Bovine respiratory disease (BRD) is the leading cause of morbidity in feeder and stocker cattle, resulting in large impacts on economics of stocker and feedlot operations. One of the most effective means of controlling BRD is the mass administration of antimicrobials (AM) at arrival or “metaphylaxis”, potentially leading to increased antimicrobial resistance (AMR). Mannheimia haemolytica (MH), the most commonly isolated bacterial pathogen in BRD cases in feedlot cattle, has been shown to have integrative-conjugative elements (ICE), which are mobile genetic elements that have the ability to integrate themselves in the host genome. Notably, these ICE have been shown to contain multiple antimicrobial resistance genes (ARG) conferring resistance to antimicrobial classes used for BRD treatment. ICE have also been shown to be transferred between different genera. Since these ICE contain genes for resistance to multiple AM classes, administration of one AM could increase pressure for bacteria to transfer ICE for resistance to multiple drug classes; therefore, mass administration of AM may lead to increased isolation of multidrug resistant (MDR) MH and increase presence of resistance genes in the metagenome. Many NGS studies to date have used low numbers of cattle or pooled samples due to cost. Pooling is an acceptable strategy to increase number of units sampled, however sequencing depth per individual sample is decreased, and there is little evidence comparing pools to individual samples. In a trial involving high risk stocker cattle, tulathromycin metaphylaxis was associated with increased isolation of MDR MH, and this was associated with ICE related genes. Using pooled DNA extracted from NPS in these animals, which were shown to be acceptable for group level comparisons, metaphylaxis also increased ARG richness and diversity in these heifers; however, BRD treatment and time had a greater effect on the mircrobiome and resistome. Further work is needed to improve MH strain classification. These finding highlight the complexity of AMR research, because though tulathromycin had a clear effect on odds of isolation of MDR MH, metaphylaxis’ effects on the resistome and microbiome were more complex, and time and BRD contributed to greater change.

Vascular Endothelial Growth Factor (VEGF) has been shown to be an absolute requirement for ovarian follicle development. Although VEGF is commonly regarded primarily as an angiogenic factor, granulosa cells are a major site of VEGF synthesis in the follicle and they express VEGF receptors (VEGFR1 and VEGFR2). Further, the development of the dominant follicle is characterised by a substantial increase in granulosa cell expression of VEGF and its receptors. In spite of this, potential non-angiogenic effects of VEGF in these follicles have not been elucidated. The objective of the three studies described in this thesis was to use an in vitro bovine granulosa cell model to investigate the roles of VEGF during development of the dominant follicle. In addition, in light of evidence in other cell types, potential interactions between VEGF signalling and heterotrimeric protein signalling in these follicles were also investigated. In the first study, granulosa cells were obtained from healthy follicles with diameters of 4 to 8 mm (corresponding to just before the selection of a dominant follicle during a follicular wave) or 9 to 14 mm (encompassing all developmental stages of a dominant follicle) and exposed to a range of VEGF concentrations (1 to 100 ng/ml) encompassing concentrations found naturally in bovine dominant follicles. VEGF at 1 ng/ml, but not at higher concentrations (P > 0.1), induced significant proliferation of bovine granulosa cells from 4 to 8 mm follicles (P = 0.024) and increased the proliferative response of these cells to FSH (P = 0.045). VEGF also induced a dose-dependent increase in ERK1/2 activation by granulosa cells from 4 to 8 mm follicles (P < 0.03) but did not have any effect on expression of the steroidogenic enzyme, CYP11A1, by these cells (P > 0.1). VEGF, at a dose of 1 ng/ml (P = 0.003), but not at higher doses (P > 0.1), induced an increase in COX-2 expression by granulosa cells from 9 to 14 mm follicles. In addition, LH stimulation of both ERK phosphorylation (P < 0.05) and COX-2 expression (P < 0.05) in granulosa cells from 9 to 14 mm follicles were prevented (P > 0.1) by specific inhibition of VEGFR2, indicating that VEGF may mediate COX-2 responses to LH in these cells. The second study sought to examine the expression of heterotrimeric G-protein á subunits and PLCâ isoforms by real-time PCR and westen blotting in bovine granulosa cells throughout follicle development to identify specific molecular components of heterotrimeric G-protein pathways that may functionally interact with intracellular VEGF signals. Results showed that GNAS, GNA11 and GNAI2 were all expressed at significantly (P < 0.05) higher levels in granulosa cells of pre-ovulatorysize follicles (10.0 to 13.9 mm) than in cells from smaller follicles (2.0 to 5.9 mm and 6.0 to 9.9 mm). In addition, all PLCB isoforms except PLCB2 were expressed in bovine granulosa cells with PLCB3 being more abundant than PLCB1 and -4. Levels of PLCB3 in granulosa cells from pre-ovulatory-size follicles were much higher (>16-fold; P < 0.005) than in smaller follicles. Immunocytochemical analysis revealed that PLCB3 was located primarily in the cytoplasm, whereas PLCB1 was distributed primarily in the nucleus. These results identified Gs, Gq/11, Gi2 and PLCâ3 as candidates for cross-talk between VEGF and heterotrimeric G-protein signalling during the development of the dominant follicle. The potential involvement of these molecules on VEGF-induced responses in granulosa cells from 9-14 mm follicles was investigated in the third study by determining the effects of specific inhibitors of Gi (pertussis toxin, PTX) or Gq/11 (YM-25489) or PLCB3 siRNAs on VEGF-induced p-ERK. Results showed a 2.3 fold mean increase in p-ERK in response to VEGF in the absence of G protein inhibitors (P < 0.0001) but a VEGF response that was completely or partially abolished, respectively, in the presence of PTX (P > 0.8) or YM-25489 (1.6-fold mean increase relative to untreated controls; P = 0.039). LH induced a 1.6 fold increase in p-ERK1/2 (P < 0.02) and this response was prevented by pre-incubation with PTX (P > 0.4) or YM-25489 (P > 0.5). In contrast, similar EGF-induced phosphorylation of ERK (about 5-fold relative to controls) occurred in the absence (P < 0.003) or presence of PTX (P < 0.003) or YM-25489 (P < 0.003). Transfection of granulosa cells with 3 siRNAs targeting PLCB3 that had been previously validated by western blotting and immunocytochemistry had no effect (P = > 0.7) on phosphorylation of ERK in response to VEGF, LH or EGF in granulosa cells. In conclusion, taken together, these results suggest novel roles of VEGF in stimulating granulosa cell proliferation and expression of COX-2 in bovine dominant follicles and implicate VEGF in synergising and/or mediating the effects of gonadotrophins in these cells. In addition, these results indicate a requirement for Gi2 and Gq/11 in VEGF activation of ERK1/2 and induction of the above responses in granulosa cells.

