Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The site of substrate binding and reduction within the Mo-nitrogenase is provided by a metallocluster, the FeMo cofactor, located in the a-subunit of the MoFe protein. The FeMo cofactor's polypeptide environment appears to be intimately involved in the delicate control of the MoFe protein's interactions with its substrates and inhibitors (Fisher K et al., 2000c). In this work, the a-subunit 278-serine residue of the MoFe protein was targeted because (i) a serine residue at this position is conserved both in the Mo-nitrogenase from all organisms examined and in the alternative nitrogenases (Dean, DR and Jacobson MR, 1992); (ii) its hydroxyl group hydrogen bonds to the Sg of the a-subunit 275-cysteine residue that directly ligates the FeMo cofactor; and (iii) its proximity to the a-subunit 277-arginine residue, which may be involved in providing the entry/exit route for substrates and products (Shen J et al., 1997). Altered MoFe proteins of A. vinelandii nitrogenase, with the a278Thr, a278Cys, a278Ala and a278Leu substitutions, were used to study the interactions of H+, C2H2, N2 and CO with the enzyme. All strains, except the a278Leu mutant strain, were Nif+. From measurement of the Km for C2H2 (C2H4 formation) for the altered MoFe proteins, the a278Ala and a278Cys MoFe proteins apparently bind C2H2 similarly to the wild type, whereas the a278Thr and the a278Leu MoFe proteins both have a Km ten-times higher than that of the wild type. Unlike wild type, these last two altered MoFe proteins both produce C2H6. These results suggest that C2H2 binding is affected by substitution at the a-278 position. Moreover, when reducing C2H2, the a278Ala and a278Cys MoFe proteins respond to the inhibitor CO similarly to the wild type, whereas C2H2 reduction catalyzed by the a278Thr MoFe protein is much more sensitive to CO. Under nonsaturating concentrations of CO, the a278Leu MoFe protein catalyzes the reduction of C2H2 with sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity between at least two C2H4-evolving sites. This phenomenon was previously observed with the a277His MoFe protein, in which the a-subunit 277-arginine residue had been substituted (Shen J et al., 1997). Together, these data suggest that the MoFe protein has at least two C2H2-binding sites, one of which may be located near the a277-278 residues and, therefore, most likely on the Fe4S3 sub-cluster of the FeMo cofactor. Like the wild type, N2 is a competitive inhibitor of the reduction of C2H2 by the a278Thr, a278Cys and a278Ala MoFe proteins. Apparently, the binding of N2 in these altered MoFe proteins is similar to that with the wild type MoFe protein, suggesting that the aSer278 residue is not directly involved in N2 binding and reduction. Previous work suggested that both a high-affinity and low-affinity C2H2-binding site were present on the MoFe protein (Davis LC et al., 1979; Christiansen J et al., 2000). Our results are generally consistent with this suggestion. Currently, there is not much information about the proton donors and how the protons necessary to complete all substrate-to-product transformations are transferred. The dependence of activity on pH (activity-pH profiles) has provided useful information about the nature of the groups involved in proton transfer to the FeMo cofactor and the bound substrate. Approximately bell-shaped activity-pH profiles were seen for all products from catalysis by all the MoFe proteins tested whether under Ar, in the presence of C2H2 as a substrate, or with CO as an inhibitor. The profiles suggested that at least two acid-base groups were required for catalytic activity. The pKa values of the deprotonated group and protonated group were determined from the pH that gave 50% maximum specific activity. These pKa values for the altered a278-substituted MoFe proteins and the a195Gln MoFe protein under various assay atmospheres were compared to those determined for the wild type. It was found that the pKa value of the deprotonated group was not affected by either substitution or changing the assay atmosphere. The wild type MoFe protein has a pKa (about 8.3) for the protonated group under 100% argon that was not affected very much by the substitution by Cys, Ala and Leu, whereas the Thr substitution shifted the pKa to about 8, which was the same as that of the wild type MoFe protein in the presence 10% CO. The pKa values for the protonated group for all the altered MoFe proteins were not changed with the addition of 10% CO. These results suggest that the aSer278 residue, through hydrogen bonding to a direct ligand of the FeMo cofactor, is not one of the acid-base groups required for activity. However, this residue may "fine-tune" the pKa of the responsible acid-base group(s) through interaction with the aHis195 residue, which has been suggested (Dilworth MJ et al., 1998; Fisher K et al., 2000b) to be involved in proton transfer to substrates, especially for N2 reduction. The activity-pH profiles under different atmospheres also support the idea that more than one proton pathway appears to be involved in catalysis, and specific pathway(s) may be used by individual substrates. / Ph. D.

