Specific AA have been demonstrated to activate signaling pathways that regulate<br />translation initiation and to stimulate protein synthesis in mammary tissue. The<br />objectives of this research were to determine the response to Ile, Leu, Met, and Thr in<br />cellular signaling and "-S1 casein fractional synthesis rates (CFSR). An experiment was<br />developed as a composite design. The experiment was replicated in tissue corresponding<br />to 5 cows. Mammary tissue slices (0.12 ± 0.02 g) from lactating dairy cows were<br />incubated 4 h in treatment media enriched with 2H5 Phe. Following incubation, slices<br />were homogenized in lysis buffer and caseins were precipitated by acidification to pH<br />4.6. An aliquot of the pellet was trypsinized and 2H5 Phe enrichment in the 34-<br />NLLRFFVAPFPE-45 peptide of "-S1 casein was measured by MALDI TOF-MS and<br />used to determine CFSR (%/h). Western immunoblotting was performed to identify total<br />and site-specific phosphorylated mammalian target of rapamycin (mTOR, Ser2448),<br />eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein (rp) S6 (Ser235/236),<br />and eukaryotic initiation factor (eIF) 2" (Ser51). Addition of Ile, Leu, Met, or Thr had<br />no effect on eIF2" phosphorylation. Isoleucine positively affected mTOR, and rpS6, and<br />negatively affected eEF2 phosphorylation. Leu had a similar effect on eEF2, but not on<br />mTOR or rpS6, and these two AA inhibited each other. Thr negatively interacted with<br />Ile on mTOR and rpS6, and with Leu on eEF2. Increasing concentrations of Ile, Leu,<br />Met, and Thr caused curvilinear increases in CFSR. The maximum response to Ile, Leu,<br />iii<br />Met, and Thr was at 71, 49, 60, and 65% of DMEM concentrations, respectively. All<br />maximums were above plasma AA concentrations observed in lactating cows fed to meet<br />NRC requirements. The CFSR estimated at those maximums were similar between AA<br />(3.6 ± 0.6 %/h). Individual AA effects on CFSR did not correlate with mTOR signaling.<br />Independent CFSR responses to individual essential AA observed in this study contradict<br />the single-limiting AA theory assumed in current requirement systems. The saturable<br />responses of CFSR to these 4 AA also demonstrate the deficiencies of a fixed postabsorptive<br />AA efficiency approach for determining AA requirements for milk protein<br />synthesis. / Ph. D.

The purpose of this research was to evaluate four objectives: 1) update and evaluate predictions of essential amino acid (EAA) outflows from the rumen, 2) predict EAA use and release by the portal drained viscera (PDV) and liver (LIV) of dairy cows, 3) predict EAA use by the mammary (MAM) and non-splanchnic, non-mammary (OTH) tissues, and 4) predict milk protein production from MAM use. To evaluate the first objective, a model was constructed using previously derived equations for ruminally undegraded (RUP), microbial (MiP) and endogenous protein (EndP) flow from the rumen and refit to literature data. Corrections were included in the model to address recovery of EAA during 24-h acid hydrolysis. Upon initial evaluation, all EAA, except Leu, were over predicted and slope bias (P < 0.01) was present for all except Met and Leu. Because of the bias, residuals were regressed on the EAA from each protein flow and adjustments were made to the protein flows. The added adjustments removed all mean bias for the EAA; however, a small slope bias was introduced for Lys and Thr. To evaluate the second objective, equations of Hanigan et al. (2004b) were tested and modifications were made to determine which equation form best represented EAA use by the tissue. Upon initial evaluation of the PDV model of Hanigan et al. (2004b), significant slope bias was present and addressed by deriving alternative forms of the equation. Initial predicted EAA use displayed a mean bias ranging from 0.15 to 45 % and a slope bias ranging from 0.02 to 76% mean square error. The alternative equation forms derived reduced the overall mean and slope bias and improved other fit statistics (RMSE, CCC). To evaluate the third objective, previously derived equations from Hanigan et al. (1998b) were tested using literature data and modifications were made to address deficiencies for each EAA. Upon initial evaluation of the MAM model, significant mean and slope bias was present and was further addressed by derivation of alternative equation forms. Initial evaluation of the OTH model displayed significant mean and slope bias for majority of the EAA ranging from 0.3 to 26 % for mean and 46 to 61 % for slope. For the last objective, several models, both linear and non-linear were evaluated to determine which EAA have a significant impact on milk protein. All models derived has prediction errors below 18-20 % which is comparable or a s light improvement as compared to previous literature data (Moraes et al., 2018). Overall, the equations evaluated show promise in accurately predicting dietary EAA from the time of absorption to their use within the tissues (PDV, LIV, MAM, and OTH) and further impact on milk protein production.

