Seasonal effects on the endocrine pattern of semi-captive female Asian elephants (Elephas maximus): timing of the anovulatory luteinizing hormone surge determines the length of the estrous cycle

Better breeding strategies for captive Asian elephants in range countries are needed to increase populations; this requires a thorough understanding of their reproductive physiology and factors affecting ovarian activity. Weekly blood samples were collected for 3.9 years from 22 semi-captive female Asian elephants in Thai elephant camps to characterize LH and progestin patterns throughout the estrous cycle. The duration of the estrous cycle was 14.6+/-0.2 weeks (mean+/-S.E.M.; n=71), with follicular and luteal phases of 6.1+/-0.2 and 8.5+/-0.2 weeks, respectively. Season had no significant effect on the overall length of the estrous cycle. However, follicular and luteal phase lengths varied among seasons and were negatively correlated (r=-0.658; P<0.01). During the follicular phase, the interval between the decrease in progestin concentrations to baseline and the anovulatory LH (anLH) surge varied in duration (average 25.9+/-2.0 days, range 7-41, n=23), and was longer in the rainy season (33.4+/-1.8 days, n=10) than in both the winter (22.2+/-4.5 days, n=5; P<0.05) and summer (18.9+/-2.6 days, n=8; P<0.05). By contrast, the interval between the anLH and ovulatory LH (ovLH) surge was more consistent (19.0+/-0.1 days, range 18-20, n=14). Thus, seasonal variation in estrous cycle characteristics were mediated by endocrine events during the early follicular phase, specifically related to timing of the anLH surge. Overall reproductive hormone patterns in Thai camp elephants were not markedly different from those in western zoos. However, this study was the first to more closely examine how timing of the LH surges impacted estrous cycle length in Asian elephants. These findings, and the ability to monitor reproductive hormones in range countries (and potentially in the field), should improve breeding management of captive and semi-wild elephants.     

Assessment of ovarian activity in captive goral (Naemorhedus griseus) using noninvasive fecal steroid monitoring

To date, there is no information on gonadal steroidogenic activity of female goral (Naemorhedus griseus), a threatened species of Thailand. Captive goral populations have been established to produce animals for ex situ conservation and reintroduction, but as yet none are self-sustaining. The objectives of the present study were to (1) determine the influence of season on ovarian steriodogenic function; and (2) examine the relationship between gonadal hormone excretion and sexual behaviors throughout the year. Fecal samples were collected 5 to 7 days/wk for 15 months from 8 adult females housed at Omkoi Wildlife Breeding Center in Thailand and analyzed for ovarian steroid metabolites using validated enzyme immunoassays. Observations of sexual behaviors and mating were conducted each morning for 30 min/session. Based on fecal estrogen and progestagen metabolite concentrations, the overall estrous cycle length was about 21 days, with a 2- to 3-day follicular phase and an 18- to 20-day luteal phase. Sexual behaviors, most notably tail-up, increased for 2 to 3 days during the time estrogens were elevated during mating. Fecal progestagens were elevated during luteal phases and increased further during gestation, which lasted approximately 7 months. The lactation period was 5 months, and females were anestrus for 2 to 5 of those months, with the exception of one that cycled continuously throughout. Two females conceived around 2 months postpartum and so were pregnant during lactation. Birth records over the past 21 years indicated young are born throughout the year. This combined with the hormonal data suggests that female gorals are not strongly seasonal, at least in captivity, although there was considerable variation among females in estrogen and progestagen patterns. In conclusion, fecal steroid metabolite monitoring is an effective means of assessing ovarian function in this species and will be a useful tool for breeding management and planned development of assisted reproductive techniques such as artificial insemination and embryo transfer.     

Ovarian Cycle and Effect of Social Changes on Adrenal and Ovarian Function in Pygathrix nemaeus

