Clinical implications of rhinoceros reproductive tract anatomy and histology.

Reproductive tracts or tissues from five male black rhinoceroses (Diceros bicornis), two male white rhinoceroses (Ceratotherium simum), two male one-horned Asian rhinoceroses (Rhinoceros unicornis), seven female black rhinoceroses, and six female white rhinoceroses from multiple institutions were examined to characterize their anatomy and histology. Some observations and measurements were obtained from in situ tracts of intact animals before or during necropsy. Formalin-fixed tissues were dissected and examined histologically. Retrospective reproductive data from each rhinoceros was obtained from the institutions of origin. Reproductive histology of these species was similar to that of other mammals. Male accessory gland structure varied among species, and the Asian rhinoceros epididymis was more loosely attached and had larger duct diameters than did the epididymides of the African species. Although histology was typically mammalian, rhinoceros reproductive morphology combined characteristics of several different mammals. Defining this unique morphology of rhinoceroses may help in understanding their reproductive physiology and will effect the development of appropriate reproductive techniques.     

Comparative studies of mitogen- and antigen-induced lymphocyte proliferation in four captive rhinoceros species.

Cellular immune function in four rhinoceros species was evaluated by way of in vitro lymphocyte proliferation responses to mitogenic and antigenic stimuli to establish normative data on white blood cell activity for each species and to identify species-specific differences that might help explain the predisposition of black rhinoceroses (Diceros bicornis) to disease. A cross section of the U.S. rhinoceros population encompassing all four captive species was sampled, including the Sumatran rhinoceros (Dicerorhinus sumatrensis) (n = 3); Indian rhinoceros (Rhinoceros unicornis) (n = 4); African black rhinoceros (n = 16); and African white rhinoceros (Ceratotherium simum) (n = 10). Of the four species evaluated, African black rhinoceroses exhibited the weakest (P < 0.05) lymphocyte proliferative responses to the mitogens: pokeweed (0.1 microg/ml), phytohemagglutinin (0.3 microg/ml), and concanavalin A (5.0 microg/ml). Total cell density at the end of culture was only 70% of that achieved with lymphocytes isolated from African white rhinoceroses, Indian rhinoceroses, and Sumatran rhinoceroses. However, lymphocyte response to bacterial endotoxin lipopolysaccharide was similar (P > 0.05) across species. Antigenic stimulation produced much weaker responses than mitogenic stimulation. No differences (P > 0.05) were observed among rhinoceros species in response to 1 and 10 microg/ml of Leptospira icterohemorrhagiae or Leptospira gryppotyphosa. Lymphocytes from African white rhinoceroses proliferated weakly in the presence of Aspergillus fumigatus filtrate, whereas lymphocytes from the southern black rhinoceros subspecies appeared slightly suppressed in the presence of increasing doses (0.1, 1, and 10 microg/ml) of Aspergillus filtrate. This comparative data set characterizing lymphocyte proliferation in the rhinoceros reveals several differences in immune cell responses among rhinoceros species and provides some evidence that lymphocytes of captive African black rhinoceroses are less vigorous than those of the other rhinoceros species.     

Digestion coefficients achieved by the black rhinoceros (Diceros bicornis), a large browsing hindgut fermenter

In contrast to the grazing white (C. simum) and Indian (R. unicornis) rhinoceros, the black rhinoceros (D. bicornis) is an exclusive browser. Due to the particular fermentation characteristics of browse, one would expect browsers to display both shorter ingesta retention times and lower digestion coefficients on comparable diets than grazers. In order to generate a database to test this hypothesis, we performed digestibility studies in eight black rhinoceroses (D. bicornis) from three zoological institutions, using total faecal collection for the quantification of faecal output. One to three regularly fed zoo rations of roughage, concentrates and varying proportions of browse material were used per animal. Additional data was taken from three hitherto unpublished studies as well as several published sources. When compared with horses on similar rations, black rhinoceroses achieved lower digestion coefficients for organic matter and CF. In general, an increase in dietary CF content led to a steeper decrease in organic matter and GE digestibility in black rhinoceroses than in horses. When comparing available data for rhinoceroses, browsing species showed a steeper decrease in organic matter digestibility than grazing species with increasing dietary cell wall content. Endogenous losses as determined by linear regression analysis were within the range reported for horses and Indian rhinoceroses. The results suggest that the horse is not a useful model animal for evaluating diets for black rhinoceroses energetically. In general, diets fed to captive black rhinoceroses seem to include higher proportions of concentrates than diets for other rhinoceros species, and an increase in browse or roughage would reduce digestion coefficients to levels observed in animals fed natural forage.     

