In a number of mammalian species, susceptibility to enteric coronavirus infection has been shown to be age-related. This is thought to be associated with enterocyte maturation and receptor protein expression. One of the factors that can influence differentiation and maturation of enterocytes is the availability of short-chain fatty acids (SCFA) in the intestinal lumen. These compounds are by-products of the bacterial fermentation of dietary fiber and serve as the primary energy source for enterocyte metabolism. The overall objective of this dissertation was to evaluate the effects of dietary fiber and short-chain fatty acids on enterocyte differentiation, maturation, and susceptibility to coronavirus infection in turkeys. Initial work involved the development of an indirect immunoperoxidase assay (IPA) for the identification and localization of turkey coronavirus (TCV) in paraffin-embedded, acid-ethanol fixed tissue. IPA was found to be superior to indirect immunofluorescent antibody test (IFA) for this and other diagnostic purposes. To evaluate cellular differentiation and maturation, an SDS-PAGE/immunoblot technique was developed to determine relative levels of villin expression in turkey embryos. Villin is an actin-bound cytoskeletal protein known to be expressed in increasing quantities at the apical surfaces of maturing enterocytes. Villin expression level was found to increase linearly as a function of embryo age. Villin localization was performed by IPA on paraffin-embedded, acid-alcohol fixed tissue. As enterocytes (embryos) matured, villin was found to concentrate at the apical surfaces and eventually at the basolateral membranes. Experiments were also conducted to see what effect in ovo butyrate administration would have on developing embryonic enterocytes. Butyrate has been shown to enhance differentiation of non-neoplastic and neoplastic cells in culture as well as promote healing of damaged intestinal epithelium in human. Villin expression was significantly enhanced in embryos receiving 0.2 and 0.3 M butyrate 36 hours post-administration. Butyrate appeared to enhance villin expression and therefore enterocyte maturation in a dose-dependent manner. Susceptibility of turkey embryos to TCV infection as a function of age and butyrate treatment was investigated as well as epithelial localization of TCV infection in poults. The level of TCV infection of epithelium was found to increase with embryo age between 17 and 23 days. Poults showed higher levels of infection on the distal 2/3 of villi and no evidence of infection in the intestinal crypts. Butyrate administration in 21-day-old embryos followed by TCV inoculation caused a significant increase of the number of infected cells per villus. This data suggested that butyrate might be used as a means to manipulate enterocyte susceptibility to TCV infection. In the final set of experiments, the effects of fiber-fortified poult diets containing 5% cellulose or 5% guar gum on luminal SCFA levels, enterocyte maturation, and TCV infection were investigated. SCFA levels in cecal contents were determined by gas chromatography. Enterocyte maturation was assessed by the determination of villin expression on immunoblot and the severity of TCV infection was determined by IPA and lesion score. Fiber-fortified diets enhanced SCFA production and villin expression, but contrary to embryo studies, TCV infection appeared to be reduced. In general, poults performed better on the diet containing cellulose. Mechanisms regarding the roles of dietary fiber and SCFA in enterocyte differentiation, maturation, and TCV susceptibility are proposed as well as future directions for research. The in ovo and poults system used in this research may serve as models for further investigation of the influences of host and dietary factors on enteric viral infection and recovery. / Ph. D.

