A retrospective analysis of adverse effects of an in vivo fluoroquinolone antibiotic enrofloxacin treatment on oocyte quality in the common marmoset.

Here we report a retrospective analysis of negative effects of routine enrofloxacin treatment of recurrent diarrhea on the ovary and the developing oocytes of the common marmoset, a small New World primate. The most deleterious effect on oocytes was observed about two months post treatment suggesting that the enrofloxacin effect is on early growing follicles. Manifestations of toxicity included decreased numbers of growing follicles and recovered culturable oocytes, as well as signs of early atresia of granulosa cells. In addition, increased amounts of holed stroma after treatment strongly suggested increased death of the early growing follicles. Of the oocytes judged to be of adequate quality for culture, maturation rates were not affected but fertilization of in vitro matured MII oocytes and subsequent cleavage rates were severely reduced in the enrofloxacin treated animals. Further, the arrested oocytes, which failed to mature or fertilize, showed obvious meiotic spindle abnormalities.

Body weight-associated differences in ovarian morphology in captive common marmoset (Callithrix jacchus).

In captivity, Callithrix jacchus (common marmoset) is on average heavier than their wild-living counterparts, and has a tendency to produce triplet litters rather than the normal twins. To provide initial basic information about possible weight-related differences among the ovaries, a morphometric study of follicular phase ovaries from 48 young adult marmosets has been carried out. Nearly 90% of these ovaries were found to contain some degree of luteal tissue composed of large and/or small cells. The luteal structures, follicles of all stages, and stroma were subjected to morphometric analysis, and these results were compared with body weight, circulating triglyceride, androstenedione, and total estrogens. Where only large luteal cells were present, the median body weight was the highest (only this group included animals over 500 g) compared with mixed, or only small luteal cells, or absence of luteal cells. Furthermore, in this group plasma triglycerides were significantly higher compared to other groups, suggesting possible role of triglycerides in promoting luteinisation. Plasma androstenedione was also a critical discriminating factor, and was elevated where large luteal cells were present even as a mixture with small cells suggesting the large luteal cells to be the likely major ovarian source of this hormone and its metabolites. Additionally, the ovaries with large luteal cells compared to those containing only small or no luteal cells, had lower primordial follicle reserve associated with high levels of atresia and luteinisation among growing non-ovulatory follicles, indicating an accelerated activation, but at the same time a suboptimal environment for follicular growth.

Visible implant elastomer (VIE) tagging and simulated predation risk elicit similar physiological stress responses in three-spined stickleback Gasterosteus aculeatus.

The effect of visible implant elastomer (VIE) tagging on the immediate physiological stress response was tested in female three-spined stickleback Gasterosteus aculeatus, using non-invasive waterborne cortisol analysis. Post-tagging cortisol levels were significantly higher compared with pretreatment baseline concentrations; however, when comparing post-tagging cortisol levels with cortisol levels after exposure to a simulated aerial predator, no significant differences were found. This study indicates that VIE tagging elicits a physiological stress response similar to those occurring in the everyday lives of this important biological model organism.

Critical estradiol dose optimization for oocyte in vitro maturation in the common marmoset.

The aim of the present study was to critically evaluate the effect of different concentrations of estradiol (E2) during IVM of common marmoset (Callithrix jacchus) oocytes from antral follicles. The doses tested were 0, 0.1, 1, or 10 µg/mL E2 (referred to as 0 E2, 0.1 E2, 1 E2, and 10 E2 groups). After a preincubation, the concentration of E2 in IVM drops under oil was approximately 20% of the amount added (0.02; 0.2 and 1.9 µg/mL, respectively) because of absorption into the oil. Oocyte progression to metaphase II was significantly higher in the 0.1 E2 group than that in the absence of E2. With progressively higher doses, the maturation rate tended to decrease suggesting an overdose effect. Furthermore, the total first cleavage rate was significantly higher in the 0.1 E2 group than that in the 0 E2 group and decreased progressively with further increases in E2 concentration, with the 10 E2 group showing the same low rate as without E2. The oocytes which failed to cleave, after maturation in 10 E2, showed obvious signs of overdose with the highest rates of degeneration and abnormal spindle form, and an absence of embryo progression. In contrast to these obvious negative effects on the oocyte, 10 E2 was the only group in which a significant increase in radial cumulus expansion was observed. The concentration 0.1 E2, which is 10 times lower than the most commonly used E2 dose, produced the best results in all oocyte factors evaluated. These results represent the first study for a primate species showing a strong positive effect of E2 on oocyte maturation and embryo development, but only at the optimal concentration, and emphasize the critical limits of the optimal concentration range.