Genetic selection for improved performance has necessitated the frequent update of amino acid requirements to support this improvement. The first objective of this dissertation was to investigate the tryptophan requirement of laying hens in peak production and the lysine and sulfur amino acid requirements of broiler chicks under a phase feeding scenario using different models. Digestible tryptophan requirements were estimated to be 137 for egg production, 133 for egg mass, and 133 for feed efficiency using the linear broken-line model. The lysine and sulfur amino acid requirement were estimated by conducting 3 experiments within the starter phase from 2 to 5, 5 to 8, and 8 to 11 days of age. The linear broken line estimate for digestible lysine was 1.22, 1.17 and 1.16% for BWG and 1.31, 1.21, and 1.14% for FCR in experiments 1, 2, and 3, respectively. The linear broken line estimate for sulfur amino acids was 0.82, 0.81 and 0.94% for BWG and 0.82, 0.80, and 0.90 for FCR in experiments 4, 5, and 6, respectively. The lysine requirements decreased from 2 to 5 to 8 to 11 days, however the sulfur amino acid requirements increased during that same period. This could be due to other roles that sulfur amino acids play in the body other than growth. The second objective of this dissertation was to investigate the effects of a direct fed microbial (DFM) on broilers exposed to a cyclic heat stress in 2 consecutive experiments. In this experiment, the heat stress treatment reduced body weight gain and lean tissue accretion from 0 to 35 d in both experiments. In Experiment 2, when the litter was reused BWG was increased by 36 g/bird with supplementation of DFM. Ileal digestibility at 28 d (2 hr post HS) was improved with DFM supplementation in both experiments. Serum FITC-d increased with HS at both 28 and 35 d. Serum FITC-d was generally decreased with DFM at 28 d but the response was inconsistent at 35 d. Overall, the results suggest that HS reduced broiler performance and DFM treatment improved intestinal permeability and nutrient digestibility responses to HS in both experiments but did not improve performance until built up litter was used in Experiment 2. The last objective of this dissertation was to investigate the effects of sulfur amino acids (SAA) on broilers exposed to a cyclic heat stress. As expected, HS reduced BWG and worsened FCR. The supplementation of SAA had no effect on live performance. At 28 d of age, supplementation of SAA to birds exposed to HS resulted in reduced intestinal permeability. The interaction was lost at 31 d, but HS still increased intestinal permeability (P ≤ 0.05). Potential oxidative damage was reduced by increased SAA supplementation as indicated by an increase in the reduced glutathione to oxidized glutathione ratio. These data suggest that intestinal permeability is compromised acutely to at least three days of heat exposure before the bird can adjust, but oxidative damage is more chronic building over the entire 7 d HS period. SAA might have some protective effect on both intestinal permeability and oxidative stress responses to HS. / Doctor of Philosophy / To provide low-cost meat and eggs to consumers, the poultry industry must focus on improving efficiency as well as reduce the impact of stressors within the environment. This is achieved mostly by genetic selection which has resulted in significant improvements in both egg production in laying hens and body weight in broiler chickens. To support this improvement in growth rate birds would require a higher amino acid dense diet to support the increased protein output. One objective of this dissertation was to update the requirement of three key amino acids (tryptophan, lysine, and sulfur amino acids) in both laying hens and broilers for better formulation of diets. These amino acids that were investigated are essential in poultry diets and cannot be produced by the birds. The results from the tryptophan experiment indicated that laying hens need 137 mg of tryptophan per day to maintain maximum egg production. Results from the lysine and sulfur amino acid requirement experiment in broiler chickens indicated that broilers need 1.31% lysine and 0.90% sulfur amino acids to support maximal growth, but these requirements change over time and would need to be adjusted based on the broiler's age. Heat stress in the poultry industry is a major challenge which can affect the growth rate as well as the welfare of broiler chickens. The second objective of this dissertation was to investigate different strategies to ameliorate the effects of heat stress. Supplementing direct fed microbials or probiotics in broiler diets has been proposed as one of those strategies. An experiment was conducted to investigate the effect of the supplementation of a DFM on broiler chickens exposed to heat stress. The results indicated that the DFM was able to ameliorate the negative impacts of the heat stress on nutrient digestibility and intestinal permeability but did not improve the growth of the chickens. Another experiment was conducted to evaluate another strategy to ameliorate the effects of heat stress on broiler chickens which was the supplementation of sulfur amino acids. The sulfur amino acids are not only used for protein synthesis but have other physiological roles in the body that are important specifically during heat stress. Results from this experiment indicated that sulfur amino acids were able to ameliorate the negative effect of heat stress on intestinal permeability and oxidative stress but did not improve the performance of chickens subjected to the heat stress.