Feeding diets rich in rapidly fermentable non-structural carbohydrates can lead to the development of ruminal acidosis. This study was conducted to determine if the duration of time that cattle are fed a high-grain diet affects their absorption of short-chain fatty acids (SCFA) and susceptibility to, and recovery from, ruminal acidosis. Sixteen Angus heifers (BW ± SEM, 261 ± 6.1 kg) were assigned to 1 of 4 blocks, and fed a backgrounding diet consisting of 60% barley silage, 30% barley grain, and 10% supplement (DM basis). Within block, cattle were randomly assigned to 1 of 2 treatments differing in the number of days they were fed the high-grain diet prior to an acidosis challenge: 34 d for long-adapted (LA) and 8 d for short-adapted (SA). All cattle were exposed to the same 20-d dietary transition using 5 dietary steps until achieving the final diet that contained 9% barley silage, 81% barley grain, and 10% supplement (DM basis). Data were collected during an 8-d baseline period (BASE), on the d of the acidosis challenge (CHAL), and during two consecutive 8 d recovery periods (REC1 and REC2). Ruminal acidosis was induced by restricting feed to 50% of DMI:BW for 24 h followed by an intraruminal infusion of ground barley at 10% DMI:BW. Cows were then given their regular diet allocation 1 h after the intraruminal infusion. The duration of time fed the high-grain diet did not affect ruminal pH, lactate, or SCFA concentrations (P > 0.050). However, during BASE and on the day of CHAL the SA heifers experienced greater linear (P = 0.031), quadratic (P = 0.016), and cubic (P = 0.008) between day change in the duration of time that pH was < 5.5 than LA heifers. Relative to BASE, inducing acidosis increased daily duration (531 to 1020 min/d; P < 0.001) and area (176 to 595 (min × pH)/d; P < 0.001) that pH was < 5.5. Inducing ruminal acidosis also increased the daily mean (0.3 to 11.4 mM; P = 0.013) and maximum (1.3 to 29.3 mM; P = 0.008) rumen fluid lactate concentrations relative to BASE, suggesting that an acute bout of ruminal acidosis was induced. In addition, a treatment × day interaction for the duration that pH was < 5.5 during REC1 suggests that LA cattle tended to recover from the CHAL more rapidly than SA cattle (P = 0.085). Indeed, analysis of covariance confirmed that the LA heifers experienced a quicker linear (P = 0.019) recovery over time from CHAL. The greater rate of recovery possibly resulted from the LA heifers having greater rates of both fractional butyrate (45 vs. 36 %/h; P = 0.019) and propionate absorption (42 vs. 34 %/h; P = 0.045), and tending to have greater rates, on an absolute basis, of butyrate absorption (94 vs. 79 mmol/h; P = 0.087) iii and, on a fractional basis, of total SCFA absorption (37 vs. 32 %/h; P = 0.100). Treatment × period interactions revealed that LA heifers had greater serum D-lactate concentrations (P = 0.003), and fractional rates of lactate absorption (P = 0.024) than SA heifers, during CHAL and REC1, respectively. When treatments were pooled, the absorption (%/h and mmol/h) of acetate, propionate, butyrate, and total SCFA increased between REC1 and REC2, with intermediate values for BASE (P ≤ 0.05). Corresponding to a reduction in absorption during REC1 (2 d post CHAL), saliva production (kg/h; P = 0.018) increased between BASE and REC1, with intermediate values for REC2. These results indicate that the duration of time cattle are fed a high-grain diet may stabilize rumen pH, both prior to and after an induced bout of acute ruminal acidosis, likely through increased ruminal absorptive capacity for SCFA and lactate. In addition, this study found evidence to suggest that beef cattle possess the ability to increase saliva secretion in order to compensate for decreased absorptive capacity.