Minimally invasive transabdominal collection of preimplantation embryos from the common marmoset monkey (Callithrix jacchus).

A novel, minimally invasive, transabdominal embryo collection method (transabdominal method) was developed as an alternative to a standard abdominal incision for embryo collection in the common marmoset. The abdominal incision method was used for 304 flushes using 36 female animals, whereas the transabdominal method was used for 488 flushes using 48 females; successful embryo collection rates were 48.0% and 48.4% (P > 0.05), respectively. These techniques were successfully duplicated at another institute (German Primate Center, DPZ). At that institution, successful embryo collection rates were 88.9% and 77.8% for the abdominal incision and transabdominal methods, respectively (P > 0.05), whereas the average numbers of preimplantation embryos obtained per flush were (mean ± SD) 1.91 ± 0.35 and 1.71 ± 0.14 (P > 0.05). The transabdominal method reduced animal stress, did not require incisional wound healing, and enabled successive embryo recoveries to be done much sooner. More embryos in early developmental stages (zygotes/morulae) were recovered using the transabdominal method (76.1%) than the abdominal incision method (52.6%, P < 0.01). In contrast, recovery of arrested or abnormal embryos was not significantly different between these two methods (9.8% and 8.3%). To verify developmental ability of embryos recovered by the transabdominal method, transfer of 28 normal embryos to 14 surrogate mothers yielded a nidation rate of 57%. Five females sustained term pregnancies and eight neonates were born. This novel transabdominal method will facilitate progress in marmoset developmental biology and embryology.

Developmental expression of the pluripotency factor sal-like protein 4 in the monkey, human and mouse testis: restriction to premeiotic germ cells.

SALL4 (sal-like protein 4) is a pluripotency transcription factor, which is highly expressed in embryonic stem (ES) cells and which is essential for mouse preimplantation development. In adult mouse organs, Sall4 mRNA is highly expressed in the testis and ovary, while there is only little or no expression in other organs. There is also a high expression of SALL4 in human testicular germ cell tumors. However, there is as yet no detailed analysis of SALL4 expression during mammalian testicular development. We analyzed SALL4 expression in ES cells, preimplantation embryos, and the developing and adult testis of a nonhuman primate (NHP) species, the common marmoset monkey (Callithrix jacchus). Immunofluorescence revealed SALL4 in the nuclei of marmoset ES cells and preimplantation embryos. Marmoset SALL4 isoform analysis in ES cells and newborn and adult testis by RT- PCR and Western blotting showed two different isoforms, SALL4-A and SALL4-B. Immunohistochemistry localized this transcription factor to the nuclei of primordial germ cells and most gonocytes in the prenatal and early postnatal marmoset testis. In the pubertal and adult testis SALL4 was present in undifferentiated spermatogonia. In the developing and adult human and mouse testis SALL4 expression mimicked the pattern in the marmoset. Adult testes from additional NHP species, the treeshrew, the cat and the dog also exhibited SALL4 in undifferentiated spermatogonia, indicating a conserved expression in the mammalian testis. Taking into account the importance of SALL4 for mouse development, we conclude that SALL4 may play an important role during mammalian germ cell development and is involved in the regulation of spermatogonial proliferation in the adult testis.

Upper respiratory tract disease in captive orangutans (Pongo sp.): prevalence in 20 European zoos and predisposing factors.

