Fecal Estrogen Metabolites Research Articles

Variations, validations, degradations, and noninvasive determination of pregnancy using fecal steroid metabolites in free-ranging pronghorn

Pregnancy status is a key parameter used to assess reproductive performance of a species as it represents a starting point for measuring vital rates. Vital rates allow managers to determine trends in populations such as neonate survival and recruitment; two important factors in ungulate population growth rates. Techniques to determine pregnancy have generally involved capture and restraint of the animal to obtain blood samples for determining serum hormone levels. Non-invasive pregnancy assessment, via feces, eliminates any hazards between handler and animal, as well as removes handling-induced physiological biases. Using noninvasive fecal sampling, we conducted hormone validations, investigated pregnancy rates, and determined hormone degradation rates across five subpopulations of pronghorn (Antilocapra americana) in Idaho. Samples were collected during April-May of 2018 and 2019 from adult pronghorn of known sex and age class. Metabolites of testosterone, cortisol, 17β-estradiol, and progesterone were measured in fecal samples, and concentrations of estradiol and progesterone were examined for pregnancy determination. Average fecal progesterone metabolite (FPM) levels of pregnant females were more than double compared to levels of nonpregnant females. Fecal estrogen metabolite (FEM) levels did not differ during concurrent sampling. The largest difference in FPM levels between pregnant and nonpregnant females began on 28 April. Pregnancy determination sampling showed average FPM levels for all five subpopulations were significantly different than the nonpregnant female validation group. Nonetheless, pregnancy rates for some subpopulations lacked conclusive estimates due to early fecal sampling. Fecal glucocorticoid metabolites (FGM) levels significantly differed between pregnant females and male pronghorn, but did not differ from nonpregnant females. Degradation rates of FPM and FGM differed across days, with values for FPM from Day 1 being significantly different from all subsequent days, and after Day 9 for FGM, demonstrating the requirement of fresh samples to accurately measure hormone concentrations. We concluded that a noninvasive method to diagnosis pregnancy is possible in pronghorn via progesterone metabolites if fresh samples are collected during late gestation.

Fecal progesterone and estrogen metabolite monitoring for cyclicity and pregnancy in southern tamandua (Tamandua tetradactyla).

Reproductive management of the southern tamandua (Tamandua tetradactyla) should include timed introductions for breeding to minimize aggression and pregnancy monitoring. Since serial blood sampling could cause unnecessary stress, and urinary progesterone metabolites are found in very low concentrations, this study sought to validate progesterone and estradiol enzyme immunoassays for measuring fecal progesterone metabolite (FPM) and fecal estrogen metabolite (FEM) concentrations in two females. Peaks in FEM concentrations coincided with breeding and conception, were 5-6 times higher than baseline concentrations, and were followed by clear luteal phases distinguished by FPM concentrations 5-6 times higher than baseline concentrations. FPM concentrations during the first 30-53 days of gestation overlapped with luteal phase concentrations, thereafter increasing to 8-25 times higher than baseline concentrations. FEM concentrations during the first 41-44 days of gestation remained near basal values for one female, whereas concentrations were 1.8 times higher than baseline for the second. FEM concentrations became elevated for the former by 44 days of gestation and increased further for the latter after 53 days, ultimately averaging four times higher than baseline for both females. The biphasic increase in FPM and FEM concentrations, follicular and luteal phase durations (follicular: 7 ± 1 days, luteal: 25 ± 1 days), total cycle length (41 ± 1 days), and gestation (161-165 days) documented in this study were consistent with previous reports from serum and urine analyses. Monitoring FPM and FEM is a reliable noninvasive method for tracking reproductive cycles and pregnancy in southern tamandua that overcomes the challenges associated with serum or urinary hormone analysis.

