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.

Penile vibratory stimulation in the marmoset monkey: a practical alternative to electro-ejaculation, yielding ejaculates of enhanced quality.

The availability of sufficient amounts of spermatozoa of high quality is one of the main limiting factors in reproductive research and development of reproductive technologies in marmoset monkeys (Callithrix jacchus). Penile vibrostimulation (PVS) has been successfully used in semen collection in the squirrel monkey but with poor success rate in the marmoset. We report here on an improved protocol for PVS with a success rate of almost 90%. Ejaculates obtained by PVS were of enhanced quality compared with those obtained by rectal probe electro-ejaculation (RPE). PVS ejaculates contained on average three to fourfold higher numbers of total and motile spermatozoa. Assessment of sperm kinematics using computer-assisted sperm analysis indicated that there are also functional differences between spermatozoa collected by PVS and RPE. Marmoset spermatozoa in samples obtained by RPE swim in a more convoluted manner compared with those obtained by PVS.

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.

Quantitative assessment of testicular germ cell production and kinematic and morphometric parameters of ejaculated spermatozoa in the grey mouse lemur, Microcebus murinus.

Germ cell production and organization of the testicular epithelium in a prosimian species, the grey mouse lemur, Microcebus murinus, was investigated to extend knowledge of comparative primate spermatogenesis. In addition, semen samples collected from adult male lemurs (body weight 53-92 g; n = 16) by rectal probe electroejaculation were evaluated using computer-assisted morphometric and kinematic analysis of spermatozoa. Epididymidal spermatozoa were collected from six animals after hemicastration; the testes were weighed and prepared for stereological analysis and flow cytometry. The relative testis mass (as a percentage of body weight) ranged between 1.17 and 5.6%. Twelve stages of testicular seminiferous epithelium as described for macaques were applied and only a single stage was observed in most of the seminiferous tubule cross-sections. On average (mean SD), a single testis contained 1870 +/- 829 x 10(6) germ cells and 35 +/- 12 x 10(6) Sertoli cells. Germ cell ratios (preleptotene:type B spermatogonia = 2, round spermatid:pachytene = 3; elongated spermatid:round spermatids = 1) indicated high spermatogenic efficacy. Sperm head dimensions and tail lengths of the ejaculated and epididymidal spermatozoa were similar. Percentages of defects (neck/mid-piece and tail) were low ( 10%) and similar for ejaculated and epididymidal spermatozoa. Spermatozoa were highly motile, characterized by extensive lateral head displacement, but relatively low progressive motility. In conclusion, the grey mouse lemur has unusually large testes with a highly efficient spermatogenic process and large sperm output. These features, together with the high proportion of morphologically normal and highly motile spermatozoa in the ejaculates, indicate that Microcebus murinus is a species in which sperm competition after ejaculation is likely to occur. The predominantly single spermatogenic stage system seems to be an ancestral feature among primates.

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.

Quantitative analysis of spermatogenesis and apoptosis in the common marmoset (Callithrix jacchus) reveals high rates of spermatogonial turnover and high spermatogenic efficiency.

Spermatogenesis is characterized by the succession in time and space of specific germ cell associations (stages). There can be a single stage (e.g., rodents and some macaques) or more than one stage (e.g., chimpanzee and human) per tubular cross section. We analyzed the organization of the seminiferous epithelium and quantified testicular germ cell production and apoptosis in a New World primate, the common marmoset (Callithrix jacchus). Tubule cross sections contained more than one stage, and the human six-stage system could be applied to marmoset spermatogenesis. Stereological (optical disector) analysis (n = 5) revealed high spermatogenic efficiency during meiosis and no loss of spermatids during spermiogenesis. The conversion of type A to type B spermatogonia was several-fold higher than that reported for other primates. Highest apoptotic rates were found for S-phase cells (20%) and 4C cells (15%) by flow cytometric analysis (n = 6 animals); histological analysis confirmed spermatogonial apoptosis. Haploid germ cell apoptosis was <2%. Marmoset spermatogenesis is very efficient and involves substantial spermatogonial proliferation. The prime determinants of germ cell production in primates appear to be proliferation and survival of spermatogonia rather than the efficiency of meiotic divisions. Based on the organizational similarities, common marmosets could provide a new animal model for experimental studies of human spermatogenesis.

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.

Non-invasive collection of ejaculates from the common marmoset (Callithrix jacchus) using penile vibrostimulation.

