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Copies of 16 publications from the thesis, authored and co-authored by the author, included as appendix. / Includes bibliographical references (leaves 195-220). / 1 v. (various pagings) : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines 3 hypotheses: that the lipid levels and fatty acid composition of meat produced in Australia may vary between cattle genotypes, ages, sexes, seasons, and anatomical sites of sampling; that genetic variation may be sufficiently large to warrant selective crossbreeding of dams to different sire genotypes as an improvement strategy in the proportion of monounsaturated fatty acids in beef; and, that genotype differences in bovine tissue may exist when phospholipid and triacylglycerol fractions of total lipids are analysed separately, regardless of fatness of the cattle. Genetic variation in fatty acid composition of 7 different cattle breads were examined in experiments using non-lactating cows, yearling steers, yearling heifers and weaner calves. Breed differences were found in the muscle and adipose tissues when phospholipid and triacylglycerol fractions were analysed separately regardless of the fatness of the cattle. Differences in age, sex, season and anatomical site were also significant. The study concludes that breed differences in fatty acid composition are related to fatness and stage of maturity such that early-maturing cattle are fatter, contain higher proportions of unsaturates, and have softer fats with low melting points than lean, late-maturing cattle. It is recommended that total lipids be separated into triacylglycerol and phospholipid fractions and analysed separately. Desturation indexes could be used as a biochemical marker for beef breeding decisions, and genetic parameters presented used for future selection indices for fatty acids in carcass quality assessment. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1999

The objective of this study was to evaluate the variability of growth curves of dairy heifers and estimate genetic parameters. 15066 records taken from birth until 26 months (808 days) on 2754 heifers of Quebec were considered. The pedigree file comprised 10123 animals. The Mixed procedure of SAS with ordinary polynomials was used for simple phenotypic analyses, fitting fixed linear, quadratic and cubic regressions of body weight (in kilograms) on age (in months) as well as random intercept, and random linear and quadratic regressions for each animal. The Wombat program (Meyer, 2007), with Legendre polynomials was used to estimate the genetic parameters by fitting fixed herd-year-season of birth and quartic regression of body weight on age in days, as well as random regressions for quadratic additive genetic and cubic permanent environmental effects. Heritability estimates of body weight ranged from 0.22 at around 70 days to 0.45 at around 210 days. Heritabilities of body weight at birth and 808 days were 0.35 and 0.32, respectively. The additive genetic correlations between body weights at different ages ranged from -0.37 to 1.00. In general, the genetic correlations were higher than the permanent environmental and phenotypic correlations.

It is well known that DNA methylation in gene promoter regions inhibits gene transcription and that tissue-specific gene expression is partially under the control of this transcription regulatory mechanism. In this study, bovine mammary gland tissues were collected from individual animals in lactating and non-lactating stages to investigate the DNA methylation patterns in the kappa-casein gene and alpha-lactalbumin gene core promoter regions using the bisulphite treatment in combination with polymerase chain reaction (PCR) sequencing. Different methylation status of each sample was classified into three categories, namely methylation at known transcription factor binding domains, methylation at core promoter non-binding domains and the absence of cytosine methylation. Real-time quantitative PCR was used to quantify the transcription levels of the kappa-casein and alpha-lactalbumin genes from the collected samples. A comparative method was used and fold-change values were calculated based on the comparison of the normalized threshold values of samples from different physiological stages as well as on various methylation patterns observed in their core promoter regions. Statistical analyses showed that the expressions of the kappa-casein and alpha-lactalbumin genes were significantly different in lactating and non-lactating mammary gland tissues. The methylation observed in the core promoter region of bovine alpha-lactalbumin gene was found to be associated with its gene expression. On the other hand, the methylation found in the core promoter region of bovine kappa-casein gene did not have any effect on its gene transcript levels.

The growth hormone receptor gene (GHR) has been previously documented to be a good candidate gene for detection of a quantitative trait locus (QTL) which influences milk production in Holstein cattle. In this study, the promoter region of the GHR gene and microsatellite markers AGAL29 and BM5004 were studied. Their effects on milk yield (MY), fat yield (FY), protein yield (PY), fat percentage (FP) and protein percentage (PP) were examined. DNA was isolated from 1746 used by the artificial insemination (AI) industry representing 26 half-sibling families. Three polymorphisms in the GHR gene were genotyped (GHRAlu, GHRAcc and GHR Stu) along with both microsatellites. The markers were analyzed in a cross-family analysis. The model included a population mean, a fixed grandsire effect, a fixed allele effect and a random residual error. The data was also analyzed using a nested model in a granddaughter design to investigate a possible consistency in the allelic effect in individual families. Lastly, the data was analyzed using the haplotypes of GHRAlu and GHR Acc, using the same model as the cross-family analysis. It included an analysis of a fixed haplotype effect instead of a fixed allele effect. (Abstract shortened by UMI.)

