Relationship between gonadal steroid hormones and vulvar bleeding in southern tamandua, Tamandua tetradactyla

This study aimed at demonstrating the profiles of circulating gonadal steroid hormones during the estrous cycle and pregnancy in a southern tamandua (Tamandua tetradactyla). Additionally, this study clarified the relationship between vulvar bleeding and hormonal changes. The concentrations of serum progesterone (P(4)) and estradiol-17β (E(2)) were determined by enzyme immunoassays. Serum P(4) and E(2) concentrations changed cyclically and the estrous cycle length (± SD) based on the E(2) cycles was 44.3 ± 4.5 days. Vulvar bleeding started to be seen at the decreasing of P(4). The cycle length for vulvar bleeding was 43.3 ± 4.2 days. Interval from the first day of bleeding to the peak of E(2) concentration was 23.1 ± 3.1 days. Serum P(4) during pregnancy remained high and E(2) increased 8 weeks after conception and remained high until parturition. The female delivered normally after a 165 day-pregnancy period and reared the offspring well. Approximately 3 weeks after parturition, serum E(2) and P(4) cycles resumed. Visual bleeding may be useful as a real-time indicator for understanding the ovarian cycle of southern tamanduas, and estrus could be expected approximately 3 weeks after the first bleeding.     

Diet composition of an escaped captive‐born southern tamandua (Tamandua tetradactyla) in a nonnative habitat in Asia

Studies on the role of natural predatory instincts in captive‐born mammalian myrmecophagy are rare. Consequently, researchers rely extensively on case reports to learn more about the contexts in which predatory behavior occurs among such animals. In this study, we recorded an uncommon case of a captive‐born southern tamandua (Tamandua tetradactyla) that accidentally escaped from a zoo into a nonnative habitat in Asia. The southern tamandua was found alive 3 months later. Two fresh fecal samples were obtained, and the diet composition was examined. Three termite species (one family, three genera), and 14 ant species (four subfamilies, nine genera) were identified in the fecal samples. The studied southern tamandua preyed on terrestrial and arboreal ants and termites, as the wild populations of its species do. Ants of the subfamily Myrmicinae and termites of the subfamily Nasutitermitinae were the most abundant prey items in the samples, which is consistent with related reports on the wild populations. Soldier ants constituted <1% of the prey items in the fecal samples, suggesting that the southern tamandua likely avoided preying on ants of the soldier caste. Fungus‐growing termites Odontotermes (Isoptera: Macrotermitinae), which are not native to neotropical regions, were also ingested by the southern tamandua. This study provides information on how a captive‐born mammalian myrmecophagy applies its natural feeding instincts in nonnative natural settings.     

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). Am. J. Primatol. 41:103–115, 1997. © 1997 Wiley-Liss, Inc.     

The reproductive biology of female Père David's deer (Elaphurus davidianus)

Data for this study came from breeding records of 27 Père David's (Elaphurus davidianus) hinds maintained in large pastures and from estrous records of four hand-reared nulliparous hinds. The mean estrous cycle length ranged from 17.5 to 19.6 days. Standing estrus resembled that of other cervids, except that a low, moaning vocalization was given in response to contact, and activity (as measured by pedometers) did not increase. Mean gestation length was 183.38 ± SD 6.11 days (n = 21), and nearly all females conceived in the second and third years. The median interbirth interval was 362 days. The median birth date was April 8, and 80% of the births occurred over a 9.5-week period. Multiparous hinds gave birth an average of 20.5 days earlier in the season than primiparous hinds. There was no dimorphism in birth weight. The results are discussed in light of comparative data for other species.     

Nutrition of the Tamandua: I. Nutrient composition of termites (Nasutitermes spp.) and stomach contents from wild tamanduas (Tamandua tetradactyla)

