Successful ovulation induction and laparoscopic intrauterine artificial insemination in the clouded leopard (Neofelis nebulosa)

Exogenous gonadotropins and a laparoscopic intrauterine artificial insemination (AI) technique were assessed for effectiveness in the clouded leopard (Neofelis nebulosa), a species difficult to breed in captivity due to severe mate incompatibility. Fourteen hormone trials using 10 female clouded leopards were performed to evaluate the ability of 50, 100, or 200 i.u. pregnant mares' serum gonadotropin (PMSG) and 75 or 100 i.u. human chorionic gonadotropin (hCG) to induce folliculogenesis and ovulation, respectively. Laparoscopic evaluation of ovarian activity was conducted at 29–48 hr after hCG administration. Time of ovulation in PMSG/hCG-treated clouded leopards was approximately 38–39 hr after hCG. Excessive follicular development was observed using the high hormone dosages (200 i.u. PMSG/100 i.u. hCG), whereas the lower dosages avoided ovarian hyperstimulation. Previous ovulation sites and mature corpora lutea were detected upon laparoscopic examinations in two of the 10 females housed alone, indicating that this species occasionally spontaneously ovulates. Five females were inseminated by depositing electroejaculated, washed sperm transabdominally into the proximal aspect of each uterine horn. One postovulatory female, previously treated with 100 i.u. PMSG and 75 i.u. hCG and inseminated in utero with 88 × 10⁶ motile sperm at 45 hr post-hCG, produced a pregnancy and two live cubs after an 89 day gestation. These results demonstrate: (1) an exquisite ovarian sensitivity to exogenous gonadotropins in clouded leopards; and (2) that artificial insemination has the potential of resulting in offspring in this species. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Studies on Asian elephant reproduction at the Washington Park Zoo

Studies of Asian elephant reproduction at the Washington Park Zoo in the areas of the estrous cycle, semen collection, bull elephant management and health care, pregnancy, and pheromones have yielded sufficient information to attempt repeated artificial insemination (AI) at the time of ovulation. While no pregnancy has been achieved to date, with the information now at hand AI can be expected to become a practical technique for breeding elephants in captivity.     

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

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

Successful artificial insemination in the giant panda (Ailuropoda melanoleuca) at ueno zoo

Successful breeding of the giant panda (Ailuropoda melanoleuca) following artificial insemination was achieved at the Ueno Zoo in 2 consecutive years (1985 and 1986). The first cub, born in June 1985, unfortunately died 43 hours after birth from being crushed by the mother panda; the second cub, born in June 1986, has been growing in good health. Electroejaculation and artificial insemination procedures were performed after immobilization with diazepam (0.1 mg/kg) and ketamine HCL (4.0–5.0 mg/kg). Semen of the male panda was collected by electroejaculation using a rectal probe with a diameter of 2.0 cm and with eight rings as electrodes. Stimulation of the male was given with 3 V (30–40 mA) over a 5-sec period with 5-sec intervals. The female panda exhibited estrus between late February and early March in 1985 and also between mid-january and early February 1986. Increased excretion of urinary total estrogen showed coincidentally at maximum behavioral estrus, and a gradual rise of pregnanediol level was followed by artificial insemination. The gestational length for the first pregnancy was 110 days and that of the second 121 days.     

Pathology and diseases of great apes at the National Zoological Park

Knowledge of the diseases of great apes in captivity is essential for captive management of self-sustaining populations. This survey of medical and pathology records of orangutans, gorillas, and one chimpanzee at the National Zoological Park was conducted to provide a data base for improving health care of captive apes. Strongyloidiasis, balantidiasis, and entamoebiasis were recurrent problems in adult and juvenile apes of all species. Cardiac fibrosis also was prevalent in middle-aged apes and was a major cause of mortality. Bacterial infections were prevalent in perinatal orangutans and resulted in the death of two. For gorillas, rheumatoid arthritis associated with mycoplasma infections, and infertility were major problems. Because the pathogenesis of many of these lesions is unknown, survival of great ape populations in captivity may depend on future research on these problems.     

