Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Eight cecally fistulated geldings were used in a randomized 4 x 4 Latin square design to observe the effect varying levels of dietary starch had on cecal environment. The 4 treatment rations contained 2 g starch/kg BW (Diet 2), 4 g/kg BW (Diet 4), 6 g/kg BW (Diet 6), or 8 g/kg BW (Diet 8). The rations were comprised of a commercial pelleted feed to meet 2 g starch/kg BW in each treatment, with ground corn used to fulfill the remaining starch requirements in each diet. Soybean meal was added to ensure diets were iso-nitrogenous, and cottonseed hulls were used to equalize dry matter intake. A 21 day adaptation period was allowed before cecal contents were sampled. Samples were drawn 4 hours after the morning meal and were immediately tested for pH. Samples were used to count total anaerobic bacteria and lactic acid bacteria, as well as determine methane activity, ammonia activity, volatile fatty acids, and in vitro dry matter digestibility (IVDMD). Stoichiometric calculations were performed to give an indirect measure of fermented hexose, methane, and carbon dioxide. Diet did not influence dry matter intake (DMI), however it did have an effect on starch intake (P < 0.0001) and caused a linear increase in starch consumption as the amount of offered starch increased (P < 0.0001). Diet did not influence the pH of the cecum (P > 0.05), although a tendency for a linear decrease (P < 0.06) in pH from 6.92 ? 6.58 occurred when dietary starch increased. Total anaerobic bacteria and lactic acid bacteria were unaffected by treatment diets (P > 0.05). Propionate production was affected by dietary treatment (P < 0.05), causing a quadratic increase (P = 0.04) from 8.26 to 14.13 mM as starch in the diets increased. Diet did not affect the production of acetate, butyrate, or ammonia (P > 0.05). Results found that stoichiometric calculations and IVDMD values were not affected by diet (P > 0.05). These results show that starch intake influenced the production of fermentative by-products, which caused decreases in pH, although there was no observed increase in the bacterial populations of the cecum.

Eight cecally fistulated geldings were used in a randomized 4 x 4 Latin square design to observe the effect varying levels of dietary starch had on cecal environment. The 4 treatment rations contained 2 g starch/kg BW (Diet 2), 4 g/kg BW (Diet 4), 6 g/kg BW (Diet 6), or 8 g/kg BW (Diet 8). The rations were comprised of a commercial pelleted feed to meet 2 g starch/kg BW in each treatment, with ground corn used to fulfill the remaining starch requirements in each diet. Soybean meal was added to ensure diets were iso-nitrogenous, and cottonseed hulls were used to equalize dry matter intake. A 21 day adaptation period was allowed before cecal contents were sampled. Samples were drawn 4 hours after the morning meal and were immediately tested for pH. Samples were used to count total anaerobic bacteria and lactic acid bacteria, as well as determine methane activity, ammonia activity, volatile fatty acids, and in vitro dry matter digestibility (IVDMD). Stoichiometric calculations were performed to give an indirect measure of fermented hexose, methane, and carbon dioxide. Diet did not influence dry matter intake (DMI), however it did have an effect on starch intake (P < 0.0001) and caused a linear increase in starch consumption as the amount of offered starch increased (P < 0.0001). Diet did not influence the pH of the cecum (P > 0.05), although a tendency for a linear decrease (P < 0.06) in pH from 6.92 ? 6.58 occurred when dietary starch increased. Total anaerobic bacteria and lactic acid bacteria were unaffected by treatment diets (P > 0.05). Propionate production was affected by dietary treatment (P < 0.05), causing a quadratic increase (P = 0.04) from 8.26 to 14.13 mM as starch in the diets increased. Diet did not affect the production of acetate, butyrate, or ammonia (P > 0.05). Results found that stoichiometric calculations and IVDMD values were not affected by diet (P > 0.05). These results show that starch intake influenced the production of fermentative by-products, which caused decreases in pH, although there was no observed increase in the bacterial populations of the cecum.