XERORADIOGRAPHIC ANATOMY OF THE EQUINE DIGIT AND METACARPOPHALANGEAL REGION

The purpose of the project was to use xeroradiography to provide a reference for radiographic anatomy of the equine digit and metacarpophalangeal region. The left foredigits and metacarpophalangeal joints of a mature horse and a 30-day-old foal were radiographed, using xeroradiographic techniques. Fifteen xeroradiographs, illustrating standard projections of each area, were selected and appropriately labeled to demonstrate normal radiographic anatomy of these regions. These xeroradiographs have been used successfully for several years to teach radiographic anatomy of these areas to first-year veterinary students at North Carolina State University.     

Radiometric assessment of hexose monophosphate shunt capacity in erythrocytes of rhinoceroses.

OBJECTIVES: To measure metabolic rates of the hexose monophosphate shunt (HMPS) in erythrocytes of rhinoceroses, and to test the hypothesis that low concentrations of endogenous ATP in erythrocytes impair HMPS capacity, thereby increasing susceptibility to oxidant-induced hemolysis. ANIMALS: 13 black and 3 white rhinoceroses, free-ranging in several regions of southern Africa, and 1 Sumatran rhinoceros in US captivity. PROCEDURE: HMPS fluxes were measured in rhinoceros erythrocytes with carbon-labeled glucose in the presence and absence of known HMPS activators. RESULTS: Compared with values for human erythrocytes, mean basal state HMPS fluxes were appreciably lower (22 to 46%) in all 3 rhinoceros species studied. Shunt activators increased HMPS rates approximately 5-fold over basal rates in rhinoceros erythrocytes, compared with increases in humans of 10-fold with ascorbate and 15-fold with methylene blue. Stimulated HMPS rates in human erythrocytes were quantitatively 5- to 10-times greater than those observed in rhinoceros erythrocytes. Overall HMPS catabolic rates were completely independent of intracellular ATP concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: HMPS glycolytic and recycling rates and responses to activators are inherently low in erythrocytes from 3 species of rhinoceros, likely contributing to (but not solely responsible for) the high susceptibility of black rhinoceroses to oxidant-induced hemolysis. Slow erythrocyte HMPS capacities were independent of intracellular ATP concentrations, invalidating a current hypothesis regarding the pathogenesis of hemolytic anemia in captive black rhinoceroses. Limitations in HMPS capacities emphasize the importance of protecting rhinoceroses from exposure to drugs, chemicals, toxins, foodstuffs, and other conditions known to increase production of oxidizing metabolites, reactive oxygen species, and free radicals.     

Acute intravascular hemolytic anemia in the black rhinoceros: hematologic and immunohematologic observations

To investigate the syndrome of acute intravascular hemolytic anemia in the black rhinoceros (Diceros bicornis), laboratory techniques used in the differential diagnosis of hemolytic anemias were performed on blood samples from 6 black rhinoceroses: 3 nonrelated healthy rhinoceroses, 1 rhinoceros with iron deficiency anemia, and 2 rhinoceroses with intravascular hemolysis. Osmotic fragility, erythrocyte membrane protein composition, hemoglobin electrophoresis, and hemoglobin stability did not distinguish between healthy and affected (anemia or hemolysis) rhinoceroses. Polyclonal antiglobulin reagents were prepared in rabbits, using whole rhinoceros serum and purified rhinoceros immunoglobulin G. These reagents were nonreactive against erythrocytes of the healthy and iron-deficient rhinoceroses. Reactions with RBC from the rhinoceros with fatal hemolytic anemia indicated increased membrane coating by the third component of complement; this was not evident in a second rhinoceros that survived a hemolytic event.     

Osteochondral fragmentation of the palmarolateral/plantarolateral aspect of the distal phalanx in four horses: A novel location

