Optimisation and validation of an in vitro bioassay as a tool for measuring luteinising hormone in several species of mammals

Understanding the underlying reproductive physiology of threatened and native species is crucial when developing conservation management strategies, especially in situations where ex-situ captive breeding programs are crucial to a species short-term survival. However, research into the reproductive biology of threatened species is hampered by the fact that it is difficult to measure the gonadotrophins, luteinising hormone (LH); and follicle-stimulating hormone (FSH), the chief controllers of reproduction in vertebrates. This project, therefore, aims to develop and validate a simple, rapid LH bioassay which is capable of measuring LH concentrations from a diverse range of mammalian species. The bioassay uses a clonal murine Leydig tumour cell line as an indirect method for measuring LH in mammals. The cell line responds to LH stimulation in vitro by producing the steroid progesterone, which is much easier to measure across species. It is envisioned that this assay would be a useful tool in situations where a high volume of samples from various species require analysis, such as in a zoo or wildlife breeding facility. Firstly an optimisation of the conditions of the assay was conducted; specifically cell density/well and incubation time required for cells to produce progesterone was optimised to improve the sensitivity of the assay. An optimal cell density of 9,375 cells/well was discovered to have greatest sensitivity to physiological levels of LH after an optimal incubation period of 120 minutes. Using these optimised conditions, pituitary LH preparations from eight species of mammals (human, porcine, monkey, rabbit, bovine, canine, equine and, rat) produced good dose-response curves demonstrating the practicality of this assay to measure LH from diverse species. The sensitivity of the bioassay differed for each species, although there was no obvious phylogenetic relationship between species and sensitivity. The sensitivity ranged from 0.0625 – 1 ng/100 μl (NB: 100 μl of sample in each well) for most of the species tested, well within the normal physiological range, with only the bovine being slightly higher at 4 ng/100 μl, but still within the physiological range. There was no cross-reactivity with FSH in the bioassay. Therefore, it was concluded that the assay might be suitable for the analysis of samples from diverse mammalian species. As standard available immunoassays are also often unsuitable for measurement of LH in marsupial species, this precludes assessment of the stage of their oestrous cycle. The application of the LH bioassay was investigated for its capability to act as an indirect method for measuring LH in marsupials, which has not been conducted previously as marsupial LH has often been described to be structurally and antigenically different from LH produced by mammals. The bioassay detected an increased circulating LH pre- and 25 minutes post- exogenous GnRH challenge in western grey kangaroos (P<0.05) and black-flanked rock wallabies (P<0.05), and also detected decreased LH levels in black-flanked rock wallabies treated with a contraceptive prior to GnRH challenge (P<0.01). Results obtained by this bioassay method were validated by comparison to those derived from an adapted enzyme immunoassay (EIA). There was no significant difference in change in LH concentrations as detected by either method in the western grey kangaroo or the control group of black-flanked rock wallabies, however, there was a significant difference in change in LH concentration between the two methods when assessing black-flanked rock wallabies on a contraceptive (P