Orientador: Reinaldo Fernandes Cooke / Resumo: Vacas leiteiras passam por muitas mudanças fisiológicas e metabólicas durante o período de transição (Gummer, 1995; Drackely, 1999). Essas prejudicam o sistema imune, expondo os animais a distúrbios metabólicos e doenças infecciosas (Mallard et al., 1998). Omingen-AF® (OMN) é um produto comercial que demonstrou potencial de modular a resposta imune de animais de produção (Wang et al., 2009). Com o objetivo de avaliar o potencial de OMN como suplemento nutricional para vacas leiteiras durante o período de transição, este estudo comparou parâmetros produtivos, fisiológicos e de saúde em vacas leiteiras suplementadas ou não com OMN durante o período de transição. Trinta e oito vacas mestiças Holandês-Gir prenhas, multíparas e não-lactantes foram classificadas por peso corporal (PC) e escore de condição corporal (ECC) e designadas a receber (OMN; n=19) ou não (CON; n=19) 56 g/vaca/dia de OMN iniciando 35 dias antes da data estimada para o parto. Omnigen foi suplementado individualmente às vacas sobre o concentrado oferecido durante o período da manhã. Antes do parto, o PC e ECC das vacas foi aferido semanalmente e amostras de sangue foram coletadas a cada 5 dias, iniciando no d-35 em relação à data prevista para a parição. Após o parto e até 46 dias em leite (DEL), o PC e ECC foram aferidos também semanalmente. O registro da produção de leite individual e a coleta de amostras de leite para análise de sólidos totais e Contagem de Células Somáticas (CCS) e de amostras de sangue de ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Submitted by Alice Poggi Brandão null (alicepbrandao@gmail.com) on 2016-12-30T00:28:42Z No. of bitstreams: 1 Dissertação Mestrado Alice.pdf: 1977086 bytes, checksum: 6fcceacacc9b5a4bac4bdb99a6c2b5d5 (MD5) / Rejected by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido está sem a ficha catalográfica. A versão submetida por você é considerada a versão final da dissertação/tese, portanto não poderá ocorrer qualquer alteração em seu conteúdo após a aprovação. Orientamos que indique o número de processo do financiamento FAPESP nos agradecimentos. Corrija estas informações e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-01-03T16:31:54Z (GMT) / Submitted by Alice Poggi Brandão (alicepbrandao@gmail.com) on 2017-01-04T15:44:55Z No. of bitstreams: 1 Dissertação Mestrado Completa.pdf: 2053776 bytes, checksum: 0d72a35c8158fb28fb236bdfa3476e1c (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-01-06T12:39:56Z (GMT) No. of bitstreams: 1 brandao_ap_me_bot.pdf: 2053776 bytes, checksum: 0d72a35c8158fb28fb236bdfa3476e1c (MD5) / Made available in DSpace on 2017-01-06T12:39:56Z (GMT). No. of bitstreams: 1 brandao_ap_me_bot.pdf: 2053776 bytes, checksum: 0d72a35c8158fb28fb236bdfa3476e1c (MD5) Previous issue date: 2016-12-19 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Vacas leiteiras passam por muitas mudanças fisiológicas e metabólicas durante o período de transição (Gummer, 1995; Drackely, 1999). Essas prejudicam o sistema imune, expondo os animais a distúrbios metabólicos e doenças infecciosas (Mallard et al., 1998). Omingen-AF® (OMN) é um produto comercial que demonstrou potencial de modular a resposta imune de animais de produção (Wang et al., 2009). Com o objetivo de avaliar o potencial de OMN como suplemento nutricional para vacas leiteiras durante o período de transição, este estudo comparou parâmetros produtivos, fisiológicos e de saúde em vacas leiteiras suplementadas ou não com OMN durante o período de transição. Trinta e oito vacas mestiças Holandês-Gir prenhas, multíparas e não-lactantes foram classificadas por peso corporal (PC) e escore de condição corporal (ECC) e designadas a receber (OMN; n=19) ou não (CON; n=19) 56 