Estrus induction in white rhinoceros (Ceratotherium simum)

The estrous cycle length in the white rhinoceros (Ceratotherium simum) is either 4 or 10 wk. The cause(s) for this variation as well as the poor fertility rate in captivity remains under debate in this species. Most captive adult white rhinoceros undergo long anovulatory periods without luteal activity which are considered a major reason for their low reproductive rate. In this study, the synthetic progestin chlormadinone acetate (CMA) was tested in combination with hCG or the GnRH analogue deslorelin for its efficiency to induce ovulation in fourteen females without luteal activity and in three, regular cycling females. HCG (N = 12), injectable GnRH analogue (N = 8) and GnRH analogue implants (N = 15) were given to induce ovulation after CMA treatment. Treatment success was determined using both transrectal ultrasonography and progesterone metabolite EIA analysis. A preovulatory sized follicle (3.5 ± 0.1 cm) or a corpus luteum (5.1 ± 0.7) was present on the ovary one day after induction in 93.1% of the treatments. Despite this high rate of ovarian response, ovulation rate differed between the study groups. The ovulation rate for hCG, injectable GnRH analogue and GnRH analogue implants was 66.7%, 62.5% and 93.3%, respectively. Ovulation rate in cyclic females treated with GnRH implants was 100% (6/6) compared with 89% (8/9) in females without luteal activity receiving the same treatment. The length of the estrous cycle when induced with hCG was 4 wk (85.7%). The estrous cycle when induced with GnRH analogue was predominantly 10 wk long. Two females without luteal activity treated with GnRH became pregnant. In conclusion, CMA in combination with GnRH analogue implants was highly effective to induce ovulation in white rhinoceroses and thus can contribute to efforts aimed at increasing natural mating and reproductive rates in the captive white rhinoceros population.     

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.     

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.     

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.     

Age estimation of the White rhinoceros (Ceratotherium simum)

Age estimation criteria for the southern White rhinoceros ( Ceratotherium simum simum ) are presented both for free-ranging live animals and for cranial material. These are based on: (i) size appearance and horn development of live animals; (ii) stages of tooth eruption; (iii) tooth wear classes; (iv) attrition in height of the first molar tooth; (v) counts of cementum lines visible in tooth sections. Selected measurements are presented for live animals, skulls and horns.
For live animals, eight size classes are distinguished, seven of these covering immature animals up to ten years of age. Sixteen tooth wear classes are established, based on eruption and surface wear of maxillary dentition. Chronological ages were assigned from individually known animals followed in the field, and from skulls from animals for which exact records of age were available, or which could be assigned to an age category from appearance at death. Cementum line counts corresponded approximately with age in years, despite difficulties in interpreting lines. Some variability was observed, possibly related to nutritional conditions.
The maximum cementum line count obtained indicates a longevity of at least 40 years. Full body weight and socio-sexual maturity are attained by males between 10 and 15 years of age, while females first give birth between six and eight years of age. Sequences and times of tooth eruption are similar to those reported for the Black rhinoceros ( Diceros bicornis ).
Comparative cranial and body measurements are presented for the northern subspecies ( Ceratotherium simum cottoni ).     

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.     

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.     

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.     

Reproductive cycle length and pregnancy in the southern white rhinoceros (Ceratotherium simum simum) as determined by fecal pregnane analysis and observations of mating behavior