Four horses presenting for lameness were diagnosed with unilateral osteochondral fragmentation (OCF) of the palmarolateral/plantarolateral aspect of the distal phalanx within the distal interphalangeal joint (DIPJ). Histological evaluation of one case supported a diagnosis of osteochondritis dissecans (OCD), with patient age and history from two cases suggesting a traumatic origin. Lesion appearance on conventional radiography, computed tomography (CT), nuclear scintigraphy and magnetic resonance imaging (MRI) are described. Fragmentation was best identified on dorsal 65° proximal-palmaro/plantarodistal oblique (D65°PrPDiO/D65°PrPlDiO) and dorsal 65° proximal-palmaro/plantarodistal lateral oblique (D65°Pr45°L-PDiMO/D65°Pr45°L-PlDiMO) radiographic projections of the foot, but articular pathology appeared more severe on cross-sectional imaging modalities. In all cases, lameness was refractory to conservative management. Arthroscopic evaluation of the DIPJ was performed in three horses, although the lesion was inaccessible in two. In one horse, access to the lesion was possible due to increased joint laxity, presumably due to concurrent soft tissue injury. One horse was euthanased after failed conservative management, one was pasture sound following palmar digital neurectomy 12 months after initial presentation, one returned to racing and one was lost to follow-up. Osteochondral fragmentation at this location has not previously been described, treatment options are limited and the prognosis appears to be poor.     

Gross anatomy and ultrasonographic images of the reproductive system of the Sumatran rhinoceros (Dicerorhinus sumatrensis)

The Sumatran rhinoceros (Dicerorhinus sumatrensis) is the smallest of all the rhino species. It is one of the rarest mammals in the world and is in imminent danger of extinction. A study was carried out on seven wild-caught females, three wild-caught males and one captive born female Sumatran rhinoceros at the Sumatran Rhinoceros Breeding Centre in Sungai Dusun, Selangor, Malaysia, beginning 1990. As a result of the paucity of scientific information on the reproductive biology of the Sumatran rhinoceros, this study was conducted to obtain information, which could assist in the captive breeding of this endangered and near extinct species. The anatomy of the reproductive system was based on two post-mortem specimens and transrectal real-time ultrasonography in six adult females. Genitalia of the Sumatran rhinoceros were similar to those of other species of rhinoceroses. The cervix consisted of several folds, the uterus was bicornuate with a short body and prominent horns and the ovaries were completely covered by the fimbriated end of the fallopian tube. The internal genitalia could be imaged by ultrasonography. The testes were located within a pendulous scrotum. Two lateral projections were located at the base of the penis. A well-defined process glandis was present at the tip of the penis. The accessory sex glands and the testes could be imaged by ultrasonography.     

Piroplasm parasites of white rhinoceroses (Ceratotherium simum) in the Kruger National Park, and their relation to anaemia

As part of a larger survey to map the geographical distribution of Babesia and Theileria parasites in the southern African rhinoceros population, white rhinoceroses were sampled during routine immobilisations in the Kruger National Park. Polymerase chain reaction (PCR) and reverse line blot (RLB) hybridisation assays were used to screen for the presence of piroplasms and complete blood counts were used to assess associated changes in clinical parameters. Of the 195 rhinoceroses sampled, 71 (36.4 %) tested positive for the presence of Theileria bicornis, with no significant change in the haematological parameters measured, while 18 (9.2 %) tested positive for Theileria equi. None of the rhinoceroses sampled tested positive for Babesia bicornis, a parasite associated with mortalities in black rhinoceroses.     

Understanding South African private landowner decisions to manage rhinoceroses

With increased poaching pressures, rhinoceroses have become a financial liability due to expensive anti-poaching security costs required to protect this species. Nonetheless, approximately one-third of South Africa’s rhinoceros population is protected on private lands. In a time when the future existence of rhinoceroses is uncertain, it is important to determine (a) the considerations included in private sector decisions to participate in rhinoceros conservation and (b) how increased conservation of rhinoceroses on private lands may be attained. We conducted semi-structured interviews with private wildlife ranchers and reserve managers in South Africa to answer these questions. Respondents cataloged financial (e.g., security costs, tourism revenues) and nonfinancial (e.g., psychological stresses, emotional attachment) factors that influence their decisions to keep rhinoceroses on their lands. Despite significant costs of rhinoceros ownership, rhinoceros owners were willing to engage in conservation efforts because of the nonfinancial benefits they derive from protecting rhinoceroses.     

Semen collection in rhinoceroses (Rhinoceros unicornis, Diceros bicornis, Ceratotherium simum) by electroejaculation with a uniquely designed probe.

