Comparative analysis of gonadal and adrenal activity in the black and white rhinoceros in North America by noninvasive endocrine monitoring

Patterns of fecal reproductive steroid metabolites and adrenal corticoids were characterized for 12‐ to 24‐month periods in black (n = 10 male, 16 female) and white (n = 6 male, 13 female) rhinoceroses at 14 institutions. All black rhinoceros females exhibited at least some ovarian cyclicity on the basis of fecal progestogen analysis (range, 2–12 cycles/yr). However, cycles often were erratic, with many being shorter (<20 days; 18% of cycles) or longer (>32 days; 21%) than the average of 26.8 ± 0.5 days (n = 104 cycles). Five females exhibited periods of acyclicity of 2–10‐month duration that were unrelated to season. One complete and seven partial pregnancies were evaluated in the black rhinoceros. Fecal progestogens increased over luteal phase concentrations after 3 months of gestation. Females resumed cyclicity within 3 months postpartum, before calves were weaned (n = 5). Approximately half of white rhinoceros females (6 of 13) showed no evidence of ovarian cyclicity. Of the cycles observed, 5 were “short” (32.8 ± 1.2 days) and 24 were “long” (70.1 ± 1.6 days). Only two females cycled continuously throughout the study. One had both long (n = 9) and short (n = 2) cycles, whereas the other exhibited long cycles only (n = 5). Fecal estrogen excretion was variable, and profiles were not useful for characterizing follicular activity or diagnosing pregnancy in either species. Males of both species showed no evidence of seasonality on the basis of fecal androgen profiles. Androgen metabolite concentrations were higher (P < 0.05) in the black (27.6 ± 6.9 ng/g) than in the white (16.8 ± 3.1 ng/g) rhinoceros. An adrenocorticotropin hormone challenge in four black rhinoceros males demonstrated that the clearance rate of corticoid metabolites into feces was ～24 hours. Fecal corticoid concentrations did not differ between males and females, but overall means were higher in the black (41.8 ± 3.1 ng/g) than in the white (31.2 ± 1.7 ng/g) rhinoceros. In summary, fecal steroid analysis identified a number of differences in hormonal secretory dynamics between the black and white rhinoceros that may be related to differences in reproductive rates in captivity. Most black rhinoceros females exhibited some cyclic ovarian activity. In contrast, few white rhinoceroses demonstrated evidence of regular estrous cyclicity, and those females that were active had comparatively long cycles. Results also suggest that fecal corticoid concentrations reflect adrenal activity and may be species specific. Continued studies are needed to determine whether fecal corticoid measurements will be useful for understanding the cause of inconsistent gonadal activity in these two species. Because all but three (15.8%) of the white rhinoceroses evaluated in this study were less than 20 years of age compared to 73.1% (19 of 26) of the black rhinoceroses, the impact of age on reproductive and adrenal activity also needs to be evaluated further. Zoo Biol 20:463–486, 2001. © 2002 Wiley‐Liss, Inc.     

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.     

Fat soluble vitamins in blood and tissues of free-ranging and captive rhinoceros

Several disease syndromes in captive rhinoceroses have been linked to low vitamin status. Blood samples from captive and free-ranging black (Diceros bicornis) and white rhinoceros (Ceratotherium simum) and tissue samples of captive individuals from four rhinoceros species were analysed for vitamins A and E. Circulating vitamin A levels measured as retinol for free-ranging versus captive black and white rhinoceros were 0.04 (+/- 0.03 SD) vs. 0.08 (+/- 0.08) and 0.07 (+/- 0.04) vs. 0.06 (+/- 0.02) microgram/ml, respectively. Circulating vitamin E levels measured as alpha-tocopherol were 0.58 (+/- 0.30) vs. 0.84 (+/- 0.96) and 0.62 (+/- 0.48) vs. 0.77 (+/- 0.32) microgram/ml, respectively. In contrast to earlier findings, there was no significant difference in vitamin E concentration between captive and free-ranging black rhinoceros. When the samples of captive black rhinoceros were grouped into those taken before 1990 and after 1990, however, those collected before 1990 had significantly lower (P < 0.001) vitamin E levels (0.46 +/- 0.83 microgram/ml) and those collected in 1990 or later significantly higher (P < 0.001) vitamin E levels (1.03 +/- 1.04 micrograms/ml) than the captive population as a whole. This is probably due to increased dietary supplementation. There were significant differences in circulating vitamin concentrations in black rhinoceroses from different regions in the wild. Serum 25-hydroxy (OH) vitamin D3 averaged 55.7 ng/ml in free-ranging rhinoceroses; no carotenoids were detected in any blood samples. Captive black and white rhinoceroses appear to be adequately supplemented in vitamin A and E. Captive Indian rhinoceroses (Rhinoceros unicornis) had significantly lower vitamin A concentrations in blood (P < 0.001) and higher vitamin A concentrations in liver tissue samples (P < 0.001) than other rhinoceros species. Equine requirements are not recommended as a model for rhinoceros vitamin requirements.     