BACKGROUND: Upper respiratory tract disease (URTD) is a significant cause of morbidity in captive orangutans (Pongo abelii, Pongo pygmaeus), and the pathogenesis is often unknown. METHODS: The prevalence of respiratory disease in captive European orangutans (201 animals; 20 zoos) and possible predisposing factors were investigated. RESULTS: Bornean orangutans (P. pygmaeus) showed chronic respiratory signs significantly more often (13.8%) than Sumatran (P. abelii; 3.6%), and males (15.8%) were more often afflicted than females (3.9%). Hand-reared animals (21%) developed air sacculitis more often than parent-reared animals (5%). Diseased animals were more often genetically related to animals with respiratory diseases (93%) than to healthy animals (54%). None of the environmental conditions investigated had a significant effect on disease prevalence. CONCLUSION: Results suggest a higher importance of individual factors for the development of URTD than environmental conditions. Bornean, male and hand-reared orangutans and animals related to diseased animals need increased medical surveillance for early detection of respiratory disease.

Social capital and physiological stress levels in free-ranging adult female rhesus macaques.

Social animals with greater access to social support, i.e. higher levels of social capital, may be able to cope better with the challenges they face in their day-to-day lives, and this may be reflected in lower physiological stress levels. Here, we examine the relationship between social capital and fecal glucocorticoid (GC) levels in pregnant free-ranging adult female rhesus macaques. In addition to social capital measures based on direct connections between social partners, which have been examined previously, we use social network analysis to generate measures of social capital based on indirect connections (i.e. connections between pairs of individuals which result from their mutual direct connection to a third party). We consider social capital based on three different types of affiliative association: grooming, the exchange of affiliative vocalizations and proximity. After controlling for variables known to affect GC output in primates (e.g. month of pregnancy), GC levels of females were significantly predicted by a social network measure of indirect connectedness in the proximity network, proximity reach, in interaction with dominance rank. High ranking females had significantly lower GC levels in months in which they had low levels of proximity reach (i.e. in months in which their proximity networks were smaller and therefore more focused). The results of our study add to a growing body of evidence which suggests that social capital may be an important means by which gregarious animals cope with day-to-day challenges. Our study also joins a small body of recent research which has demonstrated that indirect connections may be important factors in the lives of social animals.

Seasonal and social influences on fecal androgen and glucocorticoid excretion in wild male long-tailed macaques (Macaca fascicularis).

Whereas it is well known that in strictly seasonal breeding primates (income breeders), alike other vertebrates, males show pronounced changes in testicular and adrenal hormone levels concurrent with reproductive activity, hormonal patterns in males of non-strictly seasonal breeding primate species (capital breeders) and their relation to seasonal and social correlates remain largely unknown. In the present study, we examined the annual pattern of fecal androgen and glucocorticoid excretion and their relationship to environmental (rainfall, temperature) and social factors (number of cycling females, male aggression and copulation rates, male dominance rank) in a group of wild long-tailed macaques (Macaca fascicularis), a species with a moderate degree of reproductive seasonality and classified as capital breeder. The study was carried out in the Gunung Leuser National Park, North Sumatra, Indonesia over a period of ten months encompassing the conception and the birth season. Our results show that male long-tailed macaques exhibit a distinct annual variation in both androgen and glucocorticoid levels. Androgen (but not glucocorticoid) levels were significantly elevated during the conception period in association with elevated rates of male-male aggression and copulatory activity, both strongly related to the number of cycling females in the group. Neither glucocorticoid nor androgen levels were related to male dominance rank or to the environmental parameters investigated. Interestingly, levels of both hormones started to increase in the late birth season and thus 1-2 months prior to the mating season, suggesting that male long-tailed macaques go through pre-breeding hormonal changes in preparation for prospective challenges. Our data thus provide the first evidence that males of a non-strictly seasonal breeding species/capital breeder show endocrine patterns generally similar to those found in strictly seasonal/income breeders.

Effects of human pituitary FSH isoforms on mouse follicles in vitro.

