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Lactose-ureide was investigated as a potential means to deliver a known dose of ¹⁵N label directly to the colon in order to non-invasively quantify urea salvage. Two lactose-ureides, labelled with either ¹³C or ¹⁵N on the urea moiety, were synthesised and analysed for purity. Following this, a clinical trial was marginally adequate in healthy adult individuals who had accommodated to a diet marginally adequate in protein were given oral doses of the lactose-ureides, and the excretion of label was followed. Results from the lactose [¹³C]ureide administration demonstrated that little of the dose was excreted on breath before 6 h, with about 80% undergoing fermentation and hydrolysis over a 48 h period, leading to the conclusion that lactose-ureide is a suitable vehicle to non-invasively deliver label to the colon intact. An analysis of urine and stool allowed a quantification of the fate of lactose [¹⁵N¹⁵N]ureide, demonstrating that about 5% was excreted as urinary [¹⁵N¹⁵N]urea, 30% as urinary [¹⁴N¹⁵N]urea, and 22% in the stool fraction. It was concluded that, of the ¹⁵N label available to the body over half was retained on average. The discovery of label associated with lysine in the bacterial fraction of stool further suggests that de novo formed essential amino acids are available to the host. Additionally, a methodology was developed in order to directly address the potential functional significance of lysine formed de novo to the body, by attempting to separate the lysine fraction of apoliprotein B-100 hydrolysates in a quantity sufficient to determine changes in ¹⁵N enrichment using c-IRMS. An accurate detection of changes in ¹⁵N enrichment as low as 0.002 APE in samples containing 3.3 ug lysine-N was achieved, and a distinction was made between the fate of lysine, lysine-N, and lysine-¹⁵N through the analytical system. A method has been developed which allows the salvage of urea-N to be non-invasively quantified in normal adults.

Characterizing the function of a single gene can be achieved by investigating how the gene and its protein product act in a cell or by observing how a cell responds when the gene is removed or knocked out. The ladder, called reverse functional genetics, has been greatly bolstered by the development and implementation of genetic engineering tools. Most recently, CRISPR-Cas9 genetic engineering has enabled functional genetics in a wide variety of organisms due to its ease of use and superior efficiency in gene mutagenesis compared to older technologies. Functional genetics studies in marine diatoms, photoautotrophic eukaryotic algae, have too been aided by the development of CRISPR-Cas9 genetic engineering. This study optimized the CRISPR-Cas9 tool in the pennate diatom Phaeodactylum tricornutum for bacterial-mediated conjugation cell transformation methodology. Conjugation in diatoms occurs by the exchange of an artificial chromosome or episome from E. coli to Phaeodactylum. Here, a cloning scheme was developed to permit the construction of CRISPR-Cas9 episomes that can target multiple genomic loci simultaneously. Nitrogen metabolism of marine diatoms contained unique features that gives them a competitive advantage in oceanic food webs. One feature is the compartmentalization of two glutamine-glutamate metabolic cycles (GS-GOGAT) within the chloroplast and mitochondria, respectively. This study utilized the CRISPR-Cas9 episome tool to functionally analyze a key GS-GOGAT enzyme, glutamine synthetase 2 (GS2), in Phaeodactylum by producing mutant cell lines devoid of GS2. It was found that while cell growth was not compromised, GS2 mutants accumulated lipids, a precursor to biodiesel. Lastly, the CRISPR-Cas9 episome was used to investigate this phenomenon of lipid accumulation in a nitrogen-gene mutant. To do so, a knockout library of nitrogen metabolism genes was produced. Also, the library episomes were fashioned with a novel lipid body fluorescent protein reporter that, by microscopy and flow cytometry based detection methods, was indicative of lipid production in Phaeodactylum mutants. Here, nitrogen limitation and lipid biosynthesis was uncoupled in a Phaeodactylum GS2 mutant. The expansion to high-throughput mutagenesis of a nitrogen gene library will support research into diatom biofuels.

