Doctor of Philosophy (PhD) / Reproductive physiology plays a vital role in population growth and vitality. Baseline data on reproductive physiology and a comprehensive knowledge of breeding biology are essential to conservation management. Great whales have been hunted from the 16th century to the present day. Although many populations are increasing there are populations with low or declining reproductive rates. In 2001 it was recommended to the International Whaling Commission that new techniques be developed to assess the internal physiology of great whales. This study, based on this recommendation, aims to develop analytical methods to assess reproductive hormones in cetacean blow samples and determine the feasibility of its use with free-swimming great whales. A method for the assessment of steroid hormone concentrations using liquid chromatography-mass spectrometry (LC-MS) was developed and validated. These methods were then used to determine testosterone and progesterone concentrations in saliva and blow of bottlenose dolphins. The stability of testosterone and progesterone was found to be a major issue. Without inhibitors, hormone concentrations increased by up to 65% over three hours at 21oC. Storing samples at low temperatures (-20oC or -80oC) slowed but did not cease the rate of change. The addition of inhibitors, manganese chloride and amoxycillin potassium/clavulanate, improved the stability of testosterone and progesterone. It is proposed that when using dolphin saliva and blow samples to measure reproductive hormones the samples are extracted as soon as possible after collection to prevent degradation. This study highlighted the need to address steroid hormone stability prior to any longterm biological program, to ensure that changes seen in hormone concentration are due to biological activity rather than storage. A technique to collect blow samples from free-swimming great whales was developed. This technique, in conjunction with the specially developed LC-MS methods allowed for the determination of testosterone and progesterone concentrations in humpback whale blow. The techniques developed in this study to determine reproductive hormones in cetacean saliva and blow have applications for both captive and wild population studies. In captive institutions, saliva and/or blow can be used to monitor reproductive cycling in both females and males. As it is noninvasive it can be used on a daily basis with minimal stress to the animals. The use of blow sampling has the capacity to improve our understanding of reproductive cycling in great whales as it can be used to sample animals in both the breeding and feeding areas. This technique may allow us to now examine whether reproductive dysfunction is playing a role in the slow recovery of critically endangered species such as the North Atlantic right whale.

Doctor of Philosophy (PhD) / Reproductive physiology plays a vital role in population growth and vitality. Baseline data on reproductive physiology and a comprehensive knowledge of breeding biology are essential to conservation management. Great whales have been hunted from the 16th century to the present day. Although many populations are increasing there are populations with low or declining reproductive rates. In 2001 it was recommended to the International Whaling Commission that new techniques be developed to assess the internal physiology of great whales. This study, based on this recommendation, aims to develop analytical methods to assess reproductive hormones in cetacean blow samples and determine the feasibility of its use with free-swimming great whales. A method for the assessment of steroid hormone concentrations using liquid chromatography-mass spectrometry (LC-MS) was developed and validated. These methods were then used to determine testosterone and progesterone concentrations in saliva and blow of bottlenose dolphins. The stability of testosterone and progesterone was found to be a major issue. Without inhibitors, hormone concentrations increased by up to 65% over three hours at 21oC. Storing samples at low temperatures (-20oC or -80oC) slowed but did not cease the rate of change. The addition of inhibitors, manganese chloride and amoxycillin potassium/clavulanate, improved the stability of testosterone and progesterone. It is proposed that when using dolphin saliva and blow samples to measure reproductive hormones the samples are extracted as soon as possible after collection to prevent degradation. This study highlighted the need to address steroid hormone stability prior to any longterm biological program, to ensure that changes seen in hormone concentration are due to biological activity rather than storage. A technique to collect blow samples from free-swimming great whales was developed. This technique, in conjunction with the specially developed LC-MS methods allowed for the determination of testosterone and progesterone concentrations in humpback whale blow. The techniques developed in this study to determine reproductive hormones in cetacean saliva and blow have applications for both captive and wild population studies. In captive institutions, saliva and/or blow can be used to monitor reproductive cycling in both females and males. As it is noninvasive it can be used on a daily basis with minimal stress to the animals. The use of blow sampling has the capacity to improve our understanding of reproductive cycling in great whales as it can be used to sample animals in both the breeding and feeding areas. This technique may allow us to now examine whether reproductive dysfunction is playing a role in the slow recovery of critically endangered species such as the North Atlantic right whale.

Common observations of reduced gonad size and spawning inhibition in wild and laboratory raised fish exposed to pulp mill effluents indicate that reproductive neuroendocrine signalling pathways may be upset. This thesis supported the neuroendocrine disruption of reproduction hypothesis by identifying potential disruptors and targets where these impacts may occur. A mechanistic study of the in vivo fathead minnow (FHM) spawning assay used by industry to assess effluent quality showed that ovulation, but not milt production, was impaired. This finding supported the hypothesis that the neuroendocrine cascade that triggers ovulation may be disrupted. I hypothesized that neuroactive constituents previously described in effluents were originating in wood feedstocks and neuroactive extracts of hardwood and conifer feedstocks were identified. Phytochemicals associated with effluents were neuroactive. Structurally similar phenolic phytochemicals showed monoamine oxidase (MAO) inhibition, and resin acid diterpenes displayed glutamic acid decarboxylase (GAD) inhibition. Inhibitors of these enzymes may have impacts on the control of reproduction since MAO metabolizes dopamine, an inhibitor of the neuroendocrine reproductive axis, while GAD synthesizes -aminobutyric acid (GABA), a stimulator of this axis. Bioassay-guided fractionations of effluents and wood feedstocks identified that medium polar extracts of primary- and secondary-treated effluents and balsam fir feedstock contained high GAD inhibitory activity. This activity was associated with chemically complex fractions rather than single active principles. Advanced metabolomic comparison of medium polar extracts of feedstock and treated effluent identified 15 common plant metabolites, demonstrating that phytochemicals entering the mill in wood are surviving pulp production and effluent treatment processes and may be responsible for observed GAD inhibition. Discriminant metabolomics analysis identified 4-acetylpyridine, a novel compound to be described in effluents, as well as two other tentatively identified compounds, as chemical markers of GAD inhibitory effluent fractions. Five tentatively identified chemical markers and (+)-lariciresinol were found in inhibitory balsam fir feedstock fractions. Neuroendocrine pathways that control reproduction in fish, such as dopamine and GABA pathways, are also important drug targets for the treatment of neurological disorders in mammals; therefore these results also have implications for the development of natural health products from phytochemicals and tree extracts common to Canadian forests. By using an interdisciplinary approach (phytochemistry, neuroendocrinology, ecotoxicology), I was able to explore the various implications of my research on the fields of natural health products chemistry and aquatic toxicology.