Electroejaculation in rhinoceroses has historically yielded inconsistent results, with the collection of high-quality, sperm-rich samples rare. The goal of this study was to develop a reliable method of electroejaculation in the rhinoceros by designing a rectal probe that appropriately fits the anatomy of this taxon and refining the procedure. A curved probe handle ending in an oblate, ellipsoid head was built using readily available supplies. A combination of rectal massage, penile massage, and electrical stimulation with a specially designed probe was employed in attempts to collect semen on 14 occasions from greater one-horned rhinoceroses (Rhinoceros unicornis; n = 4), black rhinoceroses (Diceros bicornis; n = 2) and a southern white rhinoceros (Ceratotherium simum; n = 1). During 13 of the 14 attempts, ejaculates were collected in multiple fractions. All but one of the ejaculates contained spermatozoa, and seven ejaculates contained good-quality fractions of semen (-60% sperm motility; > or =20 x 106 spermatozoa/ml) suitable for sperm banking and assisted reproduction procedures. Mean (+/-SEM) values for volume, pH, osmolality, and total sperm number for ejaculates containing good-quality fractions (98.2 +/-21.8 ml, 8.5+/-0.1, 290.4+/-6.7 mOsm, and 37.1+/-12.0 x 10(9), respectively) did not differ (P > 0.05) from those containing only poor-quality samples. Urine and/or erythrocyte contamination was not uncommon in fractions of both ejaculate types. Males producing good-quality samples ranged in age from 7 to 34 yr. None of the samples contained > or =75% morphologically normal spermatozoa. Electroejaculation with a uniquely designed probe consistently produced ejaculates in the rhinoceros. However, the production of high-quality samples continued to be challenging, occurring in only 50% of collection attempts. Regardless, the technology has progressed to a stage at which good-quality semen samples can be produced for sperm banking and assisted reproduction, and thereby can be integrated into intensive rhinoceros management strategies for the ultimate survival of this taxon.     

Beta-globin chain hemoglobin polymorphism and hemoglobin stability in black rhinoceroses (Diceros bicornis)

To evaluate the syndrome of acute intravascular hemolytic anemia in black rhinoceroses (Diceros bicornis), the hemoglobin of this species was evaluated by use of isopropanol- and heat-stability tests and was further characterized by electrophoretic studies. Samples were obtained from 22 apparently healthy captive North American black rhinoceroses, though 3 of the study animals had survived previous hemolytic events, and 3 others were parents of 3 offspring that had suffered hemolysis. The eastern African (Diceros bicornis michaeli) and the southern African subspecies (D b minor) were represented. Comparative samples were also obtained from 2 white (Ceratotherium simum) and 1 Indian (Rhinoceros unicornis) rhinoceroses. The hemoglobin of all 3 species appeared stable when tested by use of the heat and isopropanol methods. Thus, an unstable hemoglobin does not appear to be involved in the hemolytic crises of captive black rhinoceroses. Black rhinoceros hemoglobin had a striking polymorphism. Thirteen of the samples from black rhinoceroses had a single hemoglobin band, based on results of alkaline electrophoresis. Nine had, in addition to this major band, a slow (more cathodic) minor band that comprised about 10% of the total hemoglobin. Further studies indicated that the major band and the slower minor band may contain globin chains analogous to human beta- and delta-chains respectively; these bands have been tentatively designated B and C. Phenotypes B and BC are common, in a ratio of 4:3. A genetic mechanism is proposed that assumes beta b and beta c gene loci and that beta c-locus-expressed (beta c+) and beta c-locus-inhibited (beta c degrees) are common alleles for the beta c-locus.(ABSTRACT TRUNCATED AT 250 WORDS)     

MAGNETIC RESONANCE IMAGING OF THE EQUINE DIGIT WITH CHRONIC LAMINITIS

Chronic laminitis is a severe disease affecting the equine digit. It was hypothesized that magnetic resonance (MR) imaging would improve visualization of structures within the foot and pathology associated with chronic laminitis. This study aimed to describe the MR imaging findings in chronic laminitis, compare different pulse sequences for visualization of pathology, and to compare MR imaging with standard radiography. Twenty (10 forelimb, 10 hindlimb) cadaver limbs from 10 horses clinically diagnosed with chronic laminitis (group L) and 10 limbs without laminitis (group N) were used. Lateromedial radiographs and sagittal and transverse MR images of the foot were obtained. Radiographs and MR images were evaluated for anatomic definition and evidence of pathology. Dorsal hoof wall thickness and angle of rotation and displacement distance of the distal phalanx were measured. Comparisons were made between group L and N, forelimb and hindlimb within each horse, and MR imaging and radiography. Features consistently noted with MR images in group L, but not detected using radiography, included laminar disruption, circumscribed areas of laminar gas, laminar fluid, and bone medullary fluid. Other findings seen only on MR images included increased size and number of vascular channels, alterations in the corium coronae, and distal interphalangeal joint distension. Magnetic resonance imaging allowed better definition of laminar gas lines and P3 surface irregularity observed on radiographs. Based on measurements, group L had a greater angle of rotation, distal displacement, and dorsal hoof wall thickness than group N; forelimb hoof wall thickness was greater than hindlimb; and distal displacement and hoof wall thickness measurements were smaller using MR imaging than radiography, but had a similar pattern. It is concluded that there are features of chronic laminitis consistently observed using MR imaging and that these may be additional to features observed radiographically.     

