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All extant rhinoceros and elephant species are endangered in the wild; yet urgently needed captive breeding to stabilise world populations of some species turns out to be a substantial challenge. One key issue in captive breeding is the unwanted high proportion of male offspring, a serious problem particularly in very small populations. If manipulating offspring sex was a feasible, successful and safe option, a higher number of females could be produced. This would accelerate population growth, thereby significantly improving the viability of populations and thus the conservation of these impressive animals. The ability to select sex would be especially useful in species close to extinction such as the northern white rhinoceros (Ceratotherium simum cottoni) with only two remaining female breeding candidates, worldwide. To date, the only reliable method to select offspring sex is the Beltsville sperm-sorting technology in combination with artificial insemination (AI) or in vitro fertilisation (IVF). This technique is based on flow cytometric separation of X and Ychromosome bearing spermatozoa in relation to their DNA content.
The aim of this study was to determine the feasibility of spermatozoa from elephants and rhinoceroses for flow cytometrical sex-sorting and to conduct basic investigations on sperm sex-sorting in these megaherbivores by exploring and establishing species-specific sorting conditions. First, the theoretical sortability of the spermatozoa was established through determination of the sperm sorting index (SSI). This index is calculated by multiplying the difference in relative DNA content between X and Y- chromosome bearing spermatozoa (as calculated by the flow cytometer) with the profile area of the sperm head. The resulting SSI values indicated that spermatozoa from the African savannah elephant (Loxodonta africana) deliver best preconditions for successful sex-sorting in the flow cytometer, followed by those of the Asian elephant (Elephas maximus). The general sortability of spermatozoa from the examined rhinoceros species [black (Diceros bicornis), white (Ceratotherium simum) and Indian rhino (Rhinoceros unicornis] was shown to be very similar among each other and lower when compared to the elephant species or to livestock.
Second, basic parameters for flow cytometric sex-sorting of spermatozoa from the black and the white rhinoceros and the Asian elephant were determined. Species-specific semen extenders, suitable for sex-sorting, and an appropriate DNA staining protocol (suitable amount of stain, incubation time and temperature) were developed. Sperm sex-sorting of rhinoceros spermatozoa thereby turned out to be challenging. Using the methods developed here, successful sex-sorting spermatozoa into specific X and Y- chromosome bearing populations produced high purity (94 %) but the sperm quality after sorting (sperm integrity: 42.0 ± 5.4 %; sperm motility: 11.5 ± 6.1 %) and the sorting efficiency (300 – 700 sperm/s) are still low. The high viscosity of rhinoceros ejaculates strongly interferes with DNA staining and sorting. The main component of the viscous fraction, a glycosilated protein with a molecular weight of 250 kDa molecular weight (most likely originating from the bulbourethral gland), was characterised via gel electophoresis and mass spectrometry. Investigating the liquefaction of the seminal plasma, the addition of the enzymes α-amylase and collagenase was shown to significantly decrease the viscosity without affecting sperm motility or integrity. In initial trials of enzyme addition to rhinoceros sperm samples that were too viscous for a processing by the flow cytometer, viscosity was reduced sufficiently enough to sort, indicating that enzyme treatment might allow better use of rhino ejaculates for sex-sorting.
The sex-sorting protocol developed for the Asian elephant provided very good results for post-sorting sperm quality (sperm integrity: 64.8 ± 3.2 %; motility: 70.8 ± 4.4 %) and purity (94.5 ± 0.7 %) and a reasonable sorting efficiency (1,945.5 ± 187.5 sperm/s). A successful protocol for cryopreservation of Asian elephant spermatozoa was also developed (post-thaw sperm integrity: 52.0 ± 5.8 %) by optimising the cryopreservation protocol (sperm handling pre- and post-cryopreservation, composition of cryomedium) and employing the directional freezing technology. Best post-thaw sperm quality was achieved using a two-step dilution of freshly collected and centrifuged spermatozoa in Blottner’s Cryomedia (285 mOsmol/kg) containing 16 % of egg yolk and a final glycerol concentration of 7 % before freezing. Spermatozoa were slowly cooled to 4 – 5°C and cryop reserved in large volumes of 2.5 or 8 ml at a concentration of 150 x 106 sperm/ml.
In the Asian elephant, the results of these studies have already permitted the use of sexsorted spermatozoa for AI. The sorting protocol appears to be technically reliable. However, AI using sex-sorted Asian elephant sperm has not yet produced any gestation. Regarding all the rhinoceros species, the poor post-sort sperm motility and integrity as well as the sorting efficiency have not yet enabled the application of sex-sorted spermatozoa to be used for AI at present, but their use in IVF may still be feasible.
This is the first study to explore the potential of flow cytometric sex-sorting of spermatozoa from several species of rhinoceroses and elephants. In terms of its practical value, the developed protocols are ready to be applied in elephants, whereas in the rhinoceros species further research is likely to be required. The results demonstrate the potential of the developed techniques and provide a promising base for the future use of the technology in the conservation management of the endangered megaherbivores.