Tannin-binding salivary proteins in three captive rhinoceros species

Tannin-binding salivary proteins (TBSP) are considered to be counter-defences acquired in the course of evolution by animals whose natural forage contains such tannins. As tannins mostly occur in browse material but not in grasses, it is assumed that grazers do not have a need for TBSP. Whereas it has been shown in several non-ungulate species that TBSP can be induced by dietary tannins, their presence or absence in ungulates has, so far, been shown to be a species-specific characteristic independent of dietary manipulations. We investigated saliva from three rhinoceros species from zoological gardens fed comparable, conventional zoo diets. As expected, saliva from white rhinoceroses (Ceratotherum simum, grazer) had lower tannin-binding capacities than that from black rhinoceroses (Diceros bicornis, browser). Surprisingly, however, Indian rhinoceroses (Rhinoceros unicornis), commonly regarded as grazers as well, displayed the highest tannin-binding capacities of the three species investigated. It is speculated that this discrepancy might be a result of an evolutionarily recent switch to a grass-dominated diet in Indian rhinoceroses, and that the black rhinoceros, which is closer related to the white rhinoceros than the Indian species, has evolved an inducible mechanism of TBSP production. In separate trials during which the tannin content of the diets of black rhinoceroses was increased by the addition of either tannic acid or quebracho, the tannin-binding capacity of black rhinoceros saliva was increased to levels within the same range as that of Indian rhinoceroses on the conventional diets. While induction trials in white and Indian rhinoceroses remain to be performed for a full understanding of salivary anti-tannin defence in rhinoceroses, these results are the first report of an induced salivary response to increased dietary tannin levels in an ungulate species.     

Serum alpha-tocopherol, all-trans retinol, total lipids and cholesterol in the black rhinoceros (Diceros bicornis)

1. Mean concentration of serum alpha-tocopherol (Vitamin E) in 28 free-living black rhinoceroses sedated during translocation in Zimbabwe was 1.92 (SD, 0.43) mg/l. 2. Alpha-tocopherol was not detectable (less than 0.15 mg/l) in five captive black rhinoceroses held at London Zoo. 3. Circulating levels of all-trans retinol (Vitamin A) were not different between the two groups. 4. The low level of alpha-tocopherol in captive rhinoceroses suggests a risk of acute haemolytic anaemia.     

Elevated free tyrosine in rhinoceros erythrocytes

Red blood cells of African black rhinoceroses (Diceros bicornis) are highly sensitive to oxidant-induced hemolysis and they possess a number of enzymatic and biochemical features that differ radically from other mammals. Here we show concentrations of free tyrosine in rhinoceros red blood cells which can approach levels as high as 1 mM, 50-fold higher than in human red blood cells. Elevated levels of tyrosine are also observed in red blood cells of other members of the order Perissodactyla such as the horse and zebra. Captive black rhinoceroses have significantly lower levels of red blood cell tyrosine than black rhinoceroses in the wild. Tyrosine transport studies indicate that black rhinoceros red blood cells have lost the ability to transport tyrosine as efficiently as human red blood cells.     