Noninvasive monitoring of female reproductive hormone metabolites in the endangered European mink (Mustela lutreola)

This study examined the reproductive physiology of female European mink (Mustela lutreola) to augment the available information on estrus, ovulation, and pregnancy with the long-term goal of supporting ex situ breeding management of this highly endangered species. Fecal reproductive hormone metabolites were measured using EIAs for estrogen and 20-oxo-pregnane metabolites. Seasonal hormone profiles were established. A comparison of hormone fluctuations in pregnant and nonpregnant females showed that both estrogen and 20-oxo-pregnane metabolites were significantly elevated during gestation, which is 42 days in length. Delayed implantation or embryonic diapause does not occur in this species. Litter size was correlated with 20-oxo-pregnane levels but not with estrogen concentrations. During lactation, 20-oxo-pregnane metabolite levels remained higher than in nonpregnant females. The breeding season was characterized by peaks in vaginal cornified cells and fecal estrogen metabolite levels. Up to four peaks in estrogen levels were identified and confirmed that European mink are seasonally polyestrous. The results of 20-oxo-pregnane measurements indicated that hCG can be applied to induce ovulation. With the establishment of this noninvasive method, we present a new tool to support population management of this species.

The use of altrenogest to avoid hyperestrogenism after eCG-hCG ovulation induction in southern tigrina (Leopardus guttulus)

The goal of this study was to optimize an ovulation induction protocol for use with artificial insemination in the southern tigrina (Leopardus guttulus). The specific aims were to report the efficacy of using altrenogest, an oral progestin (Regumate, MSD Animal Health, Merck Animal Health), to suppress ovarian activity and prevent follicular hyperstimulation and hyperestrogenism after the administration of exogenous eCG and hCG. To monitor ovarian responses, fecal estrogen and progestogen metabolites were quantified by enzyme immunoassay in females before and after intramuscular administration of 200-IU eCG and 150-IU hCG in two trials, 4 months apart. During the first trial, there was no use of altrenogest, only the eCG-hCG ovulation induction protocol. In the second trial, the ovulation induction protocol was preceded by the administration of oral altrenogest for 14 days (minimum of 0.192 mg per kg per day). Altrenogest administration resulted in a suppression of follicular activity in three out of six females before eCG-hCG administration on the basis of lower mean estrogen concentrations (P < 0.05). It also resulted in four out of six females presenting lower fecal estrogen metabolite concentrations (P < 0.05) after ovulation induction, and two out of six individuals showed a reduction (P < 0.05) in postovulatory fecal progestogen metabolite concentrations, all when compared to the same female's cycles without the progestin. Fecal estrogen metabolite concentrations were closer to baseline in 50% of these individuals after altrenogest and eCG-hCG treatments when compared to basal concentrations before gonadotropins without the use of altrenogest. This study reported that use of altrenogest in southern tigrina can suppress ovarian activity and avoid hyperestrogenism after administration of eCG and hCG treatment. However, not all females responded uniformly, so more studies are needed to increase the efficacy of ovulation induction for use with artificial insemination in this species.

Hormonal Correlates of Paternal Care Differences in the Hylobatidae

Only one of the 15 species of monogamous hylobatids, the siamang (Symphalangus syndactylus), demonstrates direct paternal care in the form of infant-carrying, providing a unique model for examining hormonal correlates of paternal care differences between siamangs and gibbons. We used behavioral data and fecal hormone analysis to investigate (1) differences in monthly percent father-infant proximity in relation to monthly fecal androgen metabolite concentrations from infant birth to the late postpartum period between siamangs and gibbons, (2) the pattern of change in fecal androgen and fecal estrogen metabolite concentrations during the 8-week peripartum period between siamangs and gibbons, and (3) the change in mean fecal glucocorticoid metabolite concentrations at 1-month postpartum from individual baseline between siamangs and gibbons. Father-infant proximity increased as androgen concentrations decreased over the postpartum period in siamangs but not in gibbons. Androgen concentrations increased around birth in siamangs during the 8-week peripartum period, but exhibited a decreasing trend around birth in gibbons. Estrogen concentrations increased from pre- to postpartum in siamangs during the 8-week peripartum period, but exhibited a decreasing trend from pre- to postpartum in gibbons. The difference in mean glucocorticoid metabolite concentrations from baseline was greater in siamangs than gibbons. Our data suggest a relationship between specific steroid hormone patterns and differences in paternal care among the hylobatids, warranting further investigation of such proximate mechanisms.

Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat ( Prionailurus bengalensis euptilurus)

Although the Tsushima leopard cat ( Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy.

Reproductive Endocrinology of a Small Tropical Bat (FemaleSaccopteryx bilineata;Emballonuridae) Monitored by Fecal Hormone Metabolites

We tested the suitability of fecal sexual steroid hormone metabolites for use in endocrinological studies of free-ranging, small bats. We monitored estrogen and 20-oxo-pregnane metabolites in fecal samples collected daily from 19 free-ranging female Saccopteryx bilineata during the assumed mating season. Median fecal estrogen metabolite levels equaled 0.09 ng/g for the pre- and postovulatory periods and 4.2 ng/g for the follicular phase of the estrous cycle in female S. bilineata. Median fecal 20-oxo-pregnane metabolite levels were 50.9 ng/g in preestrous, 88.8 ng/g in estrous, and 190.2 ng/g in postestrous females. Reliability of fecal steroid metabolite analysis was supported by results of video observations. Two successful copulations for a single male–female pair were recorded; mating coincided with peak fecal estrogen levels (7.0 ng/g) in the copulating female. In contrast, 2 observed mating attempts in a 2nd male–female pair occurred approximately 19 days after the female's physiological estrus and fecal estrogen metabolites in the rejecting female were only 0.2 ng/g. Fecal 20-oxo-pregnane levels were almost 3 times lower in fecal samples collected from the copulating female than those from the rejecting female (157 ng/g versus 410 ng/g). All females that were still present in the colony 6 months later gave birth to a single offspring between late May and early June. Gestation length averaged (mean ± SE) 169 ± 2 days. During the 20 days preceding parturition, fecal 20-oxo-pregnane metabolites were approximately 100 times higher than during the mating period. Fecal 20-oxo-pregnane metabolites of lactating females were comparable to those during the mating season. Noninvasive fecal steroid analysis can be a valuable tool for monitoring reproductive events in colonies of free-ranging small-sized bats.

Fecal steroid monitoring for assessing gonadal and adrenal activity in the golden eagle and peregrine falcon

We examined the efficacy of noninvasive monitoring of endocrine function via fecal steroid immunoassays in the golden eagle and peregrine falcon. High-pressure liquid chromatography analyses of fecal glucocorticoid metabolites (fGCM) revealed that minor percentages of immunoreactive fGCM co-eluted with [(3)H]corticosterone in both sexes of the eagle (2.5-2.7%) and falcon (7.5-11.9%). In contrast, most fecal estrogen metabolites in eagle and falcon females co-eluted with radiolabeled estradiol-17beta ([(3)H]; 57.6, 64.6%, respectively) or estrone ([(3)H]; 26.9, 4.1%, respectively). Most fecal progestin metabolite immunoreactivity in the female eagle (24.8%) and falcon (21.7%) co-eluted with progesterone ([(14)C]). Most fecal androgen metabolite immunoreactivity in eagle (55.8%) and falcon (63.7%) males co-eluted with testosterone ([(14)C]). Exogenous adrenocorticotropin hormone induced increased fGCM excretion above pre-treatment in both species, but only significantly (P < 0.05) in the eagle. Both species showed increased fGCM after saline administration, suggesting the detection of 'handling stress.' Both species exhibited enterohepatic and renal recirculation of administered steroids as demonstrated by biphasic and triphasic excretion patterns. Thus, noninvasive fecal hormone monitoring is a valid and promising tool for assessing gonadal and adrenal status in rare and threatened birds-of-prey.