It is important to evaluate periodic fluctuations in environment or climate recorded through time to better understand the nature of Earth's history as well as to develop ideas about what the future may hold. There exist numerous proxies by which these environmental patterns can be demonstrated and analyzed through various time scales; from sequence stratigraphic bundles of transgressive-regressive cycles that demonstrate eustatic changes in global sea level, to the geochemical composition of a skeleton that records fluctuations in ocean temperature through the life of the biomineralizing organism. This study examines some of the methods by which we can analyze environmental fluctuations recorded at different time scales. The first project examines the methods by which extrabasinal orbital forcing (i.e. Milankovitch cycles) can be tested in the rock record. In order to distinguish these patterns, computer generated carbonate rock records were simulated with the resulting outcrops tested using common methods. These simulations were built upon eustatic sea level fluctuations with periods similar to what has been demonstrated in the rock record, as well as maintaining the many factors that affect the resultant rock composition such as tectonics, subsidence, and erosion. The result demonstrated that substantially large sea level fluctuations, such as those that occur when the planet is in an icehouse condition, are necessary to produce recognizable and preservable patterns that are otherwise overwhelmed by other depositional factors. The second project examines the temporal distribution of the bivalve Semele casali from Ubatuba Bay, Brazil by using amino acid racemization (AAR) calibrated with ¹⁴C radiometric dates. This data set is one of the largest ever compiled and demonstrates that surficial shell assemblages in the area have very long residence times extending back in time 10,000 years. The area has had very little change in sea level and the AAR ratios which are highly temperature dependent could be calibrated across sites varying from 10 to 53 meters in water depth. Long time scales of dated shells provide us with an opportunity to study climate fluctuations such as El Niño southern oscillation. The third project describes a newly developed method for estimating growth rates in organisms using closely related species from similar environments statistically analyzed for error using a jackknife corrected parametric bootstrap. As geochemical analyses get more precise while using less material, data can be collected through the skeleton of a biomineralizing organism, thus revealing information about environmental shifts at scales shorter than a year. For such studies, the rate of growth of an organism has substantial effects on the interpretation of results, and such rates of growth are difficult to ascertain, particularly in fossilized specimens. This method removes the need for direct measures of growth rates and even the most conservative estimates of growth rates are useful in constraining the age ranges of geochemical intra-skeletal studies, thus elucidating the likely time period under analysis. This study assesses the methods by which periodic environmental fluctuations at greatly varying time scales can be used to evaluate our understanding of earth processes using rigorous quantitative strategies. / Ph. D.

Functional amyloids found throughout nature have demonstrated that amyloid fibers are potential industrial biomaterials. This work introduces a new 'template plus adder' cooperative mechanism for the spontaneous self-assembly of micrometer sized amyloid fibers. A short hydrophobic template peptide induces a conformation change within a highly α-helical adder protein to form β-sheets that continue to assemble into micrometer sized amyloid fibers. This study utilizes a variety of proteins that have template or adder characteristics which suggests that this mechanism may be employed throughout nature. Depending on the amino acid composition of the proteins used the mixtures form amyloid fibers of a cylindrical (~10 μm diameter, ~2 GPa Young's modulus) or tape (5-10 μm height, 10-20 μm width and 100-200 MPa Young's modulus) morphology. Processing conditions are altered to manipulate the morphology and structural characteristics of the fibers. Spectroscopy is utilized to identify certain amino acid groups that contribute to the self-assembly process. Aliphatic amino acids (A, I, V and L) are responsible for initiating conformation change of the adder proteins to assemble into amyloid tapes. Additional polyglutamine segments (Q-blocks) within the protein mixtures will form Q hydrogen bonds to reinforce the amyloid structure and form a cylindrical fiber of higher modulus. Atomic force microscopy is utilized to delineate the self-assembly of amyloid tapes and cylindrical fibers from protofibrils (15-30 nm width) to fibers (10-20 μm width) spanning three orders of magnitude. The aliphatic amino acid content of the adder proteins' α-helices is a good predictor of high density β-sheet formation within the protein mixture. Thus, it is possible to predict the propensity of a protein to undergo conformation change into amyloid structures. Finally, Escherichia coli is genetically engineered to express a template protein which self-assembles into large amyloid fibers when combined with extracellular myoglobin, an adder protein. The goal of this thesis is to produce, manipulate and characterize the self-assembly of large amyloid fibers for their potential industrial biomaterial applications. The techniques used throughout this study outline various methods to design and engineer amyloid fibers of a tailored modulus and morphology. Furthermore, the mechanisms described here may offer some insight into naturally occurring amyloid forming systems. / Ph. D.