Gamete rescue in the African black rhinoceros (Diceros bicornis)

Mortality rates are high among captive African black rhinoceroses (Diceros bicornis), due to increased susceptibility to disease. The ability to rescue genetic material from individuals that die unexpectedly represents a practical approach to assist ex situ conservation efforts. The objectives of the present study were to attempt postmortem oocyte recovery from ovaries of African black rhinoceroses (N = 6) and to test the efficacy of equine protocols for rhinoceros oocyte IVM and IVF using cryopreserved rhinoceros sperm. The interval from ovary removal to oocyte recovery was 25.3 ± 13.9 h (mean ± SD). Ovaries were transported at 4 °C or 22 °C and effects of temperature on postmortem oocyte competence was evaluated. Numbers of oocytes collected per female averaged 15.8 ± 6.9. In total, 95 oocytes were recovered. Of these, 85 were inseminated using homologous sperm and 10 were inseminated using heterologous sperm. Overall, substantial numbers of viable oocytes were retrieved from African black rhinoceros ovaries 1 to 2 days postmortem from ovaries stored at ambient temperature. A proportion of these oocytes matured and underwent penetration and fertilization by heterologous or homologous frozen-thawed rhinoceros sperm. The reproductive competence of postmortem oocytes was further demonstrated by development of a single two-cell embryo. Despite the need for further refinements, gamete rescue in the rhinoceros has promise for producing rhinoceros embryos, as well as testing sperm functions in vitro.     

Ovarian superstimulation, transrectal ultrasound-guided oocyte recovery, and IVF in rhinoceros

Numerous reports on reproductive pathology in all rhinoceros species illustrate the abundance of female infertility in captive populations. In infertile rhinoceroses, oocyte collection and embryo production could represent the best remaining option for these animals to reproduce and to contribute to the genetic pool. We report here on superstimulation, repeated oocyte recovery, and attempted in vitro fertilization (IVF) in white and black rhinoceroses. Four anestrous rhinoceroses (two white, two black) with unknown follicular status were treated with gonadotropin-releasing hormone analogue, deslorelin acetate, for 6 to 7 d. Number and size of follicles in superstimulated females was significantly higher and larger compared with those in nonstimulated anestrous females (n = 9). Ovum pick-up was achieved by transrectal ultrasound-guided follicle aspiration. Up to 15 follicles were aspirated per ovary. During six ovum pick-ups, a total of 29 cumulus-oocyte complexes (COCs) were harvested with a range of 2 to 9 COCs per collection. No postsurgical complications were noted on the rhinoceros ovaries using this minimally invasive approach. Various in vitro maturation (IVM) and IVF protocols were tested on the collected COCs. Despite the low total number of COCs available for IVM and IVF in this study, we can report the first rhinoceros embryo ever produced in vitro. The production of a 4-cell embryo demonstrated the potential of transrectal ultrasound-guided oocyte recovery as a valuable tool for in vitro production of rhinoceros embryos from otherwise infertile females.     

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.     

Allozyme variation and differentiation in African and Indian rhinoceroses

We studied variation at 25 to 31 allozymic loci in African and Asian rhinoceroses. Four taxa in three genera were examined: African Ceratotherium simum simum (northern white rhinoceros), C. s. cottoni (southern white rhinoceros), Diceros bicornis (black rhinoceros), and Rhinoceros unicornis (Indian rhinoceros). Extremely small amounts of intraspecific variation were observed in sample sizes of 2 to 10 presumably unrelated individuals per taxon: P = .00-.10, H = 0.00-0.02. We examined demographic bottlenecks and sampling errors as possible reasons for the low levels of detectable variation. The very small intraspecific genetic distance (D = 0.005) between the two living white rhinoceros subspecies is far less than the distance that has been reported for other mammal subspecies. The mean D value of 0.32 +/- 0.11 between the two African genera was also less than expected given the divergence time of greater than 7 million years suggested by the fossil record. Rhinoceroses may be evolving more slowly at the structural gene loci than are some other mammal groups. The estimate of D = 1.05 +/- 0.24 for the African-Indian split supports this idea, as the lineage diverged at least 26 million years ago. Our results contribute to the currently available scientific information on which management decisions aimed toward saving endangered rhinoceroses should be based.     

Haematological studies on wild black rhinoceros (Diceros bicornis)-evidence of an unstable haemoglobin

Baseline haematological data were obtained through routine analyses of blood samples from 31 wild black rhinoceroses captured in the Mana Pools National Park, Zimbabwe. Additional tests showed that the haemoglobin of this population is unstable; this observation helps explain the attacks of acute intravascular haemolysis documented in captive animals.     