To assist captive breeding of the endangered red-shanked douc langur (Pygathrix nemaeus), basic knowledge on female reproductive physiology is important. We aimed 1) to characterize the pattern of fecal estrogens and progestogens during the ovarian cycle and 2) to use the information to provide reliable data on ovarian cycle characteristics. Moreover, we examined the potential impact of a change in group composition on ovarian activity and adrenal function, the latter being measured by fecal glucocorticoid excretion. We collected fecal samples during 2 mo from 5 adult females of 3 groups under undisturbed conditions and from the same 5 adult females and 2 juvenile females and 2 adult males during 3 mo during which there were changes in housing and group composition. During the undisturbed period, measurement of fecal 5-P-3OH, a major progesterone metabolite, and total estrogens revealed in all adult females a cyclic pattern, from which we documented a cycle length of 26.4 ± 1.8 days, comprising a follicular phase of 13.1 ± 2.9 days and a luteal phase of 14.8 ± 1.2 days. In contrast, during the period of social changes, 3 of the 5 females showed clear signs of cycle irregularity as indicated by extended follicular phases, abbreviated luteal phases and/or anovulation. Moreover, the period of social changes was associated with an overall significant elevation in fecal glucocorticoid excretion. Our data provide the first detailed information on the endocrine characterization of the ovarian cycle in Pygathrix nemaeus and suggest that social changes have the potential to impair ovarian function, likely as a result of increased activation of the HPA-axis due to stressful situations. However, because of relatively small sample size, particularly concerning the latter finding, more data are needed to confirm these results.     

Fecal analysis of ovarian cycles in female black-handed spider monkeys (Ateles geoffroyi)

An enzyme immunoassay (EIA) was applied to characterize the reproductive endocrinology of adult female black-handed spider monkeys (Ateles geoffroyi). Analysis of paired urine and fecal samples, collected from two females housed at San Diego Zoo, confirmed that the EIAs employed provided quantitative measurements of ovarian sex steroid hormones. Fecal metabolite levels were significantly correlated with those in urine, confirming that feces are a valid source of steroid metabolites in this species. The excretion of these metabolites in feces lagged urinary excretion by 1-2 days. The ovarian cycle profiles of the two captive females and five free-ranging females are comparable, with an average length of approximately 20-23 days. Cyclical bleeding, as previously reported, was observed in one of the two captive females. Pregnancy was detected in four free-ranging females, and early fetal loss for one female was indicated by hormonal data.     

Reproductive endocrine responses to photoperiod and exogenous gonadotropins in the pallas' cat (Otocolobus manul)

Fecal samples were collected for 14–26 months from three male and six female Pallas' cats (Otocolobus manul) to examine gonadal steroidogenic activity in response to changes in photoperiod and treatment with exogenous gonadotropins. Females exhibited a seasonal anestrus from May–December, excreting consistently low concentrations of fecal estrogens (overall mean, 50.2±8.5 ng/g). During the breeding season (January–April), baseline fecal estrogen concentrations were higher, averaging 128.4±18.9 ng/g, with peak concentrations ranging from 455.8–909.6 ng/g. Interpeak intervals in estrogen excretion ranged between 7 and 21 days, with an average estrous cycle length of 14.3±1.7 days. Two females became pregnant after natural mating, with overall luteal progestogen concentrations averaging ～40 μg/g throughout gestation. Fecal estrogens increased in mid‐gestation, peaking just before birth. Induction of follicular development with eCG (100–300 IU, i.m.) resulted in an increase in fecal estrogens (peak range, 263.1–1198.1 ng/g), followed by a postovulatory increase in fecal progestogens (overall mean, 41.1±11.9 μg/g) after hCG (75–150 IU, i.m.). Despite apparently normal ovarian responses, none of the females conceived after artificial insemination (AI). The gonadotropin‐induced nonpregnant luteal phase lasted 49.8±5.3 days (range, 30–60 days), whereas gestation lasted ～70 days. In the male Pallas' cat, fecal androgens were elevated from November–April (overall mean, 352.3±30.3 ng/g) compared with nadir concentrations during the rest of the year (82.1±3.3 ng/g). Entrainment of seasonality to photoperiod was demonstrated by stimulation of gonadal steroidogenic activity in cats exposed to increasing artificial light during natural (nonbreeding season) and artificially induced short‐day photoperiods. In summary, reproduction in Pallas' cats is highly seasonal and photoperiod‐dependent. Females exhibit elevated baseline and peak fecal estrogen concentrations for 3–4 months during late winter/early spring. Testicular steroidogenic activity precedes the rise in female estrogen excretion by about 2 months, presumably to ensure maximal sperm production during the breeding season. Zoo Biol 21:347–364, 2002. Published 2002 Wiley‐Liss, Inc.     

