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.     

Seminoma in a southern black rhinoceros (Diceros bicornis minor): diagnosis, surgical management and effect on fertility

A testicular mass was identified by ultrasonography performed during a routine reproductive evaluation of an adult male southern black rhinoceros ( Diceros bicornis minor ). Histological examination of a testicular biopsy supported a presumptive diagnosis of testicular neoplasia. Hemi-castration was performed to excise the affected testis and a pathological diagnosis of a seminoma was made. Assessment of semen suggested reduced fertility as a consequence of the neoplastic process, but hemi-castration prevented further growth and metastasis of the tumour and ensured the animal's breeding potential. This is the second documented case of a seminoma in a rhinoceros species and the first case in a black rhinoceros.     

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.     

Afrotropical Culicoides: a redescription of C. (Avaritia) kanagai Khamala & Kettle, 1971, reared from elephant dung in the Kruger National Park, South Africa

The discovery of Culicoides kanagai in South Africa represents a new record for this species. The female is redescribed, and the male is described for the first time. Culicoides (A.) dasyops Clastrier, 1958 is shown to be closely related to it but C. (A.) alticola is only superficially related. Short notes on the larval habitat of C. kanagai, the dung of the African elephant, Loxodonta africana, are given. The dung of both the white rhinoceros, Ceratotherium simum, and the black rhinoceros, Diceros bicornis, is considered to be a possible alternative site for the immatures of C. kanagai.     

The correct name of the South-central black rhinoceros is Diceros bicornis keitloa (A.Smith, 1836)

In South Africa, the black rhinoceros (Diceros bicornis) is divided into two subspecies, the South-western in the west and the South-central in the east. The exact boundary between the ranges of these subspecies is uncertain, but has been defined to coincide with the administrative border between the Northern Cape and North West provinces. It is current practice to refer to the South-central black rhinoceros as Diceros bicornis minor, which has Zululand as the type-locality. This needs adjustment, because an earlier valid scientific name was given to a rhinoceros killed near Zeerust in the western part of North West province. In line with the rules of zoological nomenclature, the South-central black rhinoceros should be known as Diceros bicornis keitloa.     

Reproductive anatomy and physiology of the stallion.

Examination of the stallion's reproductive tract involves assessments of external and internal anatomy. External examinations are performed by visual inspection, palpation, or ultrasonography and include the scrotum, testes, epididymides, penis, and prepuce. Internal examinations may be performed by rectal palpation, transrectal ultrasonography, or endoscopy and include the accessory sex glands, pelvic urethra, and inguinal rings. A fertile stallion must produce, transport, store, and deliver viable spermatozoa to the mare. The physiologic processes involved include neuroendocrine control, spermatogenesis, sperm maturation, semen delivery, and fertilization.     

Rectal prolapse associated with a healed pelvic fracture in a pregnant free-ranging African black rhinoceros (Diceros bicornis). Part 2: surgery and necropsy

The oedematous and traumatised protruding section of the rectal tissue of an adult free-ranging female African black rhinoceros (Diceros bicornis) was surgically amputated. Immediately before completion of surgery, the rhinoceros died of anaesthetic-related cardiac arrest. At necropsy a deformed pelvis and sacrum associated with a healed fracture of the left ileal wing were noted. New bone formation in and around the left ventral sacral foramina may have resulted in neuropathy of particularly the 3rd and 4th left ventral sacral nerves, which (in the horse) supply the majority of the nerve fibres innervating the caudal rectum and anus. The cause of the injury is not known, although back injuries, presumably sustained during mating by bulls, have been recorded in white rhinoceros. An encounter with elephants could also have been responsible for the injury in this case.     

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.     

Recovery of Arcobacter spp. from nonlivestock species

The genus Arcobacter encompasses campylobacter-like organisms that grow in air at 25 degrees C. Arcobacter has been detected or isolated from clinically healthy livestock as well as aborted fetuses and has been presumptively identified as either Campylobacter or Leptospira, based on its growth in selective semisolid media. Because reports from nonlivestock species are limited, this study examined nine presumptive isolates of Arcobacter spp. from an alpaca (Vicugna pacos), black rhinoceros (Diceros bicornis), white rhinoceros (Ceratotherium simum), gorilla (Troglodytes gorilla), gazelle (Eudorcas thomsoni), rhea (Rhea americana), and aborted equine fetuses. Seven of these nine phenotypically identified isolates of Arcobacter were confirmed by a multiplex polymerase chain reaction assay. The remaining two isolates were subsequently identified as Arcobacter skirrowii (Case 5) and Campylobacter jejuni (Case 6) by sequence analysis of a 527-base pair fragment of the 16S rRNA gene. Together, these cases underscore the challenges to a clinical laboratory of identifying Arcobacter in cases which mimic vibrionic abortion or leptospirosis.     