Arboreal termites (Nasutitermes spp.) and stomach contents from tamanduas (Tamandua tetradactyla) were collected in central Venezuela during the mid part of the dry season (March) of 1993 and 1994. Nutritional analyses were performed on each caste (workers n = 3), soldiers (n = 5), and alates [n = (1]), on mixed caste samples (n = 1), and on stomach contents from live (n = 5) and roadkill (n = 5) tamanduas. The chemical analysis, expressed on a dry matter (DM) basis, of termite workers, which constituted the majority of the nest populations, showed the highest crude protein (CP) (67%) and the lowest DM (25%) and fat (2%) values. Ash content varied from a low of 4% in alates to a high of 7% in soldiers. The alates contained substantially higher DM (41%) and fat (40%), which was reflected in a higher caloric value (6.88 kcal/g) (gross energy) [GE], and relatively less CP (49%). Among the macrominerals, potassium (K) was consistently the highest, with an overall mean value of 0.54%, while the calcium (Ca) and phosphorus (P) levels showed overall means of 0.26% and 0.67%, respectively. Iron (Fe) was the highest among the trace minerals but highly variable (soldiers, 1,000 ppm; alates, 246 ppm; workers, 394 ppm). Differences in the concentrations of vitamin A and E were found among termites castes, with soldiers showing the highest values (20 and 85 μg/g for retinol and α-tocopherol, respectively). Acid detergent fiber (ADF) was lower in the alates (13%) and workers (27%) compared to the soldiers (35%). Alates' fat was more saturated (39%), while soldiers and workers had a much higher polyunsaturated fatty acid (PUFA) concentration. In general, similar nutrient profiles were found between the tamandua stomach contents and the overall mean composition of Nasutitermes spp. However, stomach contents had much higher ADF, ash, and Fe concentrations (31%, 14%, and 2,748 ppm) than termites (25%, 5%, and 652 ppm) but lower CP, fat, GE, and Ca values (51%, 11%, 4.58 kcal/g, and 0.11% vs. 58%, 15%, 6.01 kcal/g, and 0.26% in termites). The relatively low concentrations of Ca in both stomach contents and termites may be indicative of a low requirement in Myrmecophaga compared to other mammalian species. Diets consumed by free-ranging tamanduas contained on a DM basis 51% CP, 11% fat, 14% ash, 31% ADF, 4.58 kcal/g GE, 0.11% Ca, 0.41% P, 2.52 μg/g retinol, and 44.3 μg/g α-tocopherol. Duplication of these nutrient profiles might greatly benefit captive health and reproduction of this species. © 1996 Wiley-Liss, Inc.     

Monitoring luteal function in the lion-tailed macaque (Macaca silenus) through urinary progesterone metabolite measurements

A direct radioimmunoassay for measuring urinary 20-hydroxyprogesterone cross-reactivity to monitor and assess luteal function and detect pregnancy in the lion-tailed macaque (Macaca silenus) is described. Urine samples were collected daily during ten nonconceptive and five conceptive ovarian cycles of five dult female lion-tailed macaques. Urine was analyzed for concentrations of 20α-hydroxypro-gesterone cross-reactivity, estrone conjugates, and creatinine. The strength of the luteal phase in normal nonconceptive cycles (n = 8) is characterized by a maximum sevenfold increase (day 9) in mean 20α-hydroxyprogesterone cross-reactivity over follicular phase levels; the duration, by a 13-day sustained elevation of mean 20α-hydroxyprogesterone cross-reactivity levels. Pregnancy is detectable from 20α-hydroxyprogesterone cross-reactivity values approximately 20 days after the periovulatory estrone conjugate peak (n = 4). Apparent anovulation (n = 1), extended follicular phase (n = 1), and early abortion (n = 1) also are detectable using 20α-hydroxyprogesterone cross-reactivity measurements.     

Fecal endocrine monitoring of reproduction in female snow leopards (Uncia uncia)

Although the snow leopard (Uncia uncia) is a common endangered felid species in zoos, little is known about the complex endocrine interactions controlling ovarian function and conception in this species. The goal of this work was to characterize ovarian activity throughout the estrous cycle, nonpregnant luteal phase (pseudopregnancy), and gestation in female snow leopards. This goal was accomplished using an enzyme immunoassay to measure fecal concentrations of estrogen metabolites (E) and progesterone metabolites (P). Fecal samples were collected from 12 female snow leopards (ages 18 months to 18 years) during one to three breeding seasons. In each breeding season, the majority of females (78%, 88%, and 100%, respectively) began to exhibit ovarian activity in December or January. The estrous cycle, defined by the first day of estrus (E ≥ 2 × basal concentration) to the first day of the subsequent estrus, was 12.7 ± 0.6 days (n = 145 cycles). Estrus lasted 4.3 ± 0.4 days with mean concentrations of fecal E during the follicular phase (1661 ± 139 ng/g feces) increasing 3.2-fold above basal concentrations (515 ± 32 ng/g feces). No spontaneous ovulations were observed in any of the cycling females. Nonpregnant luteal phases were observed in eight females that bred but did not become pregnant. The length of the nonpregnant luteal phase ranged from 11 to 72 days (45.7 ± 5.7 days; n = 10) with mean concentrations of fecal P during the luteal phase (12.46 ± 1.7 μg/g feces) increasing 6.2-fold above basal concentrations of P (2.01 ± 0.2 μg/g feces). Three of the females in the study became pregnant and gave birth after a gestation of 93 (n = 2) and 95 (n = 1) days. Fecal P concentrations during pregnancy increased to 11.64 ± 1.3 μg/g feces, or 5.8-fold above basal concentrations. The results of this study provide a comprehensive characterization of reproductive endocrinology in snow leopards, and confirm that fecal hormone monitoring is an effective way to monitor female snow leopards throughout the breeding season.     