The effects of human FSH glycoforms on mouse follicle development and function in vitro were analysed, and an attempt was made to relate markers of follicular maturation to the expression of immunolocalized connexin (Cx) 43 and Cx26-based gap junctions. Three FSH fractions comprising discrete pI ranges [7.10-5.99 (pool I), pI 5.62-4.95 (pool II) and <3.75 (pool III)] were studied. Pool I produced the strongest effect on preantral granulosa cell proliferation and oestradiol production, and was highly effective for stimulating antral formation; this isoform also evoked a peripheral distribution of Cx43-containing gap junctions. Pool II was effective in promoting preantral granulosa cell proliferation but required higher FSH doses. This particular isoform provoked a more central distribution of Cx43-containing gap junctions, which was associated with a lower oestradiol production and less effective antral formation. Pool III was the least active for all markers of follicle development, and this was associated with minimal induction of Cx43-based gap junctions. The effects of the three FSH isoform pools on Cx26 expression were similar. The pattern of differences strongly suggests that FSH isoforms have complementary and specific actions on developing follicles, and that a shifting stage specific balance of isoforms is required for optimal follicle development.

Comparison of different enzyme-immunoassays for assessment of adrenocortical activity in primates based on fecal analysis.

Most studies published to date that used fecal glucocorticoid measurements to assess adrenocortical activity in primate (and many nonprimate) species applied a specific cortisol or corticosterone assay. However, since these native glucocorticoids are virtually absent in the feces of most vertebrates, including primates, the validity of this approach has recently been questioned. Therefore, the overall aim of the present study was to assess the validity of four enzyme-immunoassays (EIAs) using antibodies raised against cortisol, corticosterone, and reduced cortisol metabolites (two group-specific antibodies) for assessing adrenocortical activity using fecal glucocorticoid metabolite (GCM) measurements in selected primate species (marmoset, long-tailed macaque, Barbary macaque, chimpanzee, and gorilla). Using physiological stimulation of the hypothalamo-pituitary-adrenocortical (HPA) axis by administering exogenous ACTH or anesthesia, we demonstrated that at least two assays detected the predicted increase in fecal GCM levels in response to treatment in each species. However, the magnitude of response varied between assays and species, and no one assay was applicable to all species. While the corticosterone assay generally was of only limited suitability for assessing glucocorticoid output, the specific cortisol assay was valuable for those species that (according to high-performance liquid chromatography (HPLC) analysis data) excreted clearly detectable amounts of authentic cortisol into the feces. In contrast, in species in which cortisol was virtually absent in the feces, group-specific assays provided a much stronger signal, and these assays also performed well in the other primate species tested (except the marmoset). Collectively, the data suggest that the reliability of a given fecal glucocorticoid assay in reflecting activity of the HPA axis in primates clearly depends on the species in question. Although to date there is no single assay system that can be used successfully across species, our data suggest that group-specific assays have a high potential for cross-species application. Nevertheless, regardless of which GC antibody is chosen, our study clearly reinforces the necessity of appropriately validating the respective assay system before it is used.

Patterns of anogenital swelling size and their endocrine correlates during ovulatory cycles and early pregnancy in free-ranging barbary macaques (Macaca sylvanus) of Gibraltar.

In macaques and other cercopithecoid primates, large anogenital swellings (AS) are generally found only in those species in which reproduction is not seasonally restricted. In this respect, the Barbary macaque is unusual because while it shows a marked degree of reproductive seasonality, it also exhibits a striking, exaggerated swelling of the circumanal region and labia. Information on the characteristics of AS in female Barbary macaques is limited in that it is largely based on semiquantitative assessments of swelling size, and there are no data on endocrine parameters associated with AS during ovulatory cycles or early pregnancy. In the present study, we combined quantitative measurements of four swelling size parameters (AS width, height, and depth, and labial width) using a video-imaging technique with fecal estrogen and progestagen determinations in free-ranging females of the Gibraltar Barbary macaque population to 1) characterize the pattern of AS throughout the mating season and early gestation, and 2) examine the relationships among changes in swelling size and endocrine parameters. The patterns of all four swelling parameters correlated significantly with one another, although measures of AS depth and labial width were difficult to obtain. Using the product of AS height and width, the data demonstrate that the occurrence of AS is highly seasonal, with pronounced cyclical changes during the mating season and early pregnancy. Furthermore, the swelling cycles are characterized by progressive size increases from the early to the late follicular phase, in association with an elevated estrogen:progestagen (E:P) ratio, with ovulation occurring during the maximum swelling phase. The results also demonstrated a conspicuous postconception increase in swelling between days 18-30 of gestation. The postconception swellings were on average 80% of the size of that of the conception cycles, and were preceded by a large increase in fecal estrogen levels and the E:P ratio. This is the first study to characterize swelling patterns and their endocrine correlates during ovarian cycles and early pregnancy in naturally reproducing female Barbary macaques. The data provide a solid basis for further studies to explore sociosexual behavioral patterns and the functional significance of AS in this species.