FACTORS RELATED TO FECAL ESTROGENS AND FECAL TESTOSTERONE IN CALIFORNIA SPOTTED OWLS

Abstract We estimated concentrations of fecal reproductive steroid metabolites in free-ranging California Spotted Owls (Strix occidentalis occidentalis) during the breeding season. We collected fresh fecal samples (n = 142) from 65 individual owls in the Sierra Nevada during April–August of 2001. We developed and validated radioimmunoassay procedures to quantify fecal estrogen metabolites and fecal testosterone metabolites. We used an information-theoretic approach to identify factors that might influence fecal estrogen (E), fecal testosterone (T), and fecal estrogen:testosterone ratio (E:T ratio) levels during the owl's breeding season. We hypothesized that factors related to sampling procedures, owl characteristics (sex, reproductive status), and habitat might influence fecal reproductive steroid levels. Our analyses suggested that sampling factors and owl characteristics, but not habitat variables, were related to fecal reproductive steroid levels in Spotted Owls. Our most supported models explained <3...

Reproductive endocrine patterns in captive female and male red wolves (Canis rufus) assessed by fecal and serum hormone analysis

AbstractReproductive steroid profiles in female (n=13) and male (n=5) red wolves (Canis rufus) were characterized in fecal samples collected during the breeding season (December—May) and over a 1 year period, respectively. Blood samples from females (n=12) also were collected during the periovulatory period for luteinizing hormone (LH) and steroid analysis. High performance liquid chromatography (HPLC) of fecal extracts determined that estradiol and estrone constituted the major and minor forms, respectively, of fecal estrogen metabolites. Although native progesterone was present, pregnane metabolites predominated as the major forms of fecal progestins. HPLC analysis of fecal extracts from males revealed no native testosterone, but rather the predominance of more polar androgen metabolites. Based on hormone profiles and/or pup production, females were classified as pregnant (n=3), ovulatory‐nonpregnant (n=9), or acyclic (n=3). Longitudinal monitoring of females indicated no pregnancy‐specific differences in concentrations of either fecal progestagen or estrogen metabolites compared to ovulatory‐nonpregnant individuals; however, baseline progestagen concentrations were consistently elevated in acyclic females. There was good correspondence between serum and fecal steroid concentration during the periovulatory period. A rise in serum estrogens preceded the ovulatory LH surge which was then followed by a significant progesterone rise during the luteal phase. In males, changes in fecal androgen metabolite concentrations coincided with photoperiod fluctuations, increasing in late autumn and reaching peak concentrations during mid‐ to late winter just before the start of the breeding season. Collectively, these results serve as a database of ovarian and testicular endocrine events in this species, which can be utilized in population management and application of assisted reproductive technologies. Zoo Biol 21:321–335, 2002. © 2002 Wiley‐Liss, Inc.

Non-invasive detection and monitoring of estrus, pregnancy and the postpartum period in pygmy loris (Nycticebus pygmaeus) using fecal estrogen metabolites.

Estrone-conjugates (E1C) were measured in the feces of six female pygmy lorises (Nycticebus pygmaeus) during estrus (n = 12), pregnancy (n = 4) and the postpartum period (n = 3). Noninvasive feces collection permitted frequent sampling throughout estrus and pregnancy, without disturbance of animals. The estrous period was defined as an increase in fecal E1C levels above an average of 70 ng/g feces with peaks above 100 ng/g feces obtained in consecutive fecal samples collected over a 6- to 11-day period between the end of July and the first third of October. Comparison of the periovulatory profile of E1C and the stage of labial opening of the vagina revealed a high agreement (P < 0.001). In all pregnant females, an E1C rise was found approximately 47 days postestrus, the source of which may be the growing fetal placental unit. Estimated gestation lengths ranged between 187 and 198 days (n = 4).

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.