Fecal Progesterone Metabolites Research Articles

Non-invasive assessment of oestrous cycles and evaluation of reproductive seasonality in the female wild black rhinoceros (Diceros bicornis minor).

Female wild black rhinoceroses in Zimbabwe were monitored non-invasively using faecal progesterone metabolite analysis and observation of reproductive behaviour. A postpartum period of reproductive inactivity of at least 4 months, followed by a period of 4-7 months of oestrous cyclicity, was detected in six multiparous females. Three-quarters of the oestrous cycles (n = 21) had a total duration (mean +/- SEM) of 26.8 +/- 1 days. Other types of cycle were characterized either by an extended luteal phase, lasting on average twice as long as the normal cycle, or by an extended follicular phase. These extended cycles may have resulted from early embryo loss and heat stress. Female rhinoceroses did not conceive before 8 months after giving birth and some females (n = 2) most likely aborted after 3.0-3.5 months of gestation. The detected period of cyclic oestrus occurred between May and March in females (n = 9), and there was a 3 month extended interoestrous interval in nulliparous females during the period of decreasing daylengths that can be presumed to be the period of poorest fertility for the black rhinoceros under tropical latitudes. In contrast, the period of optimum fertility in the Southern hemisphere coincided with the late spring and early summer, and corresponded to the early rainy season. As a result, a higher incidence of births was detected in the late rainy season, providing the lactating female with the most suitable environment in terms of nutritional requirements.

The effect of level of feed intake on progesterone clearance rate by measuring faecal progesterone metabolites in grazing dairy cows

The objective of the present study was to determine the effect of level of feed intake of pasture on P4 clearance rates in dairy cows. Twelve non-lactating Holstein–Friesian cows aged 4–9 years were randomly allocated to a restricted or ad libitum group. The ad libitum group had unrestricted access to irrigated pasture, whereas the restricted group had access for only 2 h per day. Each animal was drenched orally twice daily with a chromic oxide capsule to allow daily feed intake to be estimated from faecal output (FO). Endogenous progesterone (P4) production was eliminated by subcutanously implanting a capsule containing 6 mg of a potent GnRH-agonist (deslorelin) into the ear of each animal 3 weeks before inserting a CIDR device containing 1.9 g P4 into the vagina. Two luteolytic PGF2α were given 10 days later. Each device was removed after 11 days and residual P4 measured. Daily plasma samples were assayed for P4. Faecal samples were also taken daily and assayed for pregnanes (FP4M) containing a 20-oxo-, a 20α- or a 20β-OH group with EIAs. The average daily dry matter (DM) intake of pasture was higher for cows in the ad libitum group (15.9 versus 6.3 kg DM, P=0.001). Their plasma P4 concentrations were lower (1.08 versus 1.71 ng/ml, P=0.05), even though the average residual P4 content of the used CIDR devices was not affected by feed intake (1.20 versus 1.25 g, P>0.05). The concentrations of FP4M were not affected by level of feed intake (20-oxo-: 3.3 versus 1.7, 20α-: 3.5 versus 3.7, 20β-: 2.1 versus 3.2 μg/g DM). Daily excretion rates of 20-oxo- and 20α- were higher in ad libitum cows (20-oxo-: 17.8 versus 4.3 mg per day, P=0.05; 20α-: 18.2 versus 8.9 mg per day, P=0.001), but daily yield of faecal 20β- was not affected by feed intake (11.9 versus 8.6 mg per day, P=0.5). These results show that there was a negative relationship between feed intake and plasma P4 concentrations in these CIDR-treated GnRH-downregulated Holstein cows. Concentrations of FP4M were not affected by level of feed intake or FO, but daily excretion rate of FP4M was associated with the volume of faeces.