Use of salivary steroid analyses to assess ovarian cycles in an Indian rhinoceros at the National Zoological Park

Saliva samples were collected from a female Indian rhinoceros (Rhinoceros unicornis) housed at the National Zoological Park, and ether-extracted for analyses of androgen, estrogen, and progestin metabolites to assess ovarian cycles. Analyses of both salivary androgens and estrogens were found to reliably monitor follicular activity. Although the temporal patterns of the two steroids were significantly correlated (r=0.62; P<0.05), they differed slightly. Salivary androgens increased earlier during the follicular phase, although both peaked at the same time in association with behavioral estrus. Based on salivary androgen profiles, the mean duration of the follicular phase was 11.4 days (ranged=7–15 days; n=17 cycles). Estrous cycle length, as measured by the time between consecutive androgen peaks, was 47.4±3.4 days (range=37–86 days). Salivary progestin measurements were effective for monitoring luteal function. The mean duration of the luteal phase was 15.5±1.5 days (range=10–23 days). In sum, assays were identified for measuring salivary steroids to assess ovarian function in Indian rhinoceroses. However, not all of the assay systems tested were effective, perhaps because of interfering matrix effects. Mate introductions in the Indian rhinoceros often require careful monitoring, and a technique for monitoring hormones in saliva could be an alternative to urine or fecal analyses for identifying estrus and timing breeding in this species. Such a technique would be particularly useful for situations in which it is difficult to collect uncontaminated urine and feces. Zoo Biol 23:501–512, 2004. © 2004 Wiley-Liss, Inc.     

Ultrasonographic characterization of ovarian events and fetal gestational parameters in two southern black rhinoceros (Diceros bicornis minor) and correlation to fecal progesterone

A tremendous potential exists for the application of transrectal ultrasonography as a tool to enhance the captive management of endangered species. Reproductive study of two southern black rhinoceros (Diceros bicornis minor) females was performed daily to every other day for a approximately 60 day period to document ovarian changes, and three times weekly in early pregnancy to once monthly in late pregnancy in order to characterize changes in fetal parameters throughout gestation. All ovarian and fetal anatomical structures were measured in millimeters. The mean (+/- SD) length of the estrous cycle or interovulatory period was 26 +/- 1.4 days (n=2 cycles). Follicular growth rate of a dominant follicle was approximately 3 mm/day once the follicle reached 35 mm in diameter. Ovulation was observed to occur at a mean (+/- SD) follicular diameter of 49.5 +/- 2.6 mm (n=4) and within 48 to 72 h after observed estrus (n=2). Large ovarian structures [mean (+/- SD) diameter of 71.7 +/- 2.9 mm; n=3], considered analogous to equine anovulatory hemorrhagic follicles, were observed to form in the winter months and suggest seasonal periods of reduced fertility. Fecal progesterone assays confirmed ultrasonographic events. Although preliminary, the results of fetal sexing are presented and compared to the horse. Our data indicate that fetal eye or fetal foot diameter measurements can be used to accurately predict gestational age from about 2 months to term, providing useful information to managers of both captive and wild rhino populations. The ability to identify and quickly release animals in late term pregnancy in the wild and thereby reduce-abortions and neonatal mortalities in holding bomas is one potential practical conservation benefit of the fetal age predictive models.     

Monitoring ovarian function in scimitar-horned oryx (Oryx dammah) by measurement of fecal 20α-progestagen metabolites

The feasibility of monitoring ovarian function in scimitar-horned oryx (Oryx dammah) by measurement of fecal 20α-progestagens was investigated. Fecal samples were collected daily or on alternate days over a 4–11 month period from five oryx during natural (n = 4) or synthetic PGF_2α (cloprostenol)-controlled (n = 1) cycles. Of the four oryx undergoing natural cycles, three had regular access to a vasectomised male, and mating dates were recorded. Ultrasonography was used to monitor changes in reproductive tract morphology in the female administered with cloprostenol. Neutral steroids were extracted from feces with methanol:petroleum ether (2:1 v/v) after first removing phenolic steroids with potassium hydroxide (1 M). The concentration of 20α-progestagens in the methanol phase was measured by enzymeimmunoassay. Excretion of 20α-progestagens in all females followed a cyclic pattern corresponding to the follicular and luteal phases of the ovarian cycle. Concentrations of fecal 20α-progestagens were on average twentyfold greater during the luteal phase compared with the follicular phase. Mean (±SD) ovarian cycle length, based on fecal progestagen profiles, was 24.4 ± 2.2 days with mean (±SD) luteal and follicular phase lengths of 18.7 ± 2.8 and 5.7 ± 1.6 days, respectively. Mating by a vasectomized male occurred when 20α-progestagen concentrations were still elevated or declining. Similarly, fecal progestagens did not return to follicular phase concentrations for 4–5 days after administration of cloprostenol, and a 4 day delay was observed between ovulation, as visualized by ultrasound scanning, and a rise in fecal 20α-progestagens. These data suggest a time lag of approximately 4 days between reproductive events and changes in fecal 20α-progestagen concentrations. We conclude that measurement of immunoreactive 20α-progestagen concentrations in feces has limited application for predicting ovulation or accurately timing inseminations because of delay in steroid excretion, but will enable noninvasive monitoring of ovarian cycles in scimitar-horned oryx for fertility assessment and for determining the outcome of artificial insemination programs. © 1995 Wiley-Liss, Inc.     