Non-invasive assessment of adrenocortical function in the male African elephant (Loxodonta africana) and its relation to musth.

Adult male elephants periodically show the phenomenon of musth, a condition associated with increased aggressiveness, restlessness, significant weight reduction and markedly elevated androgen levels. It has been suggested that musth-related behaviours are costly and that therefore musth may represent a form of physiological stress. In order to provide data on this largely unanswered question, the first aim of this study was to evaluate different assays for non-invasive assessment of adrenocortical function in the male African elephant by (i) characterizing the metabolism and excretion of [3H]cortisol (3H-C) and [14C]testosterone (14C-T) and (ii) using this information to evaluate the specificity of four antibodies for determination of excreted cortisol metabolites, particularly with respect to possible cross-reactions with androgen metabolites, and to assess their biological validity using an ACTH challenge test. Based on the methodology established, the second objective was to provide data on fecal cortisol metabolite concentrations in bulls during the musth and non-musth condition. 3H-C (1 mCi) and 14C-T (100 microCi) were injected simultaneously into a 16 year old male and all urine and feces collected for 30 and 86 h, respectively. The majority (82%) of cortisol metabolites was excreted into the urine, whereas testosterone metabolites were mainly (57%) excreted into the feces. Almost all radioactive metabolites recovered from urine were conjugated (86% 3H-C and 97% 14C-T). In contrast, 86% and >99% of the 3H-C and 14C-T metabolites recovered from feces consisted of unconjugated forms. HPLC separations indicated the presence of various metabolites of cortisol in both urine and feces, with cortisol being abundant in hydrolysed urine, but virtually absent in feces. Although all antibodies measured substantial amounts of immunoreactivity after HPLC separation of peak radioactive samples and detected an increase in glucocorticoid output following the ACTH challenge, only two (in feces against 3alpha,11-oxo-cortisol metabolites, measured by an 11-oxo-etiocholanolone-EIA and in urine against cortisol, measured by a cortisol-EIA) did not show substantial cross-reactivity with excreted 14C-T metabolites and could provide an acceptable degree of specificity for reliable assessment of glucocorticoid output from urine and feces. Based on these findings, concentrations of immunoreactive 3alpha,11-oxo-cortisol metabolites were determined in weekly fecal samples collected from four adult bulls over periods of 11-20 months to examine whether musth is associated with increased adrenal activity. Results showed that in each male levels of these cortisol metabolites were not elevated during periods of musth, suggesting that in the African elephant musth is generally not associated with marked elevations in glucocorticoid output. Given the complex nature of musth and the variety of factors that are likely to influence its manifestation, it is clear, however, that further studies, particularly on free-ranging animals, are needed before a possible relationship between musth and adrenal function can be resolved. This study also clearly illustrates the potential problems associated with cross-reacting metabolites of gonadal steroids in EIAs measuring glucocorticoid metabolites. This has to be taken into account when selecting assays and interpreting results of glucocorticoid metabolite analysis, not only for studies in the elephant but also in other species.

Characterization of urinary and fecal metabolites of testosterone and their measurement for assessing gonadal endocrine function in male nonhuman primates.