Deaths from exertional myopathy at the National Zoological Park from 1975 to 1985

A retrospective study was conducted to determine the incidence of deaths from exertional myopathy (EM) in the hoofstock collection at the National Zoological Park (NZP) from 1975 to 1985. The diagnosis of EM was based on history, clinical signs, clinicopathological findings, gross and microscopic lesions. Only bovids, cervids, and equids were included in the study. Data were derived from the medical and pathological records of the NZP. There were 10 cases of EM during this period, but only seven deaths were directly attributable to EM. Five deaths occurred after immobilization. Two deaths occurred after improper transport, but the anesthetic history was not known in these cases. Pathologic changes consistent with EM were incidental findings in an animal killed by dogs, and in two animals euthanized because of suspected infection with Parelaphostrongylus tenuis. Only cervids and bovids were affected. There was no age or sex predilection for developing EM and deaths occurred throughout the year. Deaths associated with immobilization occurred most often after anesthesia with xylazine and etorphine. These are the drugs used most frequently in bovid and cervid anesthesia at the NZP. The overall incidence of deaths from EM after immobilization was 0.25% for the 10-yr period.     

Results of the third reproductive assessment survey of North American Asian (Elephas maximus) and African (Loxodonta africana) female elephants

A written survey assessed reproductive status of female Asian and African elephants in AZA/SSP facilities in 2008, and data were compared to surveys conducted in 2002 and 2005. Results showed that ovarian acyclicity rates across the surveys remained unchanged for Asian (13.3, 10.9 and 11.1%) and African (22.1, 31.2 and 30.5%) elephants, respectively (P > 0.05), but were higher overall for African compared to Asian elephants (P < 0.05). In 2008, the percentages of Asian and African elephants with irregular cycles (14.3 and 15.8%) and irregular + no cycles (25.4 and 46.4%) was similar to 2005 (7.6 and 11.8%; 18.5 and 43.0%), but were increased compared to 2002 (2.6 and 5.2%; 16.0 and 27.3%), respectively (P < 0.05). For both species, ovarian acyclicity increased with age (P < 0.05). Reproductive tract pathologies did not account for the majority of acyclicity, although rates were higher in noncycling females (P < 0.05). Bull presence was associated with increased cyclicity rates (P < 0.05) for Asian (92.5 vs. 58.3%) and African (64.9 vs. 57.8%) elephants compared to females at facilities with no male, respectively. Cyclicity rates were higher for Asian (86.8 vs. 65.2%) and African (67.9 vs. 56.7%) elephants managed in free compared to protected contact programs (P < 0.05), respectively. Geographical facility location had no effect on cyclicity (P > 0.05). In summary, incidence of ovarian cycle problems continues to predominantly affect African elephants. Although percentages of acyclicity did not increase between 2005 and 2008, 42.2% Asian and 30.2% African females were no longer being hormonally monitored; thus, reproductive cycle abnormalities could be worse than current data suggest.     

Locomotion of free-ranging golden lion tamarins (Leontopithecus rosalia) at the National Zoological Park

Locomotor behavior and substrate use of cage-reared golden lion tamarins (Leontopithecus rosalia), newly released and free-ranging on the grounds of the National Zoological Park, were studied to determine if locomotion changed following exposure to naturalistic conditions. The animals employed a predominantly quadrupedal locomotor profile, incorporating leaping and vertical climbing to a lesser degree. There was no clear evidence of a change in locomotion due to the high degree of variability in these behaviors. The locomotor repertoire of the free-ranging group differed from that of groups occupying unenriched but relatively large conventional enclosures, indicating that whereas locomotion is plastic with respect to environment, substrate characteristics influence locomotor behavior and may promote stereotypical behavior. However, due to anatomical constraints, the locomotor repertoire tended to be less variable than substrate use. Similar behaviors were used in moving through a variety of habitat features in spite of strong associations between specific locomotor styles and substrate classes. © 1994 Wiley-Liss, Inc.     