g/vaca/dia de OMN iniciando 35 dias antes da data estimada para o parto. Omnigen foi suplementado individualmente às vacas sobre o concentrado oferecido durante o período da manhã. Antes do parto, o PC e ECC das vacas foi aferido semanalmente e amostras de sangue foram coletadas a cada 5 dias, iniciando no d-35 em relação à data prevista para a parição. Após o parto e até 46 dias em leite (DEL), o PC e ECC foram aferidos também semanalmente. O registro da produção de leite individual e a coleta de amostras de leite para análise de sólidos totais e Contagem de Células Somáticas (CCS) e de amostras de sangue de 0 a 7 DEL foram realizados diariamente. De 9 a 21 DEL, amostras foram coletadas em dias alternados e de 26 a 46 DEL, a cada 5 dias. Todas as amostras de sangue, do pré e pós-parto, foram avaliadas para cortisol, ácidos graxos não-esterificados (AGNE), insulina, glicose, haptoglobina, cortisol e IGF-1. Nos dias 30 e 46 após o parto vacas foram avaliadas para endometrite através da técnica do cytobrush, baseada na porcentagem de células polimorfonucledas (PMN) no total de uma contagem de 100 células (PMN + células endometriais). No dia 48,7 ± 1,6 após o parto, 9 vacas de cada tratamento receberam uma injeção de lipopolissacarídeo (LPS) (0,25μg/kg de PC) e amostras de sangue foram coletadas a cada hora de -2h até 8h após a injeção às 0h; a cada 12h das 12 às 72h e a cada 24h das 96 às 120h. Amostras de sangue coletadas durante o desafio de LPS foram analisadas para haptoglobina e de fator de necrose tumoral alfa (TNF-α). Não foi observado efeito de tratamento sobre o PC (P ≥ 0,15), ECC (P ≥ 0,55), concentrações séricas de cortisol, AGNE, insulina, glicose, haptoglobina, cortisol e IGF-1. Vacas recebendo OMN apresentaram maior (P = 0,02) produção de leite (30,3kg/d vs. 27,1kg/d) e maior (P = 0,04) porcentagem de células PMN na população celular endometrial (12,2% vs. 3,9%) quando comparadas com vacas recebendo CON. Após a administração do LPS, vacas do grupo OMN apresentaram concentração sérica média superior (P = 0,04) de haptoglobina (212 μg/mL vs. 94 μg/mLpara vacas dos grupos OMN e CON, respectivamente) assim como maior concentração plasmática de TNF-α (P ≤ 0,04) nos momentos 1 (1,34 vs. 2,35 ng/mL para OMN e CON respectivamente), 2 (0,79 vs. 2,01 ng/mL, para OMN e CON respectivamente) e 3h (0,41 vs. 1,34 ng/mL, para OMN e CON respectivamente) em relação à injeção de LPS. Em conclusão, a suplementação de Omnigen-AF® durante o período de transição elevou a concentração plasmática de marcadores da resposta imune inata e aumentou a produção de leite em vacas leiteiras. / Dairy cows undergo several physiological and metabolic changes during the transition period. (Grummer, 1995; Drackley, 1999). These changes impair the immune function and cows become more susceptible to infectious and metabolic diseases (Mallard et al., 1998). Omnigen-AF® (OMN) is a commercial feed ingredient that has been shown to modulate the immune response in livestock animals (Wang et al., 2009). Aiming to evaluate the potential of OMN as a feed additive for transition dairy cows, this study compared physiological, health, and productive parameters in dairy cows supplemented or not with OMN during the transition period. Thirty-eight non-lactating, multiparous, pregnant Holstein × Gir cows were ranked by body weight (BW) and body condition score (BCS), and assigned to receive (OMN; n = 19) or not (CON; n = 19) 56 g/cow daily (as-fed basis) of OMN beginning 35 d prior to expected calving date. Omnigen was individually offered to cows as top-dressing into the morning concentrate feeding. Before calving, cow BW and BCS were recorded weekly and blood samples collected every 5 d beginning on d -35 relative to expected calving date. After calving