Short chain fatty acids (SCFA) synthesized in the rumen from carbohydrate fermentation are an essential energy source for ruminants. Current literature supports that SCFA are absorbed across the rumen epithelium via passive diffusion or protein-mediated transport, however, the rate and degree to which these pathways adapt to a change in diet fermentability is unknown. Furthermore, Na+ flux is partially determined by SCFA absorption, and thus is a key indicator of functional changes in the rumen epithelium. The objectives of this study were to determine the time required for a change in SCFA and Na+ absorption across the bovine rumen epithelium and to evaluate the rate and degree to which absorption pathways adapt to an increase in diet fermentability relative to changes in surface area. Twenty-five weaned Holstein steer calves were blocked by body weight and randomly assigned to either the control diet (CON; 91.5% hay and 8.5% vitamin/mineral supplement) or a moderately fermentable diet (50% hay; 41.5% barley grain, and 8.5% vitamin/mineral supplement) fed for 3 (G3), 7 (G7), 14 (G14), or 21 d (G21). All calves were fed at 2.25% BW at 0800 h. Reticular pH was recorded every 5 min for 48 h prior to killing (1000 h). Ruminal tissue was collected for Ussing chamber, barrier function, surface area measurements, and gene expression. Net 22Na+ flux (JNET-Na; 80 kBq/15 mL), the rate and pathway of mucosal to serosal 3H-acetate (JMS-acetate; 37 kBq/15 mL) and 14C-butyrate (JMS-butyrate; 74 kBq/15 mL) flux, and serosal to mucosal flux of 3H-mannitol (JSM-mannitol; 74 KBq/15 mL) and tissue conductance were measured. Half of the chambers assigned to measure JMS-acetate and JMS-butyrate were further assigned to 1 of 2 acetate and butyrate concentration treatments: 10 mM (Low) and 50 mM (High). Furthermore, JSM-mannitol flux was also measured during an acidotic and hyperosmotic challenge (CHAL) and recovery (REC) to measure barrier function of ruminal tissue. Mean reticular pH, which was positively correlated with ruminal pH (R2 = 0.5477), decreased from 6.90 for CON to 6.59 for G7 then increased. Net Na+ flux increased 125% within 7 d. Total JMS-acetate and JMS-butyrate increased from CON to G21, where passive diffusion was the primary SCFA absorption pathway. Total JMS-acetate and JMS-butyrate were greater when incubated in High vs. Low. Effective surface area of the ruminal epithelium was not affected by dietary treatment. Increased JSM-mannitol, tissue conductance, and increased expression of IL-1β and TLR2 (tendencies) with increased days fed the moderate grain diet indicated reduced rumen epithelium barrier function. Furthermore, the CHAL treatment reduced barrier function, which was not reversible during REC. This study indicates that a moderate increase in diet fermentability increases rumen epithelium absorptive function in the absence of increased SA, but reduces barrier function. Data from this study also suggests that absorption and barrier function follow different timelines, posing a challenge for ruminant diet adaptation to moderately to highly fermentable diets.

The regulation of short chain fatty acid metabolism has been examined. Metabolism of acetoacetate, and short chain fatty acids such as butyrate and valerate, is predicated upon the expression of genes of the ato operon. Acetoacetate induces expression of a CoA transferase (encoded by the atoDA genes) and expression of a thiolase (encoded by the atoB gene). Metabolism of saturated short chain fatty acids requires the activities of the transferase and thiolase and enzymes of 6-oxidation as well. Spontaneous mutant strains were isolated that were either constitutive or that were inducible by valerate or butyrate instead of acetoacetate.

Os ácidos graxos de cadeia curta (AGCC) são produzidos pela microbiota intestinal e possuem papéis anti-inflamatórios e ação inibitória sobre histona deacetilases. A lesão renal aguda (LRA) é caracterizada por uma inflamação renal que influencia a função do rim. Este projeto avaliou se o tratamento com os AGCC impactaria nos desfechos inflamatórios da LRA em camundongos. Foi observado que o tratamento com AGCC, protege da LRA. Esta melhora foi associada a uma menor inflamação e menor taxa de apoptose. Além disso, o tratamento com acetato diminuiu a atividade de histona deacetilase. Administrando bactérias produtoras de acetato, também foi possível observar uma proteção da LRA, junto de uma menor inflamação sistêmica. Esta proteção do AGCC na LRA foi também observada em modelo de LRA secundária à sepse In vitro, o tratamento com AGCC modularam tanto células imunes como células renais sob estímulos inflamatórios e de hipóxia. AGCC modulam processos inflamatórios no rim via ações epigenéticas ou não, podendo ser uma promissora ferramenta na proteção da LRA. / Short-Chain Fatty Acids (SCFA) are produced by the intestinal microbiota and have anti-inflammatory and histone deacetylases inhibitors properties. Acute kidney injury (AKI) is characterized by renal inflammation that may impair kidney function. This project evaluated whether treatment with SCFA inflammatory impacts the outcomes of AKI in mice. It was observed that treatment with SCFA protected the AKI. This improvement was associated with less inflammation and lower apoptosis rate. In addition, treatment with acetate decreased the activity of histone deacetylase. Giving bacteria producing acetate, was also observed protection from AKI, along with a lower systemic inflammation. This protection of the AGCC in AKI was also observed in sepsis model. In vitro, SCFA treatment modulated both immune cells and renal cells under hypoxia and inflammatory stimuli. SCFA modulate inflammatory processes in the kidney via epigenetic actions or not, may be a promising tool in the protection of the AKI.