The aims of the present study were (i) to provide basic comparative data on the time course, route, and characteristics of excreted [14C]testosterone (T) metabolites in three nonhuman primates: the common marmoset (Callithrix jacchus), the long-tailed macaque (Macaca fascicularis) and the chimpanzee (Pan troglodytes) and (ii) to use this information to help validate the measurement of urinary and fecal testosterone metabolites for assessing androgen status in Anthropoid primates. Radiolabeled 14C-T (10-30 microCi) was injected intravenously into one adult male of each species and the excreta collected over the next 5 days. Peak radioactivity in urine was detected within 2h and accounted for 67% (Mf), 80% (Cj) and 91% (Pt) of the total radioactivity recovered. The time course of excretion of radioactivity in feces showed a higher variation between species (4-26 h to peak values). In all three species, the majority (>90%) of urinary metabolites were excreted as conjugates whereas the proportion of conjugated metabolites in feces was substantially lower and more variable. High pressure liquid chromatography (HPLC) analysis of urinary and fecal extracts revealed multiple peaks of radioactivity in all three individuals, but each with a distinctive pattern. Native T was excreted in only small amounts into the urine, whereas it was virtually absent in the feces of all three individuals. Three C17 group-specific enzymeimmunoassays using antisera against testosterone, 5alpha-androstane-17alpha-ol-3-one and androsterone were evaluated for their ability to discriminate immunoreactive androgen levels between intact males, castrated males and females based on measurements in urine and feces. In the marmoset, all assays (except for T in feces) clearly discriminated between test groups; in the chimpanzee significantly higher levels of androgen immunoreactivity in intact versus castrated males were measured in urine, but not feces. In the macaque, only the 5alpha-androstanolone measurement in feces discriminated between groups. Data on the results of a radiometabolism study using 3H-DHEA (a weak adrenal androgen) in a long-tailed macaque suggested that co-measurement of metabolites derived from T and DHEA in the assays tested might explain the difficulties in discriminating gonadal status in the two Old World primate species. Collectively, the data show that T metabolism in primates is highly complex and that no single method for noninvasive assessment of androgen status can be used for application across species. The importance of a proper validation of the methodology for each species is emphasised.

Loss of oestrus, concealed ovulation and paternity confusion in free-ranging Hanuman langurs.

Ovarian cycles in catarrhine primates are uniquely characterized by prolonged periods of sexual activity in which the timings of ovulation and copulation do not necessarily correspond. According to current hypotheses of primate social evolution, extended sexuality in multi-male groups might represent part of a female strategy to confuse paternity in order to reduce the risk of infanticide by males. We test this hypothesis by examining mating behaviour in relation to timing of ovulation and paternity outcome in a multi-male group of free-living Hanuman langurs. Using faecal progestogen measurements, we first document that female langurs have extended receptive periods in which the timing of ovulation is highly variable. Next, we demonstrate the capacity for paternity confusion by showing that ovulation is concealed from males and that copulations progressively decline throughout the receptive phase. Finally, we demonstrate multiple paternity, and show that despite a high degree of monopolization of receptive females by the dominant male, non-dominant males father a substantial proportion of offspring. We believe that this is the first direct evidence that extended periods of sexual activity in catarrhine primates may have evolved as a female strategy to confuse paternity.

Demonstration of 2-unsaturated C19-steroids in the urine of female Asian elephants, Elephas maximus, and their dependence on ovarian activity.

Air-borne volatile substances have been demonstrated to signal oestrus, induce ovulation and synchronize ovarian activity in different mammals. An oestrous-related pheromone of the female Asian elephant (Elephas maximus) is known to induce behavioural responses in elephant bulls. Additional data revealed that timing of oestrus in females with close social relationships tends to be synchronized. Therefore, urine from female Asian elephants might be expected to contain luteal phase-dependent volatile substances, which may function as additional chemical signals in this species. The aim of the present study was to identify such compounds and to investigate their pattern of excretion throughout the ovarian cycle. Urine samples were collected three times a week during the follicular phase and one to three times a week during the luteal phase from five adult female Asian elephants from a total of 13 non-conception cycles and one conception cycle, including the first 72 weeks of pregnancy. A simple headspace solid-phase microextraction method has been developed for quantification of urinary volatile substances and analysis was performed by gas chromatography. The comparison of urine collected during the follicular and the luteal phase indicated the presence of two luteal phase-dependent substances. Mass spectrometry was used to identify one substance as 5alpha-androst-2-en-17-one and a second substance as the corresponding alcoholic compound 5alpha-androst-2-en-17beta-ol. The 5alpha-androst-2-en-17beta-ol and -17-one profiles reflected cyclic ovarian activity with clear (10-20-fold) luteal phase increases. Furthermore, measurements of both compounds were correlated positively with the concentration of urinary pregnanetriol and indicated cycle duration (15.1 +/- 1.2 weeks) similar to that obtained from pregnanetriol measurements (15.2 +/- 1.6 weeks). The results demonstrate the presence of two luteal phase-specific steroidal volatile compounds in elephant urine. One of the substances, 5alpha-androst-2-en-17-one, has been demonstrated in human axillary bacterial isolates. The measurement of both volatile substances in elephant urine can be used for rapid detection of the stage of the ovarian cycle, as the analysis can be completed within 2 h.