and until 46 DIM, BW and BCS were recorded weekly. Individual milk production was recorded and milk samples were collected daily for total solids and SCC analyses. Blood was sampled daily from 0 to 7 DIM, every other day from 9 to 21 DIM, and every 5 d from 26 to 46 DIM. All blood samples were evaluated for cortisol, non-esterified fatty acids (NEFA), insulin, glucose, haptoglobin and IGF-1. On 30 and 46 DIM, cows were evaluated for endometritis via cytobrush technique, based on the % of polymorphonuclear (PMN) cells within 100 total cell count (PMN + endometrial cells). On 48.7 ± 1.6 DIM, 9 cows/treatment received a lipopolysaccharide (LPS) injection (0.25 μg/kg of BW), and blood was sampled hourly from -2 to 8 h, at 12-h intervals from 12 to 72 h, and at 24-h intervals form 96 to 120 h relative to LPS administration at hour 0. Blood samples collected during the LPS challenge were analyzed for haptoglobin and tumor necrosis factor alpha (TNF-α). No treatment differences were detected on BW (P ≥ 0.15), BCS (P ≥ 0.55), serum concentrations of cortisol, NEFA, insulin, glucose, haptoglobin, cortisol, and IGF-I. Cows receiving the OMN diet had greater milk yield (30.3 vs. 27.1 kg/d, P = 0.02) and percentage of PMN cells in endometrial cell population (12.2 vs. 3.9%, P = 0.04) compared with CON cows. After LPS administration, OMN cows had greater (P = 0.04) mean serum haptoglobin (212 vs. 94 µg/mL, for OMN and CON, respectively), as well as greater (P ≤ 0.04) plasma concentrations of TNF-α at 1, (1.34 vs. 2.35 ng/mL for OMN e CON respectively), 2 (0.79 vs. 2.01 ng/mL, for OMN e CON respectively) and 3h (0.41 vs. 1.34 ng/mL, for OMN e CON respectively) relative to LPS injection. In conclusion, supplementing Omnigen-AF® during the transition period increased plasma concentrations of innate immunity markers and increased milk yield of transition dairy cows. / FAPESP: 2015/05536-1

Staphylococcus aureus causes chronic mastitis in bovines that is difficult to treat with current therapeutics. The goal of this research is to provide information about and improve innate immune responses to infection. Infection can result in host cell apoptosis or programmed cell death. Many pathogens can inhibit apoptosis; thereby acquiring a replicative niche, a reprieve from immune responses, and an escape from treatments. We hypothesize that S. aureus inhibits apoptosis in dendritic cells (DC). To investigate our hypothesis, DC were infected with live S. aureus (LSA), γ-irradiated S. aureus (ISA), or Streptococcus agalactiae (Strep ag.) for 2 hours. Stimulations of DC included ultraviolet light (UV) and lipoteichoic acid (LTA). Results indicate that γ-irradiated S. aureus can inhibit UV-induced apoptosis by upregulating LTA. This research provides information about S. aureus infections, but further research is needed to improve responses to this type of infection. One way to improve innate immune responses to infection is by supplementing bovines with OmniGen-AF®, a probiotic that restores neutrophil function during immunosuppression. To determine the mechanism by which OmniGen-AF® functions, wildtype, MyD88 KO, and TLR4 KO mice were fed either normal chow or supplemented with OmniGen-AF® for two weeks. Mice were immunosuppressed with dexamethasone and challenged with LTA. LTA overcame immunosuppression in a TLR4-depenent manner regardless of supplementation with OmniGen-AF®. Overall this research supplies knowledge about S. aureus inhibition of apoptosis in DC and S. aureus LTA activation of PMN regardless of immunosuppression or supplementation with OmniGen-AF®. / Master of Science

