P. Nykänen-Kurki1, H. Saloniemi2, K. Kallela2 and I. Saastamoinen2
1 agricultural Research Centre, South Savo Research Station, 50600 Mikkeli, Finland2 college of Veterinary Medicine, Department of Animal Hygiene, 00581 Helsinki, Finland
Introduction
Material and methods
Results
Discussion
References
Phyto-oestrogens have been reported to cause fertility disorders in domestic animals (Kallela, 1964). In certain cases they improved animal production (Refsdal, 1976), or they had a prophylactic effect against some hormone related human malignancies (Adlercreutz et al., 1991). As there are few reports on the phyto-oestrogen content of white clover in Finland, the content and the biological effect of isoflavones (daidzein, formononetin, genistein, biochanin-A) and coumarine (coumestrol) was determined at different growing stages of white clover.
White clover varieties (Undrom and Sandra from Sweden, Jögeva 4 from Estonia and Tammisto from Finland) were sampled at the South Savo Research Station (61°40'N), Undrom also at the Karelia (62°14'N), Kainuu (64°06'N) and North Ostrobothnia (64°40'N) Research Stations in 1991. Samples were cut twice from the spring growth: at the pasture stage of rapid growth and at the silage stage in the beginning of flowering; once from the first aftermath in full blossom; and once from the second aftermath during declined growth rate. The swards were fertilized at the rate of N 12, P 60 and K 108 kg/ha in spring.
After grinding, samples were allowed to stand for 30 minutes at + 37°C for hydrolysis of conjugated phyto-oestrogens (Francis and Millington, 1967). Until chemical analysis by liquid chromatography and biological study in immature rats at the College of Veterinary Medicine, samples were stored in absolute ethanol in a refrigerator. Daidzein, genistein, formononetin and biochanin-A were determined by a UV detector, coumestrol by fluorometry. The biological study was based on the increase in weight of 21-day-old rat uterus by phyto-oestrogens (Saloniemi et al., 1993).
The oestrogenic isoflavone contents in white clover varieties were low. The isoflavones consisted mainly of formononetin (90-95 %) and genistein (5-10 %), and their contents did not differ between varieties. Some coumestrol was found in the autumn (Table 1). The uterine weight of control rats averaged 21 mg, in test groups from 29 mg to 66 mg. The oestrogenic effect of white clover on rat uterus was clearly positive (Figure 1). By visual observations no leaf diseases were recognized in white clover at samplings.
Table 1. Phyto-oestrogen content of white clover by chromatography.
|
Growing stage |
Research Station |
Sampling date |
Variety |
Height cm |
Content of DM % |
Isoflavones % in DM |
Coumestrol ppm 10 DM |
|
Pasture Rapid growth |
South Savo |
17.6. |
Jögeva 4 |
22 |
11.5 |
0.02 |
0* |
|
|
|
Sandra |
21 |
11.7 |
0.02 |
0* |
|
|
|
|
Tammisto |
20 |
11.9 |
0.02 |
0* |
|
|
|
|
Undrom |
21 |
11.5 |
0.02 |
0* |
|
|
Karelia |
19.6. |
Undrom |
18 |
8.1 |
0.06 |
5.8 |
|
|
Kainuu |
24.6. |
Undrom |
19 |
11.8 |
0.04 |
2.8 |
|
|
North Ostrobothnia |
26.6. |
Undrom |
27 |
15.1 |
0.01 |
0* |
|
|
Silage Beginning of flowering (1. cut) |
South Savo |
4.7. |
Jögeva 4 |
34 |
10.7 |
0.02 |
0* |
|
|
|
Sandra |
39 |
10.8 |
0.02 |
0* |
|
|
|
|
Tammisto |
33 |
10.7 |
0.02 |
0* |
|
|
|
|
Undrom |
33 |
10.4 |
0.02 |
0* |
|
|
Karelia |
4.7. |
Undrom |
30 |
11,0 |
0.03 |
2.2 |
|
|
Kainuu |
9.7. |
Undrom |
29 |
12.4 |
0.03 |
1.6 |
|
|
North Ostrobothnia |
11.7. |
Undrom |
30 |
12.0 |
0.02 |
4.2 |
|
|
First aftermath (2. cut) |
South Savo |
6.8. |
Jögeva 4 |
40 |
10.4 |
0.02 |
0* |
|
|
|
Sandra |
39 |
11.3 |
0.02 |
6.8 |
|
|
|
|
Tammisto |
38 |
10.6 |
0.02 |
0* |
|
|
|
|
Undrom |
40 |
10.2 |
0.02 |
0* |
|
|
Karelia |
22.8. |
Undrom |
29 |
11.6 |
0.04 |
1.8 |
|
|
Kainuu |
22.8. |
Undrom |
24 |
12.8 |
0.04 |
0* |
|
|
North Ostrobothnia |
20.8. |
Undrom |
27 |
9.6 |
0.03 |
8.7 |
|
|
Second aftermath (3. cut) |
South Savo |
2.9. |
Jögeva 4 |
21 |
11.1 |
0.02 |
0* |
|
|
|
Sandra |
21 |
12.2 |
0.03 |
6.8 |
|
|
|
|
Tammisto |
17 |
12.6 |
0.03 |
8.9 |
|
|
|
|
Undrom |
19 |
12.4 |
0.03 |
5.9 |
* - less than one 1 ppm (limit of detection)
Figure 1. Effect of white clover on weight of immature rat uterus.