Made available in DSpace on 2015-03-26T13:55:24Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1753652 bytes, checksum: 23724be0973af46287264619b5b0e719 (MD5) Previous issue date: 2014-07-10 / This is a data survey from the main commercial mineral supplements for dairy and beef cattle currently available in the Brazilian market. 307 products have been listed, from eight of the leading companies operating in Brazil. The products have been listed in six categories: ready-to-use for beef cattle ; ready-to-use for dairy cattle ; units to produce beef cattle concentrate ; units to produce dairy cattle concentrate ; premix , which must be added to NaCl; and protein salts n order to evaluate their minerals, vitamins, ionophores, yeast, urea and crude protein composition, and compare the mineral levels with the Brazilian legislation (IN12, 2004). The ready to use products do not fulfill the legal specifications for phosphorus level in all products. The same happens in some "premix" products in 1:1 dilution with NaCl, when indicated for dairy cattle. This mineral level is 64.9 % higher in products to produce dairy cattle concentrate , when compared with the same kind of products for beef cattle. The P average for protein salts category is 28.5 g/kg and in order to meet the legal specifications, the ingestion must be considered. When it comes to micro minerals the premix category does not comply with the specifications in Cu , Mn , Zn and Se in 1:1 dilution for dairy cattle. The ready to use category meets all minerals specifications for dairy cattle but it does not for beef cattle, only part of the mineral range. Dairy cattle products to produce concentrate have higher viimicrominerals levels than those used for beef cattle, and the protein salts have lower levels compensated by higher ingestion. The legislation does not detail all animal categories. This fact is associated with some other factors as the large territory and massive herd make the Brazilian market gather around the conditions to group an extensive range of products. Therefore, the current legislation itself is not able to mold products for each mineral supplements situation that can occur in all Brazilian production systems. / Foi realizado um levantamento de dados dos principais suplementos minerais voltados para a bovinocultura leiteira e de corte disponíveis atualmente no mercado brasileiro. Foram listados 307 produtos, de oito das principais empresas desse segmento no Brasil. Os produtos foram elencados em seis categorias: produtos prontos para utilização para gado de corte ; produtos prontos para utilização em gado leiteiro ; núcleos para fabricação de concentrado para gado de corte , núcleos para fabricação de concentrados para gado leiteiro; premix ou produtos a serem misturados ao NaCl; e proteinados. Objetivou-se agrupar as composições de minerais, vitaminas, ionóforos, levedura, ureia e proteína bruta, bem como comparar os níveis minerais dos produtos com a legislação pertinente (IN 12, 2004). Os níveis de P não atendem às especificações da legislação em todos os produtos prontos para uso. Na categoria premix , a diluição em 1:1 com o NaCl não atende à totalidade dos produtos quando a indicação é para bovinos leiteiros. Para os núcleos de fabricação de concentrado, os níveis desse mineral são 64,9% mais altos nos produtos destinados à pecuária leiteira do que nos produtos para corte. Nos proteinados, a média do P ficou em 28,5 g/kg, e seu atendimento às especificações do MAPA depende do consumo. Os teores de microminerais na categoria premix não atendem às especificações para os microminerais Cu, Mn, Zn e Se em diluição de 1:1 para bovinos de leite. Nos produtos prontos para uso em bovinos leiteiros, os níveis mínimos de micros atendem às vespecificações do MAPA; já nos produtos para bovinos de corte, esses níveis não são atendidos em todos os micros. Os núcleos para rações também possuem médias mais altas nos produtos para leite, em comparação aos de corte, e os proteinados possuem médias baixas fato compensado por maiores consumos. Observou-se que a legislação não discorre detalhadamente sobre todas as categorias de animais e que outros fatores, como grande extensão territorial e rebanho massivo, criaram situações para que o mercado brasileiro reunisse uma gama muito extensa de produtos. Assim, a legislação atual, por si só, não é capaz de moldar os produtos destinados à mineralização bovina para atender a todas as situações que ocorrem nos diversos sistemas de produção brasileiros.