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.     

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.     

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.     

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.     

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.     

The Microbial Community in the Feces of the White Rhinoceros (Ceratotherium simum) as Determined by Barcoded Pyrosequencing Analysis

As a non-ruminant herbivore, the white rhinoceros has the ability to utilize fibrous plant matter through microbial fermentation in the hindgut. So far, there has been no report using molecular techniques to study the gut microbiota of the white rhinoceros. We used barcoded pyrosequencing to characterize 105,651 sequences of 16S rRNA genes obtained from fecal samples from five white rhinoceroses. Results showed that Firmicutes and Bacteroidetes were the predominant phyla in the samples, which were comprised largely of unclassified bacteria. The microbiota of one animal treated with drug therapy differed from those in other healthy animals, and was dominated by Aerococcus -related bacteria. The core microbiota in the healthy rhinoceros were dominated by phyla Firmicutes and Bacteroidetes, represented by the Ruminococcaceae, Lachnospiraceae, Rikenellaceae and Prevotellaceae families. The present work provides a phylogenetic framework for understanding the complex microbial community of the rhinoceros; however, further studies are required to link the distinctive microbiota with their digestive role in the hindgut of the white rhinoceros.     

Fluid and particle retention times in the black rhinocerosDiceros bicornis, a large hindgut-fermenting browser

The mean retention time (MRT) of ingesta in the gastrointestinal tract is one of the major determinants of herbivore digestive physiology. We examined MRTs of fluids and particles in the gastrointestinal tract of six adult captive black rhinocerosesDiceros bicornis on conventional zoo diets. Fluid MRT ranged from 25–45 h and averaged 31 h. Particle MRT ranged from 28–59 h and averaged 38 h. In the six animals, both fluid and particle MRT declined as relative dry matter intake (g/kg metabolic body mass) increased. Black rhinoceroses, which are large hindgut-fermenting browsers, retained ingesta for a shorter period relative to their body size than grazing equids or grazing rhinoceros species. Our findings support the hypothesis that browsing hindgut fermenters have relatively shorter MRTs than grazing hindgut fermenters.     

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.     

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 ).     

Cross-species chromosome painting in the Perissodactyla: delimitation of homologous regions in Burchell's zebra (Equus Burchellii) and the white (Ceratotherium Simum) and black rhinoceros (Diceros Bicornis)

Conserved chromosomal segments in the black rhinoceros, Diceros Bicornis (DBI, 2n = 84), and its African sister-species the white rhinoceros, Ceratotherium Simum (CSI, 2n = 82), were detected using Burchell's zebra (Equus Burchellii, EBU, 2n = 44) chromosome-specific painting probes supplemented by a subset of those developed for the horse (Equus Caballus, ECA, 2n = 64). In total 41 and 42 conserved autosomal segments were identified in C. Simum and D. Bicornis respectively. Only 21 rearrangements (20 fissions and 1 fusion) are necessary to convert the Burchell's zebra karyotype into that of the white rhinoceros. One fission distinguishes the D. Bicornis and C. Simum karyotypes which, excluding heterochromatic differences, are identical in all respects at this level of resolution. Most Burchell's zebra chromosomes correspond to two rhinoceros chromosomes although in four instances (EBU18, 19, 20 and 21) whole chromosome synteny has been retained among these species. In contrast, one rhinoceros chromosome (DBI1, CSI1) comprises two separate Burchell's zebra chromosomes (EBU11 and EBU17). In spite of the high diploid numbers of the two rhinoceros species their karyotypes are surprisingly conserved offering a glimpse of the putative ancestral perissodactyl condition and a broader understanding of genome organization in mammals.     

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.     

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.     

Locomotion and bone strength of the white rhinoceros, Ceratotherium simum

Measurements have been made, of lengths and of geometric properties of cross-sections, of the long bones of the legs of a young white rhinoceros of about 750 kg body mass. These are considered in conjunction with data from film of white rhinoceros trotting and galloping. The stresses developed in the bones in running are rather low, in comparison with other large mammals, suggesting that rhinoceros skeletons may be built to unusually high factors of safety. The long, relatively straight legs of elephants (whose bones experience higher stresses) are contrasted with the shorter, less straight legs of the other graviportal mammals.     

