The metabolism and physiological effects of phyto-oestrogens in livestock.

Abstract

Proc. Aust. Soc. Anim. Prod. (1974) 10; 122 THE METABOLISM AND PHYSIOLOGICAL EFFECTS OF PHYTO-OESTROGENS IN LIVESTOCK R.I. COX* and A.W. BRADEN* I. INTRODUCTION The oestrogenic activity of certain cultivars of subterranean clover (Trifolium subterraneum, L.) first became known through the observation of serious infertility in ewes in the early 1940's in Western Australia (Bennetts, Underwood and Shier-1946). The manifestations in commercial flocks of the syndrome - 'clover disease' - have been dealt with in the preceding paper (Lightfoot 1974). The deleterious effects of phyto-oestrogens have been a major economic problem in livestock husbandry in Australia and to a lesser extent elsewhere (reviewed: Moule, Braden and Lamond 1963; Bickoff 1968; Braden and McDonald 1970; Lightfoot 1974). The main species affected by phyto-oestrogens is the sheep. The effects of clover disease have been much alleviated by agronomic developments and managerial changes (Lightfoot 1974), although there are some inherent restrictions to these approaches at present. After ingestion, phyto-oestrogens have been shown to undergo metabolic changes in the animal, often to compounds of very different oestrogenicity to that of the original plant constituents. Thus, in reducing clover disease by agronomic means, the hazardous cultivars have had to be identified not only in terms of the occurrence of specific phyto-oestrogens, but have had to be related to phyto-oestrogen metabolism in the animal. This is clearly seen in the observations that led to formononetin being established as the major plant constituent responsible for clover disease in sheep (Millington, Francis and McKeown 1964), because of its metabolism to equol (Batterham, et al. 1965; Shutt and Braden, 1968). Although control of clover disease would seem to be most easily effected by alteration of the pasture species available to grazing animals it is possible that some control or prevention of clover disease could be achieved through measures operative in the animal (e.g. through the manipulation of genetic, metabolic or immunophysiological mechanisms) and that these may be a useful complement to agronomic measures. This paper reviews recent work on the phyto-oestrogens in relation to their occurrence, metabolism within the animal, and the physiological mechanisms by which they produce infertility; it is not intended as a comprehensive review of the field. Some of the potential means of preventing infertility by treatment of the animal will be considered. II. PHYSIOLOGICAL MECHANISMS IN THE INFERTILITY CAUSED BY PHYTO-OESTROGENS \ The main feature of the syndrome of clover disease in sheep is a permanent depression of ewe fertility that appears after the sheep have been grazing highly oestrogenic pastures for several years. Fertility and sperm production in the ram are unaffected (Braden, Mattner and Carter 1974). The pathological condition, cystic glandular hyperplasia, is almost always found in the cervix and uterus of ewes affected by clover disease (Bennetts, Underwood and Shier 1946; Barrett et al. 1961; Davies and Nairn 1964). It appears to persist for long periods after the Ian Clunies Ross Laboratory, C.S. I.R.O. Division of Animal Physiology, Prospect, P.O. Box 239, Blacktown, N.S .w. f 2148. 122 ewes are taken off oestrogenic pastures (Underwood and Shier 1951; Turnbull, Braden and George 1966). The investigations made on sheep from flocks in which clover disease had been induced experimentally concur in indicating that the main cause of the infertility is a failure of many of the eggs to be fertilized (Turnbull, Braden and George 1966; Lightfoot, Croker and Neil 1967; Fels and Neil 1968; Kaltenbach and Davies 1970). The establishment of a reservoir of sperm in the cervix imxnediately after mating is necessary for normal fertility in sheep (Mattner 1966). In sheep affected by clover disease the number of sperm entering the cervix is considerably lower than normal and a major factor in this failure appears to be the increased amount and fluidity of the cervical mucus in affected sheep (Smith 1970). As a consequence, the number of sperm found in the oviducts is very low and the chances of an egg being fertilized are much reduced. Evidence for a higher than normal loss of embryos in the first 30-60 days after mating in ewes affected by clover disease is equivocal (Turnbull, Braden and George 1966; Fels and Neil 1968; Kaltenbach and Davies 1970). Certain assoc~iated pathological conditions contribute to the infertility and also to the increased death rates found in ewes grazing highly oestrogenic pastures. Hydrops uteri and uterine infections