Laboratory scale Cheddar type cheese were made from 411 milk samples originating from Ayrshire, Brown Swiss, Canadienne and Jersey with different phenotypes of kappa-casein (kappa-CN) and beta-lactoglobulin (beta-LG). From the milk input, cheese and whey outputs, the cheese yield, cheese composition and cheesemaking efficiency and other parameters were determined. The overall 37% moisture adjusted cheese yield and cheese yield efficiency were 11.19g, 10.19g, 11.97g, 11.46g and 98.91%, 90.10%, 90.76% and 90.92% for Ayrshire, Brown Swiss, Canadienne and Jersey respectively. The milk of the four respective breeds contained 12.91, 12.69, 13.50, 14.57% total solids; 3.97, 3.58, 4.40, 4.61% fat; 3.61, 3.73, 3.81, 4.00% protein. In Ayrshire, the combination BB/BB and BB/AA (kappa-CN/beta-LG) were associated with higher 37% moisture adjusted cheese yield with the values of 12.51 and 12.83 g/100 g milk respectively. The cheese composition for these two types of milk were 62.28 and 63.96% total solids, 24.22 and 20.40% protein; 32.75 and 37.92% fat. For Brown Swiss, type AA/BB was associated with higher cheese yield (11.18g) with composition of 62.15% total solids, 22.54% protein, 33.23% fat. The phenotype with the highest cheese yield for Canadienne is BB/AB (12.45g) with cheese composition of 63.61% total solids, 23.27% protein and 63.61% fat. In Jersey, the phenotype combination with higher cheese yield (14.59g) is BB/BB. The cheese composition corresponding to this phenotype was 58.64% total solids, 22.96% protein and 29.59% fat. Phenotypes associated with better coagulating properties for Ayrshire, Brown Swiss, Canadienne and Jersey were BB/AB, BB/BB, BB/BB and BB/BB for kappa-CN/beta-LG respectively.

Neutrophil potential phagocytic capacity (NPPC), measured on 25 AI Canadian Holstein bulls, was investigated for evidence of association with production and type traits, SCC, and survival in dairy cows. Bulls were ranked based on different degrees of NPPC (Uptakes of 0, 1, 2, and 3 or more latex beads), using the solutions coming from an animal model. A total of 42,103 first lactation records, collected from 1985 through 1993 in 2,919 Quebec dairy herds, were used to obtain EBV's for SCC and for log SCC (LogSCC) for 697 sires. Correlations between NPPC measurements and somatic cell EBV's were null. Canadian official ETA's for type traits related with mammary system had a tendency of being positively correlated with higher NPPC and negatively with Uptake-0. Canadian official ETA's for production traits were negatively correlated with higher NPPC and positively with Uptake-0. A total of 17,202 first lactation records of daughters of the 25 AI bulls were used to study the effect of NPPC and log SCC on survival in dairy cows. Survival after first lactation was more closely related to sires' NPPC-EBV's than to LogSCC-EBV.