Introdução: Ácidos graxos de cadeia curta (AGCC) são compostos que contêm de 1 a 6 átomos de carbono. Estudos mostraram que possuem efeitos imunomoduladores, antiproliferativos e pró-apoptóticos, via ativação de receptores acoplados à proteína G ou via controle epigenético, agindo na histona acetil transferase (HAT) e na histona deacetilase (HDAC). O acetato é o AGCC encontrado em maiores concentrações nos cólons e no sangue, sendo também um intermediário em diversas reações metabólicas. Apesar disso, foi pouco estudado até o momento. As células da microglia são os macrófagos residentes no sistema nervoso central e desempenham importante papel em diversas doenças. Objetivo: estudar os efeitos do acetato na microglia esclarecendo seus efeitos na produção de mediadores inflamatórios e na viabilidade celular. Conclusão: O acetato estimula a produção de TNF-&alpha; e melhora a viabilidade de culturas da microglia ativadas por LPS. A melhora da viabilidade celular ocorre pela indução de autofagia. O mecanismo responsável pela indução da autofagia é epigenético, sendo completamente independente da ativação de receptores GPR. / Introduction: short-chain fatty acids (SCFA) are compounds containing from 1 to 6 carbon atoms. Studies have shown that these compounds have immunomodulatory, anti-proliferative and pro-apoptotic effects via activation of G-protein coupled receptors or via epigenetic control acting on histone acetyl transferase (HAT) and histone deacetylase (HDAC). Acetate is the SCFA found in highest concentrations in the colon and blood. It is also an intermediate in many metabolic reactions. Nevertheless, it was little studied so far. The microglial cells are resident macrophages of the central nervous system and play an important role in several diseases. Objective: To study the effects of acetate on microglia and clarify its effects on inflammatory mediators production and cell viability. Conclusion: Acetate stimulates TNF-&alpha; production and improves cell viability of microglial cultures activated by LPS. The improved cell viability occurs by autophagy induction. The mechanism responsible for induction of autophagy is epigenetic, being completely independent of the GRP receptor activation.

Os ácidos graxos de cadeia curta (AGCC) são produzidos pela microbiota intestinal e possuem papéis anti-inflamatórios e ação inibitória sobre histona deacetilases. A lesão renal aguda (LRA) é caracterizada por uma inflamação renal que influencia a função do rim. Este projeto avaliou se o tratamento com os AGCC impactaria nos desfechos inflamatórios da LRA em camundongos. Foi observado que o tratamento com AGCC, protege da LRA. Esta melhora foi associada a uma menor inflamação e menor taxa de apoptose. Além disso, o tratamento com acetato diminuiu a atividade de histona deacetilase. Administrando bactérias produtoras de acetato, também foi possível observar uma proteção da LRA, junto de uma menor inflamação sistêmica. Esta proteção do AGCC na LRA foi também observada em modelo de LRA secundária à sepse In vitro, o tratamento com AGCC modularam tanto células imunes como células renais sob estímulos inflamatórios e de hipóxia. AGCC modulam processos inflamatórios no rim via ações epigenéticas ou não, podendo ser uma promissora ferramenta na proteção da LRA. / Short-Chain Fatty Acids (SCFA) are produced by the intestinal microbiota and have anti-inflammatory and histone deacetylases inhibitors properties. Acute kidney injury (AKI) is characterized by renal inflammation that may impair kidney function. This project evaluated whether treatment with SCFA inflammatory impacts the outcomes of AKI in mice. It was observed that treatment with SCFA protected the AKI. This improvement was associated with less inflammation and lower apoptosis rate. In addition, treatment with acetate decreased the activity of histone deacetylase. Giving bacteria producing acetate, was also observed protection from AKI, along with a lower systemic inflammation. This protection of the AGCC in AKI was also observed in sepsis model. In vitro, SCFA treatment modulated both immune cells and renal cells under hypoxia and inflammatory stimuli. SCFA modulate inflammatory processes in the kidney via epigenetic actions or not, may be a promising tool in the protection of the AKI.