Relationship between ovarian cycle phase and sexual behavior in female Japanese macaques (Macaca fuscata)

We conducted behavioral observations simultaneously with fecal sample collection on eight nonlactating females 2-3 times per week, October 1997-March 1998, to examine the relationship between ovarian hormones and the sexual behavior of female Japanese macaques (Macaca fuscata) during the mating season. We analyzed samples by enzyme immunoassay for fecal hormone levels. Hormone profiles of estrone-glucuronide (E1) and pregnanediol-glucuronide (PdG) were used to separate ovarian cycles into three phases (follicular, periovulatory, and luteal). Hormonal profiles indicate average cycle lengths of 27.6 +/- 4.2 days (+/- SD; n = 26). Average lengths of the luteal and follicular phases were 12.3 +/- 3.8 days (+/- SD) and 8.3 +/- 3.4 days (+/- SD), respectively. We observed female Japanese macaques engaging in sexual activity throughout the ovarian cycle, with the highest rates occurring during the follicular and periovulatory phases as compared to the luteal phase. The attractivity of female Japanese macaques increased significantly during the follicular and periovulatory phases of the ovarian cycle, when E1 levels are peaking and PdG levels drop to baseline. In addition, females displayed a significant increase in proceptive behavior during the follicular and periovulatory phases. Grooming bouts, as well as proximity between female and male macaques, also increased significantly during the follicular and periovulatory phases. We conclude that fluctuating levels of ovarian hormones in different phases of the cycle are significantly associated with variable rates of copulatory and pericopulatory behaviors in these Japanese macaque females.     

Comparative analysis of gonadal and adrenal activity in the black and white rhinoceros in North America by noninvasive endocrine monitoring

Patterns of fecal reproductive steroid metabolites and adrenal corticoids were characterized for 12‐ to 24‐month periods in black (n = 10 male, 16 female) and white (n = 6 male, 13 female) rhinoceroses at 14 institutions. All black rhinoceros females exhibited at least some ovarian cyclicity on the basis of fecal progestogen analysis (range, 2–12 cycles/yr). However, cycles often were erratic, with many being shorter (<20 days; 18% of cycles) or longer (>32 days; 21%) than the average of 26.8 ± 0.5 days (n = 104 cycles). Five females exhibited periods of acyclicity of 2–10‐month duration that were unrelated to season. One complete and seven partial pregnancies were evaluated in the black rhinoceros. Fecal progestogens increased over luteal phase concentrations after 3 months of gestation. Females resumed cyclicity within 3 months postpartum, before calves were weaned (n = 5). Approximately half of white rhinoceros females (6 of 13) showed no evidence of ovarian cyclicity. Of the cycles observed, 5 were “short” (32.8 ± 1.2 days) and 24 were “long” (70.1 ± 1.6 days). Only two females cycled continuously throughout the study. One had both long (n = 9) and short (n = 2) cycles, whereas the other exhibited long cycles only (n = 5). Fecal estrogen excretion was variable, and profiles were not useful for characterizing follicular activity or diagnosing pregnancy in either species. Males of both species showed no evidence of seasonality on the basis of fecal androgen profiles. Androgen metabolite concentrations were higher (P < 0.05) in the black (27.6 ± 6.9 ng/g) than in the white (16.8 ± 3.1 ng/g) rhinoceros. An adrenocorticotropin hormone challenge in four black rhinoceros males demonstrated that the clearance rate of corticoid metabolites into feces was ～24 hours. Fecal corticoid concentrations did not differ between males and females, but overall means were higher in the black (41.8 ± 3.1 ng/g) than in the white (31.2 ± 1.7 ng/g) rhinoceros. In summary, fecal steroid analysis identified a number of differences in hormonal secretory dynamics between the black and white rhinoceros that may be related to differences in reproductive rates in captivity. Most black rhinoceros females exhibited some cyclic ovarian activity. In contrast, few white rhinoceroses demonstrated evidence of regular estrous cyclicity, and those females that were active had comparatively long cycles. Results also suggest that fecal corticoid concentrations reflect adrenal activity and may be species specific. Continued studies are needed to determine whether fecal corticoid measurements will be useful for understanding the cause of inconsistent gonadal activity in these two species. Because all but three (15.8%) of the white rhinoceroses evaluated in this study were less than 20 years of age compared to 73.1% (19 of 26) of the black rhinoceroses, the impact of age on reproductive and adrenal activity also needs to be evaluated further. Zoo Biol 20:463–486, 2001. © 2002 Wiley‐Liss, Inc.     