Made available in DSpace on 2017-07-10T18:13:19Z (GMT). No. of bitstreams: 1 Romulo Batista Rodrigues.pdf: 1330851 bytes, checksum: 4991e2d69fa6f7d58830a99378844e76 (MD5) Previous issue date: 2016-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The valine is part of the branched chain amino group together with leucine and isoleucine, and has an important structural role and anabolic. Two experiments were conducted in order to determine the requirement of valine for fingerlings and juveniles of tilapia, by assessing the growth performance, and analysis of chemical composition of housing, hematological and biochemical blood, and white muscle morphometric. In the first experiment were used 270 tilapia fingerlings whit initial average weight of 1.57 ± 0.05 g total initial length of 4.16 ± 0.46 cm, distributed in completely randomized design with six treatments and three replications, using 18 boxes with 250 liter of capacity during the experimental period of 79 days. In the second experiment were used 216 tilapia juveniles with initial average weight of 21.40 ± 0.42g and initial total length of 10.07 ± 1.00 cm, distributed in 18 boxes whit 500 liters of capacity during the experimental period of 77 days. The treatments consisted of diets with increasing concentrations of valine, being for fingerlings (0.82, 0.86, 0.98, 1.04, 1.10 and 1.26% valine) and juvenile (0.54, 0.63, 0.72, 0.81, 0.90 and 0.99% valine). Feed has been held four times a day to apparent satiation. Evaluated the growth performance (weight gain, feed conversion, daily weight gain, protein efficiency ratio, condition factor, survival, protein retention efficiency, visceral fat, liver somatic index, specific growth rate and uniformity of the lot) for fingerlings and juveniles; fish proximate composition (moisture, crude protein, lipids and ash) for fingerlings and juveniles; hematological and biochemical indices of the blood (hemoglobin, erythrocytes count, hematocrit percent, total leukocytes, total thrombocytes, plasma protein, glucose, triglyceride and cholesterol) for juvenile and distribution of muscle fibers to fingerlings and juveniles. There was no statistical difference (P>0.05) for the performance variables, chemical composition and histology between the different treatments evaluated to fingerlings. For juvenile tilapia, on the performance were observed significant differences (P<0.05) between treatments for weight gain, daily weight gain and feed conversion. For hematological and biochemical parameters of blood, there were significant differences (P<0.05) between treatments for hemoglobin, total triglycerides and total cholesterol. Significant differences in the chemical composition of fish and frequency of muscle fibers were observed. It is concluded that the nutritional requirements are met with valine 0.82% valine in the diet (2.65% valine crude protein) for minnows and 0.81% of valine in the diet (2.90% valine crude protein) for juvenile Nile tilapia. / A valina faz parte do grupo de aminoácidos de cadeia ramificada, juntamente com a leucina e isoleucina, e possui importante papel estrutural e anabólico. Foram realizados dois experimentos com o objetivo de determinar a exigência nutricional de valina para alevinos e juvenis de tilápias do Nilo, por meio da avaliação do desempenho zootécnico, e das análises de composição química da carcaça, perfil hematológico e bioquímico do sangue, e da morfometria do músculo esquelético. No primeiro experimento, foram utilizados 270 alevinos de tilápia com peso médio inicial de 1,57 ± 0,05 g e comprimento total inicial de 4,16 ± 0,46 cm, distribuídos em um delineamento inteiramente ao acaso com seis tratamentos e três repetições, utilizando 18 caixas com 250 litros de capacidade, durante período experimental de 79 dias. No segundo experimento, foram utilizados 216 juvenis de tilápia com peso médio inicial de 21,40 ± 0,42 g e comprimento total inicial de 10,07 ± 1,00 cm, distribuídos em um delineamento inteiramente ao acaso com seis tratamentos e três repetições, utilizando 18 caixas com 500 litros de capacidade, durante período experimental de 77 dias. Os tratamentos consistiam em dietas com concentrações crescentes de valina, sendo para alevinos (0,82; 0,86; 0,98; 1,04; 1,10 e 1,26 % de valina na dieta) e para juvenis (0,54; 0,63; 0,72; 0,81; 0,90 e 0,99 % de valina na dieta). A alimentação foi realizada quatro vezes ao dia até a saciedade aparente. Avaliou-se o desempenho zootécnico (ganho em peso, conversão alimentar aparente, ganho em peso diário, taxa de eficiência proteica, fator de condição, sobrevivência, eficiência de retenção proteica, gordura visceral, índice hepatossomático, taxa de crescimento específico e uniformidade do lote) para alevinos e juvenis; a composição centesimal dos peixes (umidade, proteína bruta, extrato etéreo e cinzas) para alevinos e juvenis; os índices hematológicos e bioquímicos do sangue (taxa de hemoglobina, contagem de eritrócitos, percentual de hematócritos, leucócitos totais, trombócitos totais, proteína plasmática, glicose, triglicerídeos e colesterol) para juvenis e a distribuição das fibras musculares para alevinos e juvenis. Não houve diferença estatística (P>0,05) para as variáveis de desempenho, composição centesimal e histologia entre os diferentes tratamentos avaliados para alevinos. Para os juvenis de tilápia, no desempenho zootécnico observaram-se diferenças significativas (P<0,05) entre os tratamentos para o ganho em peso, ganho em peso diário e conversão alimentar aparente. Para os parâmetros hematológicos e bioquímicos do sangue, observaram-se diferenças significativas (P<0,05) entre os tratamentos para taxa de hemoglobina, triglicerídeos totais e colesterol total. Não foram observadas diferenças significativas para a composição centesimal dos peixes e frequência das fibras musculares. Conclui-se que as exigências nutricionais de valina são atendidas com 0,82% de valina na dieta (2,65% de valina da proteína bruta) para alevinos e 0,81% de valina na dieta (2,90% de valina) para juvenis de tilápias do Nilo.