Abstract: RNA helicases function in all aspects of RNA metabolism, regulating a diverse range of cellular processes by rearranging RNA structures and mediating RNA-protein interactions. Prokaryotic RNA helicases have been shown to have roles in RNA degradation, ribosome biogenesis and translation, with their effects on these processes enhanced during abiotic stress. The model cyanobacterium Synechocystis sp. PCC 6803 encodes a single DEAD-box RNA helicase, CrhR. CrhR expression is known to be regulated by a variety of abiotic stresses, sensed through the redox poise of the photosynthetic electron transport chain; however, the cellular role of CrhR is unknown. Evidence is presented that is consistent with CrhR performing roles in translation and RNA processing/degradation. CrhR co-precipitates with actively translating polysomes and components of the RNA degradosome, an interaction that is RNA-dependent. CrhR was also found to localize to the thylakoid membrane space in Synechocystis. The helicase also enhances processing of its own dicistronic transcript, at a site with sequence and structural features sufficient for cleavage by recombinant RNase E in vitro. CrhR participation in translation and RNA processing/degradation, combined with prior transcriptomic and proteomic studies, suggest CrhR regulates expression of genes involved in photosynthesis, energy metabolism, carbon metabolism, and protein stability and turnover. Related to these crucial metabolic pathways, it is shown that CrhR is regulated in response to nitrogen availability. Similar to other abiotic stresses, crhR mutation affects growth rate and abundance of photosynthetic pigments differentially in response to various sources of fixed nitrogen. Cyanobacterial nitrogen metabolism is interconnected with photosynthetic energy transfer, enhanced reduction of which regulates CrhR expression. This links the role of CrhR during nitrogen stress with the other characterized abiotic stresses that are sensed through the redox poise of the photosynthetic electron transport chain. Conservation of CrhR throughout the majority of cyanobacterial diversity was also shown. These CrhR-like helicases form a separate clade, defined by a characteristic highly conserved sequence motif in the C-terminus that is unique to cyanobacterial DEAD-box proteins. It is believed that this motif may function in substrate specificity or auto-regulation of CrhR abundance. In this thesis, a function for CrhR in regulating maintenance of photosynthesis and thylakoid membranes, particularly energy transfer, through the effects of CrhR on RNA maturation, stability and translation is proposed. This function, unique to cyanobacterial DEAD-box RNA helicases, appears to be conserved throughout cyanobacteria that encode CrhR-like helicases.

Atmospheric carbon-dioxide (CO2) enrichment is largely the cause of current global warming. Hence, in the future, organisms will experience the interactive effects of elevated CO2 (eCO2) and chronic warming rather than their individual effects. Though individual effects of eCO2 or chronic warming on plant responses have been studied in some detail, interactive effects of eCO2 and chronic warming on plant responses such as nitrogen (N) relations (uptake, translocation, assimilation) and leaf hyponasty (upward bending of leaves) have been rarely studied. Therefore, the goals of my dissertation work included (1) investigation of eCO2 plus chronic warming on plant N relations, using tomato (Solanum lycopersicum L. cv. Big Boy) and wheat (Triticum aestivum L. cv. Glenn) followed by a meta-analytic review, and (2) investigation of eCO2 plus chronic warming on leaf hyponasty and subsequent effects of hyponasty on plant growth, using tomato and other economically-important species. These goals were achieved by growing plants in a full-factorial experimental design, using two levels of CO2 (ambient vs. elevated) and two temperature regimes (near-optimal vs. supra-optimal) in controlled-environment growth chambers. In all experimental trials conducted, eCO2 plus warming inhibited tomato vegetative growth, whereas warming alone inhibited growth to a smaller extent, and eCO2 alone increased growth. One potential reason for inhibition of plant growth at eCO2 plus warming