Pregnancies following long luteal phases in southern white rhinoceros (Ceratotherium simum simum)

All extant species in the Rhinocerotidae family are experiencing escalating threats in the wild, making self-sustaining captive populations essential genetic reservoirs for species survival. Assisted reproductive technologies (ARTs) will become increasingly important for achieving and maintaining ex situ population sustainability and genetic diversity. Previous reports have shown that a large proportion of captive southern white rhinoceros (SWR) females are irregularly cyclic or acyclic, and that cycling females display two different estrous cycle lengths of approximately 30 or 70 days. It has been suggested that the longer estrous cycle length is infertile or subfertile, as no term pregnancies have been observed following long cycles. Here we report the achievement of two pregnancies following long luteal phases, using ovulation induction and artificial insemination with either fresh or frozen-thawed semen. One female SWR conceived on the first insemination attempt and gave birth to a live offspring. A second female conceived twice in consecutive long cycles although the first embryo was resorbed by 33 days post-insemination. A pregnancy from this female's second insemination is ongoing with expected parturition in November 2019. Whether prolonged estrous cycles in SWR are subfertile or infertile in natural breeding situations remains unclear. However, our findings demonstrate that the application of ARTs following prolonged cycles can result the successful establishment of pregnancies in SWR. Therefore, with ARTs, female SWR otherwise considered nonreproductive due to long estrous cycles may still have the potential for representation and contribution to the ex situ population.     

Relationship between androgens, environmental factors and reproductive behavior in male white rhinoceros (Ceratotherium simum simum)

We conducted a longitudinal study of the endocrine activity of free-range male white rhinos. An enzyme immunoassay to measure androgens in the feces was developed and validated to show that it can be used to study testicular activity. We identified two fecal metabolites similar to testosterone and dihydrotestosterone. Several lines of evidence suggest that these metabolites clearly reflect testicular activity. Firstly, the stimulation of testicular activity with synthetic GnRH caused a 156% increase in androgen metabolite concentrations in the feces 1 day after treatment. Secondly, androgen metabolite concentrations increased with sexual maturity in rhinos, and finally there was a correlation between testosterone concentrations in plasma and androgen metabolite concentrations in feces. Using the method that we developed, it was possible to establish whether a relationship exists between androgen metabolite concentrations, the behavior and environmental factors. Adult territorial males (n = 5) had elevated androgen metabolite concentrations during months of high rainfall (September-February) compared to months of little or no rainfall (March-August). The increase in concentrations coincided with the beginning of the rainy season, suggesting a seasonal trend in reproduction. This trend was confirmed by behavior observations showing both a higher frequency of conceptions within the first 4 months of increased androgen metabolite concentrations, and a higher number of inter-sexual conflicts, reflecting the initial aggression between the sexes during the consort period. It was also evident that males accompanying a receptive female had higher fecal androgen metabolite concentrations compared to being alone. The elevated levels were likely induced by female presence.     

Social and spatial relationships in captive southern white rhinoceros (Ceratotherium simum simum)

Although critical to the conservation of white rhinoceros, captive breeding has proven challenging because of the poor and irregular reproductive health of many captive rhinos, and social interactions may play a significant role. This research investigated the social and spatial relationships of two captive groups of southern white rhinoceros (Ceratotherium simum simum) by examining the frequency of companion changes, the number of space maintenance vocalizations made per hour by each reproductively mature female, and dominant/subordinate interactions. The observed captive rhinos did not change their companionships during the study. They exhibited space maintenance vocalizations and display greater than once per hour, particularly when feeding. Females housed with four calves on 0.033 km² exhibited space maintenance vocalizations more frequently (X±SE = 6.19±0.199/hr) than females housed with one calf and more space (0.06 km², X±SE = 0.55±0.182/hr) and females housed without calves and more space (0.65 km², X±SE = 1.90±0.086/hr). Wider separation of food piles and of females with young calves is suggested to reduce the interpreted spatial stress. The presence of a large number of rhinos in restricted captive space resulted in the formation of herds with dominance hierarchies that were enforced during competition for food and shade. The most subordinate rhino in each of the herds exhibited unusual behaviors such as dung‐kicking and nonestrus urine squirting, and neither has ever reproduced. Suppression of subordinate rhinos might lead to social stress that could negatively impact reproductive success. Zoo Biol 26:487–502, 2007. © 2007 Wiley‐Liss, Inc.     