Noninvasive Stress and Reproductive Measures of Social and Ecological Pressures in Free‐Ranging African Elephants

Abstract: The African elephant ( Loxodonta africana) experienced a poaching‐related 60% population decline between 1979 and 1988 that was inordinately concentrated on adults. This, coupled with political pressures to delist the elephant, has created a need for noninvasive physiological measures that can quantify the long‐term effects of past mortality patterns of this long‐lived species. We collected fresh fecal samples from 16 female elephants in three different groups over 23 months at Tarangire National Park, Tanzania, and analyzed them for fecal progesterone and cortisol metabolites. Social and ecological measures were collected concurrently. Fecal progesterone metabolite measures corresponded significantly with stage of gestation, and appear to be able to confirm pregnancy in female elephants from as early as 3 months of gestation. We found that progesterone metabolite concentrations were significantly lower during the dry season than during the wet season after controlling for stage of gestation. Fecal cortisol metabolite concentrations showed the opposite seasonal pattern, being significantly higher in the dry season and inversely correlated with rainfall across seasons. Fecal cortisol metabolite concentrations also increased with group size and were correlated positively with dominance rank in the largest group. Our results suggest that measures of progesterone and cortisol metabolites in feces provide indices of reproductive function and physiological stress that can quantify both natural and human disturbances in African elephants. These measures are ideally suited for monitoring the long‐term effects of social disruption from poaching and a variety of other management concerns.

Excretion rate of progesterone in milk and faeces in lactating dairy cows with two levels of milk yield.

This study was conducted to measure the effect of the level of daily milk yield on the excretion rate of progesterone (P4) in milk and faeces in high-producing (HP) and low-producing (LP) lactating dairy cows. A GnRH-agonist was implanted to block endogenous production of P4. A CIDR device was inserted into the vagina and left in place for 11 days. The average and peak milk yields were greater in HP cows (P < 0.0001). Mean plasma concentrations of P4 were also similar in both groups (P = 0.44), even though the average mass of P4 delivered from a CIDR device was higher with HP cows (P = 0.02). Average milk P4 concentration was similar in both groups (P = 0.81), so that average daily excretion of P4 in the milk was greater with HP cows (P = 0.05). The concentrations (P = 0.83) and daily yields (P = 0.4) of total faecal progesterone metabolites were not affected by level of milk yield. These data show that the concentrations of plasma and milk P4, and the concentration and yield of P4 metabolites are not affected by the levels of daily milk yield.

Fecal Progesterone, Estrogen, and Androgen Metabolites for Noninvasive Monitoring of Reproductive Function in the Female Indian Rhinoceros, Rhinoceros unicornis

This investigation aimed to establish noninvasive methods for endocrine monitoring of estrous cycles and pregnancy in the Indian rhinoceros. Fecal samples were collected 1–3 times per week from nonpregnant and pregnant captive females (n = 7). Enzyme immunoassays for fecal progesterone, androgen, and estrogen metabolites, respectively, were tested for their ability to determine follicular and luteal phases and to characterize endocrine profiles during pregnancy. Antibodies used were raised against pregnanediol (20α-OH-pregnanes), 20-oxo-pregnanes, epiandrosterone (17-oxo-androstanes), and total estrogens. Androgens and estrogens were found to be reliable indicators of the follicular phase, whereas 20α-OH- and 20-oxo-pregnanes were reliable indicators of luteal function. Progesterone metabolites were also reliable indicators of pregnancy, whereas 17-oxo-androstanes and estrogens were basal throughout gestation. Estrous cycles were regular throughout the year, with an average cycle length of 43.4 ± 1.5 (n = 27) days; the length of the follicular phase, as indicated by elevated estrogen levels, was 15.9 ± 1.0 days, whereas the luteal phase, as indicated by elevated 20-oxo-pregnane levels, was 19.1 ± 0.4 days. Fecal pregnane values were already increasing while follicular estrogen values were still decreasing. The length of the diestrus, indicated by basal steroid levels between declining 20-oxo-pregnanes and subsequently increasing estrogens, was 11.4 ± 1.2 days. Pregnane levels increased from the 3rd month of gestation onward and levels exceeded luteal phase concentrations ∼10 times by the 7th month of gestation onward. HPLC separation of immunoreactive fecal metabolites indicated the presence of estrone, estradiol-17β, and several 17-oxo-androstanes, 20α-OH-pregnanes, and 20-oxo-pregnanes. Concentrations of a peak with an elution profile similar to that of pregnanediol increased as pregnancy progressed. Postpartum fecal estrogen and 17-oxo-androstane concentrations in one animal indicated follicular development comparable to the follicular phase of the estrous cycle, but this was not followed by a subsequent luteal phase. In conclusion, estrous cycle and pregnancy in Indian rhinoceroses can be monitored using fecal steroid analysis. Pregnane metabolites were reliable indicators of the corpus luteum and pregnancy, whereas fecal 17-oxo-androstanes and estrogens were indicators of the follicular phase.