DNA methylation has been shown to be involved in switching a number of genes on or off in particular cells. The relationship between DNA methylation and $ beta$-casein gene expression in the mammary tissue of lactating cows and mammary epithelial cells was examined. A positive correlation existed between hypomethylation of two MspI/HpaII sites in the body and one MspI/HpaII site in the 3$ sp prime$ end of the $ beta$-casein gene and its expression. In addition to these sites, hypomethylation of a distal MspI/HpaII site and HindIII sensitivity at a HindIII site also correlated with gene expression. Five DNase I hypersensitive sites were located within a 8 kb fragment. These sites designated as H1 to H5 were mapped approximately $-5, -1.3, -0.2,$ 1.7 and 2.5 kb with respect to the start site of transcription, respectively. The H2 and H3 sites were within a 1790 bp sequence that has been reported to contain a responsive element for prolactin and extracellular matrix dependent regulation and the binding site for mammary gland specific factor. / To study the dynamic changes in hypomenthylation at the MspI/HpaII sites and HindIII sensitivity, mammary tissues from pregnant heifers were evaluated. Site specific demethylation was observed depending on the stage of gestation. Demethylation of two MspI/HpaII sites (denoted M2 and M4) occurred during the early gestation, progressed slowly until mid-pregnancy, and rapidly during the last part of pregnancy. During the early stages of gestation, changes in the HindIII sensitivity in the coding domain of the $ beta$-casein gene also took place. Despite changes in HindIII sensitivity, the second HindIII site remained resistant to HindIII. By the fifth stage of gestation, the third MspI/HpaII site (M3) became less methylated and during this time the H2 site became more sensitive to HindIII. Northern analysis confirmed that demethylation of the M3 site and the acquisition of HindIII sensitivity at the H2 site was correlated with $ beta$-casein transcription. / Although $ alpha$-lactalbumin and $ beta$-casein genes are structurally and evolutionarily unrelated, they likely share common regulatory features, since both are expressed in the mammary gland during lactation. To investigate this possibility, methylation of the $ alpha$-lactalbumin gene was examined. In vivo studies revealed hypomethylation of the bovine $ alpha$-lactalbumin gene at two MspI sites and a cluster of two HhaI sites during the first and second stage of gestation, respectively. Furthermore, hypomethylation events occured only in the functional gene and not in pseudogenes, and the hypomethylation pattern was established prior to gene expression. / Taken together, the present finding suggest that DNA hypomethylation is necessary for the expression of two mammary-specific milk protein genes, $ beta$-casein and $ alpha$-lactalbumin. Hypermethylation within the body of these genes may silence these genes in non-expressing tissues and in non-epithelial cells within the mammary gland during lactation.

Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-07-19Bitstream added on 2014-06-13T20:26:10Z : No. of bitstreams: 1 rascado_ts_dr_botfmvz.pdf: 519776 bytes, checksum: 01ba22778e4b1e2e1003285f9b0b7014 (MD5) / Este experimento objetivou analisar o padrão de expressão do mRNA de SOX2, STAT3 e GBX2 em embriões bovinos produzidos in vitro nos estádios de blastocisto (E7) e blastocisto eclodido (E10). O RNA foi extraído de embriões em cada fase do desenvolvimento embrionário (n=7) e da massa celular interna (MCI) e epiblasto isolados por imunocirurgia de 20 embriões (n=20). A expressão do mRNA foi obtida por transcriptase reversa seguida da reação em cadeia da polimerase em tempo real (RT-qPCR), quantificada pelo método da curva padrão e normalizada pela média geométrica de GAPDH, YWHAZ e SDHA. Os dados de cinco replicatas foram analisados por ANOVA seguido de comparações, aos pares, pelo teste de Tukey. A expressão relativa do mRNA de SOX2 foi significativamente maior em blastocistos (E7) do que em blastocistos eclodidos (E10) (P<0,05), sendo que a expressão na MCI foi 40X maior do que a obtida no blastocisto inteiro (P<0,05). A expressão relativa do mRNA de STAT3 3 não diferiu entre blastocistos (E7) e blastocistos eclodidos (E 10) (P>0,05). Não houve diferença entre blastocisto eclodido (E10) e epiblasto para SOX2 e STAT3 (P>0,05). Comparando-se o nível relativo de mRNA de GBX2 entre blastocistos (E7) e MCI e blastocisto eclodido (E10) e epiblasto não foi detectada diferença (P>0,05), sendo que MCI e epiblasto corresponderam a aproximadamente 90% da expressão observada nos embriões em suas respectivas fases de desenvolvimento. Portanto, com o desenvolvimento do blastocisto, há a tendência do aumento dos níveis de mRNA de STAT3 e SOX2 nas células pluripotentes do epiblasto; o nível de mRNA de GBX2 se mantém constante em blastocistos, com alta expressão em células pluripotentes / This experiment aimed to analyze the pattern of expression of the mRNA of SOX2, STAT3 and GBX2 in in vitro produced bovine embryos at stages of blastocyst (E7) and hatched blastocyst (E10). The RNA was extracted from embryos at each stage of embryonic development (n= 7) and from the inner cell mass (ICM) and epiblasts isolated from 20 embryos by immunosurgery. The expression of mRNA was obtained by reverse transcriptase-polymerase chain reaction (real-time qPCR), quantified by the standard curve method and normalized by geometric mean of genes YWHAZ, SDHA and GAPDH. The data from five replicates were analyzed by ANOVA followed by comparisons, in pairs by Tukey test. The relative expression levels of SOX2 mRNA was significantly higher in blastocysts (E7) than in hatched blastocysts (E10) (P<0.05), whereas that the expression in MCI was 40X greater than the expression obtained in blastocyst (P< 0.05). The relative expression of STAT3 mRNA did not differ between blastocysts (E7) and hatched blastocysts (E 10) (p>0.05). There was no difference between blastocyst hatched (E10) and epiblasts for SOX2 and STAT3. By Comparing the relative level of GBX2 mRNA between blastocysts (E7) and ICM and blastocyst hatched (E10) and epiblasts no difference was detected (P>0.05), ICM and epiblasts corresponded to approximately 90% of expression observed in embryos in their respective stages of development. Therefore, with the development of the blastocyst, there is a tendency for increased levels of STAT3 and SOX2 mRNA in the pluripotent cells of epiblasts; the level of GBX2 mRNA is constant in blastocysts with high expression in pluripotent cells