Penile vibrostimulation (PVS), a noninvasive repeatable method, has been shown in the squirrel monkey to yield semen of higher quality than rectal probe electro-ejaculation (RPE). The present study aimed at establishing the conditions for PVS to collect ejaculates from marmoset monkeys. Ten adult males were trained on the appropriate handling before each was subject to six to 12 PVS tests. Ejaculation was stimulated using a FertiCare personal vibrator fitted with a 2 cm x 0.5 cm i.d. glass tube. The stimulus was repeatedly applied over a frequency of 75-95 Hz and amplitude of 1-2 mm for up to 20 min. Ejaculates were analyzed for volume, total sperm number, sperm concentration, and proportion of living and motile sperm. Ejaculates were obtained in 31 of 88 PVS tests; 87.1% of the ejaculations occurred at 80-85 Hz frequency and 1-1.5 mm amplitude. In 18 tests ejaculates were produced within 49.7 seconds. Ejaculates were characterized by (mean values): volume 31.9 microl, total sperm number 34.2 x 10(6)/ejaculate, concentration 1,154.2 x 10(6) sperm/ml, live sperm 74.6%, motile sperm 59.6%. Total number and concentration of spermatozoa were significantly enhanced in singly living males. PVS yielded three to four times more spermatozoa than comparable previously published values for RPE. Enhancing the success rate by preselecting males for responsiveness may render PVS the sperm collection method of choice in marmoset monkeys.

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.

Patterns of urinary and fecal steroid excretion during the ovarian cycle and pregnancy in the African elephant (Loxodonta africana).

The aims of the present study were to (i) determine the relative abundance of the 5alpha-reduced progestins 5alpha-pregnane-3-ol-20-one (5alpha-P-3OH) and 5alpha-dihydroprogesterone (5alpha-DHP) and progesterone (P4) in African elephant feces and to establish improved fecal progestin assays for monitoring ovarian function; and (ii) describe longitudinal profiles of urinary and fecal progestin and estrogen metabolites during pregnancy. Matched urine and fecal samples were collected weekly from six adult females throughout 18 nonfertile cycles and two complete pregnancies (89 and 93 weeks duration). Fecal samples were lyophilized and extracted with 80% methanol in water and immunoreactive 5alpha-P-3OH, 5alpha-DHP, and P4 and (for pregnant females only) estrone (E1) and estradiol (E2) determined by enzyme immunoassay. Urine samples were hydrolyzed, ether-extracted, and assayed for 5alpha-P-3OH, E1, and E2. HPLC cochromatography of fecal extracts with various radioactive progestin tracers confirmed the presence of large amounts of both 5-reduced progestins (5alpha-P-3OH > 5alpha-DHP) but not of P4. 5-Reduced progestins (but not P4) were excreted in a cyclic pattern and levels were significantly correlated with urinary 5alpha-P-3OH. Fecal 5alpha-P-3OH showed the more pronounced and consistent luteal-phase elevation and a better correspondence to urine with respect to timing of the luteal-phase rise. Fecal and urinary 5-reduced progestins increased gradually during early pregnancy to maximum values around week 40-45. Levels gradually declined during the second half of pregnancy, reaching baseline values 2 days before parturition. Urinary estrogens did not show any cyclic pattern during the preconception period and levels remained low during the first 30 weeks of gestation. Thereafter, there was a rapid 10- to 20-fold increase to maximum values at mid-pregnancy, followed by a gradual decline to birth. There was no mid-pregnancy elevation in fecal estrogens, but there was a modest increase in E1 during the second half of gestation.

Efficacy and effects of short- and medium-term contraception in the common marmoset (Callithrix jacchus) using melengestrol acetate implants.

This study examines the effect of melengestrol acetate (MGA) implants on reproductive function and various biochemical parameters, ovarian activity, and uterine morphology in ten female common marmosets implanted for either 6-8 or 19-21 months. Measures of body weight, concentrations of urinary glucose and blood liver enzymes were taken. Ovarian activity was assessed by analysis of urinary progestin levels and ultrasound examinations of the ovaries. Ultrasonography was also used to evaluate uterine morphology. MGA was highly effective in preventing pregnancies in the study animals. No changes in biochemical parameters were found; however, seven females developed a substantial weight gain during the study. Follicular development was not suppressed, as indicated by the presence of antral follicles, luteinized structures, and elevated urinary progestin levels. The uteri of the MGA-treated subjects were moderately enlarged with a thickened endometrium that showed a marked change in structural appearance indicative of hypertrophy and decidualization. After implant removal these changes quickly disappeared and all females ovulated within 3 weeks and conceived within 4 months post-treatment. MGA appears to be an acceptable contraceptive in the marmoset, although non-steroidal methods should be evaluated as possible potential alternatives.