Ciliated intestinal protozoa of black (Diceros bicornis michaeli) and white rhinoceroses (Ceratotherium simum simum) in Kenya

The aim of this study was to survey ciliated intestinal protozoa of the black rhinoceros ( Diceros bicornis michaeli ) and white rhinoceros ( Ceratotherium simum simum ) in Kenyan National Parks. Faecal samples from 28 rhinoceroses that were chemically immobilized for translocation were opportunistically collected. Presence of ciliates was assessed using faecal floatation and sedimentation techniques. The ciliates were identified using cellular morphological features. Ophryoscoleciidae, Cycloposthiidae and Blepharocrythiidae were the three ciliate families represented. Ophryoscoleciidae had nine genera, Cycloposthiidae six genera and Blepharocorythiidae 1 genus. The dominant ciliate genus in all the rhinoceroses that were sampled was entodinium . It was found that the nutrient composition of the diet influences the diversity and numbers of intestinal ciliates, which in turn regulates the nutrient available to the animal. This interplay of nutrient composition of diet, ciliate diversity and nutritional benefits to the host has been used as an index to assess the nutritional state of ruminants. Because of the occurrence of rumenal ciliates in the hindgut fermentative chamber of the rhinoceros, such an index can be used to guide the formulation of feed mixtures for rhinoceros in captivity and remote nutritional assessments of rhinoceros both in free-range and in captivity.     

Metabolism of oestradiol-17 beta and progesterone in the white rhinoceros (Ceratotherium simum simum)

14C-Labelled oestradiol-17 beta and progesterone (50 mu Ci each) were injected i.v. into an adult female white rhinoceros and all urine and faeces collected separately over the next 4 days. The total recovery of injected label was 61%, 25% being present in the urine and 36% in the faeces. Of the radioactivity recovered, 69% was excreted on Day 2 of the collection period. Repeated extraction of samples obtained on Day 2 showed that, of the radioactivity in faeces, 92.4% was associated with unconjugated steroids whereas in the urine the proportion of conjugated and unconjugated steroids were similar (41.2% and 51.4% respectively). After phenolic separation of urinary steroids, HPLC followed by derivatization and recrystallization techniques identified progesterone as the major component of the unconjugated portion with 4-pregnen-20 alpha-ol-3-one as the principal metabolite in the conjugated fraction. Pregnanediol was not present. Oestrone appeared to be the most abundant oestrogen metabolite with smaller but significant amounts of oestradiol-17 beta and oestradiol-17 alpha in the unconjugated and conjugated fractions respectively. Small amounts of progesterone were found in the faecal extract in which the radioactivity consisted mainly of oestradiol-17 alpha and oestradiol-17 beta. The results have established the major excreted metabolites of oestradiol-17 beta and progesterone in the white rhinoceros and the development of more appropriate assay methods for monitoring ovarian function in African rhinoceroses should now be possible.     

Combined serial ultrasonography and fecal progestin analysis for reproductive evaluation of the female white rhinoceros (Ceratotherium simum simum): Preliminary results

Ultrasonographic examinations of one multiparous 33-year-old female southern white rhinoceros (Ceratotherium simum simum) resulted in documentation of the animal's estrous cycle, elucidation of the timing of ovulation in relation to estrus, and ultrasonographic evidence of endometritis and associated early embryonic death. The preovulatory follicle was observed to change in shape from spherical to pear-shape (n = 3) and to reach a mean follicular diameter of ˜30 mm (n = 4) in the 48 hr preceding estrus. An ovulation site in the location of the preovulatory follicle was observed to occur within 24 hr postbreeding on one occasion. Both pregnancies monitored in this female in 1995 were lost by day 28 postovulation, with collapse of the embryonic vesicle documented via ultrasound. Ultrasonographic evidence of endometritis was observed in this female and was characterized by small quantities of anechoic intrauterine fluid collections (5–20 mm diameter) in late diestrus (n = 4, mean day observed was 20.5 days postovulation, with a range of 18–24 days). Fecal samples collected at the time of ultrasound were evaluated via radioimmunoassay for progesterone metabolites. A substantial rise in fecal progestins was not identified until 7–9 days postovulation. This study illustrates the value of combining the complementary techniques of ultrasonographic “mapping” of events with fecal hormone assay to enhance the accuracy of reproductive monitoring. Zoo Biol 16:445–456, 1997. © 1997 Wiley-Liss, Inc.     