Reproductive cycle length and pregnancy in the southern white rhinoceros (Ceratotherium simum simum) as determined by fecal pregnane analysis and observations of mating behavior

Pregnancy and the reproductive cycle were monitored in 13 captive southern white rhinoceros (Ceratotherium simum simum) by measuring progesterone metabolites in fecal extracts and by observing behavior patterns. Fecal hormones were measured using a monoclonal antibody produced against 4‐pregnen‐11‐o1‐3,20‐dione hemisuccinate:BSA. Several subjects exhibited flat or erratic endocrine profiles, but we found evidence for 17 reproductive cycles in five females. Mating behavior coincided with nadirs in pregnane concentrations. These cycles appeared to fall into two general categories: those of approximately 1 month in duration (Type I: X¯ ± SEM = 35.4± 2.2 days; n = 10) and those lasting approximately 2 months (Type II: 65.9± 2.4 days; n = 7). Interluteal phase lengths were similar for the two cycle types, but Type II cycles were characterized by extended luteal phases lasting more than twice as long as Type I luteal phases. Because Type I cycles predominated in our data and because evidence suggests that some Type II cycles may be aberrant, we argue that these approximately monthly cycles represent the typical reproductive cycle for this species. Three females became pregnant during the course of the study. We were able to detect pregnancy by approximately 3 months post‐breeding, as indicated by sustained pregnane concentrations markedly higher than nonpregnant luteal phase concentrations. These data help to characterize important reproductive events of this species and should be useful for captive breeding efforts for this threatened species. Zoo Biol 18:111–127, 1999. © 1999 Wiley‐Liss, Inc.     

Reproductive steroid hormones and ovarian activity in felids of the Leopardus genus

Reproductive endocrine patterns were characterized in female ocelots (Leopardus pardalis; n = 3), tigrinas (Leopardus tigrinus; n = 2), and margays (Leopardus wiedii; n = 2) housed in captivity in southern Brazil. Females were maintained as singletons and exposed to natural fluctuations in photoperiod. Cyclic changes in ovarian steroids were monitored by analyzing estrogen and progestogen metabolites in fecal samples collected five times weekly for 14 to 18 months. Based on intervals between fecal estrogen peaks, mean (± SEM) duration of the estrous cycle was 18.4 ± 1.6 days for the ocelots (range, 7–31 days; n = 75 cycles), 16.7 ± 1.3 days for the tigrinas (range, 11–27 days; n = 23 cycles), and 17.6 ± 1.5 days for the margays (range, 11–25 days; n = 32 cycles). Fecal progestogen analyses combined with two laparoscopic observations of the ovaries confirmed that ocelots and tigrinas did not ovulate spontaneously. In contrast, non‐mating–induced luteal phases of 40.1 ± 6.3 days in duration (range, 30–60 days) were observed frequently in both margays. There was no evidence of gonadal seasonality in margays in either follicular or luteal activity. In ocelots, cyclic changes in estrogen excretion were observed during each month of the year; however, only one female cycled continuously. In the other two ocelots, periods of acyclicity of several months’ duration were observed. It was not possible to conclude whether tigrinas were aseasonal because estrous cyclicity was observed in only one of two individuals. In the female that cycled, a 3‐month period of acyclicity was observed in the late fall/early winter. These data demonstrate similarities among three felid species of the genus Leopardus, including evidence they are polyestrous but experience unexplained periods of ovarian inactivity. Only the margays differed by exhibiting occasional spontaneous, non‐mating–induced ovulations. Historically, these species have not bred well in captivity. However, it is hoped that understanding the biological similarities and differences among them could lead to improved management strategies that may one day result in increased reproductive success. Zoo Biol 20:103–116, 2001. © 2001 Wiley‐Liss, Inc.     