Changes in follicle-stimulating hormone and follicle populations during the ovarian cycle of the common marmoset.

The common marmoset (Callithrix jacchus) belongs to the family Callitrichidae, the only anthropoid primates with a high and variable number of ovulations (one to four). An understanding of folliculogenesis in this species may provide some insight into factors regulating multiple follicular growth in primates. The aims of this study were to characterize in detail changes in the antral follicle population at different stages of the ovarian cycle, to characterize the marmoset FSH profile, and to relate cyclic changes in FSH to changes in follicle sizes and circulating estradiol concentrations. Fifty-five pairs of ovaries were collected (32 of which were at five distinct stages of the cycle) from adult marmosets, and antral follicles were manually excised and separated into four size groups. Daily urinary FSH and plasma estradiol and progesterone concentrations from Day 0 of the follicular phase to 2 days postovulation were measured in 22 marmosets using enzyme immunoassays. The FSH profile revealed two distinct peaks, on Days 2 and 6, during the 10-day follicular phase, with a marginal periovulatory increase on Days 9 and 10. Estradiol levels rose significantly (P: < 0.05) above baseline (Days 1-4) on Day 5 and continuously increased to a peak on the day preceding ovulation (Days 8 and 9). Follicle dissection revealed a high (mean = 68) and variable (range, 14-158) total number of antral follicles >0.6 mm. The number of antral follicles significantly declined (P: < 0.001) with age. The number of preovulatory follicles (>2 mm) was positively correlated with the number of antral follicles (P: < 0. 001) and tended to be negatively related to age (P: = 0.06). The number of antral follicles did not vary significantly with stage of the ovarian cycle, although the follicle size distribution was cycle-stage dependent (P: < 0.05). Follicles >1.0 mm appeared only in the follicular phase, and preovulatory follicles (>2.0 mm) appeared only at the end of the follicular phase (Days 7-9). The Day 2 FSH peak corresponded to emergence of a population of medium-size antral follicles, and the Day 6 peak was consistent with rising estradiol levels and appearance of the preovulatory follicles. These results suggest that some aspects of marmoset folliculogenesis are comparable to those in Old World primates, including the absence of multiple follicular waves and the appearance of an identifiable dominant follicle in the midfollicular phase. However, the midphase FSH peak, multiple dominant follicles, and abundance of nonovulatory antral follicles differ strongly from the pattern in Old World primates and humans. The findings are discussed in relation to the regulation of growth of multiple ovulatory follicles and provide the basis for further studies on factors influencing the dynamics of follicular growth and development in this species.

Androgen levels and female social dominance in Lemur catta.

Morphological and behavioural traits which improve agonistic power are subject to intrasexual selection and, at the proximate level, are influenced by circulating androgens. Because intrasexual selection in mammals is more intense among males, they typically dominate females. Female social dominance is therefore unexpected and, indeed, rare. Ring-tailed lemurs (Lemur catta) are sexually monomorphic primates in which all adult females dominate all males. The goal of our study was to test the prediction that female dominance in this species is associated with high androgen levels. Using two captive groups, we collected data on agonistic behaviour and non-invasively assessed their androgen concentrations in faeces and saliva by enzyme immunoassay. We found that adult female L. catta do not have higher androgen levels than males. However, during the mating season there was a twofold increase in both the androgen levels and conflict rates among females. This seasonal increase in their androgen levels was probably not due to a general increase in ovarian hormone production because those females showing the strongest signs of follicular development tended to have low androgen concentrations. At the individual level neither the individual aggression rates nor the proportion of same-sexed individuals dominated were correlated with their androgen levels. We conclude that female dominance in ring-tailed lemurs is neither based on physical superiority nor on high androgen levels and that it is equally important to study male subordination and prenatal brain priming effects for a complete understanding of this phenomenon.

