The biotechnological potential for manipulating offspring sex in the rhinoceros and the elephant

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