Direct effects of the prostaglandins E2 and f2alpha on progesterone release by the corpus luteum of the marmoset monkey (Callithrix jacchus) studied by in vitro microdialysis.

The effects of the prostaglandins (PG) E2 and F2alpha on progesterone secretion in luteal tissue (32 corpora lutea) explanted from the mid-luteal ovary of the marmoset monkey (n=13) were investigated using an in vitro microdialysis system. Consecutive applications of 1, 10 and 100 microg/ml PGE2 resulted in a significant increase in secretion of progesterone at the maximum dose of 100 microg/ml, which was shown to be the stimulatory dose in both long-period and 20-min pulse (time to collect one fraction) applications. The response varied individually between 1.4- and 3. 4-fold above the baseline concentrations. Application of 500 microg/ml PGF2alpha led to similar hormone responses. In contrast, lower doses of PGF2alpha (0.5, 5 and 50 microg/ml) resulted in significantly increased levels of secretion of progesterone, to approximately 1.4-fold baseline values, only after the application was terminated (echo effect). Responses were less variable when a short pulse of 20 min duration was applied, instead of long applications of 1-2 h. On the basis of the passage rates measured for tritiated PGF2alpha, transfer through the dialysis membrane was assumed to be in the range of 1% for both PGs. Ultrastructurally, luteal cells lying in a sheath of five to seven cell layers around the dialysis tubing appeared intact and were interconnected by gap junctions. Vesiculation of the smooth endoplasmic reticulum was more prominent after PG treatment, indicating a stimulation of cellular synthesis/secretory activities that was in accordance with the stimulatory action of both PGs on progesterone release under these in vitro conditions.

Endocrinology of the ovarian cycle and pregnancy in the Asian (Elephas maximus) and African (Loxodonta africana) elephant.

In reviewing the literature, this paper assesses the current level of understanding of the hormonal control of the ovarian cycle and pregnancy in the elephant. Data are compiled from two genera, Elephas maximus and Loxodonta africana, recognizing differences where known to exist. Measurements of various steroid and peptide hormones, but most notably immunoreactive progesterone (iP), indicate an overall cycle length of 15-16 weeks, comprising an 8-11-week luteal phase and a shorter inter-luteal (follicular) period of 4-6 weeks. Oestrogen related phenomena suggest the occurrence of two (possibly more) waves of follicular development with a 3-week periodicity, although oestrogen levels are low and fluctuate without clear pattern. The inter-luteal period is characterized by two discrete LH peaks also 3 weeks apart, but only the second is associated with an iP rise. Ovulation rate is variable and additional luteal structures lacking ovulation points are probably formed each cycle. The post-ovulatory period is associated with elevated progestogen levels, mainly 5alpha-reduced compounds, while progesterone itself is a minor secretory product. A revised model for the elephant ovarian cycle is presented. Detailed information on the endocrinology of pregnancy is lacking. Elevated progestins beyond the normal luteal phase indicate that conception extends the functional lifespan of the CL, with maximum steroidogenic activity between 3-15 months. The nature of the luteotrophic support is not known and placental gonadotrophins have not been conclusively demonstrated. Progestins fluctuate at or slightly above luteal phase values throughout pregnancy. There is a marked increase in prolactin levels after 16-24 weeks and a relationship with oestrogen secretion may exist since circulating and urinary levels show a progressive increase from 6-8 months. A placental contribution to progestin secretion is likely, although direct evidence is lacking. Considerable enlargement of foetal gonads during the second half of pregnancy in African elephants suggests a possible steroidogenic function, but none has been defined. The endocrine events leading to parturition are unknown. In the Asian elephant, a fall in iP precedes parturition; oestrogen levels decline and cortisol increases at the time of birth. The paper concludes with a brief selection of priorities for future research.

Cryopreservation of non-human primate sperm: priorities for future research.