Orientador: Alba Regina Monteiro Souza Brito / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T00:26:41Z (GMT). No. of bitstreams: 1 Ferreira_AndersonLuiz_D.pdf: 1659590 bytes, checksum: a72e12af76082feafe5a240c07b04574 (MD5) Previous issue date: 2009 / Resumo: Apesar da amplitude de possibilidades terapêuticas, ainda não existe um tratamento ideal para as doenças inflamatórias intestinais (DII), com perfil adequado de eficácia e segurança. Por essa razão, é de grande interesse estudar agentes com poucos efeitos colaterais, que preferencialmente façam parte da dieta, no tratamento/prevenção dessas doenças. Neste contexto, surgiu o interesse em estudar uva, fruta com alto teor de flavonóides e fibras dietéticas, ambas benéficas à saúde. Sabe-se que substâncias antioxidantes e ácidos graxos de cadeia curta (AGCC), principalmente o ácido butírico proveniente da fermentação de fibras dietéticas, podem estar envolvidas na prevenção e tratamento de doenças intestinais. O ácido butírico é utilizado como substrato para a reparação do tecido inflamado e inibe fatores pró-inflamatórios; portanto, quantificar a produção desse ácido é imprescindível, dado que quanto maior for sua produção, maior será a eficiência da dieta para a recuperação do tecido. Para tanto, foi realizada uma análise in vitro que demonstrou que a concentração de 0,5 g de Uva foi a que produziu maiores quantidades de butirato na análise por cromatografia de íons. Após a escolha da concentração da fruta, seguiu-se o estudo in vivo utilizando-se a dose de 0,5 g.kg-1. A princípio, verificou-se a ação antioxidante avaliando os níveis de GHS além das atividades das enzimas antioxidantes GSSG-Rd, GSH-Px e SOD em ratos com colite induzida por ácido trinitrobenzenosulfônico (TNBS). Os animais tratados com Uva apresentaram altos níveis de GSH; as atividades da GSSG-Rd, GSH-Px permaneceram próximas às do grupo sem colite. A atividade da SOD, que também foi drasticamente diminuída no grupo TNBS, foi restabelecida no grupo tratado. O Tratamento com Uva foi capaz de exercer efeito antinflamatório intestinal, já que os animais tratados apresentaram menor escore macroscópico da lesão, além de diminuir alguns mediadores pró-inflamatórios, como (COX-2, NF-_B e molécula de adesão intestinal MadCAM-1) e a atividade da MPO. A diminuição do dano cólico pela Uva pode ser explicada, em parte, pela diminuição da produção de interleucinas pró-inflamatórias (IL-6 e IL-12) que funcionam como fatores quimiotáxicos da inflamação. Por outro lado, a Uva aumentou a produção da citocina antinflamatória IL-10, sendo que esse aumento foi maior que os valores normais obtidos para o grupo salina. Conclui-se, que a Uva mostrou habilidade em modular enzimas antioxidantes, além de aumentar a produção de IL-10 o que, provavelmente, reduziu a produção de citocinas pró-inflamatórias e, consequentemente, a expressão de mediadores inflamatórios. / Abstract: Despite the range of therapeutic possibilities, there is still no ideal treatment for inflammatory bowel disease (IBD), with the right profile of efficacy and safety. For this reason, it is of great interest to study agents with little side effect, which preferably form part of the diet and that treats / prevents such diseases. In this context, emerged the interest in studying Grape, fruit with high content of flavonoids and dietary fiber, two compounds that improve the healthy. It is known that antioxidants and short chain fatty acids (SCFA), especially butyric acid, obtained from the fermentation of dietary fiber, may be involved in the prevention and treatment of intestinal diseases. The butyric acid is a substrate for the repair of inflamed tissue and inhibits pro-inflammatory factors; for that reason, to quantify the production of this acid is mandatory, given that the higher the production, the greater the efficiency of the diet for tissue recovery. Therefore, an in vitro analysis showed that the concentration of 0.5 g of Grape