Estrous expression during a fixed-time artificial insemination protocol enhances development and interferon-tau messenger RNA expression in conceptuses from Bos indicus beef cows

Expression of estrus (EST) near the time of fixed-time artificial insemination (FTAI) increases pregnancy success in beef females. This outcome has been associated with improved pregnancy establishment and maintenance, although research is still warranted to validate this theory. Hence, this experiment compared ovarian, uterine and conceptus factors associated with pregnancy establishment in Bos indicus beef cows according to estrous expression during a FTAI protocol. One hundred lactating multiparous Nelore cows received a 2 mg injection of estradiol benzoate and an intravaginal progesterone (P4) releasing device on day −11, a 12.5 mg injection of prostaglandin F_2α on day −4, P4 device removal in addition to 0.6 mg injection of estradiol cypionate and 300 IU injection of equine chorionic gonadotropin on day −2, and FTAI on day 0. An estrous detection patch was attached to the tailhead of each cow on day −2, and estrous expression was defined as removal of >50% of the rub-off coating from the patch at FTAI. Overall, 39 cows expressed EST, 55 did not express EST (NOEST), and six cows lost their patch and were discarded from the experiment. Ovarian ultrasonography was performed at FTAI, and on days 7 and 15 of the experiment. Blood samples were also collected on days 7 and 15. Only cows without a corpus luteum (CL) on day 0, and with a CL on days 7 and 15 remained in the experiment (EST, n=36; NOEST, n=48). On day 15, cows were randomly selected within each group (EST, n=29; NOEST, n=30) for conceptus collection via transcervical flushing, followed by endometrial biopsy in the uterine horn ipsilateral to the CL. Within cows not assigned to conceptus collection, blood samples were collected for whole blood RNA extraction (day 20) and pregnancy status was verified by transrectal ultrasonography (day 30). Diameter of dominant follicle on day 0 and plasma P4 concentrations on day 7 were greater (P⩽0.02) in EST v. NOEST cows. Conceptus length and messenger RNA (mRNA) expression of prostaglandin E synthase and interferon-tau were greater (P⩽0.04) in EST v. NOEST cows. Moreover, EST cows diagnosed as pregnant on day 30 had greater (P<0.01) blood mRNA expression of myxovirus resistance 2 on day 20 compared with NOEST. In summary, estrous expression near the time of FTAI enhanced pregnancy establishment factors in B. indicus cows, including conceptus development and mRNA expression of interferon-tau.     

Reproductive evaluation of elephants culled in Kruger National Park, South Africa between 1975 and 1995

To reduce elephant densities and preserve biological diversity, 14,629 elephants were culled from Kruger National Park, South Africa (1967–1999). Data were catalogued between 1975 and 1996 on 2737 male and female elephants, including pregnancy and lactational status for 1620 females (≥5 years of age) and, uterine and/or ovarian characteristics for 1279. This study used these data to investigate the effects of age and precipitation on reproduction. The youngest age of conception was 8 years (n = 6) and by 12 years of age all females were sexually mature. From the age of 14 years, the percentage of reproductively active females (pregnant and/or lactating) was >90%; however, this percentage declined when females reached 50 years of age. Overall, one-tenth of females were nonreproductive (not pregnant or lactating) at any given time, mostly in the youngest (<15 years) and oldest (>50 years) age classes. Eighteen (3.3%) of the nonpregnant females had reproductive tract pathologies, including endometrial, uterine or ovarian cysts. There was a seasonal distribution of mating activity that correlated with the rainy season. As has been demonstrated in other populations of free-ranging African elephants, most of the females in Kruger National Park were reproductively active; however, age and climate affected reproductive activity.     

Reproductive,behavioral, and endocrine characteristics of the Dall's sheep (Ovis dalli dalli)

Failed interspecific embryo transfer between Dall's sheep (Ovis dalli dalli) and domestic ewes (Ovis aries), and a paucity of physiological data available for the Dall's sheep, provided incentive for developing a reproductive database in Dall's sheep. Urine samples were collected on a regular basis from a captive herd of Dall's sheep during the breeding to lambing interval over a 3 year period. To provide comparative endocrine data during pregnancy, samples also were collected during a single gestation period from five Suffolk ewes. All urine samples were analyzed for pregnanediol-3-glucuronide (PdG) and estrone conjugates (EC). Behavioral observations from Dall's sheep were made each year during the rut and lambing periods to calculate estrous cycle and gestation lengths. Placentas from Dall's sheep (n = 20) and Suffolk ewes (n = 10) were collected, the total number of cotyledons counted, and the length, width, area, and weight of eight cotyledons/horn from each placenta calculated. Endocrine and behavioral results indicated that the Dall's sheep is seasonally polyestrous and monovulatory, with a mean estrous cycle length of 18.2 ± 0.3 days, a mean luteal phase of 11.8 ± 0.5 days, and an average gestation length of 171.6 ± 0.9 days. While hormonal patterns for pregnancy generally were similar between the two species, there was a pronounced difference in the magnitude of hormone concentrations, particularly PdG, and an absence of a marked preparturition EC rise in domestic sheep. Cotyledon numbers, weight, and area were smaller (P < 0.05) in the Dall's sheep, compared to Suffolk ewes. These data suggest that unsuccessful interspecies embryo transfer attempts may have been due to failed fetal/maternal communications resulting from hormonal and/or placental differences. © 1996 Wiley-Liss, Inc.     

