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.     

Intestinal Ciliated Protozoa of African Rhinoceros: Two New Genera and Five New Species from the White Rhino (Ceratotherium simum Burchell, 1817)1

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 × 10⁵/ml digesta) in the ascending colon. Exceedingly small numbers were observed in the descending colon, indicating temporary accommodation only.     

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.     

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.     

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 ).     

Intestinal ciliate composition found in the feces of the Cypriot wild donkey,Equus asinus Linnaeus, 1758

Species composition and distribution of large intestinal ciliates was investigated in the feces from 13 Cypriot wild donkeys, free-living in the Karpaz national park, Northern Cyprus. We identified 16 ciliate genera and 22 species. This is the first report on intestinal ciliates in Cypriot wild donkeys, and no endemic species were observed. The genus Cycloposthium occurred in all animals. The mean number of ciliates was 3.0±2.5×10⁴ cells ml^?1 of feces and the mean number of ciliate species per host was 6.5±4.8. Characteristics of the wild donkey ciliates was almost identical to those reported in other equids from various regions around the world. We thus conclude that there is no pronounced geographic variation in the intestinal ciliate fauna of equids.     

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.     

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.     

Intestinal ciliate composition found in the feces of the Turk rahvan horse Equus caballus, Linnaeus 1758

Species composition and distribution of large intestinal ciliates were investigated in the feces from 15 Turk rahvan horses, living in the vicinity of Izmir, Turkey. Twenty-two ciliate genera consisting of 36 species were identified. This is the first report on intestinal ciliates in Turk rahvan horses and no previously unknown species were observed. The mean number of ciliates was 14.2 ± 13.9 × 10⁴ cells ml⁻¹ of feces and the mean number of ciliate species per host was 9.9 ± 7.1. No ciliates were observed in 2 horses. Bundleia and Blepharocorys were considered to be the major genera since these ciliates were constantly found in high proportions. In contrast, Paraisotricha, Didesmis and Gassovskiella were only observed at low frequencies. The ciliates found in this survey had almost the same characteristics as those described in previous reports, suggesting that there was no significant geographic variation in the intestinal ciliate fauna of equids.     

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.     

A new family, genus, and seven new species of Entodiniomorphida (Protozoa) from the gut of African rhinoceros

This report deals with a group of ciliated protozoa with short ciliary bands found mainly in the cecum of black rhinoceros, Diceros bicornis (linnaeus, 1758), and white rhinoceros, Ceratotherium simum (Burchell, 1817) from southern Africa. A new genus, Rhinozeta, based on the sum total of the characteristics of seven new related species is described. The species described are R. rhinozeta n. sp., R. triciliata n. sp., R. caecalis n. sp., R. addoensis n. sp., R. cristata n. sp., R. multiplatus n. sp., and R. unilaminatus n. sp. The specific features of the new genus make it incompatible with any of the known families of the Order Entodiniomorphida containing the ciliates present in the digestive tract of herbivorous mammals. This merits the creation of a new family, the Rhinozetidae.     

A New Family, Genus, and Seven New Species of Entodiniomorphida (Protozoa) from the Gut of African Rhinoceros

ABSTRACT. This report deals with a group of ciliated protozoa with short ciliary bands found mainly in the cecum of black rhinoceros, Diceros bicornis (Linnaeus, 1758), and white rhinoceros, Ceratotherium simum (Burchell, 1817) from southern Africa. A new genus, Rhinozeta , based on the sum total of the characteristics of seven new related species is described. The species described are R. rhinozeta n. sp., R. triciliata n. sp., R. caecalis n. sp., R. addoensis n. sp., R. cristata n. sp., R. multiplatus n. sp., and R. unilaminatus n. sp. The specific features of the new genus make it incompatible with any of the known families of the Order Entodiniomorphida containing the ciliates present in the digestive tract of herbivorous mammals. This merits the creation of a new family, the Rhinozetidae.     

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 rumen ciliate community composition in domestic sheep, deer, and cattle, feeding on varying diets, by means of PCR-DGGE and clone libraries

The structure and variability of ciliate protozoal communities in the rumens of domestic New Zealand ruminants feeding on different diets was investigated. The relative abundance of ciliates compared with bacteria was similar across all samples. However, molecular fingerprinting of communities showed ruminant-specific differences in species composition. Community compositions of cattle were significantly influenced by diet. In contrast, diet effects in deer and sheep were weaker than the animal-to-animal variation. Cloning and sequencing of almost-full-length 18S rRNA genes from representative samples revealed that New Zealand ruminants were colonized by at least nine genera of ciliates and allowed the assignment of samples to two distinct community types. Cattle contained A-type communities, with most sequences closely related to those of the genera Polyplastron and Ostracodinium. Deer and sheep (with one exception) harboured B-type communities, with the majority of sequences belonging to the genera Epidinium and Eudiplodinium. It has been suggested that species composition of ciliate communities may impact methane formation in ruminants, with the B-type producing more methane. Therefore, manipulation of ciliate communities may be a means of mitigating methane emissions from grazing sheep and deer in New Zealand.     

Intestinal ciliate composition found in the feces of racing horses from Izmir, Turkey

Species composition and distribution of intestinal ciliates were investigated in the feces from 15 racing horses living near Izmir, Turkey. Thirty-seven species belonging to 21 genera were identified. Although no new species were observed, this is the first report on intestinal ciliates in racing horses living in Turkey. The mean number of ciliates was 26.4 ± 13.9 × 10(4) cells ml(-1) of feces and the mean number of ciliate species per host was 18.8 ± 7.1. No ciliates were observed in one horse. Bundleia and Polymorphella were found to be the two dominant genera, occurring in high proportions. In contrast, Didesmis and Prorodonopsis were only observed at a low frequency. Bundleia nana, Blepharoconus hemiciliatus, Paraisotrichopsis composita, Prorodonopsis coli and Spirodinium equi were newly recorded from Turkey.     

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.     

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.     

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.