Follicular waves during the oestrous cycle in Nili-Ravi buffaloes undergoing spontaneous and PGF2alpha-induced luteolysis

The objective of this study was to characterize the follicular waves and associated ovarian events during spontaneous and PGF(2alpha)-induced oestrous cycles in Nili-Ravi buffaloes. In Exp. 1, (n=13 oestrous cycles) follicular and luteal development was monitored by ultrasonography and jugular blood samples were collected simultaneously on alternate days. Of 12 oestrous cycles, 9 (75%) had two waves of follicular activity and only 3 (25%) had three waves. The mean (+/-S.E.M.) length of the oestrous cycle was shorter (P<0.05) in buffaloes with two waves than in those with three waves (21.2+/-0.1 days versus 22.8+/-0.1 days). In Exp. 2, follicular dynamics were compared in buffaloes undergoing spontaneous (n=12 oestrous cycles) and PGF(2alpha)-induced (n=9) regression of the corpus luteum (CL). The dynamics of ovulatory follicular growth during the 3 days before oestrus were similar (P>0.05) in buffaloes undergoing spontaneous and PGF(2alpha)-induced luteolysis. These results show that (1) the majority of buffaloes had a two wave pattern of follicular growth and emergence of a third wave was associated with a longer luteal phase, and (2) follicular dynamics during the 3 days before oestrus were similar in buffaloes undergoing spontaneous and PGF(2alpha)-induced luteolysis.     

Parasitism in captive and reintroduced red wolves

Fecal examinations revealed that at least 10 of 21 (48%) captive red wolves (Canis rufus) and eight of 12 (67%) free-ranging red wolves were infected with intestinal parasites. No captive wolves and only one of seven reintroduced wolves had dirofilariasis. Ticks were collected from 10 of 21 (48%) captive wolves and nine of 12 (75%) free-ranging animals. Ivermectin administered at a dosage of 50 micrograms/kg of estimated body weight every 30 to 60 days apparently prevented or ameliorated parasitism in red wolves.     

Comparative endocrine investigations in three bear species based on urinary steroid metabolites and volatiles

In order to improve breeding of in situ populations of bears, a comprehensive study of reproductive physiology in Brown (Ursus arctos), Spectacled (Tremarctos ornatus) and Giant panda bears (Ailuropoda melanoleuca) was performed. The objective was to perform non-invasive analyses of urinary and fecal steroid metabolites. In addition, we investigated the presence of reproduction-related urinary volatile substances of these bears that might trigger the reproductive behavior. Urinary estrogen concentrations, routinely used to monitor follicular activity in Giant panda, were inappropriate for monitoring follicular activity in Spectacled bear. In addition, no estrogen peak related to mating activity was observed in Brown bear. Further contrasting Giant panda, although urinary pregnanediol analyses failed to indicate luteal activity in either Spectacled or Brown bears, urinary (Spectacled bear) and fecal (Brown bear) concentrations of progesterone were an appropriate indicator of luteal activity. The Giant panda had volatile components (medium-chain fatty acids) in their urine that increased simultaneously with the seasonal increase of estrogens. These fatty acids were also detected in the Brown during estrus and Spectacled bear. Further studies on the behavioral relevance of these fatty acids are required to determine if they are pheromones.     

Activity of alkaline phosphatase in uterine flushings of dairy cows affected with ovarian cysts or endometritis

Both uterine horns of 14 dairy cows with ovarian follicular cysts, and four animals affected with purulent endometritis were flushed via catheter using 30 ml phosphate buffered saline, following evisceration at a local abattori. Activity in the flushing media of alkaline phosphatase (ALP) and aspartate aminotransferase (GOT) were examined. Ovaries were prepared for light microscopy. Amount and morphological integrity of luteinized tissue found on the ovaries were reflected by correspondent levels in ALP activity, which was higher in the media taken from the ipsilateral to the luteal tissue situated uterine horns (651 +/- 228 vs 244 +/- 62 u/l, n = 3). Only cows having relatively large amounts of luteal tissue on the cystic ovaries (as in luteinized follicular cysts) exhibited very high ALP activity in uterine flushings (2693 +/- 1348 u/l, n = 2). Results suggest the existence of local relationships between luteal tissue in the ovary and the ipsilateral uterine horn in cows with ovarian follicular cysts.     