Ten cows were used in 4 x 4 Latin squares with an extra period for estimation of carry-over effects to study plasma amino acid responses to jugular infusion of amino acids at early, mid, and late lactation. Methionine (M), methionine + lysine (ML), and methionine + lysine + phenylalanine (MLP) in a balance with glutamic acid (C) were infused at 25% of the amino acid content of pretreatment milk via the jugular vein. Rations were formulated at 16, 15, and 14% crude protein and 15, 18, and 21% crude fiber for 30, 120, and 240 day lactational groups. Carry-over effects were present only in arterial blood at 240 days. Differences in milk production were not significant, although production increased on M, ML, and MLP at 30 days. Plasma amino acid responses of nonruminants were utilized to aid in evaluation. Arterial and venous plasma amino acid concentrations were lowest on ML at 30 days, while amino acid uptake was significantly increased by M. At 120 days plasma responses were inconclusive and non-significant. The amino acid uptake trend followed that observed at 30 days. At 240 days plasma amino acid concentrations were lowest with ML, while uptake was increased on MLP. Essential amino acids were ranked in potential orders of limitation by amino acid extraction and utilization for milk protein by the mammary gland. Order of limitation differed between calculation methods but was similar for all three stage.s of lactation. Orders of limitation were as follows: Amino acid extraction - methionine~ lysine, arginine, leucine, phenylalanine, isoleucine, threonine, valine, and histidine. Amino acid utilization - phenylalanine, threonine or histidine, valine, leucine, methionine, lysine, isoleucine, and arginine. Since there was no substantial change in order of limitation with infusion, either protein intakes were above requirements or amino acids not infused are candidates for limitation. However, among amino acids infused, the parameters observed suggested lysine or methionine at 30 days, methionine at 120 days, and no apparent choice at 240 days. / Master of Science

Ultraviolet light damages genomic material by inducing the formation of covalent bonds between adjacent pyrimidines. Cis-syn cyclobutane pyrimidine dimers (CPD)constitute the most abundant primary lesion in DNA. Photolyase, a light-activated enzyme, catalytically repairs these lesions. Although many steps in the photolyase-mediated repair process have been mapped, details of the mechanism remain cryptic. Difference FT-IR spectroscopy was employed to obtain new mechanistic information about photorepair. Purified oligonucleotides, containing a central diuracil, dithymidine, or cyclobutane thymidine dimer, were monitored using vibrational methods. Construction of difference infrared data between undamaged and damaged DNA permitted examination of nucleic acid changes upon formation of the CPD lesion; these experiments indicated that C=O and C-H frequencies can be used as markers for DNA damage. Furthermore, in purified photolyase containing isotopically-labeled aromatic amino acids, we observed that tryptophan residues in photolyase underwent structural changes during photorepair. These data indicate that electron transfer during DNA repair occurs through-bond, and that redox-active, aromatic residues form the pathway for electron transfer. / Master of Science

As productive performance is improved by breed selection, amino acid requirements may change to support this higher performance in poultry. The first objective of this dissertation was to update the valine and tryptophan requirement of small-framed laying hens and the lysine requirement of young broilers using empirical dose-response methods. The tryptophan requirement was estimated as 155.8 mg/d for egg mass, 153.2 mg/d for egg production and 140.4 mg/d for feed conversion ratio using a linear broken line model. For valine, the requirement was highest for egg mass, 597.3 mg/d, followed by egg production, 591.9 mg/d and feed conversion ratio (FCR), 500.5 mg/d. The lysine requirement of young chicks was estimated by conducting four short term experiments from 1 to 3, 3 to 5, 5 to 8 and 8 to 11 days of age, respectively. The lysine requirement from 1 to 3, 3 to 5 and 5 to 8 days of age were not able to be estimated as no dose response was observed on growth performance most likely due to an overestimation of the lysine requirement. Digestible lysine requirement from 8 to 11 days of age was 1.057%, 1.050% and 1.016% based on body weight gain, FCR and pectoralis major weight using a linear broken line model, respectively. In addition to determining amino acid requirements, research was conducted to develop a new bacterial protein meal for use in laying hens diets. The data suggested that diets containing 7.5% of the bacterial protein meal was able to at least maintain egg production in laying hens, but 15% bacterial protein meal resulted in reduced performance. The second objective of this dissertation was to investigate the effects of various concentrations of dietary vitamin D3 on pullet and laying hen performance, eggshell quality and bone health in laying hens. Pullets/hens were randomly assigned to five dietary treatments containing vitamin D3 from 1,681 to 68,348 IU/kg diet from day of hatch until 68 weeks of age. These data suggested that dietary vitamin D3 fed at 68,348 IU/kg resulted in reduced egg production, but vitamin D from 8,348 to 35,014 IU/kg diet maintained egg production, increased egg vitamin D content in a dose dependent manner, and generally increased both eggshell quality and pullet and hen bone mineral status. / Ph. D. / The goal of the poultry industry is to increase the efficiency of meat and egg production. To achieve this goal, laying hens with higher egg production and broilers with faster growth rates are genetically selected over time. By breed selection, laying hens are able to produce 2-3 additional eggs every year. The body weight of a broiler chicken raised today is approximately four times greater than one raised to the same age in 1958. This Increased egg production and body growth requires a higher nutrient intake, especially amino acids, to support protein production. One objective of this dissertation was to update the requirement of three