VALIDATION OF A NOVEL TECHNIQUE FOR CREATING SIMULATED RADIOGRAPHS USING COMPUTED TOMOGRAPHY DATASETS

Understanding radiographic anatomy and the effects of varying patient and radiographic tube positioning on image quality can be a challenge for students. The purposes of this study were to develop and validate a novel technique for creating simulated radiographs using computed tomography (CT) datasets. A DICOM viewer (ORS Visual) plug‐in was developed with the ability to move and deform cuboidal volumetric CT datasets, and to produce images simulating the effects of tube‐patient‐detector distance and angulation. Computed tomographic datasets were acquired from two dogs, one cat, and one horse. Simulated radiographs of different body parts (n = 9) were produced using different angles to mimic conventional projections, before actual digital radiographs were obtained using the same projections. These studies (n = 18) were then submitted to 10 board‐certified radiologists who were asked to score visualization of anatomical landmarks, depiction of patient positioning, realism of distortion/magnification, and image quality. No significant differences between simulated and actual radiographs were found for anatomic structure visualization and patient positioning in the majority of body parts. For the assessment of radiographic realism, no significant differences were found between simulated and digital radiographs for canine pelvis, equine tarsus, and feline abdomen body parts. Overall, image quality and contrast resolution of simulated radiographs were considered satisfactory. Findings from the current study indicated that radiographs simulated using this new technique are comparable to actual digital radiographs. Further studies are needed to apply this technique in developing interactive tools for teaching radiographic anatomy and the effects of varying patient and tube positioning.     

Acute intravascular hemolysis in the black rhinoceros: erythrocyte enzymes and metabolic intermediates

Enzymes of aerobic and anaerobic glycolysis, glutathione cycling, and nucleotide metabolism were assayed on erythrocytes from 7 healthy rhinoceroses, 2 rhinoceroses during periods of intravascular hemolysis, and 1 rhinoceros without clinical signs of illness, which was the mother of 3 offspring with intravascular hemolytic syndrome. Measurements also were made of erythrocyte concentrations of glycolytic intermediates, adenine nucleotides, and glutathione. Although comparison of results for healthy and affected rhinoceroses did not identify an enzyme abnormality as a cause for the hemolytic syndrome, the data provided information regarding the metabolic characteristics of erythrocytes from healthy rhinoceroses.     

Corticosteroid-induced suppression of in vitro lymphocyte proliferation in four captive rhinoceros species.

Captive African black rhinoceroses (Diceros bicornis) are unusually susceptible to several diseases not commonly observed in any of the other three rhinoceros species maintained in captivity. The potential role of corticosteroids (either endogenously produced or exogenously administered) in the development of these sometimes fatal diseases has been questioned. In this study, the suppressive effects of two therapeutic corticosteroids (dexamethasone and hydrocortisone) on in vitro lymphocyte proliferation was examined in four rhinoceros species, including the Sumatran rhinoceros (Dicerorhinus sumatrensis, n = 3), Indian rhinoceros (Rhinoceros unicornis, n = 4), African black rhinoceros (n = 10), and African white rhinoceros (Ceratotherium simum, n = 5). Three blood samples collected from each rhinoceros 1 mo to 1 yr apart provided replicates for the study. Both dexamethasone and hydrocortisone suppressed (P < 0.05) lymphocyte proliferation stimulated by B-cell mitogens (pokeweed and lipopolysaccharide) and T-cell mitogens (phytohemagglutinin and concanavalin A). Suppressive effects of the glucocorticoids differed (P < 0.05) depending on the mitogen used to stimulate the lymphocytes. Overall, dexamethasone was a more potent suppressor of cell proliferation when compared with hydrocortisone (P < 0.05). However, black rhinoceros cell proliferation in response to any of the four mitogens was never completely suppressed, even in cultures containing the highest steroid concentration tested (10(-3) M). The effect of the two corticosteroids differed slightly among the rhinoceros species and subspecies tested, but there was no evidence that eastern or southern black rhinoceros lymphocytes were more sensitive to the suppressive effects of corticosteroids than the other rhinoceros species.