could be the observed increase in leaf hyponasty. Warming or eCO2 alone caused modest leaf hyponasty, whereas eCO2 plus warming caused severe leaf hyponasty, which correlated with decreased leaf area and biomass. This could be explained by decreased light interception, and thus in situ photosynthesis, as leaves became more vertically-oriented. Severe hyponasty driven by eCO2 plus warming was observed only in the compound-leaved species tested, but not in the simple-leaved species tested. Tomato plants grown at eCO2 plus warming also had the lowest nitrate (NO3-) and ammonium (NH4+) -uptake rates, as well as the lowest root-to-shoot net-N translocation. Moreover, eCO2 plus warming decreased whole-plant N assimilation in tomato, which was mainly driven by the inhibition of whole-plant NO3- assimilation. However, tomato plants grown at eCO2 plus warming had higher in vitro activities of key N-assimilation enzymes (an index of assimilation capacity) and higher % carbon (C) and N levels in roots, indicating that inhibition of N assimilation was not due to the damage to these enzymes nor due to resource limitation for root functions. Thus, in tomato, eCO2 plus warming may decrease N uptake and assimilation in large part because of reduced whole-plant growth, and thus N demand, caused by leaf hyponasty. In wheat, plant growth, %N, protein concentration, and root N-uptake rates were each significantly affected only by CO2, while N- and NO3--assimilation were significantly affected only by warming. However, plants grown at eCO2 plus warming had the lowest concentrations of N and protein, and, hence, lower nutritional quality, with decreased protein concentration at eCO2 plus warming likely due to the inhibition of N assimilation. A comprehensive meta-analysis of the effects of eCO2 plus warming on plant N relations further supported the above-mentioned experimental observations. According to the meta-analysis, eCO2 alone or in combination with warming reduced shoot %N (more so at ≥300 vs. 2), while root %N was significantly reduced only by eCO2; warming alone often increased shoot %N, but mostly did not affect root %N. Though root N-uptake rate was unaffected by eCO2, eCO2 plus warming decreased N-uptake rate, while warming alone increased it. Similar to %N, protein concentration decreased with eCO2 in shoots and grain (but not roots), increased with warming in grain, and decreased with eCO2 and warming in grain. These results show that in the future, two key variables of climate change, eCO2 and chronic warming, will have the potential to interactively disrupt plant N metabolism and reduce nutritional quality of some economically important species. Therefore, when developing crops for future climates, transgenic, genetic engineering, and traditional plant-breeding approaches should focus on generating genotypes with more-resilient N uptake and assimilation, and, in the case of tomato and other compound-leaved species, genotypes that exhibit less climate-change induced leaf hyponasty.

Nitrogen is an essential element for plant nutrition. Nitrate and ammonium are the two major inorganic nitrogen forms available for plant growth. Plant preference for one or the other form depends on the interplay between plant genetic background and environmental variables. Ammonium-based fertilization has been shown less environmentally harmful compared to nitrate fertilization, because of reducing, among others, nitrate leaching and nitrous oxide emissions. However, ammonium nutrition may become a stressful situation for a wide range of plant species when the ion is present at high concentrations. Although studied for long time, there is still an important lack of knowledge to explain plant tolerance or sensitivity towards ammonium nutrition. In this context, we performed a comparative proteomic study in roots of Arabidopsis thaliana plants grown under exclusive ammonium or nitrate supply. We identified and quantified 68 proteins with differential abundance between both conditions. These proteins revealed new potential important players on root response to ammonium nutrition, such as H+ -consuming metabolic pathways to regulate pH homeostasis and specific secondary metabolic pathways like brassinosteroid and glucosinolate biosynthetic pathways.