Characterization of Estrous Cyclicity in the Sable Antelope(Hippotragus niger)through Fecal Progestagen Monitoring

Fecal progesterone metabolite monitoring techniques were validated for the sable antelope and used to characterize ovarian cycle dynamics and reproductive seasonality in a captive population at the National Zoological Park's Conservation and Research Center. Hormone was homogeneously distributed within fecal samples. Longitudinal fluctuations in fecal progesterone metabolites were consistent with typical luteal phase patterns and corresponded closely with changes in serum progesterone. The lag time from an im injection of progesterone to peak excretion in feces was 16 h. The pattern of births showed a slight peak in the summer (May–July), but year-round endocrine monitoring of six nonpregnant females showed no evidence of seasonality in ovarian activity. Females exhibited 11–14 estrous cycles per year, averaging 24.2 ± 0.9 days in length. Luteal and interluteal phases were 18.4 ± 0.9 and 5.8 ± 0.4 days in length, respectively. Although only a small number of hippotragine antelope species have been studied, data indicate that they exhibit considerable interspecific variability in estrous cycle length and reproductive seasonality and thus may be a useful group for further investigation of factors regulating fertility.

Serum and fecal steroid analysis of ovulation, pregnancy, and parturition in the black rhinoceros (Diceros bicornis)

Studies were conducted to determine: (1) if fecal hormone metabolite concentrations correlated with serum estrogen and progesterone concentrations, follicular activity and reproductive behavior in the black rhinoceros (Diceros bicornis) and (2) if threshold values of respective fecal metabolite concentrations correlated with pregnancy. Blood and fecal samples were collected, in conjunction with transrectal ultrasound and behavior observations, for an 18-month period from one black rhinoceros female. Subsequently, serial fecal samples were collected from 13 females in 10 zoos. Quantitative analysis of serum progesterone (P4) and estradiol (E2) was performed by radioimmunoassay (RIA): analysis of fecal estrogen metabolites (E) and fecal progesterone metabolites (P) were performed by enzyme immunoassay (EIA). Serum P2 concentrations identified two luteal phase patterns and two nadirs which corresponded with behavioral estrus. Fecal E patterns indicated a sharp peak which corresponded with breeding. concentrations of fecal P illustrated identifiable nadirs and several peaks which corresponded to serum P4 nadirs and luteal phases. Serum P4 concentrations were not different between the luteal phase and pregnancy. Fecal P concentrations started to rise above luteal phase concentrations approximately 150 days postbreeding and remained elevated until immediately before parturition. Serum E2 and fecal E concentrations rose and subsequently declined after parturition. In the fecal samples from seven pregnant females, fecal P concentrations were similarly elevated compared to six nonpregnant females. Results indicated that fecal steroid metabolites accurately reflected serum steroid hormone concentrations and that the measurement of P and E concentrations permitted the characterization of the estrous cycle, the diagnosis of pregnancy, and the onset of parturition. Zoo Biol 16:121–132, 1997. © 1997 Wiley-Liss, Inc.