Development of a high‐throughput,indirect antibody immobilization format chemiluminescence enzyme immunoassay (CLEIA) for the determination of progesterone in human serum

A high‐throughput and simple chemiluminescence (CL) enzyme immunoassay (CLEIA) for the determination of progesterone (P) in human serum was developed, with the highly sensitive 4‐methoxy‐4‐(3‐phosphatephenyl)‐spiro‐(1,2‐dioxetane‐3,2′‐adamantane) (AMPPD)–alkaline phosphatase (ALP) system as the CL detection system. The results showed that the indirect immobilization of rabbit anti‐progesterone polyclonal antibody (RAPA) through secondary antibody exhibited apparent advantages over direct coating in terms of antibody saving and improvement of the coating stability and uniformity. The direct analysis of P in human serum without extraction was realized by using 8‐anilino‐1‐naphthalenesulphonic acid (ANS) to displace P from its binding proteins. The effect of several relevant parameters of the immunoreaction were examined and optimized. Compared with some commercial progesterone kits, the presented CLEIA has higher sensitivity with detection limitation as low as 0.06 ng/mL. The recoveries were 95.9–101%. The coefficient of variation was <8.4% and 9.9% for intra‐ and inter‐assay precision, respectively. This method has been successfully applied to the evaluation of P in human serum. Copyright © 2008 John Wiley & Sons, Ltd.     

Characterization and correlations of behavioral and adrenocortical activities of zoo‐housed lesser anteaters (Tamandua tetradactyla)

We characterized behavioral and adrenocortical activities of Tamandua tetradactyla under human care driven by the hypothesis that they vary between males and females. We also assessed the potential association between natural or abnormal behaviors and adrenocortical activity. We kept females and males T. tetradactyla in individual, contiguous enclosures at Córdoba Zoo (Argentina), under natural photoperiod and temperature. During 29 consecutive days we monitored the animals’ behavior by recording their activity pattern every 5 min using infrared cameras (8352 records/individual). We collected all feces and measured fecal glucocorticoid metabolites (FGM) with an 11‐oxoaetiocholanolone enzyme immunoassay. We found individual differences in all behavioral variables. We detected that females exhibited lower total activity than males (23.8 ± 0.2% and 32.3 ± 0.3%, respectively; p = .005). Females were more active at night and males during the day (p < .05) and exhibited less abnormal behaviors than males (p = .05). Although we did not find sex‐related differences for average FGM, we detected individual differences (p < .0001). We found that daily FGM showed negative (?0.39) and positive (0.38) correlations with natural and abnormal behaviors, respectively (p < .0001). Thus, we consider that individual input and sex are factors to be considered in stress responses of the species in captivity. Natural and abnormal behaviors may demand different levels of adrenocortical activity. Our findings may prove useful as normative data for ex situ management of conservation programs.     

Reproductive biology and management of captive black-footed ferrets (Mustela nigripes)

A captive breeding program is being conducted with black-footed ferrets (Mustela nigripes), an endangered species. Results of 5 years of study are reported. Simple, but specialized, nontraumatic handling techniques allowed assessment of reproductive status with minimal stress, which was important in breeding management. Black-footed ferrets are sexually mature and may successfully reproduce in their 1st year. Proestrus lasts approximately 2-3 weeks. Duration of estrus in unbred females was 32–42 days; females usually bred within 20 days. Most breeding activity occurred during April. Mean gestation length was 42.7 days (±0.7, range 42–45 days), litter size averaged 3.0 kits (±1.4, range 1–6 kits), and weaned kits/litter averaged 2.4 (±1.7, range 1–6 kits). Weaning rate of kits was 80%. Sex ratio of kits was essentially 1:1. Productivity was greatest among females ?3 years of age. Rapid expansion of the captive population is possible and will be important for genetic management of the species and for achieving the primary goal of the recovery program, which is to return black-footed ferrets to the wild.     