Future rises in world population, increased demand for food production and greater concern for environmental emissions means that new strategies are required for sustainable growth of ruminant industries. Improvement of feed efficiency in cattle is a major solution to increasing production at lower costs; however, identifying between-animal variation requires markers to predict their phenotype. The studies reported in this thesis investigated a new approach to predict feed efficiency based on measuring the differential fractionation of the stable isotopes of N (¹⁴N and ¹⁵N) in plasma and milk. The main objectives were to evaluate the advantages of using nitrogen-use efficiency (NUE; g milk N/g feed N) as a measurement of feed efficiency in dairy cows, investigate the relationship between plasma and milk N isotopic fractionation (∆¹⁵N) and feed efficiency in beef and dairy cattle, and lastly to understand the genetic factors involved in these relationships. Studies were carried out in Ireland and New Zealand, using growing beef heifers and lactating dairy cows in a number of herds, diets based on grass silage or grazed grass, as well as a range of diet composition and production levels. Chapter 3 showed a highly correlated relationship between NUE and an energy based measure of efficiency (ECE) (r² = 0.90; P

The fungus Neurospora crassa, like most fungi, is very metabolically flexible. N. crassa metabolizes ammonia as a preferred nitrogen source, but has the ability to metabolize other nitrogen sources when the need arises. Furthermore, the nitrogen metabolism of N. crassa is heavily controlled by the gene nit-2. This study analyzes the protein profiles of both wild type N. crassa and a nit-2 mutant N. crassa grown on both Vogels minimal media, which contains ammonia, or Westergaards media, which contains nitrate. Protein was extracted from N. crassa tissue of each genetic composition from each growth medium and analyzed by two-dimensional gel eletrophoresis (2DGE) with the resulting gels being imaged by the PharosFX™ imaging system and analyzed by the use of PDQuestFX™ software.The study showed that protein profiles change based on the nitrogen source and whether the nit-2 gene was active. N. crassa grown under poor nitrogen had a significantly lower number of protein spots than when grown under a preferred nitrogen source for both the wild type and nit-2 mutant strains. When grown in poor nitrogen over 100 proteins were produced by the wild type that did not match the Vogels grown counterpart and there were over 60 proteins that were produced by the wild type that were not produced by the nit-2 mutant under the same condition. The identified proteins include a heat-shock protein, a translation Sh3-like protein, an enolase, an aldolase, and a mitochondrial aconitate. Spot differences also suggest a new, previously not discussed role for nit-2 as a possible repressor under normal growth conditions.

The main objective of this PhD study was to investigate the relationship between nitrogen (N) partitioning and isotopic fractionation (δ15N) in ruminants consuming diets with varying levels of N and/or water soluble carbohydrate (WSC). Nitrogen balance studies were conducted for all studies with measuring δ15N from various N sinks (e.g., milk, plasma, urine and faeces). Concentrations of blood (BUN) or milk urea N (MUN) and urinary excretion of purine derivative (PD) were also measured to provide indications to N metabolism in the whole body and rumen levels. Treatment effects were tested using ANOVA; the relationships between measured variables were analysed by linear regression analysis. Studies of lactating cows (Chapter 4) and lactating goats (Chapter 5) explored the manipulation of WSC/N effect on N-use efficiency (NUE; milk N output/N intake), whilst studies of non-lactating sheep (Chapter 3) and lactating goats (Chapter 5) explored the manipulation of WSC/N effect on urinary N output proportion to N intake (UN/NI) and urinary N output proportion to faecal N output (UN/FN). The increase in WSC/N increased NUE of lactating cows by 56 % through increasing energy intake and milk N output (MN), and decreasing N intake. On the other hand, the increase in NUE and MN were not shown in lactating goats study. It is believed the lack of response was mainly due to the high dietary N concentration and very high dietary WSC concentration in conjunction with unchanged DMI, compared with lactating cows study. No change in MCP was detected in lactating goats study; but a possibility of rumen acidosis resulted from excessive WSC intake should not be excluded. No change in UN/NI was observed