Hormonal correlates of reproductive seasonality in wild female hanuman langurs (Presbytis entellus).

To date, it is not known whether the seasonal occurrence of sexual behavior and mating in free-ranging Hanuman langurs at Ramnagar, Nepal, is correlated with seasonal changes in female ovarian function, and, if so, which factor(s) triggers the onset of the reproductive period. Using noninvasive fecal hormone analysis in combination with behavioral observations, this study was carried out to: 1) investigate and characterize seasonal patterns of ovarian cyclicity and timing of conception in wild langur females living in a highly seasonal habitat; and 2) examine the relationship between seasonal patterns of ovarian cyclicity, behavioral estrus, and female physical condition. Behavioral data and fecal samples were collected during a total period of 14 months from nine females living in a multi-male group. Physical condition of the females was assessed monthly by visual inspection, using a seven-fold scale. Ovulatory cycles and timing of conceptions were identified by the measurement of immunoreactive pregnanediol glucuronide (iPdG) in extracted feces. Hormone profiles in individual females revealed a clearly seasonal distribution in the occurrence of ovulatory cycles, which were restricted to the period from July to October. The distribution of female estrus behavior showed a similar seasonal pattern, and in total 88.2% of all estrus periods observed in the focal females were accompanied by ovulation. Onset of ovarian cycles as well as mating activities were strongly correlated with the onset of the rainy season. Females conceived, on average, in their second ovulatory cycle (pregnancy length: 211.6 +/- 3.4 days), with timing of conception being confined to the months when animals showed an improved physical condition. Collectively the present data clearly suggest that in seasonally-breeding langurs at Ramnagar, ecological conditions (rainfall, food availability, and quality) influence the onset of ovulations and timing of conceptions.

Comparative aspects of the metabolism and excretion of cortisol in three individual nonhuman primates.

A radiometabolism study is described to provide the first comparative data on the time course, route, and characteristics of excreted [3H]cortisol metabolites in three nonhuman primates: the common marmoset (Callithrix jacchus), the long-tailed macaque (Macacafascicularis), and the chimpanzee (Pan troglodytes). A low dose (40-100 microCi) of 3H-labeled cortisol was administered intravenously to one adult male of each species and the excreta collected over a 5-day period postinjection. The major proportion of radioactivity was excreted in the urine (>80%). Peak radioactivity in urine was recovered within 5.5 h following injection in all three species, while in the feces peak levels of radioactivity were recovered within 26 h postinjection. In all three species, urinary metabolites were primarily excreted as conjugates (61-87%), whereas the percentage of conjugated metabolites in feces was 50% or less. The number and relative abundance of urinary and fecal [3H]cortisol metabolites were determined by reverse-phase high-performance liquid chromatography (HPLC) and immunoreactivity of the radioactivity peaks was assessed by screening HPLC fractions with established cortisol, corticosterone, and 11-oxoetiocholanolone enzyme immunoassays (EIA), the latter being a group-specific assay for measuring 11,17-dioxoandrostanes. HPLC separation of urinary and fecal extracts revealed multiple peaks of radioactivity, several of which were common to all three species. The relative proportion of these peaks, however, differed considerably among species and between urine and feces. HPLC indicated that native cortisol was a major urinary excretory product in the marmoset, while comparatively small amounts were present in the urine of the macaque and chimpanzee. In contrast, in feces, cortisol was only detected in low amounts in the marmoset and was virtually absent in the macaque and chimpanzee. In all three species, one of the major radioactivity peaks showed a retention time comparable to 11-oxoetiocholanolone and high immunoreactivity in the 11-oxoetiocholanolone EIA. The measurement of urinary- and/or fecal-immunoreactive 11,17-dioxoandrostanes is therefore implicated for noninvasive assessment of adrenal function in Old World monkeys, New World monkeys, and great apes.