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.     

Progesterone, but not luteal estrogen, is required for the establishment of pregnancy in the new world monkey Cebus apella

The aim of this study was to examine the requirement of luteal progesterone or luteal estrogen for the establishment of pregnancy in the Cebus monkey and to test in a primate species the synergism between RU 486 and letrozole (LTZ) found in rodents for inhibiting implantation. Exposure of target tissues to either hormone was suppressed during the mid-luteal phase of mating cycles by subcutaneous administration of the antiprogestin (RU 486), the aromatase inhibitor LTZ or the antiestrogen (ICI 182780) on days 4-7 of the luteal phase. Administration of 0.1 or 0.5 mg/kg of LTZ on days 5-7 of the luteal phase caused a profound drop in the levels of E(2) in all animals, whereas administration of ICI 182780 0.2 mg/kg on days 4-6 of the luteal phase had the opposite effect. The pregnancy rate in vehicle treated cycles of the same females was (58.3%). Treatment with RU 486, 0.8 mg/kg/day on days 5-7 of the luteal phase-induced endometrial bleeding in 3/5 mated females none of which became pregnant, whereas pregnancy was confirmed in one of the two animals that did not bled. Treatment with RU 486, 0.4 mg/kg/day alone or with LTZ on days 5-7 or ICI 182780 alone, on days 4-6 of the luteal phase failed to induce bleeding, allowing the establishment of pregnancy in 50.0-66.6% of the animals in these groups. We conclude that in Cebus monkeys, progesterone but not luteal estradiol is required for the establishment of pregnancy and that RU 486 and LTZ do not exhibit in this species the synergism found in rodents.     

Fecal androgen metabolite analysis for noninvasive monitoring of testicular steroidogenic activity in felids

Limited data are available on long-term, seasonal changes in testicular steroidogenic activity in nondomestic felids, primarily because of the difficulties associated with longitudinal blood sampling (e.g., handling, restraint, anesthesia). Therefore, a noninvasive approach for assessing testicular androgen production was developed using the domestic cat (Felis catus) as a model. Two adult males were injected i.m. with 4 μCi¹⁴-testosterone to determine the time course and relative proportions of androgen metabolites excreted in urine and feces. Peak urinary radioactivity was detected 13 and 19 hr postinjection and accounted for ∼8% of the total radioactivity recovered. High performance liquid chromatography (HPLC) analysis detected multiple polar urinary metabolites, none of which eluted with the ³H-testosterone reference tracer. The majority of urinary testosterone metabolites consisted of nonenzyme-hydrolyzable, water-soluble (presumably conjugated) forms. In feces, radioactivity was detected in the first sample collected at 22 hr postinjection for both males, although peak metabolite excretion in one male was not observed until 61 hr postinjection. HPLC analysis detected several fecal metabolites consisting primarily of nonhydrolyzable, water-soluble forms (84.4 ± 0.9%) with some ether-soluble forms (15.6 ± 0.9%). None of the fecal androgen metabolites were associated with free testosterone. However, one or more of the water-soluble fecal metabolites was quantifiable using a commercially available testosterone radioimmunoassay. The biological relevance of this immunoactivity was confirmed in the domestic cat; concentrations were high in adult, intact males and nondetectable in intact females and castrated males and females. In addition, fecal androgen concentrations in a male Pallas' cat (Felis manul) exhibited seasonal fluctuations that corresponded with parallel changes in serum testosterone and ejaculate quality. These data indicate that testicular steroidogenic activity can be monitored non invasively in felids, providing a potentially valuable tool for endangered felid management to: (1) assess pubertal status, (2) determine the influence of season on reproduction, and (3) diagnose possible causes of sub- or infertility. © 1996 Wiley-Liss, Inc.     

