Effect of germination on vomitoxin level in grain.

The present investigation clarified the effect of enzymes or other substances formed during the germination process on the vomitoxin level of contaminated oats. The studies found that oats containing vomitoxin germinated very poorly; the decrease in toxins was also slight. The amount of pure vomitoxin added to toxin-free grain decreased (barley 53%, oats 40%, wheat 20%) during germination (5 d). In homogenized mixture of germinated grain (2.4 and 7 d) and toxic grain no decrease in toxin amount occurred during a 1-7 day period. In contrast, when germinating toxin free grains and toxic oats in a grain mixture the toxin level decreased at first, but later rose considerably. On the basis of these results, the utilization of germination processes for the improvement of grain containing vomitoxin is of questionable value.

The effect of storage on the estrogenic effect of red clover silage.

Pure red clover was selected for plant estrogen analyses from the fresh fodder crops of 1972 and 1973. The investigations were made on fresh red clover and on the same red clover after periods of storage of varying length. The red clover was stored in manilla bags in a silo among the ordinary silge fodder, use being of the "green solution method" (Farmos Oy). In the studies the estrogenic effect of the fodder was ascertained by means of bioassay, the criterion being the increase in murine uterine weight. The known plant estrogens were determined by thin layer chromatography and by liquid chromatography. The estrogenic effect of the red clover silage fodder of 1972 was greater in all the silage fodder samples than it was in the fresh red clover. The quantity of individual isoflavones and "transformed" estrogen too, was greater in many of the silage fodder samples than it was in the respective fresh red clover. The estrogenic effect of the silage fodder made from the red clover of 1973 varied considerably; in some samples it was greater but in most it was smaller than the estrogen effect of the fresh red clover. However, apart from a few exceptions, the quantity of individual isoflavones and of transformed estrogens was smaller than it was the fresh red clover.

The estrogenic effect of silage fodder.

The changes in the amounts of estrogens and in the estrogenic effects were compared with the changes occurring in the quality of silage fodders, which were prepared by three different methods (without any additives, by green-solution method, and with molasses) from the 1977 aftermath, composed of 50% of different kinds of grasses and 50% of red clover. The estrogenic isoflavones known (formononetin, biochanin-A, genistein and daidzein) were analysed quantitatively by liquid chromatography, the estrogenic effect by bioassay using female rats aged 21-22 days as test animals the criteria being the uterine weight, the vaginal opening, and the uterine liquid quantity. Other characteristics of the fodders were determined by 21 laboratory determinations or perceptible estimates. The correlations between the characteristics of the fodder showed that, in addition to the plant estrogens, the amount of lactic acid had significant influences on the estrogenic effect. The pH values and the amount of lactic acid had significant effects also on the changes in the amounts of estrogenic isoflavones known. The quantities of isoflavones and the estrogenic effects varied in fodders ensiled by various methods.

Combined action of plant estrogens, F-2 toxin and natural estrogens.

The investigation concerns the interaction of plant estrogens, F-2 toxin, and natural estrogen (estradiol-benzoate) as causative factors of eostrogenic changes. Plant estrogens and F-2 toxin were applicated at three and estradiol-benzoate at two dosage levels. The estrogenic effect was studied by bioassay on immature rats taking uterine weight, uterine liquid and vaginal opening as criteria. When uterine weight is taken as a criterion, plant estrogens and F-2 toxin, no matter if they are administered alone or together, regularly increase the effect of estradiol-benzoate, but when uterine fluid is taken as a criterion they always decrease the effect of estradiol-benzoate. According to the present results it seems obvious, that the mode of action of estrogenic substances occurring in plants considerably differ from that of natural estrogens. Thus the mechanisms which cause disturbances may be different from those of natural estrogens.

Decomposition of the Fusarium graminearum toxin zearalenone in storage conditions.

The studies illustrate the effects of preservatives - "Luprosil" (propionic acid) and "Gasol" (contains organic acids and some additional compounds) on the growth of Fusarium graminearum and the quality of zearalenone in contaminated grain. The laboratory conditions resembled, concerning the supply of oxygen, the circumstances in the surface layers of preserved grain mass. The mycelium growth of the fungus Fusarium graminearum was visually observed. Three successions of studies of grain cultures were carried out before addition of preservatives and after 3, 14 and 28 days of storage: 1) liquid-chromatographic examination indicating the amount of soluble zearalenone in oat-wheat culture 2) bioassay indicating the amount of insoluble zearalenone in grain (solid residue) 3) bioassay indicating the total amount of zearalenone (insoluble + soluble zearalenone) in oat-wheat-barley culture. The preservatives completely prevented the visible mycelium growth of Fusarium graminearum. Propionic acid ("Luprosil") had no reducing effect on the quantity of zearalenone. "Gasol", on the contrary, reduced the amount of the mycotoxin very clearly. The decrease in different successions of studies was after 3, 14 and 28 days from the commencement of storage as follows: 1) 60, 90 and 95% 2) 15, 17 and 90% 3) 70, 80 and 90%.

The oestrogenic Fusarium toxin (zearalenone) in hay as a cause of early abortions in the cow.

