Level of nutrition modulates the dynamics of oestradiol feedback on plasma FSH in ovariectomized ewes.

The frequency of multiple ovulations in mature, cyclic ewes is strongly influenced by the level of nutrition, but it is difficult to demonstrate concurrent changes in plasma concentrations of gonadotropins. The failure to do so might be a consequence of rapid compensation by the homeostatic feedback mechanism linking secretion by the hypothalamus/pituitary gland and ovarian hormones. Most experimental models have examined the components of the homeostatic feedback system after steady state relationships had been established. We hypothesised that the effects of nutrition might be observed more readily if the system were disrupted and then examined while equilibrium was being re-established. This hypothesis was tested in three experiments in Merino ewes by allowing gonadotropin secretion to escape feedback for 5 days after ovariectomy and then replacing ovarian hormones and examining effects of feeding regimen on the return of plasma concentrations of FSH to baseline values. In all three experiments, oestrogen replacement caused plasma concentrations of FSH to decline more rapidly (P < 0.05) in ewes fed at 0.5x maintenance, than in ewes fed at 1.4x maintenance, with groups fed at maintenance being intermediate. No effect of diet was observed on plasma FSH concentrations in the absence of oestradiol, and neither progesterone nor charcoal-treated bovine follicular fluid influenced the effect of nutrition. Plasma concentrations of oestradiol were 9.8% lower on average (NS) in ewes fed above maintenance than in the sheep fed below maintenance over the three experiments, suggesting that there may have been a reduced clearance of oestradiol which contributed to the result. We conclude that feeding regimen affects the secretion or clearance of gonadotropins in mature ewes, as in the mature ram, and that this is one mechanism by which ovulation rate may be affected.

Detection of the effects of phytoestrogens on sheep and cattle.

Cows and ewes fed estrogenic forage may suffer impaired ovarian function, often accompanied by reduced conception rates and increased embryonic loss. Males are relatively unaffected, but the mammary glands in females and castrate males may undergo hypertrophy of the duct epithelium, accompanied by secretion of clear or milky fluid. In cows, clinical signs resemble those associated with cystic ovaries. The infertility is temporary, normally resolving within 1 mo after removal from the estrogenic feed. However, ewes exposed to estrogen for prolonged periods may suffer a second form of infertility that is permanent, caused by developmental actions of estrogen during adult life. The cervix becomes defeminized and loses its ability to store spermatozoa, so conception rates are reduced, although ovarian function remains normal. Importantly, both temporary and permanent infertility in ewes often occur without observable signs and can be detected only by measurement of phytoestrogens in the diet, or measurement of their effects on the animal. Low background concentrations of dietary phytoestrogens are suggested to play an important role in prevention of disease in humans and laboratory rats, but subclinical effects of phytoestrogens in cattle have not yet been described. Effects of low concentrations of phytoestrogens on reproductive function in ruminants are likely to receive increasing attention.

Organizational and activational effects of phytoestrogens on the reproductive tract of the ewe.

Ewes exposed to phytoestrogens may display two forms of infertility, categorized as temporary or permanent. Temporary infertility results from actions of estrogen that are similar to the activational effects of estrogen in most species of mammals. The permanent infertility results from changes to the cervix which are analogous to the organizational effects of estrogen reported in other species treated during organogenesis. However, in the ewe these effects may be produced after organogenesis by prolonged treatment during adult life. It has recently become apparent that the level of nutrition and metabolic hormones influence the degree of uterus-like histological change in the cervix produced by prolonged treatment with estrogen. It is hypothesized that, under some nutritional conditions, the hormonal milieu in adult ewes may simulate hormonal patterns that are normally experienced by fetal lambs in utero, thereby allowing the cervix of the adult ewe to give an organizational response to estrogen.

Effect of diet on the clearance of estradiol-17 beta in the ewe.

The twinning rate of ewes is poorly related to plasma concentrations of gonadotropins. In this study, we tested an alternative hypothesis for the control of twinning rate, by testing whether nutritional treatment sufficient to affect twinning could alter estrogen metabolism, with a particular focus on the enterohepatic recirculation. Groups of 5 ovariectomized ewes were fed either above maintenance (supplemented) or below maintenance (restricted). The metabolism of estradiol-17 beta was examined by following the fate of a single i.v. injection of 1.45 micrograms [3H]estradiol-17 beta. In both groups, 74% of the radioactivity was recovered from the feces within 10 days, predominantly as free estradiol-17 alpha, but excretion was slower in the nutritionally restricted ewes. A further 6% of injected radioactivity was excreted in the urine, mostly within 24 h, with no effect of dietary group. Radioactivity in plasma was characterized by ion-exchange chromatography and HPLC. Within 30 min of injection, the main circulating radioactive compound was estradiol-17 alpha sulfate. This remained at a greater concentration than free steroid for the next 48 h, and was greater after 16 h (p < 0.05) in plasma of nutritionally restricted ewes than in the supplemented group. At 0.5 and 2 h, the free steroid was almost entirely estradiol-17 beta, but a polar compound, which appeared by 4 h and probably arose by recirculation from the intestine, remained the major unconjugated metabolite in plasma for the next 24 h. Plasma concentrations of this compound were higher (p < 0.05) in the restricted ewes than in the supplemented ewes during this period.(ABSTRACT TRUNCATED AT 250 WORDS)

Sexual behaviour of ewes with clover disease treated repeatedly with oestradiol benzoate or testosterone propionate after ovariectomy.

