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The American dog tick, Dermacentor variabilis, is an obligate blood feeding ectoparasite. This tick is a known vector of pathogens that affect the health of wildlife, humans, and livestock and is abundant in Manitoba. The etiological agent of bovine anaplasmosis, Anaplasma marginale, along with members of the spotted fever group rickettsiae are bacteria that are transmitted by this tick. I examined the distribution of these bacteria in Manitoba’s tick population using molecular techniques. During the eradication of an outbreak of bovine anaplasmosis in Manitoba, there was no evidence the bacterium had spilled over into the tick population. Rickettsia montanensis was detected with a mean prevalence of infection of 9.8% (range, 0.00 - 21.74% among localities) in 8 of 10 localities within the province. It was also determined that 19.9% (SE ±1.14) of adult questing ticks collected in one vector season overwintered through to the next spring.

Accurate and reliable measurement of meat quality is essential for the Australian beef industry to remain competitive in both the domestic and export markets. Recent developments of the resonator technique have lead to the commercial availability of the High Resolution Ultrasound Spectroscope (HR-US). This research project was designed to assess the potential of HR-US for the analysis of post-mortem bovine muscle. This was accomplished by; 1) establishing a suitable measurement protocol that considered sources of variability, 2) the effects of post-mortem aging on HR-US parameters, 3) analysis of thermal related changes that occur in muscle, and extracted connective tissue during heating, and 4) the use of HR-US for the measurement of the intramuscular fat. A procedure for the measurement of bovine muscle with HR-US was established. Briefly, an external semisolid cell was used as the measurement cell. The frequency range of 2000 KHz to 3000 KHz was selected as the most suitable for whole muscle analysis and all five resonance peaks within this range were analysed and used to obtain velocity and attenuation values of the meat sample. Water was used as the reference media, and measurements were conducted at 250C. Changes were made to this method during experimental work depending on the sample being run and the objectives of the study. The measurement protocol was shown to be repeatable. Factors likely to cause variation in measurements of the samples, such as water loss and freeze-thaw, were also considered when developing the operational parameters of the study. High resolution ultrasound spectroscopy was applied to measure the post-mortem changes that occur in bovine muscle. Using two muscle types, Semitendinosus and Psoas major, significant changes were observed in HR-US parameters with ageing. Significant increases in the acoustic impedance of bovine muscle with increased ageing time were attributed to degradation of the muscle structure. This was confirmed in transmission electron microscopy images where clear disruption the myofibillar structure was apparent in the muscle at 21 days post-mortem.In localised regions the Z bands and the adjoining actin fibres were totally degraded. Water loss from the muscle system had a significant influence on HR-US measurements. Thermal related changes that occur in whole bovine muscle and in isolated intramuscular connective tissue were observed with HR-US. Heat induced changes were identified in whole muscles and included the melting of the fat within samples at 48oC, coagulation of sarcoplasmic proteins between 450C and 55oC, and the shrinkage of collagen fibres at 630C. An 80% reduction in the attenuating properties of extracted connective ageing in buffer solution was observed within the first 5 days. This is attributed to the degradation of proteoglycans and the resulting disassociation of collagen fibrils. Structural changes occurring in extracted connective tissue were observed with TEM. HR-US measurements proved to be highly sensitive to identifying temperatures at which transitions occurred. Unfolding of the triple helix structure of collagen was identified in velocity transitions between 59°C and 63oC. HR-US results suggested a greater sensitivity to thermal related changes in extracted intramuscular connective tissue when compared with differential scanning calorimeter results. An increase in temperature was observed for thermal denaturation of collagen with ageing, however a reduction was also observed in the temperature range at which the denaturation process occurred. Temperature ramps conducted on extracted intramuscular bovine showed a reduction in velocity from 1613.1m/s at 250C to 1343.1 m/s at 900C equalling an overall reduction in velocity of 270m/s. A transition in the velocity trend seen at 46°C indicates the majority of the triglycerides are melted (or in liquid state) above this temperature. Results are confirmed with differential scanning calorimeter thermogram. HR-US measurements showed high sensitivity