Wild populations of many non-human primate species have declined alarmingly due to habitat destruction, hunting and genetic isolation. Captive breeding programmes to aid species survival could be enhanced by the use of assisted reproductive techniques, such as artificial insemination (AI), if a source of viable sperm was readily accessible. Cryobanks of primate sperm could provide such a supply if techniques for freezing sperm could be developed. Although sporadic attempts to cryopreserve primate sperm have been reported for some of the more frequently encountered zoo-maintained species, there is limited information available on techniques for sperm collection and storage. It is vital that adequate reporting of all cryopreservation attempts be made to avoid repetition of inappropriate methodologies and wastage of valuable genetic material from rare or endangered animals. An integrated approach to the cryobanking of non-human primate sperm is considered to be essential for species conservation. In this review, the factors affecting the success of sperm cryopreservation are outlined, existing information is compiled from previous reported attempts at cryopreservation, and suggestions are made for cryopreserving sperm in further non-human primate species. Moreover, recommendations are given for additional studies to augment existing data. It is intended that this information should serve as a guide for developing cryopreservation protocols in the future, particularly for endangered species.

Metabolism and excretion of oestradiol-17beta and progesterone in the Sumatran rhinoceros (Dicerorhinus sumatrensis).

3H-labelled oestradiol-17beta and 14C-progesterone were injected i.v. into an adult female Sumatran rhinoceros (Dicerorhinus sumatrensis) and all urine and faeces collected over 4 days. Of the injected steroid, 68% of 3H-oestradiol and 89% of 14C-progesterone were recovered. Peak excretion in urine occurred on day 1 for both steroids, and for faeces on day 2 for 14C-progesterone, and between days 2 and 3 for 3H-oestradiol. Oestradiol metabolites were predominantly (nearly 70%) excreted into the urine, while progesterone metabolites were almost exclusively (> 99%) excreted into the faeces. The majority (> 70%) of urinary excreted oestrogens consisted of water-soluble (i.e., conjugated) forms, with > 90% of these being glucuronides. In contrast, > 75% of faecal oestrogen and progesterone metabolites were excreted as ether-soluble (i.e., unconjugated) forms. HPLC co-chromatography of oestrogens in hydrolysed urine indicated only one peak of radioactivity, co-eluting with authentic oestradiol-17beta, whereas two peaks of radioactivity were found after HPLC of faecal oestrogens, the major one co-eluting with oestrone and the less prominent one with oestradiol-17beta. Progesterone was excreted as numerous metabolites into the faeces. The three most abundant of these were identified using HPLC and gas chromatography mass spectrometry (GCMS) as 5beta-pregnane-3alpha,20alpha-diol, 5beta-pregnane-3alpha-ol-20-one, and a second pregnanediol, the exact structure of which could not be deduced. Measurement of urinary oestradiol-17beta and faecal immunoreactive pregnanediol and 5alpha-pregnane-3alpha-ol-20-one in daily samples enabled the first endocrine characterization of the ovarian cycle and indicated a cycle length of approximately 25 days.

Functional significance of behavioral, morphological, and endocrine correlates across the ovarian cycle in semifree ranging female Tonkean macaques.

The role of sexual displays in mating strategies and their reliability in indicating the time of ovulation has given rise to multiple explanations in nonhuman primates. In order to discriminate among hypotheses, socio-sexual behaviors were recorded in a semifree ranging group of Tonkean macaques (Macaca tonkeana), together with sexual skin swelling volumes and measurements of urinary concentrations of estrone conjugates and pregnanediol glucuronide. A clear preovulatory peak of urinary estrogen levels occurred 2 days before a defined rise in pregnanediol glucuronide concentrations, indicating that both hormones pointed out the female's ovulatory period. The concept of estrus rightly could be applied to female Tonkean macaques since fluctuations in estrogen levels correlated with cyclic changes in genital swelling sizes and rates of female behavioral attractivity and proceptivity. Males proved to be capable of recognizing the optimal conception period as judged from the occurrence of maximal rates of following behavior, serial matings, and ejaculations during the peri-ovulatory phase. During this time, males succeeded in maintaining exclusive and enduring associations with females. However, consortships occurred precociously, with males starting to affiliate with females, follow, and mount them 1 week before the presumed time of ovulation. These long-lasting consortships appear to be a consequence of the female extended follicular phase. This presumably sexually selected character allowed females to extend conspicuous sexual displays: genital swelling and utterance of an estrous call, which might attract males' attention and arouse them. With regard to female mating tactics, the combination of reliably indicating the time of ovulation to the male and durable periods of competitor exclusion led to reject explanations assuming manipulation about paternity or long-lasting intermale competition incitement in Tonkean macaques. Competition for mates between females also turned out to be an irrelevant factor as it was very low in the species. We conclude that the main function of sexual displays is to herald the approach of ovulation toward available mates.

Artificial insemination in Callithrix jacchus using fresh or cryopreserved sperm.