amino acids (valine, tryptophan and lysine) in poultry production to provide current and accurate information to poultry producers. Valine, tryptophan and lysine are essential amino acids that cannot be synthesized by poultry in sufficient quanities and needs to be ingested from the diet. Three experiments were conducted to determine the valine and tryptophan requirement in laying hens and lysine requirement in broilers. The results of the current experiment show that a laying hen require at least 156 mg tryptophan and 597 mg valine per day to maximize egg production from 41 to 60 weeks of age. The broiler chicks need to ingest rations containing at least 1.06% lysine to support growth from 8 to 11 days of age. Bacterial protein meal is a feed ingredient that has been proposed for use in poultry diets. It is usually produced via the fermentation process by converting various substrates such as methane, methanol, or agriculture by-products into protein-rich biomass. The advantage of using bacterial protein meal in the poultry industry is to decrease feed cost and alleviate the demand on croplands. A novel bacterial protein meal, generated from waste water purification, was evaluated as a feedstuff for laying hens. Two levels of bacterial protein meal, 7.5 or 15%, were added to a regular laying hen diet to replace soybean. The results indicated that replacing soybean meal with 7.5% bacterial protein meal was a feasible solution for egg production but a 15% inclusion rate may result in a decreased egg production. During egg production, bone structural health can be reduced as laying hens age. This loss of bone structural health is due to the loss of bone mineral content, especially calcium and phosphorus, as laying hens produce the calcium rich eggshell. With age, decreased bone mineral mass will induce a higher probability of bone structural failure. Vitamin D plays an important role on calcium absorption and bone mineral deposition. In addition to benefits to skeletal health, the addition of vitamin D₃ in the diet will result in increased vitamin D₃ content in eggs used for human consumption. An experiment was conducted to evaluate the use of high concentrations of vitamin D to increase egg vitamin D content, improve eggshell quatility and increase hen skeletal health. The data suggest that adding vitamin D₃ from 8,300 to 35,000 IU/kg diet will increase egg viatimn D content, and generally improve eggshell and bone quality; however, adding vitamin D₃ at 68,000 IU/kg diet resulted in negative effects on pullet growth and subsequent egg production of adult hens.

In recent years, there has been a growing interest in feeding phytogenic products to poultry for their growth and health benefits. The objective of this experiment was to determine the effects of a phytogenic feed additive on turkey poult performance and digestive physiology over a 6-week brooding period. In total, 864 male Hybrid Converter turkey poults were obtained from a local commercial hatchery on day-of-hatch and placed into floor pens with clean pine shavings. The treatments were randomly assigned including an industry-type corn-soybean meal positive control (PC), a similar diet with a 1.5% reduction in crude protein and key amino acids (NC), or the NC with the addition of Digestarom® (PFA) included at a rate of 1 g/kg. Body weight gain (BWG), feed intake (FI), feed efficiency (FE), and mortality were recorded throughout the experiment. On day 14 poults fed the PFA had significantly higher BWG compared to the PC and NC fed birds. By day 42 birds fed the PC and PFA diets had significantly higher BWG compared to the NC, and the PC had significantly higher FE compared to the NC and PFA fed birds. No differences in FI or mortality were observed throughout the experiment. There were no significant differences in AMEn, crude protein digestibility, or nutrient transporter or pancreatic enzyme mRNA expression among treatments. However, aminopeptidase activity tended to be lower in the ileum of poults fed the PC or PFA diets compared to the NC, suggesting that the PFA may be improving protein utilization. / Master of Science / Recent regulations and customer demand have severely restricted the use of antibiotics in poultry production. Historically, antibiotics have been included in poultry diets at sub-therapeutic concentrations to improve performance and reduce disease incidence. Since this restriction, producers have experienced increased feed conversion ratio, decreased body weights, and increases in morbidity and mortality. Not only does this present an animal welfare issue, but producers experience decreased profits due to the decrease in performance associated with restricted antibiotic use. Many researchers are investigating the ability of alternative feed additives to help mitigate the lost performance this restriction has caused. One such alternative is a class of compounds known generally as phytogenic feed additives. These are herbs, spices, and associated products which can be included into the diet at relatively low concentrations. Previous research has demonstrated the ability of phytogenic feed additives to positively influence performance and gastrointestinal tract health, primarily in broiler chickens. Therefore, the objective of this thesis is to investigate the effect of a commercially available phytogenic feed additive on the performance, nutrient digestibility, and absorption in turkey poults over the first six weeks of feeding. Previous indications with this product seem to indicate that it also increases protein utilization so this product was evaluated using a reduced protein diet. By day 14, poults fed the feed additive had significantly higher body weights compared to both the positive and negative control. At the conclusion of the experiment on day 42, the birds fed the feed additive had significantly higher body weight compared to the negative control and were similar to the positive control. This suggests the ability of the feed additive to improve performance when dietary protein is reduced. Data from aminopeptidase activity in the ileum suggest a potential improvement in the efficiency of protein utilization, which may explain the performance improvements when poults were fed the phytogenic feed additive.