Ultrasonographic monitoring of fetal development in unrestrained bonobos (Pan paniscus) at the Milwaukee County Zoo

The bonobo, Pan paniscus, is one of the most endangered primate species. In the context of the Bonobo Species Survival Plan^®, the Milwaukee County Zoo established a successful breeding group. Although the bonobo serves as a model species for human evolution, no prenatal growth curves are available. To develop growth graphs, the animals at the Milwaukee County Zoo were trained by positive reinforcement to allow for ultrasound exams without restraint. With this method, the well being of mother and fetus were maintained and ultrasound exams could be performed frequently. The ovulation date of the four animals in the study was determined exactly so that gestational age was known for each examination. Measurements of biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur length (FL) were used to create growth curves. Prenatal growth of P. paniscus was compared with the data of humans and the common chimpanzee, P. troglodytes. With respect to cranial structures, such as BPD and HC, humans have significant acceleration of growth compared with P. paniscus and P. troglodytes. In P. paniscus, growth of AC was similar to HC throughout pregnancy, whereas in humans AC only reaches the level of HC close to term. Growth rate of FL was similar in humans and the two Pan species until near day 180 post‐ovulation. After that, the Pan species FL growth slowed compared with human FL. The newly developed fetal growth curves of P. paniscus will assist in monitoring prenatal development and predicting birth dates of this highly endangered species. Zoo Biol 30:241–253, 2011. © 2010 Wiley‐Liss, Inc.     

Successful artificial insemination using semen frozen and stored by an ultrafreezer in the Majorera goat breed

The semen of five Majorera breed bucks was collected and processed to reach a final concentration of 200 × 10⁶ spermatozoa/straw in the extender containing 4% of glycerol and 12% of egg yolk. Two freezing techniques were assessed: (LN) straws were frozen and stored in liquid nitrogen, and (ULF) straws were frozen and stored in the ultra-low freezer at −152 °C. Semen quality (sperm motility, acrosome integrity and abnormal sperm cells percentages) was determined for different storage times (1, 30, 90 and 365 days of cryopreservation). Thereafter, 150 Majorera goats were assigned to four experimental groups: for groups LN-1 (n = 40) and LN-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in liquid nitrogen, respectively, while for groups ULF-1 (n = 40) and ULF-6 (n = 35), the goats were transcervically inseminated with frozen-thawed semen stored for 1 and 6 months in an ultra-low freezer at −152 °C, respectively. The pregnancy rate was determined by transabdominal ultrasound scanning; in addition, the kidding rate and prolificacy were recorded at parturition. In vitro results showed that the freezing protocol did not affect sperm quality with similar values for up to 1 year of cryopreservation. The kidding rates were not significantly different between experimental groups (43.6%, 38.5%, 42.8% and 40.0% for groups LN-1, ULF-1, LN-6 and ULF-6, respectively). In all experimental groups, the kidding rate and prolificacy were significantly higher (p < 0.01) in multiparous than in nulliparous goats. Therefore, the in vitro results and fertility trials confirmed the efficiency of the ULF technique for freezing and storage of goat semen.     

Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa

Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen–thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2 ± 5.4%, motility following 30 min incubation at 37 °C: 58.5 ± 6.0% and following 3 h incubation: 21.7 ± 7.6%, intact acrosome: 57.1 ± 5.2%, normal morphology: 52.0 ± 5.8% and viability: 67.3 ± 6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed.     

