol. The method is based on selective extraction, paper chromatography, spectrophotometry and fluorometry, and use of radioactive recovery standards. A modification including gas chromatography is also described. Intraruminal administration to sheep of the synthetic isoflavones G, BA, or F gave rise within 2½ hr to detectable blood levels of the compound infused. In addition, intraruminal administration of BA and F was followed by the appearance of G and D, respectively, in blood and fat depots. Such O-demethylation at the C4 position was also observed after intramuscular injection of BA or F, and in sheep grazing subterranean clover (Trifolium subterraneum L.) and red clover (T. pratense). Plasma levels of free G above 5 µg/100 ml (excluding circulating isoflavone conjugated as glucuronide) were associated with maximal uterine growth response in ovariectomized ewes, while levels of 1–5 µg/100 ml appeared to elicit a graded uterine response; free plasma F and D levels above 0.5 �g/100 ml seem to be required for detectable uterotrophic action in the 5-day assay used. G, BA, F, and D were present in the plasma of sheep grazing subterranean clover var. Yarloop in concentrations adequate to explain the observed uterine growth response, but in sheep grazing red clover, the plasma levels seemed too low to account fully for the oestrogenic action of this pasture. No free isoflavones were detected in plasma from sheep grazing subterranean clover var. Marrar, despite the high isoflavone content of the pasture.

Adaptation by sheep to isoflavones in clover

1969 ◽  
Vol 9 (41) ◽  
pp. 569 ◽  
Author(s):  
FHW Morley ◽  
D Bennett ◽  
A Axelsen
Keyword(s):  
Subterranean Clover ◽  
Biochanin A ◽  
Uterine Weight ◽  
Pre Treatment ◽  
High Concentrations

Vaginal mucus scores and uterine weights of ovariectomized ewes that grazed subterranean clover for 13 days prior to tests were similar to those from ewes fed non-oestrogenic rations during this pre-treatment, if the leaves of test clovers contained high concentrations of formononetin (F) (Yarloop and Dinninup). No responses were obtained from subterranean clovers low in F, but high in genistein (G) or biochanin A (BA) (Clare and Bacchus Marsh) except for a group which was fed a non-oestrogenic diet during pre-treatment, and which gave a positive mucus test, but not a stgnificant uterine weight response. These results indicate that ewes can become non-responsive to G and BA, but not to F, and offer an explanation for some inconsistencies among bioassays of subterranean clovers. The task of selection and breeding of subterranean clover seems to be simplified since G and BA are unlikely to be harmful.

Observations on the removal of Oesophagostomum columbianum Curtice from sheep grazing on green oats and on pastures.

1955 ◽  
Vol 6 (4) ◽  
pp. 456 ◽  
Author(s):  
WH Southcott
Keyword(s):  
Considerable Increase ◽  
Red Clover ◽  
Subterranean Clover ◽  
Field Trials ◽  
Sheep Grazing ◽  
Factors Associated ◽  
Oesophagostomum Columbianum ◽  
Clover Pasture

Field trials in 1951 and 1952 confirmed previous observations on the evacuation of adult Oesophagostomum columbianum Curtice from sheep which graze continuously on green oats. Ingestion of phalaris-subterranean clover or red clover pasture also resulted in the evacuation of some O. columbianum but was much less effective in this regard. Evacuation of O. columbianum from sheep on green oats was not complete and was associated with softening, and lowering of the pH, of the faeces. The phenomenon occurred both with and without a considerable increase in liveweight. Some of the factors associated with oat-grazing which may influence removal of O. columbianum are discussed.

Quantitative aspects of phytooestrogen metabolism in sheep fed on subterranean clover (Trifolium subterraneum cultivar clare) or red clover (Trifolium pratense)

1970 ◽  
Vol 21 (5) ◽  
pp. 713 ◽  
Author(s):  
DA Shutt ◽  
RH Weston ◽  
JP Hogan
Keyword(s):  
Residence Time ◽  
Trifolium Pratense ◽  
Daily Intake ◽  
Red Clover ◽  
Subterranean Clover ◽  
Trifolium Subterraneum ◽  
Biochanin A ◽  
Free Form ◽  
The Mean ◽  
High Level