from both the non-lactating sheep and lactating goats studies. Little dietary effect on UN/NI in lactating goats may be explained by similar reasons (i.e., high dietary N and WSC concentration and fixed DMI) led to lack of N partitioning response to dietary treatment. Calculated microbial crude protein production per unit of fermentable ME (MCP/FME) and PD analysis suggested no improvement in MCP was achieved in non-lactating sheep study. No change in UN/FN was observed in both lactating goats and non-lactating sheep studies. The opposite effect from dry matter digestibility (increased) and N digestibility (decreased) resulted in little changes in non-lactating sheep FN when WSC/N increased. On the other hand, lack of changes in MCP synthesis resulted in no changes in UN in lactating goats study. Plasma, milk, faeces, muscle, wool and liver were enriched in δ15N compared with feed, while urine was depleted in δ15N relative to feed. It is suggested that the main reason for the enrichment of δ15N in faeces is the presence of enriched endogenous material. The majority of N in wool, liver, muscle, milk and plasma exists as true protein, which has been reported to be enriched in δ15N. In contrast, urea is the main N source of urine and is reported to be depleted in δ15N. Regression analysis demonstrated that urine and faecal δ15N were related to feed δ15N (Chapter 5), milk δ15N and plasma δ15N were related to each other (Chapter 4), and muscle δ15N - feed δ15N was related to urine δ15N - feed δ15N (Chapter 3). It is believed that these observed relationships related to N isotopic fractionation during digestion and absorption of feed components, such as WSC and N or a possible common effect of the endogenous N contribution to various N sinks from feed N. A major observation from this PhD project was the linear relationship between N isotopic fractionation and N partitioning from both non-lactating sheep (Chapter 3) and lactating cows studies (Chapter 4). Two plausible sites (i.e., liver and rumen) of N isotopic fractionation that may be related to N partitioning were discussed in Chapter 6. Overall, under the condition of these studies (high dietary N); the relationships between N isotopic fractionation and N partitioning was believed to be mainly driven by deamination or transamination in the liver, rather than MCP synthesis in the rumen. Results confirmed there is a potential of using N isotopic fractionation as an easy to conduct indicator of N partitioning. However, further research on the accuracy of using N isotopic fractionation predicting N partitioning at different feeding system (e.g., low vs high N intake); animal variations contribute to changes in N isotopic fractionation; and the contribution from different body parts (e.g., rumen, liver and intestines) are needed.

Environmental sustainability of the New Zealand dairy industry is becoming increasingly important. The typical pasture-based diets fed to New Zealand dairy cattle provide an excess of crude protein in the diet which leads to high urine nitrogen (N) concentrations. The main source of on-farm N leaching and nitrous oxide (N₂0) emissions is from urine patches deposited by grazing animals. Dietary inclusion of grape mare, a by product of the viticulture industry, contains condensed tannins and low crude protein concentrations, properties which are known to alter N partitioning in ruminants, and improve environmental sustainability. The objective of this experiment was to determine if the dietary inclusion of grape mare altered N partitioning in dairy cows fed a typical pasture-based diet. The experiment was arranged in a 2 x 4 factorial design, including four levels of grape mare supplementation (0, 2, 4, 6 kg DM/d) and two levels of polyethylene glycol (PEG; 0, 350 g/d) supplementation. Forty Friesian x Jersey crossbred multiparous, lactating cows were evenly divided into one of eight treatment groups. Supplementing grape mare to cows receiving a typical pasture-based diet reduced dietary N (g/kg DM) intake by up to 10.2% and increased dietary condensed tannin concentrations. The dietary inclusion of PEG decreased fecal N % (P < 0.001), while the supplementation with grape mare significantly lowered urine N % (P = 0.005) and urine urea concentration (P = 0.003). Cows fed grape mare also had significantly lower plasma urea (P = 0.024) and MUN (P = 0.026) concentrations. These results indicate that the dietary inclusion of grape mare is effective at increasing fecal N and reducing urine N concentrations, which will improve the environmental sustainability of pastoral-based dairy farming systems in New Zealand.