THE FATE OF BLACK RHINOCEROSES RELEASED IN NAIROBI NATIONAL PARK

Information on the black rhinoceros population of Nairobi National Park has been obtained from two-month surveys in 1967 and 1968 backed by data collected at random over the past few years. It proved impossible to identify some of the rhinoceroses whose presence was indicated by spoor and unidentified sightings, and an estimate of the total number present at the end of 1968 must vary between 27 and 33 animals. Twenty-two rhinoceroses have been brought into the Park by the Game Department Capture Unit since November 1966. The fate of these animals has been: two dead, two not seen and 18 identified in the Park 3–33 months after release. The newcomers have produced only one serious conflict with a resident rhinoceros and two collisions with motor vehicles. Despite the small size of the Park, the inadequacy of its boundaries and the legendary intolerance of the species, it would appear that the translocation operations have been a success.     

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.     

Five new species of Trichostomatida (ciliated protozoa) from the colon of wild African rhinoceroses

Five new species of Charonina Strand, 1928 were revealed, in addition to 41 species of 20 other apparent genera, in a survey of ciliated intestinal protozoan endocommensals of both black and white wild African rhinoceroses. Charonina species infected the ventral and dorsal region of the ascending colon, where the average total protozoan populations (× 10³/ml digesta fluid) were 100 and 80, respectively, in the white and 270 and 260, respectively, in the black rhinoceroses. Charonina species constituted up to 50% in the ventral and 25% in the dorsal populations. Measurements in micrometres and specific characteristics of the five species are: C. odontophora n. sp. length 70±5.7, width 32±7.2, dorso-ventral thickness 7±1.1, slender ovate-lanceolate-shaped body with frontal lobe and prong-like protrusion in oral-opening; C. tortuosa n. sp. length 87±9.3, width 42±5.3, dorso-ventral thickness 15±2.7, body-shape ovate-lanceolate without frontal lobe, oral-opening with longitudinal ridge, cytopharyngeal canal with sharp bend after emerging from oral-opening; C. dicerotis n. sp. length 67±8.6, width 37±4.0, dorso-ventral thickness 12±2.2, body-shape ovate-lanceolate without frontal lobe, oral-opening without ridge, cytopharyngeal canal curved without sharp bend; C. tenuis n. sp. length 56±10.0, width 16±4.1 at anterior end and 10±2.7 at posterior end, dorso-ventral thickness 7±1.2 at anterior end and 5±1.1 at posterior end, body-shape cone-like with longitudinal striations and frontal lobe; C. tetragona n. sp. length 58±4.7, width 26±3.2, dorso-ventral thickness 12±1.8, body-shape rectangular with frontal lobe and caudal flaps. The length, width and dorso-ventral thickness of the five species are on average in the approximate ratio of 6:3:1, thus showing them to be dorso-ventrally compressed. The body conformation of C. tetragona n. sp. closely resembles that of Didesmis quadrata Fiorentini, 1980, but is distinguished from the latter by the absence of a concrement vacuole and the presence of an elongate cytopharyngeal canal.     

One Small Step for Rhinos,One Giant Leap for Wildlife Management- Imaging Diagnosis of Bone Pathology in Distal Limb

Chronic foot disease poses a threat to the general health, represents a tremendous clinical challenge, and often is a reason for euthanasia in captive megaherbivores, among them the elephant and rhinoceros. Nevertheless, apart from the elephant, foot pathology is handled as being confined only to soft tissues whereas bone pathology is often overlooked. As a case in point, the authors selected the second largest mammal on land, the rhinoceros. We performed a computed tomographic (CT) study using the highest resolution available in veterinary world, followed by digital radiography of eight distal limbs from two white and one Indian rhinoceroses. Our study demonstrated that bone pathology in rhinoceroses’ foot is present and in large numbers, yet none of these were diagnosed ante mortem. Even when the animals were euthanized due to foot problems, the decision was based on soft tissue pathology rather than orthopedic reasons. Even more worrying is the fact that the largest number of osteopathologies was present in one of the white rhinoceroses that showed no discernable related clinical signs. This study describes for the first time the existence of bone pathology in white rhinoceros foot, in addition to the two previously described rhinoceros species - Indian and black rhinoceroses. Furthermore, the chronic foot disease reported for the Indian rhinoceros in our study was not restricted to soft tissue structures as was presumed ante mortem but included severe bone pathology. New evidence suggesting that osteopathology in rhinoceroses’ distal limb is more widespread than it was thought before could force us to rethink of radiographic diagnosis in captive megaherbivores as routine examination incorporated into their health management. The anticipated improvements in radiologic examinations in megaherbivores will increase the effectiveness of their management and husbandry and open the way for improved animal welfare and better wildlife conservation.     