Structure of white rhinoceros (Ceratotherium simum) horn investigated by X-ray computed tomography and histology with implications for growth and external form

The nasal and frontal horns of two individuals of Ceratotherium simum were examined by x-ray computed tomography (CT scanning), gross observation of sectioned horn, and light microscopy of histological sections of the horn tissue. CT scans of both sets of horns reveal a periodic banding pattern that is evident upon gross observation of sections as darker bands of tissue. The overlap of these bands in both histological and CT slices suggests the presence of both a photoabsorbent component (melanin) and a radiodense component (calcium phosphate salts, most likely hydroxyapatite or octocalcium phosphate). The distribution of these two components in the horns is hypothesized to contribute to the differential wear patterns that produce the characteristic sweeping conical shape of rhinoceros horn from what otherwise (in the absence of wear and UV exposure) would be cylindrical blocks of constantly growing cornified papillary epidermis. Although extant rhinocerotids are unique in possessing a massive entirely keratinous horn that approximates the functions of keratin-and-bone horns such as those of bovid artiodactyls, the tissue structures that make up the horn are strikingly convergent with other examples of papillary cornified epidermis found in horses, artiodactyls, cetaceans, and birds.     

Characterization of microsatellite loci in the black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum): their use for cross-species amplification and differentiation between the two species

As the population sizes of the black and white rhinoceroses continues to decline, more efforts are needed in multiple areas to help with the conservation efforts. One area being explored is the use of genetic diversity information to aid conservation decisions. In this study, we designed 21 microsatellite primers for white and black rhinoceroses, 16 and 17 of which amplified bands in the white and black rhinoceros, respectively. Out of these primers all 16 were polymorphic in the white rhinoceros and 12 of the 17 were polymorphic in the black rhinoceros. The mean number of alleles was 3.31 and 2.12, the expected heterozygosities were 0.420 and 0.372, and the observed heterozygosities were 0.436 and 0.322 for the white and black rhinoceroses, respectively. Seven of the primers produced different allele sizes and variations that distinguished between black and white rhinoceroses. Further genetic analyses with larger wild population sample sizes and markers are recommended to obtain a better understanding of the genetic structure of the black and white rhinoceros populations in order to be useful in the conservation efforts of these critically endangered species. A. Kilbourn—In memoriam.     

Factors affecting the behavior of captive white rhinoceros (Ceratotherium simum) and the accuracy of ad-hoc keeper data

Although white rhinoceros (Ceratotherium simum) are common in captivity, few behavioral studies have been conducted and there is seemingly no research for immersive exhibits where potential for visitor effects is high. Moreover, little information exists on possible effects of weather and temperature on rhino outside their native range. Here we analyze 14,501 observations of rhino in a drive-through enclosure. Data were collected by researchers (n = 12,160 datapoints) and keepers (n = 2341 datapoints) over a 4-month period. We aimed to: (1) quantify behavior using detailed researcher-collected data and contemporaneous but ad hoc keeper-collected data; (2) compare datasets statistically; (3) establish effects of visitors, temperature, and weather on behavior; and (4) assess the influence of visitors on similarity of researcher/keeper datasets. Activity budgets were similar to the wild and the single previous study from a traditional (nondrive-through) enclosure. There was some discrepancy in activity budgets between researcher and keeper data due to significant differences in recorded frequency of two rare behaviors (horn rub; social interaction) and two behaviors that could be easily confused (grazing vs. standing with head-down): recording of other behaviors matched well. Weather and temperature affected behavior, with rhino becoming more sedentary (−locomotion, grazing; +resting, standing, and sedentary eating of hay) on hot/sunny days compared to cool/wet days. The number of visitor vehicles had a fairly negligible effect but resting was lower on busy days, possibly as vigilance increased. The match between researcher/keeper datasets was lowest when visitor numbers were high, suggesting visitors might affect keeper ability to accurately record behavior.     

Gross anatomy of the reproductive tract of female black (Diceros bicornis michaeli) and white rhinoceros (Ceratotherium simum simum)

Reproductive tracts were collected from three black rhinoceros and two white rhinoceros at necropsy. All females were nulliparous, except for one primiparous white rhinoceros. The animals ranged in age from 7 days to 28 years. All nulliparous animals had a constriction in the vaginal canal, which appeared to be a hymen. The primiparous female had only remnants of a hymen. The total length of the tract averaged 102 cm in three adult animals (≥21 years old). The distance from the vulva to the external cervical os averaged 40 cm in these animals. The endometrium of a 28-year-old nulliparous black rhinoceros and a 27-year-old nulliparous white rhinoceros exhibited signs of hyperplasia, whereas this condition was not present in the other animals. It is not known if this condition was related to the fertility of these animals. The cervix of the rhinoceros was firm, and the lumen followed a very tortuous path through eccentric rings of tissue. Visual examination of the ovaries revealed the presence of surface follicles on the ovaries in both species. The overall size of the reproductive tract, especially the vagina and uterine horns, and the extremely tortuous cervical lumen will present obstacles that must be overcome when developing artificial insemination and embryo transfer proceduers for use in rhinoceros.     