Creeping bentgrass (Agrostis stolonifera L.) is a major low-cut cool-season turfgrass used worldwide. The objectives of this research were to: 1) to gain insight into the diurnal fluctuation of N metabolism and effects of cytokinin (CK) and nitrate; 2) to characterize the impacts of N and CK on creeping bentgrass under heat stress; 3) to investigate the simultaneous effects of CK and N on the antioxidant responses of heat stressed creeping bentgrass; and 4) to examine the expression pattern of the major heat shock proteins (HSPs) in creeping bentgrass during different heat stress periods, and then to study the influence of N on the expression pattern of HSPs. The transcript abundance of nitrate reductase (NR), nitrite reductase (NIR), plastidic glutamine synthetase (GS2), ferredoxin-dependent glutamate synthase (Fd-GOGAT), and glutamate dehydrogenase (GDH) and N metabolites in shoots were monitored during the day/night cycle (14/8 h). All the measured parameters exhibited clear diurnal changes, except GS2 expression and total protein. Both NR expression and nitrate content in shoots showed a peak after 8.5 h in dark, indicating a coordinated oscillation. Nitrate nutrition increased diurnal variation of nitrate content compared to control and CKHowever, CK shifted the diurnal in vivo NR activity pattern during this period. Grass grown at high N had better turf quality (TQ), higher Fv/Fm, normalized difference vegetation index (NDVI), and chlorophyll concentration at both 15 d and 28 d of heat stress than at low N, except for TQ at 15 d. Shoot NO3-, NH4+, and amino acids increased due to the high N treatment, but not water soluble proteins. High N also induced maximum shoot nitrate reductase activity (NRmax) at 1 d. CK increased NDVI at 15 d and Fv/Fm at 28 d. In addition, grass under 100 µM CK had greatest NRmax at both 1 d and 28 d. Under high N with 100 µM CK, root tZR and iPA were 160% and 97% higher than under low N without CK, respectively. Higher O2- production, H2O2 concentration, and higher malonydialdehyde (MDA) content in roots were observed in grass grown at high N. The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) in roots were enhanced by high N at 19, 22, and 24% levels, respectively, relative to low N. Twenty-eight days of heat stress resulted in either the development of new isoforms or enhanced isoform intensities of SOD, APX, and POD in roots compared to the grass responses prior to heat stress. However, no apparent differences were observed among treatments. No CK effects on these antioxidant parameters were found in this experiment. At week seven, grass at medium N had better TQ, NDVI, and Fv/Fm accompanied by lower shoot electrolyte leakage (ShEL) and higher root viability (RV), suggesting better heat resistance. All the investigated HSPs (HSP101, HSP90, HSP70, and sHSPs) were up-regulated by heat stress. Their expression patterns indicated cooperation between different HSPs and that their roles in creeping bentgrass thermotolerance were affected by N level.

Cells in all domains of life have developed complex regulatory schemes to ensure nitrogen homeostasis. The mechanisms associated with nitrogen homeostasis have been examined in a wide variety of organisms and the molecular aspects of these regulatory systems present a view of an essential global regulatory program for each organism. Recognizing that a similar complex regulatory scheme was most likely present in the Archaea, we chose to investigate the response of Haloferax volcanii to variations in the amount and the quality of its nitrogen source as a model of global regulation in the Archaea. Analysis of the recently sequenced H. volcanii genome showed that this organism encodes enzymes for the core ammonia assimilation pathways found in all organisms and several enzymes for the assimilation of nitrogen from alternative substrates. Phyletic distribution studies and phylogenetic analyses indicate that many of these alternative nitrogen assimilation pathways are absent in other Archaea and that they many have been acquired by the haloarchaea through horizontal gene transfer (HGT) from bacterial genomes. To investigate the global transcriptome response of H. volcanii to changes in nitrogen availability, a genome-wide tiled array was constructed and used to characterize the RNA populations of cells undergoing balanced growth, during growth with a poor nitrogen source and under conditions of nitrogen starvation. Changes in the RNA populations indicated that genes encoding core nitrogen assimilation pathways showed differential expression. However, the regulatory proteins common to the bacterial systems, and those described for Archaea, were absent in H. volcanii. An analysis of the RNAs identified a specific regulatory protein of the AsnC family in the negative regulation of the glnA gene and showed that the general transcription factor (GTF) genes, tbp and tfb, also exhibited differential expression. These data also uncovered the regulated expression of numerous genes encoding uncharacterized proteins. A notable example was a gene encoding a serine/threonine protein kinase, prkA, which is present only in the haloarchaea. This gene exhibited high levels of RNA during nitrogen starvation, and other conditions of nutrient limitation, suggesting that this novel enzyme plays an important role in the physiological response to starvation in the haloarchaea. Expression of the histidine utilization genes, hutUGIH, was examined in vivo as a model for the regulation of nitrogen assimilation genes that were acquired by HGT. In vivo studies established that the positive transcription regulator, HutR, regulated the hut operon genes when histidine or urocanate was provided to cells. The HutR protein is a member of the bacterio-opsin activator (BOA) family of regulators and this class of regulator has not previously been associated with nitrogen regulation in any other organism. The results of this study provide a detailed view of the response of the haloarchaeon H. volcanii to nitrogen limitation and they have uncovered associations to genes of uncharacterized functions. These results have also established the specific regulatory mode of the hut genes, which have been acquired by HGT and are functionally assimilated into the metabolism of this haloarchaeon.