to increasing concentration of bovine fat in prepared emulsions with an adjusted R2 99.46% for velocity measurements taken at 5100 kHz. Attenuation values at 8100 kHz also showed a strong linear response to increasing fat concentration in the emulsion (R2 98.77). The use of HR-US for the measurement of intramuscular bovine fat demonstrated a high sensitivity to extracted bovine fat in prepared emulsions. An increase in the intramuscular fat content of whole bovine muscles resulted in a reduction in the velocity measurements and an increase in the attenuation of the ultrasonic signal. This provides the basis for potential method for the prediction intramuscular fat in bovine muscle. The present studies have highlighted the complexities of investigations relating to meat quality and have demonstrated the diversity of data required to assess quality. Only when comprehensive data are available, can we hope to accurately determine meat quality and predict how it will vary with changes in animal production and meat processing.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

Abstract Biotechnological strategies aimed at producing male-only offspring have the potential to improve the yield of the Australian beef industry. As a proof-of-concept project, I aimed to target the primary male sex-determining gene Sry to the X chromosome in mice, to produce a transgenic XY male that would transmit Sry – and hence maleness – to both XX and XY offspring. In this project I aimed to target a 14.5 kb DNA fragment containing Sry to an X-chromosome locus that escapes X-inactivation. After considering many potential loci, a targeting strategy and construct were designed for the SMCX locus, which is well conserved between mouse, human and bovine. A targeting vector with 5kb and 3kb arms of homology was also constructed without Sry, to target the locus. Attempts to introduce the 14.5 kb Sry fragment into the construct were unsuccessful, and a smaller construct, containing only the coding sequence of the Sry gene driven by a strong promoter, is currently being made. In order to translate this transgenic approach into cattle, other facets of bovine sex determination required investigation. First, it was important to identify the necessary regulatory regions upstream of bovine SRY needed for the gene to be functional, and secondly to investigate the timing of testis development in male bovine embryos. To enable sequence comparison, I sequenced upstream of the bovine and goat SRY gene and through bioinformatic analysis identified regulatory regions common to several mammals. I identified four regions of high homology upstream of bovine SRY conserved between human, goat, and pig, but not mouse. These regions are likely to be important for the regulation of the gene in these species, as they share unique transcription factor binding sites. From this research I concluded that 9 kb upstream of bovine SRY were likely to be useful in transgenic strategies to produce sex-reversed cattle. Although I attempted to use a 15 kb bovine genomic fragment containing SRY to sex reverse XX mice, this project was unsuccessful. I also investigated the expression pattern of genes known to have a role in sex determination, including SRY, in early bovine embryos. I identified the major time points important for male sex determination, including the first appearance of the gonadal ridge from the mesonephros at day 31, the onset of SRY expression and its peak at day 39, and the appearance of testis cords at day 42, along with the pattern of expression of many other genes downstream of SRY. This information will allow future researchers to check that transgenic SRY expression is occurring at the correct time and place for it to be able to cause XX sex reversal in cattle. I also identified some of the major time points important for female sex determination, including that ovigerous cords form between CRL 37-91 in female bovine embryos. In addition I show the cellular differentiation of the cortex and medulla at this time. I have also predicted the female germ cell entry into meiosis around CRL 40 in bovine embryos through the use of qRT-PCR for STRA8 and SYCP3. This is the first detailed account of gene expression profiles in early female bovine embryos, unfortunately the data is incomplete due to an uneven distribution of embryo ages due to the difficulty of obtaining embryos from timed matings. Hopefully in the future obtaining more female embryos of the missing stages can complete the female data. This project has provided additional basic knowledge about bovine sex-determination events to ensure the possibility of making single-sex livestock a real possibility in the future. The similarity between human and bovine developmental time frames also points to cattle being a good alternative model for human development, and emphasises the need for further research in species other than mouse, with the aim of ultimately understanding our own biology.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.