E. Bamberg

Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals

Abstract Non-invasive faecal oestrogen and progesterone metabolite evaluations are well established approaches for monitoring reproductive function in a variety of mammalian species. The route of excretion of steroid hormone metabolites varies considerably among species, and also between steroids within the same species. Steroid concentrations in faeces exhibit a similar pattern to those in plasma, but have a lag time, which depending upon the species, can be from 12 h to more than 2 days. Faecal steroid metabolites in mammals are mainly unconjugated compounds. Faecal oestrogens consist predominantly of oestrone and/or oestradiol-17α or -17β. Therefore, specific oestrogen antibodies or antibodies against total oestrogens can be used for their determination. Progesterone is metabolised to several 5α- or 5β-reduced pregnanediones and hydroxylated pregnanes prior to its faecal excretion. Therefore, relevant antibodies for their determination show considerable cross-reactivities with several pregnane metabolites, whereas specific progesterone antibodies are less suitable. Faecal oestrogen evaluations have been used as reliable indicators of pregnancy in several ungulate and some primate species. They have also been used to determine the preovulatory period in carnivores, corpus luteum activity in New World primates, and to diagnose cryptorchidism in horses. Faecal progesterone metabolite analysis has been successfully used for monitoring corpus luteum function and pregnancy, abortion, seasonality and treatment therapies in an ever expanding list of species.

Excretion of corticosteroid metabolites in urine and faeces of rats

Stress enhances the production of corticosteroids by the adrenal cortex, resulting in the increased excretion of their metabolites in urine and faeces. An intraperitoneal injection of radioactive corticosterone was applied to adult, male Sprague-Dawley rats to monitor the route and delay of excreted metabolites in urine and faeces. Peak concentrations appeared in urine after 3.2 ± 1.9 h and in faeces after 16.7 ± 4.3 h. Altogether about 20% of the recovered metabolites were found in urine and about 80% in faeces. Using high-performance liquid chromatography (HPLC), several peaks of radioactive metabolites were found. Some metabolites were detected by enzyme immunoassay (EIA) using two different antibodies (corticosterone, 11β-OH-aetiocholanolone). There was a marked diurnal variation with low levels of faecal corticosterone metabolites in the evening and higher values in the morning. This diurnal variation was influenced neither by the intraperitoneal injection of isotonic saline nor by ACTH. However, the administration of dexamethasone eliminated the morning peak for 2 days.

Excretion of corticosteroids in urine and faeces of hares (Lepus europaeus).

Abstract Increased production of glucocorticoids by the adrenal cortex is found in mammals under stress. As cortisol itself is absent in the faeces, an enzyme immunoassay (11-oxoaetiocholanolone) measuring 11,17-dioxoandrostanes has already been established to measure faecal cortisol metabolites in ruminants for non-invasive monitoring of adrenocortical activity. The aim of this study was to establish route and delay of excretion of glucocorticoids in hares and to determine whether a cortisol-, corticosterone- or this new enzyme immunoassay is best suited to detect faecal glucocorticoid metabolites. In the first experiment radioactive-labelled glucocorticoids (14C-cortisol and 3H-corticosterone) were administered intravenously to two groups of three hares in metabolic cages. All voided urine and faecal samples were collected for 4 days. Metabolites of both steroids were found predominantly in the urine (91 ± 4%). Peak concentrations were observed in the first urinary sample following infusion (13 ± 6 h) and in the faeces with a delay of about 1 day (23 ± 7 h). Most of the radioactivity was not extractable with diethylether, indicating that the metabolites excreted in urine and faeces are mainly conjugated or polar unconjugated ones. This was confirmed by reverse-phase high-performance liquid chromatography separations of the metabolites, which also revealed marked differences concerning the metabolism of the two glucocorticoids injected. Compared with the cortisol and the corticosterone enzyme immunoassay, only the group-specific enzyme immunoassay for 11,17-dioxoandrostanes detected high quantities of immunoreactive metabolites. In a second experiment hares (n=20) were stressed by rousing them three times (5 min, 10 min and another 5 min) with a 20-min break in-between. Faecal samples were collected 2 days before until 4 days after stress and analysed using the 11-oxoaetiocholanolone enzyme immunoassay. After stress significantly (P < 0.001) increased 11,17-dioxoandrostane concentrations were found. Based on these results, measuring 11,17-dioxoandrostanes in faeces enables non-invasive monitoring of disturbances in hares and thus provides a tool for field investigations elucidating the role of stress in hare populations.