are the most frequently found; they have been reported in lo-25% of ewes examined after 4-6 years grazing of oestrogenic pastures (Turnbull, Braden and George 1966; Fels and Neil 1968). Histological examination of the ovaries, pituitary gland and adrenal cortex did not reveal any abnormalities in ewes with clover disease (Hearnshaw et al. 1972) although there is some evidence that the response of the hypothalama such ewes to an oestrogenic stimulus is sub-normal (Findlay et al. 1973). The oestrous cycles of infertile ewes are usually of normal .length (Underwood and Shier 1951; Lightfoot, Croker and Neil 1967; Obst and Seamark 1970) and where abnormal cycle lengths occur in some ewes they are often due to pathological conditions such as hydrops uteri (Hearnshaw et al. 1972). The mechanisms involved in the 'temporary' form of phyto-oestrogen-induced infertility have no t been as fully studied as those of the classical syndrome. The 'temporary' infertility occurs when sheep are grazing highly.oestrogenic pastures at the time of mating (Morley, Axelsen and Bennett 1964); the incidence of multiple births is depressed, but there is no evidence that this is due to a depression in the number of eggs ovulated (Morley, Axelsen and Bennett 1964; Holst and Braden 1972). The evidence available, however, suggests that the infertility may be due to a depressed fertilization rate and also to a disturbance in the rate of transport of ova through the oviducts (Hoist and Braden 1972). III. OESTROGENIC SUBSTANCES IN PASTURES A feature of both naturally occurring and synthetic oestrogens is the wide variety of structures that show oestrogenic activity (Hogg and Korman 1956; Hilgar and Palmore 1968; Emmens and Miller 1969). Plant constituents that have been identified as oestrogenic are mainly isoflavones and include formononetin, daidzein, genistein and biochanin A (Fig. 1). Of the other phyto-oestrogens the coumestans, coumestrol and 41-C-methyl-coumestrol, have been isolated from certain medics that are used as pasture plants in Australia. In the plant the phyto-oestrogens often occur as glycosides (Beck and Knox 1971). Although these phyto-oestrogens are only of the order of lo-3 to lO'%as oestrogenic as oestradiol-176 in laboratory animals they are often present in relatively high concentrations in pasture species and thus the.daily intake of sheep can be sufficient to produce biological effects. The isolation, measurement and concentrations of phyto-oestrogens in pasture plants and the factors that can affect their concentrations have been extensively investigated (reviewed: Bickoff 1968; Braden and McDonald 1970; Rossiter 1970; Wong and'Latch 1971; Lightfoot 1974). Recent findings of interest are the effects of foliar infection. Thus, Loper and Hanson (1964), Shemesh, Lindner and Ayalon (1969), Francis and Millington (1971) found an increase in coumestan content of some medics as a result of fungal infection. White clover, which normally has negligible oestrogenic activity can, after fungal infection, contain sufficient amounts of 123 coumestans to produce and Latch 1971; Saba had not been isolated the need to be on the formation. IV. oestrogenic effects in sheep grazing the pastures (Wong, Flux et al. 1972; Newton and Betts 1973). Some of these coumestans previously (Wong and Latch 1971). These findings illustrate alert for new phyto-oestrogens or situations leading to their IN THE METABOLISM OF PHYTO-OESTROGENS (a) Metabol ism in sheep THE ANIMAL In considering the biological effects of phyto-oestrogens, of particular importance are the metabolic changes they undergo in the animal (for references see Braden and McDonald 1970). Thus formononetin, which is present at levels of 102% of dry weight in a number of cultivars of subterranean and red clovers,has little or no oestrogenic activity itself, but is converted in the rumen largely to equol, which is oestrogenic. Indeed,equol is probably the oestrogen responsible for clover disease (Shutt and Braden 1968). In contrast, genistein and biochanin A, which are themselves 'oestrogenic when given parenterally, are metabolised almost completely to inactive compounds in the rumen. During grazing,ruminal degradation of these phyto-oestrogens appears to increase markedly over the first few days of feeding until their oestrogenic effects in the sheep are negligible. In contrast the ' proportion of formononetin converted to equol does not change much with time and so the oestrogenicity remains high (Lindsay and Francis 1969). With coumestans the situation appears to be intermediate: a ten-fold decrease in oestrogenic activity occurs over 8 days (Kelly 1972). Discovery of these metabolic sequences together with observations on the relative