Studies have been made of the digestion and metabolism in Merino wethers of the isoflavones in subterranean clover (Trtfolium subterraneurn cv. Clare) and red clover (T. pratense). The dietary intake of isoflavones with both clovers was about 9 g per day. With the subterranean clover, the isoflavones were predominantly genistein and biochanin A, and slight teat length increases in the wethers ingesting this clover indicated a low level of oestrogenicity. With the red clover formononetin represented 60% of the isoflavone present and the wethers on this diet exhibited maximal teat length increases indicating a high level of oestrogenicity. Less than 1 % of the daily intake of the isoflavones was excreted as such in the faeces and urine; hence most of these compounds were metabolized or retained in the sheep. The dietary isoflavones were found to disappear rapidly from the rumen, and it was estimated by using marker techniques that the removal of these compounds from the stomach (rumen, reticulum, omasum, and abomasum) was virtually complete. Equol (7,4'-dihydroxyisoflavan), a metabolite of formononetin, was the predominant phyto-oestrogen in the digesta and excreta when red clover was given. The excretion of 3.9 g/day of this compound, mainly in urine, was equivalent to 70% of the intake of formononetin. It was calculated that about 86% of the equol produced in the rumen was absorbed from that organ; the mean residence time for equol in the rumen was estimated to be 1.7 hr. The isoflavones were present in blood plasma mainly in conjugated forms. Equol predominated with both clovers. The levels of equol were much lower with the subterranean clover than with the red clover diet; the concentrations of the conjugated form were respectively 13 and 300-440 �g/100 ml. Equol in the free form, although not detectable with the subterranean clover, was present at 4-10 �g/100 ml with red clover. The data were considered to be consistent with the conclusion that equol accounts for most of the phyto-oestrogenic activity in sheep fed on clovers containing high levels of formononetin.

Studies in uterotrophic activities in strains of subterranean clover

1989 ◽  
Vol 40 (4) ◽  
pp. 871 ◽  
Author(s):  
HL Davies
Keyword(s):  
Subterranean Clover ◽  
Biochanin A ◽  
Uterine Weight ◽  
The Mean ◽  
Negative Controls

Uterotrophic responses in ovariectomized ewes when fed or grazed on Mount Barker, Yarloop or Dwalganup varieties of subterranean clover are reported. The high formononetin varieties Dwalganup and Yarloop always elicited a uterotrophic response. The low-formononetin, high-genistein and biochanin A variety, Mount Barker, stimulated a uterotrophic response, using a 5-day bioassay, when fed at 200g/day or 800g/day, but little or no response when grazed. When chopped lucerne hay was offered with 200 g of the Mount Barker the uterotrophic response was suppressed and the mean uterine weight was not significantly different from that of the negative controls (P= 0.57). The uterine weight of sheep eating 200 g/day of Yarloop and offered lucerne chaff or pellets in one experiment was lower (but not significantly lower) than the unsupplemented group. This trend may be worthy of further investigation.

Effects of pasture type on the growth and wool production of weaner sheep during summer and autumn

1984 ◽  
Vol 24 (126) ◽  
pp. 322 ◽  
Author(s):  
PT Kenny ◽  
KFM Reed
Keyword(s):  
White Clover ◽  
Crude Protein ◽  
Perennial Grass ◽  
Red Clover ◽  
Subterranean Clover ◽  
Sheep Grazing ◽  
Neutral Detergent Fibre ◽  
Dry Matter Digestibility ◽  
Feeding Value ◽  
Persian Clover

The productivity of weaner sheep grazing lucerne, white clover, red clover or Persian clover and pastures sown with subterranean clover and perennial ryegrass, cocksfoot or tall fescue, was measured in summer-autumn trials incorporating four soil types in each of three years. During summer and autumn, the mean growth rate of sheep grazing lucerne, white clover or Persian clover pastures was 50 g/d compared with 1 g/d for sheep grazing perennial grass-subterranean clover pastures. Sheep grazing strawberry clover on reclaimed swampland, and sheep in a feedlot (fed hay and lupin grain) averaged 100 and 75 g/d, respectively. Sheep grazing red clover averaged 45 g/d over years 1 and 2. In year 2, a subterranean clover-dominant pasture was included in the study. Its feeding value was similar to that of the perennial grass-subterranean clover pastures. Sheep grazing legume pastures, and sheep in the feedlot, consistently produced more wool (up to 700 g/head) than sheep grazing the perennial grass-subterranean clover pastures. The concentration of neutral detergent fibre in herbage from legume pastures was frequently lower (P< 0.05) and the concentration of crude protein was frequently higher (P< 0.05) than in herbage from grass pastures. The dry matter digestibility of legume herbage was higher (P<0. 05) than that of grass herbage on only one occasion. Measurements of crude protein and neutral detergent fibre of pasture in December accounted for 67% of the variation in liveweight gain (P<0.01). Ewe weaners grazing red clover pasture exhibited an increase (P< 0.05) in teat length of 3.9 mm in 21d of grazing. Oestrous activity of ewe weaners was related to liveweight gain (r = 0.77). The implications of the results for agricultural systems are discussed.

scholarly journals Oestrogenic Isoflavone Content in Natural Strains of Subterranean Clover (Trifolium subterraneum L.) from Sardinia

2006 ◽  
Vol 1 (7) ◽  
pp. 1934578X0600100 ◽  
Author(s):  
Aldo Tava ◽  
Luciano Pecetti ◽  
Alessia Bertoli ◽  
Efisio Piano
Keyword(s):  
Trifolium Pratense ◽  
Red Clover ◽  
Subterranean Clover ◽  
Trifolium Subterraneum ◽  
Biochanin A ◽  
Lower Amount ◽  
Positive Role ◽  
Isoflavone Concentration ◽  
Trifolium Pratense L ◽  
Natural Strains