Pregnancy diagnosis in cows and heifers by determination of oestradiol-17α in faeces

Abstract The concentration of unconjugated oestradiol-17α was measured in faeces of 21 non-pregnant animals and of 39 cows and heifers between the 10th and 25th week of gestation. After the 14th week of gestation all pregnant animals secreted significantly more oestradiol-17α in the faeces than non-pregnant animals. This is a possible method therefore for the confirmation of pregnancy not only in cows but also in heifers.

Pregnancy diagnosis in sows by estimation of oestrogens in blood, urine or faeces☆

Abstract Concentrations of oestrone sulphate in blood and of total unconjugated oestrogens in faeces were measured during the oestrous cycle and early gestation. The level of these hormones in blood and faeces was consistently higher (blood, 100%; faeces, 69–100%) between Days 24 and 30 in pregnant than in non-pregnant sows. Therefore, early pregnancy diagnosis based on the concentration of oestrogens in blood, urine and faeces was performed on Day 27, 28 or 29 after service for a total of 290 sows. Blood samples (25 μl) were taken from the tail vein of 289 sows. One hundred urine samples (1 ml) were collected by inserting a cellulose sponge into the vagina and faeces samples (2 g) were taken from the rectum of 290 sows. Pregnancy diagnosis was made on the assumption that more than 2.79 ng oestrone sulphate/ml blood, 19.7 ng oestrogens/g faeces and 65.6 ng oestrogens/ml urine indicated a pregnant animal. The efficiency of the tests was determined by comparing the diagnosis based on oestrogen concentrations with the farrowing results. The oestrone sulphate concentration in blood, as measured by enzyme immunoassay on microtitre plates, showed that 265 of the examined sows were correctly diagnosed, an accuracy of 91.7%. Seven results were false negative and 17 false positive. The determination of oestrogens in urine by radioimmunoassay (RIA) showed that 98 of 100 examined sows were correctly diagnosed and 2 were false negative. According to the oestrogen concentration in faeces, measured by RIA, 271 sows (93.5%) were correctly diagnosed, 16 were false negative and 3 false positive.

Monitoring ovarian cycle and pregnancy in the / giant anteater Myrmecophaga tridactyla by faecal progestagen and oestrogen analysis

Oestrogen and progestagen metabolites were measured in the faeces of five female giant anteaters (Myrmecophaga tridactyla), to characterise the oestrous cycle and pregnancy. Faecal samples were collected twice weekly for a minimum of 6 months, and immunoreactive progestagens and oestrogens were analysed using enzyme immunoassays (EIA). For progestagens, two antibodies that cross-reacted with 20alpha-hydroxy- or 20-oxo-progestagens were used. Both assays effectively monitored ovarian cyclicity; however, the concentrations obtained using the antibody for 20alpha-hydroxy-progestagens were higher, and the hormonal changes were more pronounced. Regular ovarian cycles were identified in three of the five females. Average (+/-SEM) length of the oestrous cycle (n=10) was 51.4+/-5.6 days. Peak concentrations of 20alpha-hydroxy-progestagens ranged from 80-660 ng/g of faeces and those of oestrogens from 20-100 ng/g. Hormone concentrations were measured during parts of two pregnancies and during four post-partum periods. The length of one gestation (from oestrous oestrogen peak until parturition) was 184 days. In the second half of gestation, progestagen concentration started to increase above luteal phase values; in the week before parturition it was approximately 20 times higher than those during the luteal phase. Concentrations of excreted oestrogens began to increase after two thirds of gestation and exceeded that of the follicular phase by approximately 2.5-fold in the week before parturition. Onset of ovarian cyclicity after parturition varied from 4-11 weeks. In conclusion, the measurement of faecal immunoreactive progestagens and oestrogens in the giant anteater indicated an ovarian cycle of approximately 7 weeks in length and provided potentially useful data for successful breeding management.