oestrogenic potency in sheep of various cultivars of subterranean clover, led during the 1960% to a complete revision of the list of 124 the cultivars deleterious to sheep. Phyto-oestrogens and their metabolites after absorption, largely from the rumen, circulate in the blood plasma. Conjugation with glucuronic acid and some further metabolism probably occurs in the liver. Rapid excretion occurs, mainly via the urine, in contrast with the largely faecal excretion route for metabolites of the endogenous steroid .oestrogens. Phytooestrogens and their metabolites circulating in blood plasma are preponderantly in the form of glucosiduronates and in this form are probably biologically inactive (Shutt, Axelsen and Lindner 1967). Less than 1% are present as the biologically active unconjugated form and small amounts appear to be present as the sulphoconjugates (Wong and Cox 1971) which can probably be converted in vivo to the active unconjugated form. In the case of coumestrol, however, relatively high proportions are present in the unconjugated and sulphoconjugated forms (Cox, unpublished). Investigations of the influence of cobalt or selenium intake on clover induced infertility have not produced definitive results. Davies (1966) did not find any improvement in the infertility of affected ewes after treatment with cobalt or selenium. On the other hand, Godwin, Kuchel and Buckley (1970) reported that selenium treatment prevented the depression in fertility seen in ewes grazing oestrogenic pastures. However, this study appears to involve 'temporary infertility' rather than the clover disease syndrome. The proportion of dietary formononetin that is excreted in the urine as equol was not found to be affected by cobalt or selenium dosage (Cox and Braden, unpublished). It is evident that the factors that modify or change the metabolism of phyto-oestrogens in ruminants have not been fully elucidated and are in need of further study. (b) Metabol ism in other species Whether phyto-oestrogens have deleterious effects in cattle remains equivocal. Some hyperoestrogenic disturbances in cattle have been attributed to phyto-oestrogens (Adler and Trainin 1960; Thain 1968; Weitzkin, Marinov and Roderig 1968) yet there have been no reports of such effects from Western Australia. A comparative study showed that there were similar concentrations of phyto-oestrogens in the plasma of sheep and cattle fed the same pastures (Braden, Thain and Shutt 1971). The metabolism of formononetin to equol was an important conversion in both species but the evidence suggested that the rates of metabolism and conjugation were faster in cattle than in sheep. There has been little study of the metabolism of phyto-oestrogens in nonruminants. Caution is required in deducing oestrogenicity in one species from data obtained in another species. Thus, genistein is oestrogenically active when fed to laboratory test animals (Biggers and Cur-now 1954; Micheli et al. 1962) whereas In poulwhen fed to sheep it is rendered virtually inactive by ruminaldegradation. try, the conversion of genistein to equol has been reported, and is well supported by the data obtained (Hertelendy and Common 1964; Cayen, Tang and Common 1965). Any species in which effects of phyto-oestrogens are being considered needs to be studied by bioassay using that species; suitable techniques for the sheep have been discussed by Braden and McDonald (1970). Discrepancies in bioassay data between species may indicate metabolic differences. A useful biochemical measure of the oestrogenicity of compounds in vitro, which may be correlated with in vivo findings, is the competitive binding reaction for the oestrogen-specific protmuterine cytosols (shutt and Cox 1972; Shemesh, Lindner and Ayalon 1972). (c) Formononetin metabolites other than equol As formononetin is largely converted to equol and the latter appears to be the major active oestrogen responsible for clover infertility, any evidence for alteration in the extent of equol formation requires attention. Several other formononetin metabolites have been noted recently (Fig. 2). About 70% of the formononetin ingested by sheep is. converted to urinary equol 125 Weston and Hogan 1970) together with a small amount of daidzein and (shutt, desmethylangolensin (Batterham et al. 1971). This general pattern has been confirmed in observations on a series of over 30 wethers fed a diet of dried, pelleted red clover (Cox and Braden 1974). However, other metabolites were found, one of which has been identified as 4'0O-methyl-equal and small amounts of angolensin appear to be present as well as an unidentified compound. The O-methyl-equol was found to be present in appreciable amounts in the urine of 9 of 30 