In this study, the occurrence of a series of early abortions in a herd of cattle kept at an experimental station in North Savo has been described, the cause of which has evidently been hay feed in which the toxin zearalenone had formed. Several early abortions occurred in this particular herd 30-90 days after artificial insemination had been performed. It was proved by means of a liquid chromatograph test that the hay contained 10 ppm zearalenone, and a biological experiment with rats gave a clear indication of its oestrogenic strength by means of the chloroform extract obtained with the hay. Early abortions ceased to occur after feeding with the suspected feed was discontinued. The cows were on heat again 2-23 days after the early abortions had occurred, though they subsequently experienced difficulties in becoming pregnant.

The effects of rumen fluid on the content of zearalenone in animal fodder.

The object of the study was to clarify whether rumination has a bearing on the decomposition of zearalenone in the metabolism of the toxin or not. The experiment was performed using rumen fluid in vitro. Rumen fluid samples containing various amounts of zearalenone were incubated in a carbon dioxide atmosphere at 38 degrees C for two days. The zearalenone content was determined by liquid chromatography after 0, 4, 24 and 48 hours incubation periods. The incubation of 4 hours decreased the toxin content by 11%, that of 24 h by 29% and that of 48 h by 37.5% on average. These results indicate that the quantity of zearalenone and the quality of rumen fluid have effects on the ratio of the toxin decomposed.

The effects of "Gasol" grain preservative dosages on the growth of Fusarium graminearum and the quantity of the toxin zearalenone.

In earlier experiments regarding the effects of preservative "Gasol" (containing various acids and additional compounds and intended for whole grain preservations) in recommended dosages, prevents the growth of Fusarium graminearum and reduces the amount of zearalenone when added to milled grain. It was proved in the tests carried out that the smaller dosages of "Gasol" were sufficient for the prevention of growth of the fungus and reduction in quantity of zearalenone. The degree to which the toxin was destroyed depended on the dosage of the preservation. A dosage twice as strong as that recommended in the instructions for practice destroyed all the toxin formed in the grain. A very small dosage of "Gasol" (1/8 of recommended dosage) prevented the growth of the fungus and only slightly reduced the amount of toxin only at the beginning of the test. Later, the growth of the fungus was more vigorous and significantly more toxin was present in the treated grain than in the grain which had never been treated with the preservatives, it has been proved that the preservative

Plant oestrogens; the cause of decreased fertility in cows. A case report.

During the stall feeding period 1982-1983, it was established that serious fertility disturbances, indicating oestrogenic stimulation, had occurred in a herd of cattle in an area supported by the College of Veterinary Medicine's ambulatory clinic. Whilst investigating the cause of these disturbances it was proven that the silage administered during this period was prepared almost entirely from pure red clover aftergrowth. The oestrogenic isoflavone content of the silage (liquid chromatograph examination) and the oestrogenic strength (bioassay) were considerably great. When feeding with the fodder was subsequently discontinued the disturbances ceased to occur and the cows became pregnant more easily. On the basis of the aforementioned incidences it was ascertained that plant oestrogens were almost certainly the cause of the fertility disturbances.

Variation of F-2 toxin production on different substrates.

The F-2 producing capacity of one Fusarium graminearum strain (strain No. 13) and of three hyphal tip transverse lines (a b and c) isolated from the original strain and of a mixture of these lines (a b and c) was studied in two successive years on different substrates: oats, barley, wheat, grain mixture and wheat bran. In the first year the original strain produced high amounts of F-2 but was heterogenous in toxin production. The F-2 producing capacity of one of the hyphal tip transverse lines (a) was high and that of two lines (b and c) and of the mixture of the three lines (a+b+c) was poor. In the following year the F-2 producing capacity of the fungal cultures had changed: the F-2 producing capacity of the original strain (No. 13) was greatly reduced, that of one hyphal tip transverse line (b) remained poor and that of two hyphal tip transverse lines (a and c) and of the mixture of the lines clearly increased. The F-2 production was changed in all the substrates and in about the same proportion. In general oats was the best substrate for F-2 production. The possible causes of the changed F-2 production are discussed. The estrogenic effect of F-2 produced in different substrates was studied by using as criteria the uterine weight, vaginal opening and liquid content in the uterus of immature female rats. The effect was in direct proportion to the amount of F-2 ingested by the rats. The substrate was without any influence on the physiological effect of F-2, and in this respect our results deviate from some earlier findings.

Phytoestrogen content and estrogenic effect of legume fodder.

This study is a summary of Finnish investigations of the phytoestrogen content of legume plants, red clover, white clover, alfalfa, and goat's rue. In addition to the chemical analyses, biological studies were performed. Uterine weight of immature rats was used as an indicator of the estrogenic effect of the fodder used. All red clover varieties studied contained estrogenic isoflavones, especially formononetin and biochanin-A. The phytoestrogen content varied from 1.0% to 2.5% of dry matter. The biological study of white clover showed a clear estrogenic effect not visible through chemical analysis. Alfalfa contains small quantities of formononetin and biochanin-A, but 25-65 ppm coumestrol in dry matter. The estrogenic effect of alfalfa was obvious in the biological study. Goat's rue did not contain any known phytoestrogens, and the biological study was completely negative.