Orientador: Fernanda da Cruz Landim / Coorientador: João Pessoa Araujo Junior / Banca: Sony Dimas Bicudo / Banca: Fabiana Ferreira Sousa / Banca: Felipe Perecin / Banca: Cláudia Barbosa Fernandes / Resumo: Este experimento objetivou analisar o padrão de expressão do mRNA de SOX2, STAT3 e GBX2 em embriões bovinos produzidos in vitro nos estádios de blastocisto (E7) e blastocisto eclodido (E10). O RNA foi extraído de embriões em cada fase do desenvolvimento embrionário (n=7) e da massa celular interna (MCI) e epiblasto isolados por imunocirurgia de 20 embriões (n=20). A expressão do mRNA foi obtida por transcriptase reversa seguida da reação em cadeia da polimerase em tempo real (RT-qPCR), quantificada pelo método da curva padrão e normalizada pela média geométrica de GAPDH, YWHAZ e SDHA. Os dados de cinco replicatas foram analisados por ANOVA seguido de comparações, aos pares, pelo teste de Tukey. A expressão relativa do mRNA de SOX2 foi significativamente maior em blastocistos (E7) do que em blastocistos eclodidos (E10) (P<0,05), sendo que a expressão na MCI foi 40X maior do que a obtida no blastocisto inteiro (P<0,05). A expressão relativa do mRNA de STAT3 3 não diferiu entre blastocistos (E7) e blastocistos eclodidos (E 10) (P>0,05). Não houve diferença entre blastocisto eclodido (E10) e epiblasto para SOX2 e STAT3 (P>0,05). Comparando-se o nível relativo de mRNA de GBX2 entre blastocistos (E7) e MCI e blastocisto eclodido (E10) e epiblasto não foi detectada diferença (P>0,05), sendo que MCI e epiblasto corresponderam a aproximadamente 90% da expressão observada nos embriões em suas respectivas fases de desenvolvimento. Portanto, com o desenvolvimento do blastocisto, há a tendência do aumento dos níveis de mRNA de STAT3 e SOX2 nas células pluripotentes do epiblasto; o nível de mRNA de GBX2 se mantém constante em blastocistos, com alta expressão em células pluripotentes / Abstract: This experiment aimed to analyze the pattern of expression of the mRNA of SOX2, STAT3 and GBX2 in in vitro produced bovine embryos at stages of blastocyst (E7) and hatched blastocyst (E10). The RNA was extracted from embryos at each stage of embryonic development (n= 7) and from the inner cell mass (ICM) and epiblasts isolated from 20 embryos by immunosurgery. The expression of mRNA was obtained by reverse transcriptase-polymerase chain reaction (real-time qPCR), quantified by the standard curve method and normalized by geometric mean of genes YWHAZ, SDHA and GAPDH. The data from five replicates were analyzed by ANOVA followed by comparisons, in pairs by Tukey test. The relative expression levels of SOX2 mRNA was significantly higher in blastocysts (E7) than in hatched blastocysts (E10) (P<0.05), whereas that the expression in MCI was 40X greater than the expression obtained in blastocyst (P< 0.05). The relative expression of STAT3 mRNA did not differ between blastocysts (E7) and hatched blastocysts (E 10) (p>0.05). There was no difference between blastocyst hatched (E10) and epiblasts for SOX2 and STAT3. By Comparing the relative level of GBX2 mRNA between blastocysts (E7) and ICM and blastocyst hatched (E10) and epiblasts no difference was detected (P>0.05), ICM and epiblasts corresponded to approximately 90% of expression observed in embryos in their respective stages of development. Therefore, with the development of the blastocyst, there is a tendency for increased levels of STAT3 and SOX2 mRNA in the pluripotent cells of epiblasts; the level of GBX2 mRNA is constant in blastocysts with high expression in pluripotent cells / Doutor