Reproduction Abstracts

Successful semen collection and preservation is vital to the development of a crocodile artificial insemination program for the purpose of intensive production or endangered species breeding. We describe a manual cloacal palpation method for the safe and repeatable recovery of semen from Crocodylus porosus along with corresponding seminal characteristics. Experiments were also conducted to investigate fundamental sperm physiology, including the effect of excessive dilution, rapid temperature change, exposure to anisotonic media, cryoprotectant toxicity and cryopreservation. Spermic samples were recovered from 30 of 31 collection attempts from a total of 24 sedated males ranging from 197 to 400 cm in body length. Mean (±S.E.M.) seminal volume, pH, osmolality, sperm concentration, percentage motile sperm and the percentage of sperm with an intact membrane measured 0.91±0.16 ml, 7.3±0.1, 335.5±9.0 mOsm/kg, 2.29±0.26×10⁹, 50.7±4.2%, and 79.9±3.6%, respectively. Sperm abnormalities included macro and microcephalic nuclei, teratoid spermatozoa, loose heads and a range of abnormal flagella. Crocodile spermatozoa were capable of being diluted 1:16 without any reduction of motility, were not affected by rapid temperature shock to 0 °C and the plasma membrane showed a remarkable tolerance to extremely hypo-osmotic media (25 mOsm/kg). Motility of crocodile sperm diluted in PBS with increasing molarities (0.068, 0.135 and 2.70 M) of glycerol, DMSO and DMA showed a corresponding decrease in sperm motility after 1 h incubation at 4 °C but there was no adverse effect on plasma membrane integrity; sperm cryopreserved in these same diluents showed only moderate levels of plasma membrane integrity post-thaw (18–26%) but no motility.