The chemical study showed a low content of phyto-oestrogens in white clover as reported earlier. Like red clover their content increased slightly in the autumn, when nights were getting cooler (Kallela et al., 1987). After infection by diseases the content of phenolic compounds increased, which could be due to a natural defence mechanism in white clover (Wong et al., 1971).
Daidzein and formononetin were active in ruminants, while biochanin-A and genistein were inactive (Petterson et al., 1984). The silage containing formononetin 0.56 % of dry matter in the spring caused fertility disturbances in dairy cows. Cows were fed silage ad libitum during the indoor feeding period (Kallela et al., 1984). Coumestrol was more effective than isoflavones, and it had a cumulative effect (Whitten et al., 1992). The minimum level of coumestans to cause fertility disorders in sheep ranged between 20 and 50 ppm (Wong et al., 1971).
None of the reasons discussed explained the imperfect correlation between biological and chemical studies. Some compounds not shown in the chemical analyses increased the oestrogenic potency. Bickoff et al. (1969) showed coumestrol to be active in mice and sheep, while some poly-hydroxycoumestans were active in mice. The contribution of coumarines in white clover warrants further study.
ADLERCREUTZ, H., HONJO, H, HIGASHI, A., FOTSIS, T., HÄMÄLÄINEN, E., HAGESAWA, T. and OKADA, H. (1991) Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet. American Journal of Clinical Nutrition. 54, 1093-1100.
BICKOFF, E.M., SPENCER, R.R., WITT S.C. and KNUCKLES, B.E. (1969) Studies on the chemical and biological properties of coumestrol and related compounds. United States Department of Agriculture, Technical Bulletin No 1408.
FRANCIS, C.M. and MILLINGTON, A.J. (1967) Varietal variation in the isoflavone content of subterranean clover: its estimation by a microtechnique. Australian Journal of Agricultural Research. 16, 557-564.
KALLELA, K. (1964) The incidence of plant oestrogens in Finnish pasture and fodder plants with special reference to their possible effects in cases of sterility of ruminants. Thesis. College of Veterinary Medicine, Helsinki.
KALLELA, K., HEINONEN, K. and SALONIEMI H. (1984) Plant oestrogens: the cause of decreased fertility in cows. A case report. Nordisk Veterinærmedicin. 36, 124-128.
KALLELA, K., SAASTAMOINEN, I. and HUOKUNA, E. (1987) Variations in the content of plant oestrogens in red clover-timothy-grass during the growing season. Acta Veterinaria Scandinavica. 28, 255-262.
PETTERSSON, H., HOLMBERG, T., KIESSLING, K-H. and RUTQVIST, L. (1984) Växtöstrogener i foder och reduktionsstörningar hos idisslare. Svensk Veterinärtidning, 36, 677-683.
REFSDAL, A.O. (1976) Fertiliteten hos kyr i relasjon til forbruk av sufor og tort stråfor i de olike fylke i Norge. Norsk Veterinærtidsskrift, 88, 597-604.
SALONIEMI, H., KALLELA, K. and SAASTAMOINEN, I. (1993) Study of the phyto-oestrogen content of goat's rue (Galega orientalis), alfalfa (Medicago sativa) and white clover (Trifolium repens). Agricultural Science in Finland (Submitted).
WHITTEN, P.L., RUSSELL, E. and NAFTOLIN, F. (1992) Effects of a normal, human-concentration, phytoestrogen diet on rat uterine growth. Steroids. 57, 98-106.
WONG, E., FLERX, D.S. and LATCH, G.C.M. (1971) The oestrogenic activity of white clover (Trifolium repens L) New Zealand Journal of Agricultural Research. 14, 639-645.