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

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

Split-sample comparison of directional and liquid nitrogen vapour freezing method on post-thaw semen quality in white rhinoceroses (Ceratotherium simum simum and Ceratotherium simum cottoni)

To increase the quality of cryopreserved sperm in white rhinoceros, the liquid nitrogen vapour (LN vapour) freezing and the multi-thermal gradient directional freezing methods were compared. Sixteen white rhinoceros (Ceratotherium simum sp.) were electro-ejaculated. Semen samples were diluted with cryoextender (Tris, lactose, egg-yolk, DMSO) and aliquoted into straws for LN vapour freezing, and glass hollow tubes for directional freezing. The sperm quality was evaluated before and after freezing by assessing the following parameters: motility, morphologic state, acrosomal integrity and plasma membrane function and integrity (i.e. sperm viability) as defined by the hypo-osmotic swelling. Directional freezing improved the sperm viability by 5.6% (p < 0.005), progressive motility score by 34.7% and sperm motility index (SMI) by 8.1% (p < 0.005) versus LN vapour freezing. When data was categorized into groups of low (<19%), moderate (20-39%) and high (>40%) percentages of morphologically normal, directional freezing (DF) resulted in 31.4% less abnormal acrosomes for the low quality group as well as 18.7% increase in intact acrosomes and 10.9% increase in motility for the high quality group compared to LN vapour freezing (LN) (p < 0.01, p < 0.03, p < 0.01, respectively). LN showed a significant reduction in sperm head volume (5.7%, p < 0.05) compared to the prefreeze; whereas, no significant reduction in head volume was demonstrated after DF. Several additives (xanthenuric acid, cytochalasin D, potassium, EDTA) to the basic cryoextender provided no significant improvement in spermatozoal survival after directional freezing. In conclusion, directional freezing proved to facilitate higher gamete survival compared to LN vapour freezing. This is especially effective in ejaculates of low sperm quality and is important in endangered species where high quality semen donors are often not accessible. These results suggest that directional freezing could be valuable particularly for species with limited freezability of spermatozoa.     

Reproductive behavior of the captive Sunda pangolin (Manis javanica Desmarest, 1822)

Understanding reproductive behavior is important for the conservation of endangered species, but research on the reproductive behavior of Sunda pangolins (Manis javanica Desmarest, 1822) is still very scarce. In this study, we used focal animal sampling and all-occurrence recording by an infrared monitor to observe the behaviors of two Sunda pangolins during a 5-day breeding period at the Pangolin Research Base for Artificial Rescue and Conservation Breeding of South China Normal University (PRB-SCNU). The behavioral characteristics and breeding strategies were analyzed, and the results were discussed together with information on other captive Sunda pangolin pairs at PRB-SCNU. The results found that there was no obvious estrus behavior in the captive female, while the male could exhibit sexual excitement and courtship behavior after a brief introduction period. Repeated copulation continued over many days after the female accepted the courtship. The average duration of copulatory behavior was 248.9 ± 148.7 s (n = 25), and all copulation occurred between 20:00 and 08:00 hr in the natural day–night photoperiod. The mating position of Sunda pangolins was lateral–ventral and was classified as the ninth or eleventh pattern under both Dewsbury's and Dixson's classification systems. This study can provide scientific guidance for the captive breeding and management of Sunda pangolins and other pangolin species, which is of great significance for ex situ conservation tactics.     

Reproductive soundness of captive southern and northern white rhinoceroses (Ceratotherium simum simum, C.s. cottoni): evaluation of male genital tract morphology and semen quality before and after cryopreservation

White rhinoceroses suffer from a low rate of reproduction in captivity. This study examines the role of male reproductive function as a contributing factor. We used ultrasonography to image accessory sex glands, testis and epididymis. Electroejaculation provided 36 ejaculates from 21 rhinoceroses. Based on the percentage of progressively motile spermatozoa, semen was categorized in three groups, high (I: >75%), intermediate (II: 50-70%) or low (III: <50%) quality. Only 52% of the males showed high semen quality. Ejaculates in the high motility category also had the highest proportion of morphologically intact spermatozoa. Both semen parameters, sperm motility and morphology, were found to positively correlate with size of the accessory sex glands. The semen category was associated with group size suggesting that the social status influenced functional reproductive parameters. Change of territorial status (n = 1) improved semen quality. Testicular fibrosis was characterized as a sign of reproductive ageing in all males older than 15 years of age (n = 13); although, this ageing process did not notably affect semen parameters. Furthermore, for the benefit of assisted-reproduction and genetic banking programs protocols for the storage of cooled semen and the cryopreservation of spermatozoa were designed using different cryodiluents. This report provides basic data for the evaluation of reproductive components and of breeding management in male rhinoceros. Our results indicate that low rate of reproduction in captivity can be attributed to reduced male reproductive fitness. Changes in management of white rhinoceroses may positively affect male reproductive function.     