Steroid metabolism and validation of noninvasive endocrine monitoring in the African wild dog (Lycaon pictus)

The purpose of this study was to validate noninvasive endocrine monitoring techniques for African wild dogs (Lycaon pictus) and to establish physiological validity of these methods by evaluating longitudinal reproductive-endocrine profiles in captive individuals. To determine the primary excretory by-products of ovarian steroid metabolism, [14C]-progesterone and [3H]-estradiol were co-administered to a female and all excreta were collected for 80 hr postinjection. Radiolabel excretion peaked ≤ 18 hr postinfusion, and progesterone and estradiol metabolites were excreted in almost equivalent proportions in urine (39.7 and 41.1%, respectively) and feces (60.3 and 58.9%, respectively). Most of the urinary metabolites were conjugated (estradiol, 94.3 ± 0.3%; progesterone, 90.4 ± 0.5%), so that immunoassays for pregnanediol-3α-glucuronide (PdG) and estrogen conjugates (EC) were effective for assessing steroid metabolites. Two immunoreactive estrogens (estradiol and estrone) and at least one immunoreactive progesterone metabolite (3α-hydroxy-5α, pregnan-20-one) were detected in feces. Urine and fecal samples were collected (1–3 times per week) for 1.5 yr from one adult female and two adult males to assess longitudinal steroid metabolite excretion. Overall correlation of urinary PdG to matched, same-day fecal progesterone metabolites immunoreactivity was 0.38 (n = 71, P < 0.05). Similarly, urinary EC was correlated (P < 0.05) with same-day fecal estrogen immunoreactivity (r = 0.49, n = 71). During pregnancy and nonpregnant cycles, copulation occurred at the time of peak (or declining) estrogen metabolites and increasing progesterone metabolites concentrations. Estrus duration was 6–9 days and gestation lasted 69 days with parturition occurring coincident with a drop in progesterone metabolites. Males exhibited seasonal trends in fecal testosterone excretion with maximal concentrations from July to September coincident with peak mating activity. Although these limited longitudinal hormone profiles should be interpreted cautiously, noninvasive gonadal steroid monitoring suggests that: (1) both female and male wild dogs may exhibit reproductive seasonality in North America, (2) females are monoestrous, and (3) peak testicular activity occurs between August and October coincident with mating behavior. From a conservation perspective, noninvasive endocrine monitoring techniques should be useful for augmenting captive breeding programs, as well as for developing an improved understanding of the physiological mechanisms underlying reproductive suppression in response to social and ecological pressures. Zoo Biol 16:533–548, 1997. © 1997 Wiley-Liss, Inc.

Use of group-specific antibodies to detect fecal progesterone metabolites during the estrous cycle of cows

Progesterone is metabolized to pregnanediones and hydroxylated pregnanes prior to its fecal excretion. Therefore, use of progesterone antibodies underestimates the actual amount of fecal metabolites. To improve the methodology of noninvasive fecal progesterone metabolite analysis, enzymeimmunoassays (EIA) using group-specific antibodies against 5-reduced 20-oxo-pregnane-C3-conjugates were developed. Fecal and milk samples were collected at 1- to 2-d intervals during the morning and evening milking throughout 1 estrous cycle in dairy cows (n = 12). Six immunoreactive metabolites were detected in the feces with high performance liquid chromatography (HPLC), eluting as 5α- and 5β-reduced pregnanes containing a 20-oxo-group (20-oxo-pregnanes). Fecal samples of 3 cows were analyzed by 3 EIAs using antibodies against 4-pregnene-6α-ol-3,20-dione 6HS:BSA (6HS-progesterone), 5α-pregnane-3β-ol-20-one 3HS:BSA and 5β-pregnane-3β-ol-20-one 3HS:BSA, respectively. The follicular and luteal phases were identifiable with each EIA. Luteal phase values and the differences between mean follicular (Days 0 to 2 and 19 to 21) and luteal phase (Days 10 to 16) values obtained with the 5-pregnane EIAs were 3- to 4-fold higher than with the 6HS-progesterone EIA. Since results with the former 2 EIAs were almost identical, the remaining samples were only analyzed by the EIA for 5β-pregnane-3α-ol-20-one. Fecal 20-oxo-pregnane concentrations were parallel to milk progesterone values, but had a lag time of about 0.5 d; the coefficient of correlation (P < 0.001) was 0.76 (y = 155.2 × + 37.2). Fecal 20-oxo-pregnane concentrations during the follicular and luteal phase were 39.5 ± 2.2 and 341 ± 15.2 ng/g feces, respectively. In conclusion, fecal 20-oxo-pregnanes are significantly correlated to milk progesterone concentrations. They consist of several metabolites and compared to a 6HS-progesterone antibody, their evaluation was improved using antibodies against 5-reduced pregnanes.