was the one that produced the greatest amount of butyrate in the analysis by ion chromatography. After choosing the concentration of the fruit, the next step was an in vivo study where was used the dose 0,5 g.kg-1. At first, was checked the antioxidant action through the analysis of the levels of GSH and the activity of antioxidants enzimas like GSSG-Rd, GSH-Px and SOD in rats with colitis induced by trinitrobenzenosulfonic acid (TNBS). Animals treated with Grape showed high levels of GSH, the activities of GSSG-Rd, GSH-Px remained close to the group without colitis. The activity of SOD, which was also drastically reduced in the TNBS group, was restored in the treated group. The treatment with Grape was able to exert intestinal anti-inflammatory effect, since the treated animals showed lower scores of the lesion and reduced some proinflammatory mediators such as MPO activity, COX-2, NF-_B and the intestinal adhesion molecule MadCAM-1 expression. The decrease in colonic damage can be explained, partially, by the action of the Grape that decreased the production of pro-inflammatory interleukins (IL-6 and IL-12) that act as chemotactic factors of inflammation. On the other hand, the treatment of Grape increased the production of anti-inflammatory cytokine, such as IL-10, and this increase was higher than normal values showed by the saline group. It can be concluded that the Grape showed ability to modulate the antioxidant enzymes and increased the production of IL-10. Probably, the increased of the levels of this interleukin resulted in a decrease in the production of proinflammatory cytokines and, consequently, in a decrease the expression of inflammatory mediators. / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Introdução: Ácidos graxos de cadeia curta (AGCC) são compostos que contêm de 1 a 6 átomos de carbono. Estudos mostraram que possuem efeitos imunomoduladores, antiproliferativos e pró-apoptóticos, via ativação de receptores acoplados à proteína G ou via controle epigenético, agindo na histona acetil transferase (HAT) e na histona deacetilase (HDAC). O acetato é o AGCC encontrado em maiores concentrações nos cólons e no sangue, sendo também um intermediário em diversas reações metabólicas. Apesar disso, foi pouco estudado até o momento. As células da microglia são os macrófagos residentes no sistema nervoso central e desempenham importante papel em diversas doenças. Objetivo: estudar os efeitos do acetato na microglia esclarecendo seus efeitos na produção de mediadores inflamatórios e na viabilidade celular. Conclusão: O acetato estimula a produção de TNF-&alpha; e melhora a viabilidade de culturas da microglia ativadas por LPS. A melhora da viabilidade celular ocorre pela indução de autofagia. O mecanismo responsável pela indução da autofagia é epigenético, sendo completamente independente da ativação de receptores GPR. / Introduction: short-chain fatty acids (SCFA) are compounds containing from 1 to 6 carbon atoms. Studies have shown that these compounds have immunomodulatory, anti-proliferative and pro-apoptotic effects via activation of G-protein coupled receptors or via epigenetic control acting on histone acetyl transferase (HAT) and histone deacetylase (HDAC). Acetate is the SCFA found in highest concentrations in the colon and blood. It is also an intermediate in many metabolic reactions. Nevertheless, it was little studied so far. The microglial cells are resident macrophages of the central nervous system and play an important role in several diseases. Objective: To study the effects of acetate on microglia and clarify its effects on inflammatory mediators production and cell viability. Conclusion: Acetate stimulates TNF-&alpha; production and improves cell viability of microglial cultures activated by LPS. The improved cell viability occurs by autophagy induction. The mechanism responsible for induction of autophagy is epigenetic, being completely independent of the GRP receptor activation.