Pregnancy and the reproductive cycle were monitored in 13 captive southern white rhinoceros (Ceratotherium simum simum) by measuring progesterone metabolites in fecal extracts and by observing behavior patterns. Fecal hormones were measured using a monoclonal antibody produced against 4‐pregnen‐11‐o1‐3,20‐dione hemisuccinate:BSA. Several subjects exhibited flat or erratic endocrine profiles, but we found evidence for 17 reproductive cycles in five females. Mating behavior coincided with nadirs in pregnane concentrations. These cycles appeared to fall into two general categories: those of approximately 1 month in duration (Type I: X¯ ± SEM = 35.4± 2.2 days; n = 10) and those lasting approximately 2 months (Type II: 65.9± 2.4 days; n = 7). Interluteal phase lengths were similar for the two cycle types, but Type II cycles were characterized by extended luteal phases lasting more than twice as long as Type I luteal phases. Because Type I cycles predominated in our data and because evidence suggests that some Type II cycles may be aberrant, we argue that these approximately monthly cycles represent the typical reproductive cycle for this species. Three females became pregnant during the course of the study. We were able to detect pregnancy by approximately 3 months post‐breeding, as indicated by sustained pregnane concentrations markedly higher than nonpregnant luteal phase concentrations. These data help to characterize important reproductive events of this species and should be useful for captive breeding efforts for this threatened species. Zoo Biol 18:111–127, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

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.     

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.     

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.     

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.     

The effect of long non-reproductive periods on the genital health in captive female white rhinoceroses (Ceratotherium simum simum, C.s. cottoni)

White rhinoceroses suffer from a low reproductive rate in captivity. Intensive efforts to propagate specifically the northern white rhinoceros have been very limited. The dismal outlook for this subspecies in the wild makes successful ex situ breeding programs paramount. In this context, this study examined 48 southern and 6 northern white rhinoceroses using ultrasound and faecal hormone analysis to elucidate causes for female reproductive failure and to determine whether long non-reproductive periods have a detrimental impact on genital health. Results showed that 76% of the nulliparous females had intact hymenal membrane indicating these females had never been bred, at an age when their wild counterparts have delivered multiple offspring. Fifty-six percent of the studied population had various reproductive pathology. Cystic endometrial hyperplasia; leiomyomas of the cervix, uterus and ovary, adenoma; para-ovarian cysts and hydromucometra represent the scope of lesions identified. The stages of the lesions in nulliparous females correlated with age (r = 0.4, P < 0.05). Due to the severity of the lesions, 28% of the study population was considered post-reproductive. Therefore, the reproductive life span in some individuals was 10-20 years shorter than expected. However, in parous females the incidence of pathological lesions was significantly lower (P < 0.0001). Seventy-eight percent females studied had erratic or absent luteal activity. The hormone data corresponded with two ultrasonographic levels of ovarian activity, active and inactive, occurring within an age range of 3-19 years and 15-38 years, respectively. This suggests the lack of ovarian activity by reproductive mid-life in non-reproducing females. The accuracy of the ultrasound findings was validated by necropsy in nine animals showing a strong positive correlation (r2 = 0.9, p < 0.001). Our data suggests that the development of reproductive pathology and ovarian inactivity in white rhinoceros is an age-related consequence of long non-reproductive periods. This asymmetric ageing process of the genital organs can be prevented with the achievement of at least one pregnancy.     

Factors influencing the diurnal behaviour of white rhino (Ceratotherium simum) in Matobo National Park,Zimbabwe

Activity periods and diurnal behaviour of southern white rhino (Ceratotherium simum simum) were studied in Matobo National Park, Zimbabwe, from August 2016 to July 2017 using camera trapping and focal group sampling, respectively. Camera trap data showed that diurnal and nocturnal movements of white rhino varied significantly with moon phase, with more frequent nocturnal photographic captures during full- and third-quarter moon phases than during new and crescent moon phases. Permutational MANOVA and canonical correspondence analysis of focal group observation data were used to test the effects of season, time of day, habitat type, distance to roads, moon phase, presence/absence of other mammals, age/sex category, individual identity and herd composition on daytime behaviour. Weather conditions accounted for the majority of variation in rhino behaviour, with more time spent feeding during cloudy and rainy weather than sunny weather. Time of day and moon phase also accounted for high variation in behaviour, followed by habitat attributes and age/sex category. Variance partitioning by sets of explanatory variables showed that weather conditions had the greatest explanatory power in the variation of rhino behaviour, followed by temporal attributes, habitat attributes and age/sex category. Our findings provide insights for white rhino population management strategies aimed at increasing the effectiveness of security patrols, minimising disturbance and improving sightings for tourists.