Retrograded starches are commonly found in foods due to the production and/or processing conditions they have received prior to consumption. These resistant starches escape digestion in the small intestine and are fermented in the colon by the microflora present, to produce gases and SCFA in varying amounts. These are utilised by the host animal as an energy source, with a low gut pH being maintained by the production of SCFA. The fermentation of carbohydrates within the colon is beneficial to the health of the gut, as the beneficial bacterial species such as Lactobacillus and Bifidobacterium spp. are maintained, and a low pH reduces the activity of potentially harmful species such as the coliforms. The production of toxic metabolites from the breakdown of proteins will be reduced if these resistant starches persist further along the colon as a carbohydrate source. This is particularly important in the distal region of the colon, where the carbohydrate source usually becomes limited. The fermentation of both native and retrograded starches from various botanical sources containing varying amounts of the major components amylose and amylopectin, was examined. In particular, the effects on bacterial fermentation of variations in the ratios of amylose and amylopectin in starch, and of treatments such as retrogradation and/or pancreatin digestion was examined.

The effects of starch and phosphorus (P) source on P partitioning and ruminal phytase activity were evaluated in eight lactating cows (113 DIM). Four cows were ruminaly cannulated. Cows were randomly assigned to treatments in a duplicated 4x4 Latin square with four, 18-d periods. Diets included dry ground corn (DG) or steam flaked corn (SF), with a no supplemental P (low P diet; 0.34% P) or supplemental purified phytic acid (PA; 0.45% P) to provide additional P from an organic source. Total collection of milk, urine, feces, and feed were sampled each period, while rumen fluid was sampled on d 18. Excretion of feces, urine, P, and N was lower in cows fed SF than in cows fed DG. Milk yield was unaffected by diet despite a lower DMI by cows fed SF. Cows fed SF tended to have a higher feed efficiency and lower milk urea nitrogen (MUN) concentration than cows fed DG. Rumen pH was unaffected by diet, but milk fat content was lower for cows fed SF. Milk yield, DMI, and feed efficiency were not affected by PA. Cows fed PA had increased P intake and excretion, but a lower milk P as a percentage of intake compared with cows fed the low P diet. An interaction of starch source and P source was observed for ruminal phytase activity. Altering dietary sources of starch and P offers opportunity to improve P availability and reduce manure nutrient excretion. / Master of Science

Doctor of Philosophy / Department of Animal Sciences and Industry / Evan C. Titgemeyer / Previous data suggest that greater amounts of postruminal protein increase small intestinal starch digestion in cattle. Duodenally and ileally cannulated steers were used in 5 studies to measure responses in small intestinal starch digestion to amino acids (AA) or casein. Flows of starch to the ileum from the diet were small. Small intestinal starch digestibility was 34.0% when raw cornstarch was continuously infused into the duodenum. Infusion of casein linearly increased (P ≤ 0.05) small intestinal starch digestibility, and small intestinal starch digestion adapted to infusion of casein in 6 d. Ethanol-soluble starch and unpolymerized glucose flowing to the ileum increased linearly (P ≤ 0.05) with increasing infusion of casein. Plasma cholecystokinin was not affected by casein infusion, but circulating levels of glucose increased linearly (P ≤ 0.05). In another study, 5 steers were fed a low-starch diet and provided continuous duodenal infusion of raw cornstarch in combination with AA or casein in order to measure response of small intestinal starch digestion. Duodenal infusion of casein increased (P ≤ 0.05) small intestinal starch digestion. When a mixture of AA with a profile similar to casein (CASAA) was infused, small intestinal starch digestion was similar (P = 0.30) to casein infusion. Infusion of only non-essential AA tended to increase (P = 0.14) small intestinal starch digestion relative to control; however, infusion of essential AA alone did not affect (P = 0.84) small intestinal starch digestion. Additionally, infusion of casein or essential AA increased ileal flows of ethanol-soluble starch, but non-essential AA alone were not different than the negative control. Duodenal infusion of Glu increased (P ≤ 0.05) small intestinal starch digestion, whereas a mixture of Phe, Trp, and Met (PTM) did not. Neither Glu nor PTM increased ileal flow of ethanol-soluble starch, but Glu and PTM provided together tended (P = 0.07) to increase ileal flows of ethanol-soluble starch. Our data suggest that Glu alone can increase small intestinal starch digestion in cattle similar to casein, but increases in small intestinal starch digestion in response to Glu are not associated with an increase in ileal flows of ethanol-soluble starch.