Cage restraints for rhinoceroses

Captive breeding programs for the rhinoceros can be enhanced by studying their reproductive physiology. To do so requires repetitive manipulations under physically controlled circumstances. To facilitate these procedures, zoos throughout the world have constructed restraint devices, or chutes. Chute designs are usually determined by the space in which they are to be situated and the budget available for their construction. In this study, eight chute designs at seven institutions were compared for efficiency in controlling rhinoceroses during ultrasonography, semen collection, and blood sampling. Procedures were conducted on 16 rhinoceroses of four species: the white rhinoceros (Ceratotherium simum simum), the black (Diceros bicornis), the greater one-horned Asian (Rhinoceros unicornis), and the Sumatran (Dicerorhinus sumatrensis). Chutes were evaluated for dimensions, fabricating materials, procedures to be attempted, and structural features such as shoulder restraints. Permanent, indoor, side-adjustable, pass-through chutes were determined to be the most efficient and convenient type because they saved time and reduced stress on the animals. Zoo Biol 17:343–359, 1998. © 1998 Wiley-Liss, Inc.     

RELATIONS AMONGST VARIOUS LINEAR MEASUREMENTS AND WEIGHT FOR BLACK RHINOCEROSES IN KENYA

Records of black rhinoceroses obtained from different areas of Kenya suggest a very close relationship between body weight and length and chest girth. The best regressions are: log weight on log length+log girth, and weight on length x girth². The square of the correlation coefficient in both cases is more than 0.98, and the relations appear to be independent of habitat and sex. In contrast the two sexes show marked differences in the relation between front and back horn length with the males tending to grow relatively longer front horns. Attempts to predict the size of the animal and the length of its horns from the spoor were less successful.     

Pregnancy diagnosis in the black rhinoceros (Diceros bicornis) by salivary hormone analysis

Saliva samples collected from 12 black rhinoceroses (Diceros bicornis) were analyzed for 20 alpha-hydroxyprogesterone-like and total estrogen immunoreactivity. Five nonpregnant, two conception, and nine pregnancy intervals were monitored. HPLC co-chromatography of immunoreactive free steroid components in saliva indicated that one of three immunoreactive progestin components eluted with 20 alpha-hydroxypreg-4-en-3-one, whereas the single major estrogen peak eluted with estradiol. Direct radioimmunoassay measurements of ether extracts for these two reproductive hormones in saliva provided accurate and consistent profiles in which pregnancy was detected 13 months prior to parturition, and parturition was predicted by approximately 1 month. Measurement of 20 alpha-hydroxypreg-4-en-3-one was more useful in pregnancy diagnosis, and estradiol measurement was useful as an indicator of impending parturition. © 1996 Wiley-Liss, Inc.     

Rhinoceros accounting in Kruger National Park,South Africa

Trends in rhinoceroses (rhinos) in Kruger National Park (Kruger) is of key concern. Poaching drives trends in the Park. Reconciling annual population estimates with yearly reports of poached carcasses fuels public critique. We account for trends in rhinos by extracting time series of estimates. Progressively modelling influences of management introductions and removals, effects of environmental variation and rhino density, direct impacts of poaching, consequences of imperfect carcass detection, and indirect impacts of deaths of dependent calves form the basis of accounting for rhinos. Models that considered all these influences explained 93% of white and 83% of black rhino population trends. In addition, the models predicted 2,515 white and 225 black rhinos, similar to estimates of 2,607 (95% CI: 2,475–2,752) and 202 (95% CI: 172–237) during 2020 respectively. The best model, however, predicts slow recovery with a white rhino population equivalent to pre-poaching achieved between 2030 and 2040. For black rhinos, recovery to pre-poaching population size would be between 2040 and 2050. The poaching onslaught in Kruger disrupted eruptive white rhino dynamics and prevented black rhinos from transitioning into eruptive dynamics. Authorities require innovative approaches within and beyond Kruger to help re-ignite rhino conservation.