Estrogen and LH dynamics during the follicular phase of the estrous cycle in the Asian elephant

Pituitary and corpus luteum hormone patterns throughout the elephant estrous cycle have been well characterized. By contrast, analysis of follicular maturation by measurement of circulating estrogens has been uninformative. This study tested the ability of a urinary estradiol‐3‐glucuronide radioimmunoassay to noninvasively assess follicular development during the nonluteal phase of the elephant estrous cycle, and to determine the relationship between estrogen production and the “double LH surge.” Daily urine and serum samples were collected throughout seven estrous cycles from three Asian elephants, and urine was collected from an additional three females, for a total of 13 cycles. Serum was analyzed for luteinizing hormone (LH), and urine was analyzed for estrogens and progestins. Elephants exhibited a typical LH pattern, with an anovulatory LH (anLH) surge occurring approximately 21 days before the ovulatory LH (ovLH) surge. The urinary estrogen pattern indicated the presence of two follicular waves during the nonluteal phase. The first wave (anovulatory) began 5 days before the anLH surge and reached a maximum concentration the day before the peak. Thereafter, urinary estrogens declined to baseline for 2 weeks before increasing again to peak concentrations on the day of the ovLH surge. Urinary progestins were baseline throughout most of the follicular phase, increasing 2–3 days before the ovLH surge and continuing into the luteal phase. These results support previous ultrasound observations that two waves of follicular growth occur during the nonluteal phase of the elephant estrous cycle. Each wave is associated with an increase in estrogen production that stimulates an LH surge. Thus, in contrast to serum analyses, urinary estrogen monitoring appears to be a reliable method for characterizing follicular activity in the elephant. Zoo Biol 22:443–454, 2003. © 2003 Wiley‐Liss, Inc.     

The daily activity of the elephant in the Rwenzori National Park, Uganda

Eighteen elephants were followed individually in the Rwenzori National Park, Uganda for varying periods and their activity noted at 4-min intervals. Continuous data extending over 24 h or more were collected from seven of these elephants. About 75% of the total time was spent in feeding and about 75% of the feeding activity consisted of grazing or feeding at ground level. There were three feeding peaks, one in the morning, another in the afternoon and the third around midnight. The feeding rate tended to increase during the day. The proportion of the total feeding time spent in browsing increased in the dry season. Walking took place mainly at dusk. The principal sleeping period occurred during the small hours of the morning with a shorter rest period in the early afternoon. The elephants defaecated about eleven times in the 24 h and drank an average of 1–3 times. The rate of progress through the bush averaged 0–5 km/h. Comparisons are made with published data concerning the activity patterns of waterbuck and warthog in the Rwenzori Park and of elephants in the Tsavo National Park, Kenya.     

Timely estrus in wild brown mouse lemur females at Ranomafana National Park, southeastern Madagascar

The small-bodied nocturnal mouse lemurs (genus Microcebus) occupy a variety of habitats in Madagascar. Gray (M. murinus) and brown (M. rufus) mouse lemurs have been widely studied both in the wild and captivity. Whereas captive studies revealed an endogenous regulation of reproduction entrained by photoperiod, field studies have suggested that reproductive activation could be affected by additional climatic, physical, or social conditions. I collected data on wild brown mouse lemur females at Ranomafana between 2004 and 2008 to determine: 1) the timing of estrus and estrous periodicities across multiple seasons, and 2) whether additional factors such as body mass, age, or rainfall are correlated with onset of reproduction. In mouse lemur females at Ranomafana, the first seasonal estrus occurs 3-4 weeks after the vernal equinox. I report ~1 month's intra-population variation in the timing of estrus and inter-annual estrous intervals with periodicities of ~365 days. There were significant differences between the onset of reproduction across years. Estrous onset was uncorrelated with body mass, but there was an apparent age effect. There was a significant negative correlation between August rainfall and onset of reproduction when 2004 data were removed from the analysis. Results from this study are consistent with the notion that timing of estrus is photoperiod-dependent. As in captivity, intra-population variation in estrous onset is ~4 weeks in length. In the wild, variation in estrous onset and polyestry (multiple reproductive opportunities per year) appear to be favored under the highly unpredictable conditions of Madagascar's environments. In the wild, variation in estrous onset and polyestry (multiple reproductive opportunities per year) appear to be favored under the highly unpredictable conditions of Madagascar's environments.