A collection of subterranean clover (Trifolium subterraneum L.) genotypes from Sardinia was evaluated for the content of oestrogenic isoflavones to assess differences in total and individual compounds. Daidzein, genistein, and their respective 4′-methoxy derivatives biochanin A and formononetin, were detected in leaves and quantified by GC/FID and GC/MS methods. A large variation among genotypes for individual compounds and for the total isoflavone concentration was observed. Total isoflavones were measured in the range 0.8-13.4 mg/g fresh weight (0.3-4.5% dry matter). Daidzein was usually present in lower amount compared to the other isoflavones. Genistein and biochanin A were found in most cases in higher concentration than formononetin. Phyto-oestrogens have been shown to have both oestrogenic and anti-oestrogenic properties, and their effects entail a positive role for the human health. T. subterraneum revealed the presence of higher concentration of isoflavones compared to other sources of these compounds, e.g. soybean (Glycine max (L.) Merrill) and red clover (Trifolium pratense L.). This study provided information on the biodiversity of the natural strains from Sardinia, emphasizing the importance of these genetic resources for their variation in the amount of total and individual isoflavones.

The acylated isoflavone glycosides from subterranean clover and red clover

1971 ◽  
Vol 24 (7) ◽  
pp. 1509 ◽  
Author(s):  
AB Beck ◽  
JR Knox
Keyword(s):  
Methyl Esters ◽  
Red Clover ◽  
Subterranean Clover ◽  
Pure Form ◽  
Biochanin A ◽  
Structural Assignment ◽  
Derivatives Of

Formononetin and biochanin A 7-O-glucosides (1a and 2a) and their malonate hemi-ester derivatives (1b and 2b) have been isolated from red clover and from the Yarloop strain of subterranean clover. Structure (2b) is a correction of an earlier structural assignment to this compound. Small amounts of the derived methyl esters (1c and 2c) are also present in both clovers. Similar derivatives of genistein have been obtained from the Yarloop strain, although only the 7-O-glucoside (3a) has been isolated in pure form. Studies on red clover and on nine strains of subterranean clover have demonstrated in all cases that the acidic malonate esters account for the predominant proportion of the glycosides.

Physiological and ecological studies on the oestrogenic isoflavones in subterranean clover (T. subterraneum L.) I. Effects of temperature

1966 ◽  
Vol 17 (1) ◽  
pp. 29 ◽  
Author(s):  
RC Rossiter ◽  
AB Beck
Keyword(s):  
Total Concentration ◽  
Subterranean Clover ◽  
Maximum Growth ◽  
Biochanin A ◽  
Ecological Studies ◽  
Leaf Material ◽  
Long Day ◽  
Effects Of Temperature

Two strains of subterranean clover – Yarloop and Mt. Barker – were grown at controlled temperatures ranging from 36/31° to 9/4°C under long-day conditions. Leaf material was analysed for the three isoflavones formononetin, genistein, and biochanin A. In the Mt. Barker strain, the formononetin concentration was low (< 0.2% dry wt.) and varied little with temperature. Otherwise, isoflavone levels generally rose from very low values at 36/31° to a peak at 15/10°, and then decreased again to 9/4°. For both strains the total concentration of the three isoflavones and also the total amount of isoflavones per leaf were highest at 15/10°. This temperature was lower than that at which maximum growth occurred. The data are discussed in relation to evidence on the effects of temperature on the formation of flavonoid (especially anthocyanin) compounds. We conclude that temperature is unlikely to be a major factor in the incidence of "clover disease".

Comparison of plasma phyto-oestrogen levels in sheep and cattle after feeding on fresh clover

1971 ◽  
Vol 22 (4) ◽  
pp. 663 ◽  
Author(s):  
AWH Braden ◽  
RI Thain ◽  
DA Shutt
Keyword(s):  
Plasma Concentration ◽  
Dairy Cattle ◽  
Blood Plasma ◽  
Red Clover ◽  
Subterranean Clover ◽  
Biochanin A

Wethers and heifers were fed on freshly cut pasture (red clover, Mt. Barker or Tallarook subterranean clover, or ryegrass) and the blood plasma concentration of the phyto-oestrogens and their metabolites estimated 5 hr, 24 hr, and 6 days after the beginning of the feeding. The heifers were allowed to graze the pastures after the first day. On the basis of increase in teat length in the wethers, red clover and Tallarook were estimated to have equivalent oestrogenicity ; Mt. Barker was much less active. Qualitatively, the metabolism of formononetin and biochanin A appeared similar in the two species, but there was evidence that the rates of metabolism and of conjugation were faster in cattle than in sheep. The findings are discussed in relation to an infertility syndrome in dairy cattle.

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