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. / The purpose of this research was to develop a system to predict the supply of essential amino acids (EAA), tissue use of the absorbed EAA, and milk protein production in dairy cows. Essential amino acid supply was predicted from previously published predictions of protein flows and revised estimates of amino acid composition of the respective proteins with corrections for amino acid recovery from acid hydrolysis. Use was predicted for the portal drained viscera (PDV), liver (LIV), mammary (MAM), and an aggregation of non-splanchnic, non-mammary tissues (OTH). Splanchnic EAA release (supply – use) was predicted with root mean squared errors of the mean observed that were less than 12%, and concordance correlation coefficients greater than 0.85 with mean and slope biases ranging, respectively, from 0 to 76% and 0 to 45% of mean squared errors. Mammary tissue EAA use had root mean squared errors ranging from 28 to 148% of the observed mean and concordance correlation coefficients ranging from -0.04 to 0.48. Arterial EAA concentrations and use by OTH was predicted as components within the overall system. OTH utilization was predicted with root mean squared errors < 6% of the observed mean and concordance correlations near 1.0. Alternative equation forms were also derived for each tissue that resulted in marginal reductions in mean and slope biases. Milk protein production was predicted from predicted mammary EAA utilization and digestible energy supply with a root mean squared error of less than 18% which is an improvement over existing field application models. Based on model performance, it was concluded that the system could be used in field application software which would allow more accurate ration formulation resulting in reduced nitrogen excretion by dairy cattle.

Tesis por compendio / [ES] Los estudios sobre nutrición proteica en conejos son escasos en comparación con otras especies, el cálculo de las necesidades es relativamente antiguo, no están ofrecidos a nivel ileal y sus peculiares hábitos alimenticios complican su conocimiento. Además, en los últimos años, se ha establecido relación directa entre este nutriente con una de las principales enfermedades que afectan esta especie (la Enteropatía Epizoótica del Conejo), y como consecuencia, el contenido proteico de las dietas se ha reducido. Por otro lado, se ha comprobado una pérdida en la efectividad de la selección por velocidad de crecimiento en las líneas paternales la cual podría estar relacionada con una mala estimación de los requerimientos proteicos (especialmente en los animales con elevadas velocidades de crecimiento). Finalmente, el nitrógeno ureico plasmático (PUN) podría ser una buena herramienta para estimar el grado de aprovechamiento proteico del pienso, pero su uso no ha sido comprobado aún en esta especie. Con todo ello, la principal hipótesis de esta tesis es que existe una mala estimación de las necesidades de aminoácidos en conejos con elevadas velocidades de crecimiento y que otra combinación de aminoácidos podría maximizar su rendimiento. Para evaluar esta hipótesis se utilizaron un total de 1464 conejos de engorde (provenientes principalmente de la línea R, línea paternal seleccionada por velocidad de crecimiento, y, en mucha menor medida, de líneas maternales con menores velocidades de crecimiento), se formularon un total de 32 dietas (donde principalmente diferían el contenido de los tres aminoácidos típicamente limitantes; lisina, aminoácidos azufrados y treonina), se realizaron 69 y 71 ensayos de digestibilidad fecal e ileal, respectivamente, se valoró la retención de nutrientes de 126 animales durante su crecimiento y por último se analizaron los niveles de PUN de más de 2700 muestras. Con el primero de los trabajos obtuvimos la confirmación del primer indicio: las dietas actuales parecían presentar deficiencias en aminoácidos para los conejos con altas velocidades de crecimiento, comprobando que aquellos animales que crecían más depositaban proporcionalmente más energía y menos proteína (esto sugería una desviación de cierta proporción de proteína como fuente de energía, siendo poco eficiente). Antes de plantearnos qué combinación de aminoácidos mejoraría este problema, tendríamos que poner a punto la técnica para detectar dichas deficiencias. En el segundo trabajo no solo comprobamos que el PUN podría ser utilizado, en esta especie, sino que se estableció la mejor metodología: A las 08:00h (bajo una alimentación ad libitum) o a las 21:00h (bajo un ayuno de 10h y un restablecimiento