A series of experiments were conducted to determine nutritional strategies to improve the efficiency of N utilization in dairy cows when feeding co-products including wheat-based (W-DDGS) and corn-wheat blend distillers grains with solubles (B-DDGS), and dried whey permeate (DWP). In Experiment 1, the objective was to determine the effects of replacing canola meal (CM) as the major protein source with W-DDGS on ruminal fermentation, microbial protein production, omasal nutrient flow, and animal performance. Cows were fed either a standard barley silage-based total mixed ration containing CM as the major protein supplement (0% W-DDGS, control) or diets formulated to contain 10, 15 and 20% W-DDGS (dry matter [DM] basis), with W-DDGS replacing primarily CM. Diets were isonitrogenous (18.9% crude protein [CP]). Inclusion of W-DDGS to the diet did not negatively affect ruminal fermentation, microbial protein production, and omasal nutrient flow. However, there was a 0.7- to 2.4-kg increase in DM intake, and a 1.2- to 1.8-kg increase in milk yield after the addition of W-DDGS in place of CM. In Experiment 2, the objective was to delineate the effects of including either W-DDGS or B-DDGS dried distillers grains with solubles as the major protein source in low or high CP diets fed to dairy cows on ruminal function, microbial protein synthesis, omasal nutrient flows, urea-N recycling, and milk production. The treatment factors were type of distillers co-product (W-DDGS vs. B-DDGS) and dietary CP content (15.2 vs. 17.3%; DM basis). The B-DDGS was produced from a mixture of 15% wheat and 85% corn grain. All diets were formulated to contain 10% W-DDGS or B-DDGS on a DM basis. Feeding up to 10% of dietary DM as B-DDGS or W-DDGS as the major source of protein did not have negative effects on metabolizable protein (MP) supply and milk production in dairy cows. However, reducing dietary CP content from 17.3 to 15.2% decreased milk production. This response was attributed to an insufficient supply of ruminally degradable protein (RDP) that suppressed microbial nonammonia N (NAN) synthesis in the rumen, thus decreasing intestinal MP supply. In Experiment 3, the objective was to determine the effects of replacing barley or corn starch with lactose (as DWP) in diets containing 10% W-DDGS on ruminal function, omasal nutrient flow, and lactation performance. The treatment factors were source of starch (barley vs. corn) and dietary inclusion level of DWP (0 vs. 6%; DM basis) as a partial replacement for starch. Diets were isonitrogenous (18% CP) and contained 3 or 8% total sugar. The starch content of the low sugar diet was 24% compared to 20% for the high sugar diet. Dry matter intake, and milk and milk component yields did not differ with diet. However, partially replacing dietary corn or barley starch with sugar up-regulated ruminal acetate and propionate absorption, and reduced ruminal NH3-N concentration, but had no effect on ruminal pH, microbial protein synthesis, omasal nutrient flow and production in dairy cows. In summary, data presented in this thesis indicate that W-DDGS and B-DDGS can be included as the major source of protein in dairy cow diets without compromising ruminal function, nutrient supply and milk production in dairy cows. Feeding medium to low CP diets, and partial replacement of starch with sugar in diets containing W-DDGS and B-DDGS can improve N utilization efficiency in dairy cows. Additionally, an upregulation of facilitated transport of acetate and propionate across epithelial cells possibly prevents the occurrence of ruminal acidosis when lactose partially replaces starch in cow diets.