Intestinal ciliated protozoa of African rhinoceros: two new genera and five new species from the white rhino (Ceratotherium simum Burchell, 1817)

This report represents the first published information on intestinal ciliated protozoa in the African white rhinoceros (Ceratotherium simum Burchell, 1817). Two new genera which do not relate to any known ciliated protozoa from the intestines of mammals and five new species are described. The ciliates were found in the colon of three of these free-living hindgut-fermenting grazers that were shot in widely spaced districts in southern Africa. Phalodinium digitalis n. gen., n. sp., Arachnodinium noveni n. gen., n. sp., Monoposthium vulgaris n. sp., M. bracchium n. sp., and M. latus n. sp. constituted between 1% and 10% of the total ciliate population (ca. 1 X 10(5)/ml digesta) in the ascending colon. Exceedingly small numbers were observed in the descending colon, indicating temporary accommodation only.     

Measurement of fecal steroids in the black rhinoceros (Diceros bicornis) using group-specific enzyme immunoassays for 20-oxo-pregnanes

The metabolism and excretion of progesterone in different animal species results in several fecal 5-reduced progesterone metabolites (pregnanes), which in recent studies were quantified using progesterone antibodies. To increase the accuracy of fecal 20-oxo-pregnane evaluations in the black rhinoceros, enzyme immunoassays (EIA) using antibodies against 5α-pregnane-3β-ol-20-one 3HS:BSA (5α-20-one EIA) and 5β-pregnane-3α-ol-20-one 3HS:BSA (5β-20-one EIA) were developed. The assays showed high crossreactivities with pregnanes containing a 20-oxo group and are referred to as group-specific; results of these assays were compared with an EIA using an antibody against 6HS-progesterone (4-ene-20-one EIA). Fecal samples of both subspecies of the black rhinoceros (Diceros bicornis michaeli, n = 5, and Diceros bicornis minor, n = 1) during pregnancy were collected 1–3 times/week. HPLC separation showed three major immunoreactive fecal 20-oxo-pregnane peaks; their elution profiles and different crossreactivities in the three EIAs provided strong evidence that these peaks are 5α-pregnane-3, 20-dione, 5α-pregnane-3α-ol-20-one, and 5α-pregnane-3β-ol-20-one. Pregnane values in the pregnant animals continuously increased between months 3–7 and were significantly (P < 0.01) elevated above the levels of nonpregnant animals (0.2 μg/g) by week 11. During months 6–13 concentrations in the 5α-20-one and in the 5β-20-one EIA (5–11 μg/g) were significantly (P < 0.01) higher than in the 4-ene-20-one EIA (1.5–3 μg/g). In conclusion, the immunoreactive fecal 20-oxo-pregnane metabolites in the black rhinoceros are determined more accurately with antibodies against pregnane-20-one-C3 conjugates, as compared with a progesterone antibody. © 1996 Wiley-Liss, Inc.     

Assessing reproductive cycles and pregnancy in cheetahs (Acinonyx jubatus) by vaginal cytology

Vaginal cytology was used to monitor ovarian cycles, two pregnancies, and three pseudopregnancies. Vaginal smears were collected two or three times per week from three adult females; smears plus blood samples were collected once per week from a fourth, adolescent female. Mean cycle lengths, based on intervals between onset of leukocyte infusions, were 11.9 ± 4.9 days (n = 43 cycles), 10.8 ± 5.1 days (n = 49), and 12.3 ± 6.3 days (n = 7) for the three females. Weekly hormone data from the adolescent female revealed a correlation between serum estradiol and percent anuclear cells, suggesting that these cells may be indicative of estrus. The fourth female experienced two sustained, 6-week increases in serum progesterone, one spontaneous and the other following follicle-stimulating hormone (FSH) administration. Leukocyte infusions continued during these periods of increased progesterone secretion. However, leukocyte infusions ceased during the two pregnancies of one adult female and during two FSH-induced pseudopregnancies of another. © 1992 Wiley-Liss, Inc.