Faecal steroid metabolites for non-invasive assessment of reproduction in common warthogs (Phacochoerus africanus), red river hogs (Potamochoerus porcus) and babirusa (Babyrousa babyrussa)

The objectives of this study were to analyse faecal steroid metabolites in African and South East Asian pig species kept in European zoos. Species studied were the warthog (Phacochoerus africanus), the red river hog (Potamochoerus porcus) and the babirusa (Babyrousa babyrussa). Faecal samples were collected 1-3 times per week from non-pregnant and pregnant captive female warthogs (n = 9), red river hogs (n = 7) and babirusas (n = 5). Enzyme-immunoassays for faecal progesterone, androgen, and oestrogen metabolites, were tested for their ability to determine follicular and luteal phases. In all three species, oestrous cycles could be monitored with 20alpha-OH- and 20-oxo-pregnane assays. In contrast, oestrogens and androgens were not useful in characterising follicular activity during the oestrous cycle in any species. Faecal 20alpha-OH- and 20-oxo-pregnane values were significantly correlated. Faecal pregnane concentrations revealed species-specific differences. Luteal phase values of 20alpha-OH-pregnanes were considerably higher than 20-oxo-pregnanes; 20alpha-OH-pregnanes were in the range of 3-10 microg/g in warthogs and red river hogs, whereas concentrations were 30-200 microg/g faeces in the babirusa. Regular oestrus cycles had a length of about 35 days in all three species studied. Results indicated a seasonal influence on the occurrence of reproductive cycles in the warthog with anoestrous periods in the European summer. The red river hog was found to be a seasonal and poly oestrous breeder; oestrus cycles started by January and continued until summer. In contrast, the babirusa showed non-seasonal ovarian cyclicity. In pregnant red river hogs, progesterone metabolites were comparable to luteal phase values of the oestrous cycle during the first 3 months of gestation, but did further increase during the last month of pregnancy. Oestrogens and 17-oxo-androstanes were significantly elevated during the second half of gestation. In summary, the reproductive biology of three exotic pig species was studied using non-invasive faecal steroid analysis and these methods were used for comparative investigations of oestrous cycles, pregnancy and seasonality.     

Monitoring fecal samples for estrogen excretion across the ovarian cycle in Goeldi's monkey (Callimico goeldii)

In captive Goeldi's monkeys, estrogen concentration was determined in fecal samples collected from 4 cycling/unmated females and 4 postpartum/mated females in order to ascertain the potential of fecal estrogen monitoring for providing basic information about reproductive status in this endangered Amazonian monkey. Subjects were fed an omnivorous diet and first-void feces were collected in the home cage at 1–3-day intervals for 30–50 days from the cycling females, and at 6–14-day intervals around the estimated time of the postpartum ovulation in each of the 4 mated females. Estimates for the duration of ovarian cycles (22–26 days) and the timing of ovulation were based on cyclic profiles of either blood progesterone or urinary pregnanediol-3α-glucuronide. Fecal estrogen values were normalized using these plasma or urinary profiles. HPLC analysis of estrogen from postpartum fecal samples demonstrated the presence of unconjugated estrone and estradiol-17β (“unconjugated estrogen”). Unconjugated estrogen was extracted and its fecal concentration estimated via EIA. The correlation (r) between plasma estrone conjugates and fecal unconjugated estrogen across nonconception ovarian cycles was 0.65 and measurement of the latter generated cyclic profiles. A range of 4–36-ng unconjugated estrogen g^?1 feces was identified for follicular phases of nonconception cycles. Fecal unconjugated estrogen first exceeded the concentration range of the follicular phase 2–5 days after ovulation; the range was 49–402 ng g^?1 feces in samples collected during the remainder of these nonconception luteal phases. Luteal phase concentrations were on average 10-fold higher than follicular phase concentrations. Each of the 4 mated females conceived at its postpartum ovulation; concentrations of fecal unconjugated estrogen excreted by 3 of these females demonstrated a marked postovulatory increase. This study demonstrates that fecal unconjugated estrogen can be applied to monitor ovarian cyclicity in Goeldi's monkey. © 1994 Wiley-Liss, Inc.     

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.