animals, and in some, the levels found approached those of equol. O-methyl-equol has also been noted to occur in renal deposits as well as in urine of sheep grazing oestrogenic pastures (Nettle and Beck, personal communication). Such a metabolite is of interest as it indicates that, at least for some sheep under some circumstances, an appreciable proportion of the formononetin may be metabolized by an alternative route involving reduction without prior demethylation. The methoxy compound is likely to be less oestrogenic than equol and hence to be of significance in relation to biological effects in the sheep. The factors that affect the extent to which formononetin is metabolized by this route have yet to be elucidated. or v. POSSIBLE FUTURE APPROACHES TO CONTROL OF CLOVER DISEASE Although agronomic methods are likely to be of increasing importance in the control of clover diseaseit is pertinent to consider the ways in which prevention also might be possible through measures operative in animals. These include genetic selection of sheep for resistance to clover disease, alteration of phyto-oestrogen metabolism, administration of oestrogen antagonists and irtununization to phytooestrogens. 126 The genotype-pasture interaction in the fertility of ewes has been discussed in a number of studies (e.g. Chang 1963; Davies, Rossiter and Mailer 1970) and recent findings in this area are noted by Lightfoot (1974). The genetic selection of resistant animals would be greatly assisted if there were some biochemical or metabolic marker for resistance. Reports from South Australia suggested that the blood haemoglobin type of the ewe was correlated with resistance to infertility on oestrogenic pastures (Obst, Seamark and McGowan 1971), but this has not been confirmed by other data (Wroth et al. 1973). The haemoglobin type of wetherswas not found to be correlated with the ratio of 4'0O-methyl-equol to equol in the urine (Cox, Ferguson and Braden, unpublished observations). The alteration of the metabolism of phyto-oestrogens in the animal is a potential method of control of clover disease. The rumen is the most accessible site in which to accomplish this, but such changes may be difficult to maintain. Such an approach would necessitate a greater understanding of the factors affecting rumen metabolism of phyto-oestrogens. Administration of inhibitory compounds would either require a potent substance with a lower oestrogenicity than the weak phyto-oestrogens, yet selective enough not to interfere appreciably with endogenous oestradiol action, or alternatively the use of other hormones which would exert antagonistic effects. This approach would seem difficult to achieve. The administration of androgens and progestagens has not yielded promising results (Anon 1964; Davies 1967). It has been shown that animals can be immunized against steroid oestrogens (Lieberman et al. 1959) and phyto-oestrogens (Bauminger et al. 1969). Immunization against endGus oestrogens can result in physiological effects in the animals through a neutralization of the hormone (Ferin et al. 1969; Caldwell et al. 1970). Low molecular weight compounds such as the phyto-oestrogens are not antigenic,but after covalent. attachment of antigenic proteins with suitable derivatives, can act as haptens. Such derivatives of a variety of phyto-oestrogens, including formonon&t&, daidzein and genistein, combined to proteins and injected into sheep have resulted in antibody formation in most of the animals and the sera have reacted specifically with the plant-oestrogens in laboratory tests (Cox et al. 1972). This work is being extended to raising antisera to the most important compounds, equol and coumestrol. Sera have not reacted appreciably with oestradiol-17B which is ' secreted by the sheep ovary, and hence it is'unlikely that the normal oestrous cycle would be affected. The antibodies have been found to persist in sheep for about a year without retreatment and a further immunization after 1 year has resulted in a marked increase in antibody titres. Preliminary biological tests in sheep immunized against genistein as a model compound indicated that the antibodies did afford some degree of protection against the biological effects of injected genistein (Cox, Wong, Little and Braden,unpublished observations). Through this immunophysiological approach it may be possible to bring about a-partial neutralization of the circulating phyto-oestrogens by decreasing the proportion of free, biologically active, phyto-oestrogen in the blood. At least some of the unconjugated phytooestrogen would be taken up by the specific antibodies and hence not be available to produce biological effects. 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