Cloning, sequencing and expression of white rhinoceros (Ceratotherium simum) interferon-gamma (IFN-gamma) and the production of rhinoceros IFN-gamma specific antibodies

Bovine tuberculosis (BTB) is endemic in African buffalo (Syncerus caffer) in the Kruger National Park (KNP). In addition to buffalo, Mycobacterium bovis has been found in at least 14 other mammalian species in South Africa, including kudu (Tragelaphus strepsiceros), Chacma baboon (Papio ursinus) and lion (Panthera leo). This has raised concern about the spillover into other potentially susceptible species like rhinoceros, thus jeopardising breeding and relocation projects aiming at the conservation of biodiversity. Hence, procedures to screen for and diagnose BTB in black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum) need to be in place. The Interferon-gamma (IFN-gamma) assay is used as a routine diagnostic tool to determine infection of cattle and recently African buffalo, with M. bovis and other mycobacteria. The aim of the present work was to develop reagents to set up a rhinoceros IFN-gamma (RhIFN-gamma) assay. The white rhinoceros IFN-gamma gene was cloned, sequenced and expressed as a mature protein. Amino acid (aa) sequence analysis revealed that RhIFN-gamma shares a homology of 90% with equine IFN-gamma. Monoclonal antibodies, as well as polyclonal chicken antibodies (Yolk Immunoglobulin-IgY) with specificity for recombinant RhIFN-gamma were produced. Using the monoclonals as capture antibodies and the polyclonal IgY for detection, it was shown that recombinant as well as native white rhinoceros IFN-gamma was recognised. This preliminary IFN-gamma enzyme-linked immunosorbent assay (ELISA), has the potential to be developed into a diagnostic assay for M. bovis infection in rhinoceros.     

Serial temperature monitoring and comparison of rectal and muscle temperatures in immobilized free-ranging black rhinoceros (Diceros bicornis)

Control of body temperature is critical to a successful anesthetic outcome, particularly during field immobilization of wild animals. Hyperthermia associated with exertion can lead to serious and potentially life-threatening complications such as organ damage (including myopathy) and death. Methods for monitoring core body temperature must accurately reflect the physiologic status of the animal in order for interventions to be effective. The goal of this preliminary study was to compare serial rectal and muscle temperatures in field-immobilized black rhinoceros (Diceros bicornis) and evaluate a possible association. Twenty-four free-ranging black rhinoceros were immobilized between February and March of 2010 in Ethosha National Park, Namibia. Pairwise comparisons showed a correlation of 0.73 (95% CI; 0.70-0.75) between rectal and muscle temperature measurements. Results from a multivariable model indicate that muscle temperature readings were, on average, 0.46 degrees C (95% CI; 0.36-0.57 degrees C) higher than rectal temperatures while adjusting for repeated measurements on the same rhinoceros, effect of duration of immobilization, and effect of ambient temperature on rhinoceroses' temperature readings. As immobilization time increased, muscle and rectal temperature values within an individual rhinoceros tended to equilibrate. The overall temperatures decreased by an average of 0.00059 degrees C/min (95% CI; -0.0047 to -0.0035 degrees C/min; P = 0.779). As the ambient temperature at time of immobilization increased by 1 degree C, the average rhinoceros temperature increased by 0.09 degrees C (95% CI; 0.06-0.11 degrees C, P < 0.0001). Higher body temperature creates a potential for cellular damage leading to complications that include myopathy. Methods for monitoring rectal, muscle, and ambient temperatures should be incorporated into anesthetic monitoring protocols for large ungulates, particularly under field conditions.     

Salmonella spp. survey of captive rhinoceroses in U.S. zoological institutions and private ranches.

A survey was mailed to 72 institutions in the USA requesting information on Salmonella spp. cultures in black rhinoceroses (Diceros bicornis), white rhinoceroses (Ceratotherium simum), and Indian rhinoceroses (Rhinoceros unicornis). Sixty-one institutions responded (85% return rate), with seven reporting positive cultures (11% prevalence rate; 10% if survey nonresponders had negative cultures). There were 17 positive cultures from 16 different animals, with nine different serotypes of Salmonella and 2 additional cultures identified to the group level.     

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)     

Germany/Australia Index of Sperm Sex Sortability in Elephants and Rhinoceros

Flow cytometric sexing of spermatozoa followed by application in artificial insemination or in vitro fertilization provides a unique opportunity to predetermine the sex of offspring and might enhance the conservation management of endangered species in captivity such as the elephant and rhinoceros. To obtain an indication of the sortability of spermatozoa from these species, the relative DNA differences between X and Y chromosome bearing spermatozoa (fresh, frozen thawed, epididymal) from three rhinoceros species [white ( Ceratotherium simum ), black ( Diceros bicornis ), Indian ( Rhinoceros unicornis )] and both elephant species, the Asian and the African elephant ( Elephas maximus, Loxodonta Africana ), were determined through separation of spermatozoa into X and Y chromosome bearing populations, using a modified high speed flow cytometer. The head profile areas of spermatozoa from all five species were measured using light microscopy. By multiplying the relative DNA differences and the head profile areas, the sperm sorting indices were calculated to be 47, 48 and 51 for white, black and Indian rhinoceros respectively. The calculated sorting index for the Asian elephant was 66. In the African elephant, we determined the highest sorting index of 76. These results indicate the practicability of flow cytometric sex sorting of spermatozoa from the tested rhinoceros species and both elephant species. The lower sorting indices in rhinos indicate that sex sorting of spermatozoa from the rhinoceros will be more challenging than in elephants.     