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.

Pregnancy detection in bighorn sheep (Ovis canadensis) using a fecal-based enzyme immunoassay.

We developed and validated an enzyme immunoassay for immunoreactive pregnanediol-3-glucuronide (iPdG) in feces to monitor reproductive status in desert and Rocky Mountain bighorn sheep (Ovis canadensis). Fecal iPdG concentrations were strongly correlated (r = 0.71) with serum progesterone concentrations in paired fecal and blood samples collected from 34 free-ranging desert bighorn sheep. In bimonthly fecal samples collected from 12 captive ewes, fecal iPdG profiles were similar between desert and Rocky Mountain bighorn sheep and we selected a pregnancy detection cutoff value of iPdG > or = 1.8 ng/mg feces. Fecal iPdG concentrations always exceeded this cutoff value when samples were collected from about day 60 of pregnancy to a few days before parturition, but values < 1.8 ng/mg (false negatives) were common for samples collected during the first 60 days of gestation. Although we tested a small number of known pregnant and non-pregnant ewes, the accuracy of the assay was 100% when two samples, collected 2 wk apart, were evaluated for any given ewe. Based on these data, this direct enzyme immunoassay for fecal progesterone metabolites has promise as a diagnostic tool to monitor hormone excretion and pregnancy in a free-ranging ungulate species.

Excretion of progesteone metabolites and estriol in faeces from pregnant women during ampicillin administration

Progesterone metabolites and estriol were determined in urine and faeces collected daily from three pregnant women (33–37 weeks) before and during ampicillin administration (2 g/day orally). Two of the three subjects showed marked changes in their faecal steroid excretion during ampicillin administration: the faecal progesterone-metabolite pattern changed from containing 69–79% unconjugated metabolites and 19–26% glucuronides under control conditions, to high steroid sulphate content (28–44%); the faecal elimination of 3β-hydroxy-5α-pregnan-20-one and 5α-pregnane-3β,20α-diol glucuronide all but ceased; two 16α-hydroxylated progesterone metabolites were detected in significant amounts in faeces during ampicillin administration but not under normal conditions. Steroid sulphate hydrolysis, epimerization of 3α,5α- to 3β,5α-steroids and 16α-dehydroxylation are all well known actions of intestinal bacteria on biliary steroids. It thus seems clear that the changes found in the faecal progesterone metabolite pattern are due to the reduction of the intestinal flora by ampicillin. Under control conditions the bulk of the faecal estriol was unconjugated. During ampicillin administration this excretion remained unchanged but in addition large quantities of conjugated estriol appeared in the faeces, apparently as a result of inhibition of bacterial deconjugation. Ampicillin administration also caused decreased urinary excretion of estriol and pregnanediol glucuronide. It seems likely that these well documented effects of ampicillin on urinary steroid excretion are caused by an interruption of the enterohepatic circulation of steroids which results from the inhibition of intestinal steroid metabolism described above.