Fecal estrogens in one primate and several ungulate species during various reproductive stages

Abstract Fecal estrogen concentrations were monitored during pregnancy and the immediate postpartum period by enzyme immunoassay (EIA) in four zoo animal species. The gorillas, sable antelopes and tapir showed a steady increase in estrogen concentrations throughout pregnancy, with maximum values recorded just before parturition and rapid decline to basal values immediately after parturition. In contrast, estrogen concentrations in the Grevys zebra peaked by mid-gestation and declined during the final stages of gestation. The determination of fecal estrogen concentrations by EIA provides a practical method for diagnosis and confirmation of pregnancy in many zoo animals but must be used cautiously, particularly if extended to species whose profiles are not clearly established.

Plasma and fecal progestagen evaluations during and after the breeding season of the female vicuna (Vicuna vicuna ).

Plasma and fecal progestagen patterns of female (n = 10) vicunas (Vicuna vicuna ) were determined about 1 to 2 mo before and until 4 mo after breeding. The vicunas were caught wild and were penned at the Lauca National Park (Chile, 4470 m above sea level) for 7 mo (December to June). Plasma and fecal samples before and during the mating period (January to March) were collected 4 to 5 times weekly, and once or twice weekly thereafter. The samples were analyzed by enzymeimmunoassays (EIA) using antibodies against progesterone and 20alpha-dihydroprogesterone. High performance liquid chromatography (HPLC) separations confirmed that progesterone and 20alpha-dihydroprogesterone predominated in the plasma, whereas in the feces several unconjugated, immunoreactive progestagen metabolites containing either a 20-oxo- or a 20alpha-OH-group occurred. The coefficients of correlation (n = 409; P < 0.01) between matched plasma and fecal samples were 0.39 and 0.53 for 20-oxo- and 20alpha-progestagens, respectively. Elevated (5 to 6 d) plasma and corresponding fecal progestagens after mating indicated cyclic corpus luteum activity in 5 of the animals. After the mating period (23.2 +/- 3.3 d), corpus luteum function in these 5 animals persisted, as it did in 3 other animals that were not observed to be mating. The persisting corpus luteum function was demonstrated by increased mean plasma and fecal progestagen concentrations (> 1 ng/ml and > 100 ng/g, respectively). Mean plasma 20alpha-dihydroprogesterone concentrations exceeded that of progesterone by about 1 ng/ml (P < 0.01). The results demonstrated that in addition to plasma progesterone, plasma 20alpha-dihydroprogesterone and noninvasive fecal progestagen evaluations are useful, valid tools for determining corpus luteum function in vicunas.

Progesterone, 17α-hydroxyprogesterone, androgens and oestrogens in bovine ovarian cysts

The concentration of steroid hormones in cystic follicular fluid was determined in cows with cystic ovaries. There was a significant difference in concentrations of the hormones from the cysts with granulosa cell layers, and the cysts without granulosa cell layers or only 2 to 3 layers with pycnotic nuclei in the granulosa cells (P < 0.01). The cystic follicles that consist of both thecal- and granulosa cell layers contained a low amount of progesterone and high levels of 17α-hydroxyprogesterone, androgens and oestrogens, not different from preovulatory follicles. In contrast, cysts that consist only of thecal cell layers contained a very high amount of progesterone, but very low levels of 17α-hydroxyprogesterone, androgens and oestrogens. That is functionally similar to the bovine corpus luteum which produces high concentrations of progesterone but has no or very low 17α-hydroxylase activity. In conclusion, the cystic follicles without granulosa cells are not capable of secreting 17α-hydroxyprogesterone, androgens and oestrogens, in spite of high levels of progesterone. It may be suggested that in these cysts there is a blockade of 17α-hydroxylase activity.

Rapid and efficient extraction of steroids from microlitre quantities of plasma using extrelut® mini-columns

Abstract The radioimmunoassay (RIA) of steroid hormones in body fluids normally involves extraction procedures which are both time consuming and costly with respect to special equipment (glassware, shakers, centrifuges etc.). The extraction stages can be omitted when direct RIA is used, but then highly specific antisera are required and only relatively high concentrations of steroids can be measured. Recently, as an alternative to conventional extraction procedures, the use was proposed [1] of columns of granular Kieselgur (Extrelut ®, Merck). We have modified such an extraction method using mini-columns of Extrelut®, and have tested the reliability and repeatability of this extraction method with respect to several steroids. Our findings are reported below.