del alimento a las 18:00h). Ahora ya, sabiendo que podría existir dicha deficiencia y que además podemos utilizar el PUN, seleccionamos de una matriz de 27 piensos (donde habían tres niveles de lisina, azufrados y treonina), aquel que optimizaba los valores de PUN y que previsiblemente mejoraría la asimilación proteica. De este experimento surge la Dieta MAB (8.1, 6.6 y 5.7 g/kg materia seca de lisina, aminoácidos azufrados y treonina, respectivamente) como candidato a sustituir a las actuales recomendaciones, Dieta MMM (8.1, 5.8 y 6.9 g/kg materia seca de lisina, aminoácidos azufrados y treonina, respectivamente). Pero hasta ahora solo podíamos decir que el MAB reduce los nivele de urea: ¿Está esto relacionado con unos mejores parámetros productivos? Finalmente en el último trabajo se comprobó la superioridad productiva de esta combinación, ya que mejoró significativamente tanto la velocidad de crecimiento como el índice de conversión. Tras la valoración digestiva, podemos aconsejar unos niveles de 5.3, 4.3 y 2.9 g/kg de materia seca para la lisina, aminoácidos azufrados y treonina a nivel ileal aparente, arrojando más información sobre la nutrición proteica de precisi� / [CA] Els estudis sobre nutrició proteica en conills són escassos en comparació amb altres espècies, el càlcul de les necessitats és relativament antic, no estan oferts a nivell ileal i els seus peculiars hàbits alimentaris compliquen el seu coneixement. A més, en els últims anys, s'ha establert relació directa entre aquest nutrient amb una de les principals malalties que afecten aquesta espècie (la enteropatia epizoótica del Conill), i com a conseqüència, el contingut proteic de les dietes s'ha reduït. D'altra banda, s'ha comprovat una pèrdua en l'efectivitat de la selecció per velocitat de creixement en les línies paternals la qual podria estar relacionada amb una mala estimació dels requeriments proteics (especialment en els animals amb elevades velocitats de creixement). Finalment, el nitrogen ureic plasmàtic (PUN) podria ser una bona eina per estimar el grau d'aprofitament proteic del pinso, però el seu ús no ha estat comprovat encara en aquesta espècie. Amb tot això, la principal hipòtesi d'aquesta tesi és que hi ha una mala estimació de les necessitats d'aminoàcids en conills amb elevades velocitats de creixement i que una altra combinació d'aminoàcids podria maximitzar el seu rendiment. Per avaluar aquesta hipòtesi es van utilitzar un total de 1464 conills d'engreix (provinents principalment de la línia R, línia paternal seleccionada per velocitat de creixement, i, en molta menor mesura, de línies maternals amb menors velocitats de creixement), es van formular un total de 32 dietes (on principalment diferien el contingut dels tres aminoàcids típicament limitants; lisina, aminoàcids ensofrats i treonina), es van realitzar 69 i 71 assajos de digestibilitat fecal i ileal, respectivament, es va valorar la retenció denutrients de 126 animals durant el seu creixement i finalment es van analitzar els nivells de PUN de més de 2700 mostres. Amb el primer dels treballs vam obtenir la confirmació del primer indici: les dietes actuals semblaven presentar deficiències en aminoàcids per als conills amb altes velocitats de creixement, comprovant que aquells animals que creixien més dipositaven proporcionalment més energia i menys proteïna (això suggeria una desviació de certa proporció de proteïna com a font d'energia, sent poc eficient). Abans de plantejar- nos què combinació d'aminoàcids milloraria aquest problema, hauríem de posar a punt la tècnica per detectar aquestes deficiències. En el segon treball no només vam comprovar que el PUN podria ser utilitzat, en aquesta espècie, sinó que es va establir la millor metodologia: A les 08: 00h (sota una alimentació ad libitum) oa les 21: 00h (sota un dejuni de 10h i a un restabliment de l'aliment a les 18: 00h). Ara ja, sabent que podria existir aquesta deficiència i que a més podem utilitzar el PUN, seleccionem d'una matriu de 27 pinsos (on havien tres nivells de lisina, ensofrats i treonina), aquell que optimitzava els valors de PUN i que previsiblement milloraria l'assimilació proteica. D'aquest experiment sorgeix la Dieta MAB (8.1, 6.6 i 5.7 g / kg matèria seca de lisina, aminoàcids ensofrats i treonina, respectivament) com a candidat a substituir les actuals recomanacions, Dieta MMM (8.1, 5.8 i 6.9 g / kg matèria seca de lisina, aminoàcids ensofrats i treonina, respectivament). Però fins ara només podíem dir que el MAB redueix els anivelli d'urea: Està això relacionat amb uns millors paràmetres productius? Finalment