Patterns of excretion of fecal estradiol and progesterone and urinary chorionic gonadotropin in Grevy’s zebras (Equus grevyi): Ovulatory cycles and pregnancy

The ovulatory cycle and pregnancy of the endangered Grevy’s zebra (Equus grevyi) was characterized by analyzing excretion patterns of fecal estradiol and progesterone. Pregnancy was further described by assay of urinary equine chorionic gonadotropin. Courtship and mating behavior were similar to that seen in the domestic horse, but cycle length was longer, ranging from 28 to 35 days. Steroid hormone profiles during pregnancy were likewise similar to the horse, but gestation length was longer (391 and 406 days) in the two mature mares. A young mare was first seen to mate when 1 year 3 months of age, at which time she conceived but then aborted at 101 days’ gestation. Conception occurred after one and possibly two more abortions. A healthy foal was born after a gestation period of 425 days, when the mare was 3 years 4 months old, the youngest age at first foaling to be reported for this species. Equine chorionic gonadotropin (eCG) was first detected between 35 and 40 days’ gestation, as in the horse, returning to baseline at about 195 days. However, in one young mare, eCG was also detectable around the time of ovulation. The eCG antibody may have cross‐reacted with luteinizing hormone, which would have been reaching peak concentrations at about that time. The general agreement of the endocrine results with observed occurrence of mating behavior and of parturition demonstrates that assay of excreted hormones can be used to document puberty, monitor ovarian cycles, and diagnose and monitor pregnancy in Grevy’s zebra mares. Zoo Biol 20:185–195, 2001. © 2001 Wiley‐Liss, Inc.     

Fecal corticoid metabolite measurement in the cheetah (Acinonyx jubatus)

This longitudinal study addresses the relationship of cortisol excretion to ovarian activity in captive female cheetahs. A radioimmunoassay was developed and validated to measure corticoid metabolite concentrations in feces. A restraint experiment was used to demonstrate that fecal cortisol is detectable following stressful episodes. In studies of 7 females, fecal cortisol output indicated that they could be placed into 3 different categories. Females of the high-in-cortisol category (∼200 ng/g feces, n = 2) were independently rated by caretakers as the most nervous individuals in the collection (n = 24). These females appeared to be compromised in their ovarian cycling, as indicated by fecal estrogen measurements. In contrast, reproducing females fell into the low and intermediate cortisol excretion categories. A non-cycling high-cortisol female had an episodic cycle following a period of relatively low (intermediate) cortisol levels, followed by resumption of acyclicity and high cortisol excretion. Stress and reproductive failure may, therefore, be associated in the female cheetah. The close proximity of conspecifics as a potential source of stress and, consequently, suppressed ovarian activity in some females is suggested by these results. Zoo Biol 16:133–147. 1997 © 1997 Wiley-Liss, Inc.     

Urinary gonadotropin and estrogen excretion during the postpartum estrus,conception, and pregnancy in the cotton-top tamarin (Saguinus oedipus oedipus)

Hormonal profiles during postpartum estrus, time of conception, and pregnancy were determined in urine samples from six cotton-top tamarins (Saguinus oedipus oedipus). Noninvasive collection techniques permitted daily sampling throughout lactation and pregnancy. Urinary estrone (E1), estradiol (E2), and both bioactive and immunoreactive luteinizing hormone/chorionic gonadotropin (LH/CG) measures revealed an interval of 19 ± 2.07 (S E M) days between parturition and the postpartum ovulatory LH peak. An increase in both E1 and E2 was seen prior to the LH peak; however, E1 and E2 continued to increase to their highest concentrations after the LH peak. Since postpartum ovulations resulted in pregnancy, neither postpartum estrus nor conception was suppressed by lactation. The length of gestation (measured from the LH peak to parturition) was 183.7 ± 1.14 (S E M) days, which is at least 30 days longer than that previously reported for other callitrichid species. Both E1 and E2 reached their maximum levels during midpregnancy but showed a rapid decline at parturition. Gestational levels of CG were first detectable approximately 20 days after the LH peak and continued to be elevated for approximately 80 days. The Sub-Human Primate Tube Test (SHPTT) for pregnancy did not detect the LH Peak and was less sensitive than other methods in detecting CG. Two RIA methods and a bioassay technique could not distinguish between LH and CG. We concluded that monitoring both estrogen and LH concentration was needed to determine when ovulation occurs in the cotton-top tamarin, since peak values of estrogen are seen after the ovulatory LH peak. Also, these tamarins were pregnant the majority of the time, indicating an unusually high fertility rate in contrast to most noncallitrichid primate species.