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.     

Artificial insemination in the anoestrous and the postpartum white rhinoceros using GnRH analogue to induce ovulation

The objective of this study was to develop AI and to achieve first time pregnancy in a nulliparous rhinoceros. For this, one 24-year-old irregular cycling female white rhinoceros was selected, which had never been mated. The endocrine function was monitored by faecal and serum pregnane analysis. Ultrasound determined the optimal day for AI by measuring follicle sizes of 2.0, 2.6, 3.0, 3.2 cm on days -6, -4, -1, 0 of the induced oestrous cycle, respectively. AI was performed and ovulation induced when a pre-ovulatory-sized follicle was present using GnRH analogue, deslorelin. Fresh semen was deposited in the uterine horn using a patented AI catheter overcoming the hymeneal membrane and torturous cervical folds non-surgically. Moreover, ultrasound monitoring of the uterine involution and ovarian activity on days 16, 26, 30 postpartum facilitated the induction of and the AI on the first postpartum oestrous in a rhinoceros using GnRH analogue. Two consecutive pregnancies were achieved by AI for the first time in the rhinoceros. Pregnancies were diagnosed by elevated serum and faecal 20-oxo-pregnane concentrations. In addition ultrasound measured biometric parameters of the two foetuses on days 86 and 133 of gestation. Two female calves were born after 490 and 502 days of gestation, yet one calf was stillborn. AI in rhinoceros might now be used as assisted reproduction technology tool to boost critically small captive rhinoceros populations.     

Feasibility of characterizing reproductive events in large nondomestic species by transrectal ultrasonic imaging

The feasibility of using transrectal ultrasonography for imaging the in situ morphology of the reproductive tract of females of several large nondomestic and endangered species was studied. Two black (Diceros bicornis) and 1 white (Diceros simus) rhinoceros, 2 Asian (Elaphus maximus) and 2 African (Loxodonta africana) elephants, 4 banteng (Bos javanicus), 1 gaur (Bos taurus), 1 giraffe (Giraffa camelopardalis), and 1 bactrian camel (Camelus bactrianus) were examined. Real-time ultrasonic images were obtained for the following structures: 1) rhinoceros—corpus luteum, ovarian follicles, uterus, cervix, and early conceptus, 2) elephants—posterior uterus and cervix, 3) banteng and gaur—corpus luteum, ovarian follicles, uterus, cervix, and conceptus, 4) giraffe—posterior uterus, placentomes, and late conceptus, 5) camel—posterior uterus, fetal fluids, and fetal membranes. Individual ovarian follicles were identified and monitored over a 34 day observational period in 1 nontranquilized white rhinoceros. Difficulties and limitations in viewing the ovaries in the elephants were attributed to operator inexperience and to the size, positioning, and demeanor of the animals. Pregnancy was detected in 1 black rhinoceros (27 days), 1 banteng cow (48 days), the giraffe (13 months), and in the bactrian camel (approximately 3½ months). Impending embryonic loss was suspected in the banteng cow because a heartbeat was not detected in the embryo proper; the cow was subsequently diagnosed nonpregnant by transrectal palpation 20 days later. It is concluded that the ability afforded by transrectal ultrasonography to detect and measure ovarian structures and changes in morphology of the tubular genitalia and conceptus provides a research methodology for the elucidation of certain aspects of reproductive biology, and a clinical modality for reproductive management and assisted fertilization programs of large nondomestic species.     

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.     

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.     

Vitamin E in captive and wild black rhinoceros (Diceros bicornis)

The mean plasma level of alpha-tocopherol (vitamin E) measured in 31 free-ranging black rhinoceros (Diceros bicornis) was significantly higher (P less than 0.001) than that in 11 captive animals (means +/- SE = 0.77 +/- 0.05 and 0.18 +/- 0.03 micrograms/ml, respectively). Vitamin E status may influence the health of captive black rhinoceros; in particular, it may be linked to hemolytic anemia commonly observed in these animals in captivity.