Assisted reproductive techniques are needed urgently to facilitate the captive breeding of many New World primate species which are endangered in the wild and to assist the effective genetic management of small colonies. A protocol was devised for artificial insemination in the common marmoset, Callithrix jacchus, using ejaculated sperm obtained by vaginal washing after copulation. A double insemination protocol was employed, with the first insemination taking place the day before ovulation was expected to occur and the second 48 h later. All six females inseminated with fresh ejaculated sperm became pregnant, delivering a total of 16 offspring at term. The gestation lengths and litter sizes were not statistically different from those observed in pregnancies following natural mating. The insemination protocol was adapted for use with cryopreserved ejaculated sperm by including an additional insemination on the day of expected ovulation, to take into account differences in the capacitation time of frozen-thawed sperm compared to fresh sperm. Three out of six females inseminated according to this triple insemination schedule, conceived, although one female subsequently resorbed twin foetuses approximately 100 days later. The remaining two pregnant females delivered four babies at term, one singleton and one set of triplets. In the final group, six females were inseminated with low doses of cryopreserved epididymal sperm using the same triple insemination protocol used for frozen-thawed ejaculated sperm. One female conceived, delivering triplets.

A local oxytocin system is part of the luteinization process in the preovulatory follicle of the marmoset monkey (Callithrix jacchus).

Luteinization is a complex differentiation process involving the interaction of extrinsic and intraovarian factors. The aim of this study was to examine the components of an intraovarian oxytocin (OT) system during the periovulatory period in the marmoset monkey, as well as the possible relationship of these components to other factors involved in the luteinization process, using immunohistochemistry and cell culture techniques. Ovaries were collected on Day 7 of the follicular phase (before the endogenous LH surge) and on Day 8 (22 h after an exogenous hCG application, but before ovulation). Before the endogenous LH increase, OT immunoreactivity was detectable at low levels in most antral follicles, where its presence was confined to antral granulosa cell (GC) layers. In contrast, immunoreactivity for the OT receptor (OTR) was localized primarily in the basal GC layer. After application of exogenous hCG, there was a marked enhancement in both the staining intensity and the number of cells positive for OT and the OTR in all GC layers of antral follicles, especially in the preovulatory follicle. Progesterone receptor and 3beta-hydroxysteroid dehydrogenase activity in GC were clearly present only when follicles were obtained after gonadotropin stimulation. Secretion of authentic OT was demonstrated from cultured GC obtained before the LH surge, with highest amounts in cells cultured from preovulatory as opposed to smaller antral follicles. OT production could be stimulated by the application of hCG to the GC cultured from preovulatory follicles, whereas the gonadotropin was without effect on GC from small follicles. FSH had no effect on OT production by GC from either follicle type. Application of OT to the cultures caused an increase in progesterone production by GC from large preovulatory follicles but was without effect on steroidogenesis by cells from small antral follicles. These results describing the presence and distribution of OT and OTR and their modulation by hCG, as well as the luteotrophic effect of OT in cultured GC from preovulatory follicles, implicate OT as a paracrine mediator in the luteinization process in the primate ovary.

Reproductive efficiency in the common marmoset (Callithrix jacchus): a longitudinal study from ovulation to birth monitored by ultrasonography.

This study examines reproductive efficiency in the common marmoset monkey, Callithrix jacchus, using sequential ultrasound examinations to establish ovulation number, implantation rate, and incidence of prenatal loss. Ultrasound was carried out with a 10 or 7.5 mHz probe in nonsedated animals, daily during the late follicular phase, approximately twice a week until day 20 after ovulation, and at days 35, 56, and 85 of pregnancy to enable visualization of gestation sacs, heartbeats, and fetal heads, respectively. Ovulatory follicles could be seen 3-4 days before ovulation and by day -2, 98% of ovulating follicles were > 2mm diameter, although almost 10% of follicles of this size disappeared without ovulating. Total number of ovulating follicles for 15 females was 45 (mean ovulation rate = 3.0, range 2-4). In the 14 animals that conceived, 41 corpora lutea were identified (mean ovulation rate = 2.9) within 10 days of ovulation. All pregnancies went to term (no abortion occurred) resulting in the birth of 37 neonates (9 triplets, 5 twins) and an average litter size of 2.64. All four losses were confined to the embryonic period (< day 85), two occurring before day 35, one between days 35 and 56, and one between days 56 and 85. In demonstrating that 90% of ovulatory follicles gave rise to live offspring, the results of this study indicate an extremely high reproductive efficiency in the marmoset monkey (when maintained under favorable captive conditions) and a rate of prenatal loss much lower than that reported for other primate species.