Salmonella surveillance in a herd of asymptomatic captive black rhinoceros (Diceros bicornis) using fecal culture and PCR.

Feces were collected from captive black rhinoceros (Diceros bicornis minor) housed at Disney's Animal Kingdom to examine the frequency of Salmonella spp. shedding in asymptomatic animals using enrichment culture and broth culture- polymerase-chain-reaction (PCR) for detection. Three samples per animal were collected during the first week of each month between February 2001 and December 2003. During the study period, six different individual animals from one herd participated in the study, including two growing calves. A total of 550 cultures, using duplicate samples at two different laboratories, and 464 PCR tests were performed. When culture and PCR results were compared by the same laboratory, similar herd prevalence was found (2.4% positive cultures compared with 2.6% positive PCR tests). However, even though tests were performed on replicate samples, not every sample that was positive by culture was positive by PCR and vice versa. These results suggest that using multiple diagnostic methods and increasing the number of samples submitted may increase the likelihood of finding an asymptomatic Salmonella shedder. Although all of the rhinos shared the same environment throughout the study period, only four out of the six animals tested shed Salmonella spp. even though a minimum of 37 fecal samples were taken from each of the negative animals. Although this study followed a small number of rhinoceros, it suggests that asymptomatic shedding probably occurs more frequently in captive black rhinoceros than was previously believed. The prevalence appears to be similar to that reported for domestic ungulates.     

Risk factors associated with a skewed natal sex ratio in captive black rhinoceroses (Diceros bicornis) in the United States.

The skew toward males in the sex ratio of calves is considered to be a problem for the captive population of black rhinoceroses (Diceros bicornis) in North America. This study used a retrospective case-control design to determine whether there was a predisposition toward birth of male calves over female calves and to examine risk factors associated with the occurrence of any detected skewed natal sex ratio in captive black rhinoceroses in the United States. The study population included captive female black rhinoceroses housed in the United States that had given birth to at least one calf of known sex. This study confirmed a skewing of the natal sex ratio toward male calves in the captive black rhinoceros population. The skewed ratio was found in calves born to wild-born dams, for which an increased time in captivity, irrespective of age, was associated with an increased likelihood of a male calf. Dams between 12 and 19 yr of age had a decreased likelihood of a male calf. The data also suggested a possible trend for the southern subspecies of black rhinoceros (Diceros bicornis minor) to be more likely to produce male calves than the eastern subspecies (Diceros bicornis michaeli). No associations were found with the sex of offspring in captive-born dams: however, this lack of association could be a result of low power in the study.     

Rectal prolapse associated with a healed pelvic fracture in a pregnant free-ranging African black rhinoceros (Diceros bicornis). Part 1: anaesthesia

Anaesthesia was required in a heavily-pregnant, adult, free-ranging African black rhinoceros Diceros bicornis with a rectal prolapse for examination and possible treatment. The animal was immobilised with 4.5 mg etorphine and 60 mg azaperone. For continued observation, the immobilised animal was transported to a boma. Additional etorphine and azaperone were administered to keep the animal anaesthetised during treatment and transport. In addition, 15 mg nalorphine was administered during this time to improve ventilation and reduce muscle rigidity. Sixty hours later, in preparation for surgery, 2.5 mg etorphine and 40 mg azaperone were administered, followed by endotracheal intubation and halothane anaesthesia. During anaesthesia, a decrease in tidal volume was observed. Venous blood-gas analysis indicated a decrease in the oxygen partial pressure, and a mixed respiratory and metabolic acidosis. Cardiac arrest was preceded by an increase in heart rate and tidal volume after 80 min of inhalation anaesthesia.     

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.     

Fungal pneumonia in a captive black rhinoceros

A captive black rhinoceros (Diceros bicornis) with a hoof abscess was treated with long-term antibiotic therapy. After 9 months of treatment, there was rapid deterioration, marked weight loss and reluctance to stand. Profuse, bilateral epistaxis developed accompanied by collapse and the animal was euthanased. Necropsy revealed pulmonary aspergillosis with concurrent Pseudomonas aeruginosa infection. Though a well-recognized disease of black rhinoceros, fungal pneumonia has not been reported in this species in Australia. The cost and efficacy of treatment have been questioned, however, prophylactic antifungal drug administration will be considered in any further cases of chronic, debilitating illness in black rhinoceros at Western Plains Zoo.     

Idiopathic distal esophageal dilation in a southern black rhinoceros (Diceros bicornis minor).

An adult female southern black rhinoceros (Diceros bicornis minor) experienced intermittent periods of regurgitation while eating, suggesting an esophageal disorder. Endoscopy for evaluation of the trachea, esophagus, and stomach revealed a 30-mm nasopharyngeal orifice with associated recess located in the caudodorsal pharynx and a 10-cm dilated segment of the distal esophagus that was presumably the cause of regurgitation. Dietary management of esophageal dilation through short-term utilization of a "soft feed" program successfully eliminated the regurgitation. This is the first report of esophageal dysfunction in a rhinoceros.