Two experiments were conducted to evaluate the differences in TNC composition of gamagrass harvested in the afternoon (PM) compared to the morning (AM). In the first experiment gamagrass hay was fed to goats with half receiving a protein supplement while the other half received none in a crossover design with a 2 X 2 factorial arrangement of treatments. In the second experiment gamagrass baleage fertilized at 2 different nitrogenous levels 50 kg N/ha (LO) and 150 kg N/ha (HI) was fed to steers in a similar experimental design with half of the steers receiving LO and the other HI. In both experiments TNC concentration including monosaccharides was increased in PM compared to AM (P < 0.001). During the ad libitum phase, goats and steers both had similar DMI of PM and AM (P < 0.88). There was a tendency (P < 0.14) for digestible DMI (DDMI) to be increased by 4 % for PM compared to AM in the goat study; however, was decreased by 9 % in the steer study. During the balance trial, there was a tendency (P < 0.09) for goats to have increased hay and total (hay + supplement) intakes of PM compared to AM, but there were no differences for steers. DDMI was 7 % greater for PM than AM in the goat study, whereas the opposite held true for the steers. DDMI was 9.5 % greater for AM than PM. Compared to AM, PM improved dry matter digestibility (DMD) (55.7 vs. 53.1% DM) when fed to goats. There was a tendency for DMD to be improved in AM (53.2 vs. 51.0 % DM) when fed to steers. In both experiments, the higher protein level (supplement and HI) improved DMD, although the supplement did so to a greater extent. Goats and steers fed supplement and HI had increased N intakes and N digestibility (P < 0.02). In both experiments, the higher protein levels increased urinary N, urine urea N, and urea N as a percent of total urinary N (P < 0.001). Compared to no supplement and LO, supplementation and HI increased plasma urea N (P < 0.05), with overall levels being higher in goats (13.24 vs. 8.61 mM) than steers (6.04 to 4.12 mM). Due to all of these factors N retention was improved with supplementation (2.3 vs. 1.2 g/d), but was similar (P < 0.89) between fertilization levels. We concluded from these results that PM forages do have increased TNC concentration and the differences are preserved through the drying process of making hay and fermentation during ensiling. However, ruminants do not always prefer the PM forages, especially when they are ensiled. N retention in goats can be improved with supplementation of low CP forages. To investigate the mechanism that resulted in an increased preference or DMI of PM forages we measured insulin and ghrelin concentrations. There was a tendency for insulin to be elevated in AM compared to PM (P < 0.12) and HI compared to LO (P < 0.07). Across all treatments, ghrelin concentration peaked at 1000 and decreased for the remainder of the day. We concluded from this data that ghrelin concentration in beef steers increases prior to feeding (animals were fed at 1000), which supports other data suggesting that it may be involved in meal anticipation. My hypothesis was that ghrelin and insulin concentrations would be affected by differences in macronutrient intake, specifically TNC. In the steer study, AM increased DMI, DDMI, and DMD, and the differences in plasma hormone concentration may suggest that regulation of intake between time of harvest may differ as well.