en l'últim treball es va comprovar la superioritat productiva d'aquesta combinació, ja que va millorar significativament tant la velocitat de creixement com l'índex de conversió. Després de la valoració digestiva, podem aconsellar uns nivells de 5.3, 4.3 i 2.9 g / kg de matèria seca per la lisina, aminoàcids ensofrats i treonina a nivell ileal aparent, llançant més informació sobre la nutrició proteica de precisió i pretenent tallar la problemàtica que ens plantejàvem a l'inici de la tesi. / [EN] Studies on protein nutrition in rabbits are scarce compared with other species, the estimation of their requirements is relatively old and no data are reported at ileal level. Moreover, their peculiar feeding behaviour, including caecotrophy, complicates more this study. In addition, in recent years, a relationship has been established between dietary crude protein and Epizootic Rabbit Enteropathy (one of the most important disease that affects this species). As a consequence, the protein content of diets has been reduced. On the other hand, there has been a loss of effectiveness of the selection for growth rate in the paternal lines, which could be related with a possible nutritional deficit in high growth rate rabbits when using the current commercial diets. Plasma urea nitrogen (PUN) could be a good indicator of an amino acid imbalance, but its use has not yet been tested in rabbits. Thus, the main hypothesis of this thesis concerns that growing rabbits selected by growth rate could have amino acid requirements different from current recommendations, and that another amino acid combination would maximize productive traits. To evaluate this hypothesis, a total of 1464 growing rabbits were used (mainly from the R line, a paternal line selected by growth rate, and, to a much lesser extent, from some other maternal lines with lower growth rates), 32 diets were formulated differing in the content of the three typically limiting amino acids (lysine, sulfur amino acids and threonine). Faecal and ileal digestibility trials were performed with 69 and 71 animals, respectively. Nutrient retention during their growing period was evaluated in 126 animals. Finally, PUN was analyzed in more than 2700 samples. From the first trial,we obtained indication about the possible existence of some limiting amino acid when current moderate protein diets are used in growing rabbits with high growth rates, as retained protein to retained energy ratio was clearly lower for animals of the paternal lines (this suggested a deviation of a certain proportion of protein as a source of energy, which is inefficient). Before considering what combination of amino acids would improve their utilization, it was necessary to evaluate the possible interest of PUN to detect deficiencies in amino acids and establish the appropriate methodology to optimize its use for this purpose in growing rabbits. In the second study we verified the applicability of PUN in rabbits and checked the highest differences between diets: from 04:00h to12:00h in animals fed ad libitum and at three hours after refeeding (21:00h) when a fasting period of 10 h was applied. At this point, knowing that amino acid deficiency could exist and that PUN could be an indicator of it, we formulated a matrix of 27 diets (in a 3x3x3 factorial design, with three levels of the three amino acids mentioned above, using the current recommendations and increasing or decreasing them by 15%), searching for the combination minimizing PUN values and, hypothetically, improving the protein assimilation. From this trial, the diet MHL (8.1, 6.6 and 5.7 g/kg dry matter of lysine, sulphur amino acids and threonine, respectively) emerged as a candidate to replace the current recommendations (diet MMM; 8.1, 5.8 and 6.9 g/kg dry mater of lysine, sulphur amino acids and threonine, respectively). However a question arises: could this new combination improve productive traits? Finally, in the last study we verified the usefulness of this combination since it significantly improved both the growth rate and the feed conversion ratio. After digestibility assessment, we propose levels of 5.3, 4.3 and 2.9 g/kg dry matter of apparent ileal digestible lysine, sulphur amino acids and threonine, respectively. These data are intended to improve current knowledge on protein nutrition and move towards precision protein nutrition. / Marín García, PJ. (2019). Lysine, sulfur amino acids and threonine requirements of growing rabbits from a line selected by growth rate [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/132186 / Compendio