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.

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.

Ectoparasitic dermatitis in free-ranging swamp wallabies (Wallabia bicolor) in New South Wales.

We report extensive dermatological lesions in three, free-ranging, adult, male swamp wallabies (Wallabia bicolor), presented to Taronga Western Plains Zoo wildlife hospital. All three animals were moderately infested with the louse species Heterodoxus ualabati, and two were concurrently infested with a previously undescribed sarcoptiform mite of the genus Diabolicoptes. Histological changes included moderate to marked compact hyperkeratosis, focal deep pyoderma and superficial dermatophytosis. Mild to moderate hyperplastic perivascular and periadnexal dermatitis with marked superficial and follicular hyperkeratosis was evident in the skin of the inguinal region and lateral thigh of the two wallabies infested with the Diabolicoptes species. This is the first report of ectoparasitic dermatitis in free-ranging swamp wallabies and the first report of mites of the genus Diabolicoptes from a macropodid.

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.

Split-sample comparison of directional and liquid nitrogen vapour freezing method on post-thaw semen quality in white rhinoceroses (Ceratotherium simum simum and Ceratotherium simum cottoni).

To increase the quality of cryopreserved sperm in white rhinoceros, the liquid nitrogen vapour (LN vapour) freezing and the multi-thermal gradient directional freezing methods were compared. Sixteen white rhinoceros (Ceratotherium simum sp.) were electro-ejaculated. Semen samples were diluted with cryoextender (Tris, lactose, egg-yolk, DMSO) and aliquoted into straws for LN vapour freezing, and glass hollow tubes for directional freezing. The sperm quality was evaluated before and after freezing by assessing the following parameters: motility, morphologic state, acrosomal integrity and plasma membrane function and integrity (i.e. sperm viability) as defined by the hypo-osmotic swelling. Directional freezing improved the sperm viability by 5.6% (p<0.005), progressive motility score by 34.7% and sperm motility index (SMI) by 8.1% (p<0.005) versus LN vapour freezing. When data was categorized into groups of low (<19%), moderate (20-39%) and high (>40%) percentages of morphologically normal, directional freezing (DF) resulted in 31.4% less abnormal acrosomes for the low quality group as well as 18.7% increase in intact acrosomes and 10.9% increase in motility for the high quality group compared to LN vapour freezing (LN) (p<0.01, p<0.03, p<0.01, respectively). LN showed a significant reduction in sperm head volume (5.7%, p<0.05) compared to the prefreeze; whereas, no significant reduction in head volume was demonstrated after DF. Several additives (xanthenuric acid, cytochalasin D, potassium, EDTA) to the basic cryoextender provided no significant improvement in spermatozoal survival after directional freezing. In conclusion, directional freezing proved to facilitate higher gamete survival compared to LN vapour freezing. This is especially effective in ejaculates of low sperm quality and is important in endangered species where high quality semen donors are often not accessible. These results suggest that directional freezing could be valuable particularly for species with limited freezability of spermatozoa.

A review of drugs and techniques used for sedation and anaesthesia in captive rhinoceros species.

Captive rhinoceros species are most frequently sedated and/or anaesthetised with the potent opioid, etorphine hydrochloride in combination with an alpha-2 adrenoreceptor agonist or the butyrophenone, azaperone. Carfentanil citrate based combinations have also been used to a lesser extent. In recent years butorphanol tartrate based combinations have been used with good success to induce neuroleptanalgesia. Sedation and anaesthesia are complicated by the large size of all rhinoceros species and their sensitivity to potent opioids. Potential complications include respiratory depression, hypoxaemia, hypertension, pulmonary shunting and ventilation/perfusion mismatch. The pharmacology of the principal drugs used for sedating/anaesthetising rhinoceros is reviewed. Techniques for sedating/anaesthetising the various species and potential complications associated with chemical restraint are discussed.