The effects of different forms of excess nitrogen (amino acids vs ammonia) on the partitioning of nutrients between milk production and body weight change in lactating ewes suckling twin lambs were investigated. Effects on nutrient intake, ewe and lamb live weight changes, milk yield and composition, and nitrogen (N) balance were assessed. Nine abomasally cannulated lactating 4 year-old Coopworth ewes with mean initial weight of 66.41 ± 5.90 kg each suckling twin lambs with initial litter weight of 5.72 ± 1.22 kg were used. All ewes were fed the same basal diets in 12 equals meals at 2-hour interval. Treatments consisted of abomasal infusions of water (control), casein (150 g/d), and ammonia (85 g/d) in two periods with a carry-over design. Lambs were fed with milk from their own mothers throughout the experimental periods and also given the same amount of 200 g/d of creep feed in the second period. Ewe dry matter intakes and live weight gain (LWG) were not different between treatments. Lamb LWG was lowest in the ammonia group (p

An experiment was conducted to determine the effects in wool growth of: (i) Pregnancy and lactation in sheep receiving daily supplements of formalin-treated casein. (ii) Formalin-treatment of the casein in pregnant and lactating sheep. (iii) Three experimental treatments were tested (i) Pregnant and lactating animals receiving a daily supplement of 45gms. of formalin-untreated casein (ii) Pregnant and lactating sheep receiving 45gms. of formalin-treated casein and (iii) Non-breeding sheep receiving 45gms. Of formalin-treated casein. (iv) Wool growth rate was determined by clipping of mid-side patches every 28 days and the components fibre diameter, fibre length growth and change in fibre numbers were measured. Dry sheep produced more wool than pregnant and lactating animals and the differences were significant or highly significant in all except the first experimental period. Formalin-treatment of casein did not have a significant effect on wool growth except in Period 1 of the experiment when sheep were in the very early stages of pregnancy. There was no significant change in numbers of active follicles during the course of the experiment and of the other two components of production, variation of fibre cross-sectional area was more important than variation of length growth in explaining changes in wool production.

Ruminant metabolism is essentially dependent upon the presence of rumen microorganisms. These can digest plant fibre, otherwise indigestible, ferment carbohydrate and hydrolyse protein and non-protein compounds, both dietary and endogenously secreted, to degradation products (Hungate 1966). Ingested protein is initially converted in the rumen to peptides and amino acids and these are degraded to ammonia and fatty acids (Hungate 1966). Ammonia is also produced from the non-protein nitrogen fraction of the diet and from endogenous nitrogen compounds. Some of these degradation products may be absorbed through the wall of the gastrointestinal tract, chiefly in the rumen, but most are used for the synthesis of microbial protein, both enzymic and structural (Weller et at. 1958, McDonald and Hall 1957). The synthesis of microbial protein is known to be closely connected to carbohydrate fermentation (Hungate 1966). The protein presented for gastric digestion in the abomasums is mainly bacterial and protozoal and is of a constant biological value (Purser & Puechler 1966). In the acid conditions of the abomasums, which is analogous to the conventional mammalian stomach, the bacterial and protozoal cells are killed proteolysis occurs; amino acids are released for subsequent absorption in the small intestine (Harrison & Hill 1961). In particular bacteria synthesize the essential amino acids of the ruminant, a function inherently lacking in the tissues of mammals (Downes 1961). The essential amino acids are absorbed in the small intestine and have a special significance for wool growth (Little & Mitchell 1967). Extensive ruminal degradation of dietary proteins is inefficient for ruminant nutrition, particularly proteins with a suitable balance of amino acids for significant nitrogen retention and wool growth, as wastage occurs through absorption of ammonia from the rumen and subsequent excretion in the urine as urea (Little & Mitchell 1967, Mcdonald 1948). Protection of dietary protein with formaldehyde prevents ruminal microbial attack and increases the essential amino acids reaching the small intestine for absorption (Ferguson, Hemsley & Reis 1967). The experimental work described here was designed to study aspects of nitrogen metabolism in sheep fed casein and formalin-treated casein supplementary to a basal maintenance diet. To attain an overall picture of nitrogen balance in the sheep, excretion of nitrogen in the faeces and urine must be determined in addition to nitrogen intake and utilisation. However the work described here is limited to the study of plasma urea nitrogen levels and amino acid levels; in effect the sequestering of dietary nitrogen into specific chemical components of the blood plasma.