Ovariectomized ewes, 14 with permanent clover disease infertility (affected ewes) and 14 controls, were injected daily with 40 micrograms oestradiol benzoate for 12 days, and run with 2 rams fitted with marking crayons. The control ewes were mated sooner (P less than 0.05) but both groups became refractory at a similar rate. In a second experiment, 20 similar affected ewes and 19 controls were injected daily with 5 mg testosterone propionate for 31 days and observed daily for 50 min with rams. Affected ewes again were slower to show female behaviour (P less than 0.05) but faster (P less than 0.05) to show aggression against the rams and other ewes. Over the 31 days, the incidence of female sexual behaviour declined at a similar rate in affected and control ewes. When examined in individual pen tests with oestrous ewes on Day 28, affected ewes showed more male-like courting behaviour than did controls (P less than 0.05). The changes in behaviour are too slight to account for the infertility but they do support the hypothesis that phyto-oestrogens can act on the ewe by some of the pathways of sexual differentiation, even after puberty.

Oestrogen receptors and metabolic activity in the genital tract after ovariectomy of ewes with permanent infertility caused by exposure to phytooestrogens.

Characteristics of the uterus anc cervix after ovariectomy of ewes with permanent phytooestrogen infertility (PPI) were compared with controls. Ewes with PPI had more oestrogen-binding sites in the cervix, but not in the uterus. There was no difference between the two groups of ewes in the binding affinity constant of receptors from the uterus or cervix. There were more keratinized cells in the vaginal epithelium of ewes with PPI, and the rates of protein and glycoprotein synthesis in the uterus and cervix were higher in ewes with PPI. These results offer further evidence that PPI in adult ewes is similar to the "persistent oestrus' syndrome in rodents oestrogenized neonatally.

A changed responsiveness to oestrogen in ewes with clover disease.

When clover-infertile ewes are subsequently exposed to non-oestrogenic pasture, they have a reduced fertilization rate, due to an inability to store spermatozoa in the cervix, and the cervical mucus has a reduced spinnbarkeit, caused by a slower response to oestrogenic stimulation. Vaginal cell keratinization and oestrous behaviour occurred more slowly after treatment of affected ewes with oestrogen. Other changes in affected ewes suggest that phyto-oestrogens have permanent mild differentiating effects on adults. Sexual behaviour is masculinized, the cervix takes on a uterine-like appearance and the genital tract becomes permanently oestrogenized. The manner in which these changes relate to the altered responsiveness to oestrogen remains to be clarified.

Toxic effects of large amounts of DL-methionine infused into the rumen of sheep.

Four Merino wethers were fed ad libitum on a roughage diet comprised of equal parts of chopped lucerne hay and oaten chaff. The sheep received continuous infusions of DL-methionine into the rumen; the daily dose increasing from 0 g (control treatment) up to 30 g in 3 g amounts at weekly intervals. Dry matter intake (DMI) fell below the control level when 24 g/day or more of the amino acid was infused, while plasma free methionine levels increase substantially when 30 g/day was given. There was no effect of DL-methionine supplementation on the bodyweight of the animals. However, when 30 g/day of DL-methionine was infused one animal died and the acute condition of 2 others necessitated their slaughter. Significant lesions included acute nephrosis and haemolytic anaemia, with milder changes in the liver and pancreas. This report indicates that sheep are unlikely to show the kind of chronic methionine toxicity seen in other species.

Changes in oestradiol-17beta binding in the hypothalami and pituitary glands of persistently infertile ewes previously exposed to oestrogenic subterranean clover: evidence of alterations to oestradiol receptors.

The binding of [3H]oestradiol-17beta to the hypothalamus and pituitary gland of clover-affected permanently infertile and control ovariectomized ewes was compared in vivo and in vitro. When [3H]oestradiol-17beta was infused into the carotid artery (10 ng/min), the total homogenate and the nuclear and protamine-precipitable cytosol fractions of hypothalami and pituitary glands from clover-affected ewes bound significantly more[3H]oestradiol than those of the controls. Cytoplasmic oestradiol-17beta receptors from the pituitary glands of clover-affected ewes showed a significantly lower apparent association constant and a higher number of binding sites/mg protein in vitro. It is suggested that the hypothalami and pituitary glands of ewes made permanently infertile by oestrogenic clover are less sensitive to feedback regulation of oestradiol-17beta at physiological levels.

Morphological changes in the organs of ewes grazing oestrogenic subterranean clover.

The morphological effects of phytooestrogen exposure were determined in 10 ewes exposed to subterranean clover for 60 days, compared with 10 controls. In a second experiment, the time course of the development of the changes was studied. Typically oestrogenic changes were observed in ovary, oviduct, uterus, cervix, vagina and mammary glands. There was an early increase in cervical mucus, followed by a decrease. The delta basophils of the pituitary became degranulated, and hyperactive in appearance. The adrenal and thyroid glands increased in weight, and thyroid epithelium increased in height. There appeared to be a temporary increase in neurophysin storage in the hypothalamus, and shrunken, hyperchromatic neurones were observed in the hypothalamus of some affected ewes. All changes were observed within three weeks of exposure.