Doctor of Philosophy / Department of Animal Sciences and Industry / Christopher D. Reinhardt / Two studies evaluated dry-rolled corn (DRC) manufacturing considerations in feedlot finishing diets. In study 1, feedlots (n = 35) participated in a survey to evaluate dry-rolled corn (DRC) processing practices, processed corn particle size distribution, and fecal starch content in finishing cattle. Average particle size of dry-processed corn, including DRC and hammermill-ground corn across all operations (n = 35) was 4,223 ± 1,265 µm with a range of 1,165 to 6,823 µm. Fecal starch content averaged 19.0 ± 6.5% with a range of 7.0 to 36.6%. Diet composition was evaluated for co-product [27.8 ± 13.4%] roughage concentration [8.9 ± 2.0%] and NDF concentration [19.3 ± 4.3%]. In study 2, cross-bred yearling steers (n = 360; initial BW = 395 ± 33.1 kg) were used to evaluate the effects of dry-rolled corn (DRC) particle size in diets containing 20% (DMB) wet distiller’s grains plus solubles (WDGS) on feedlot performance, carcass characteristics, and starch digestibility. Treatments were Coarse DRC (4,882 µm; COARSE), Medium DRC (3,760 µm; MEDIUM), Fine DRC (2,359 µm; FINE), and Steam-flaked corn (SFC, 0.35 kg/L). Final BW and ADG were not affected by treatment (P > 0.05). Dry matter intake was greater and G:F was lower (P < 0.05) for steers fed DRC vs. SFC. There was a linear decrease (P < 0.05) in DMI in the final 5 weeks on feed with decreasing DRC particle size. Fecal starch decreased (linear, P < 0.01) as DRC particle size decreased. In situ starch disappearance was lower for DRC vs SFC (P < 0.05) and increased linearly (P < 0.05) with decreasing particle size at 8 and 24-h. The final study evaluated steam-flaked corn (SFC) manufacturing practices implemented, equipment utilized, and methods used and parameters targeted to measure flake quality from commercial feedlots (n = 17). Significant variables contributing to the final multiple linear regression model using enzymatic starch availability (Enzymatic) as the dependent variable were: SFC Moisture, cooled flake density (CoolFD), throughput, roll diameter, steam cabinet temperature (Temperature), and temper time (Enzymatic = 19.4476 - (0.6927*SFCMoisture) - (2.1664*CoolFD) - (0.5060*Throughput) + (0.6281*Roll Diameter) + (0.4312*Temperature) – (0.1963*Temper Time; P < 0.15).

Several biological abnormalities exist between the ruminant and nonruminant small intestine and influences carbohydrate assimilation. Two experiments were conducted to identify potential mechanisms to improve carbohydrate utilization in cattle. Experiment 1 evaluated the effects of duodenal starch infusions with casein or glutamic acid on post-ruminal carbohydrase activities. Experiment 2 evaluated the effects of dietary fructose on visceral organ development and expression of nutrient transporters and digestive enzymes involved in carbohydrate assimilation. In experiment 1, the results suggest that small intestinal starch digestion may be improved in cattle with increased small intestinal flow of casein through increases in post-ruminal carbohydrase activities. In experiment 2, dietary fructose supply influenced nutrient utilization, visceral organ growth, and digestive enzyme mRNA expression and activity in neonatal calves.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.