This dissertation evaluated nutritional approaches such as the addition of betaine, prebiotics, probiotics, transitional metabolic substrates, and β-mercaptoacetate (MAA; a compound which inhibits β-oxidation) to the diet of lactating dairy cows to determine their impact on physiological, metabolic, hormonal and production responses during thermal stress. The first objective was to evaluate the use of an organic osmolyte, betaine to reduce the impact of heat stress (HS). Cows were fed either 0 (control; CON), 57 mg/kg BW (mid) or 114 mg/kg (high; HI) body weight (BW) betaine and subjected to thermoneutral (TN) and HS conditions. There was an increase in milk yield during TN with HI betaine over controls (P< 0.01), but the advantage was lost during HS. Plasma glucose increased during HS in HI dose cows compared to control (P < 0.01) as did plasma insulin (P = 0.01). Betaine increased milk production during TN and plasma glucose in HS, but did not improve the HS response. Objective two evaluated the use of a probiotic or direct fed microbial (DFM), Calsporin (Bacillus subtilus C-3102) to decrease the effects of HS in dairy cows. We hypothesized that feeding Calsporin prior to and during HS would reduce pathogenic strains of bacteria, maintain commensal microbes, and improve ruminal anaerobic fermentation resulting in improved milk yield (MY). Milk yield was numerically increased (1.26 kg, P = 0.11) in cows fed Calsporin during TN but was reduced under HS (-2.67 kg, P < 0.01) and milk protein content was decreased (P = 0.05). The DFM tended to decrease somatic cell count (SCC) across periods (P = 0.07). Calsporin addition to the diet did not affect respiration rates and was associated with higher rectal temperature at 1800 in HS (P = 0.02). The expression of heat shock protein 27 (HSP27) was decreased with Calsporin treatment (P = 0.03) and in both HS and TN. The fecal microbial count did not change with the exception of the Calsporin strain in treated animals (P < 0.01). The third objective was to feed OmniGen-AF (OG) to dairy cows before and during thermal stress. We hypothesized that feeding OG to HS dairy cows will improve the immune response, and decrease production losses associated with HS. Cows fed OG maintained lower SCC compared to control (P < 0.01) during the recovery period. We did not detect differences between groups in serum calcium while serum non-esterified fatty acid (NEFA) concentrations (P = 0.10) tended to be greater in OG fed cows across the Agricultural Research Center (ARC) portion including HS. Serum Adrenocorticotropic hormone (ACTH) levels were greater in OG cows (P<0.0001) across all sample days. Feeding OG reduced the HS response including serum Cortisol. The final study measured the effects of the metabolic substrate β-hydroxybutyrate (BHB) during HS on feed intake and metabolites. Under TN conditions the cows received a bolus dose of BHB and dry matter intake (DMI) and metabolites were measured. The second part of this study used a bolus of MAA to limit the up-stream production of acetyl-CoA available for ketogenesis by inhibiting ß-oxidation. We proposed that dosing lactating dairy cows with BHB would decrease DMI, increase plasma insulin, decrease NEFAs and increase skin temperature by vasodilatation. The same cows were then subjected to HS and dosed with saline and MAA on different test days. The infusion of BHB increased skin temperature (time 0.5, 1, 2, 3 and 4°C r² =0.98 with serum BHB) and decreased serum NEFA levels (P < 0.01). There was no change in mean DMI, glucose or insulin. The bolus of MAA decreased feed intake, vaginal temperature, and insulin. There was an increase in serum BHB with the initial dose of MAA and an initial decrease in serum glucose (P < 0.0001) with MAA. Serum glucose increased as insulin decreased with MAA. The infusion of BHB did not alter feed intake in this study despite high plasma levels of BHB.