M. S.

Captive gray squirrels were fed acorns or diets containing commercial tannins to determine the effects of tannins on digestion and detoxification (glucuronidation) activity. In the Acorn experiment, Northern red oak acorns (Quercus rubra), white oak acorns (Q. alba), or rat chow were fed to squirrels. Levels of phenols, condensed tannins, and hydrolyzable tannins were higher in red oak acorns than white oak acorns and were likely responsible for the lower dry matter intake, lower apparent protein digestibility, lower digestible protein and energy intakes, and higher glucuronidation activity observed in squirrels fed red oak acorns. Although apparent protein digestibility and digestible protein intakes were reduced on a white oak acorn diet, this diet did not continuously suppress dry matter intake or stimulate glucuronidation. It appears that gray squirrels may not be able to subsist on red oak acorns alone, but may require other foods to dilute tannin intake and provide additional nutrients. In the Tannin experiment, squirrels were fed rat chow containing no tannins, 4% or 8% tannic acid (hydrolyzable tannin), or 3% or 6% quebracho (condensed tannin). Apparent protein and energy digestibilities of tannic acid-containing diets were lower than the control. These reductions were likely due to the formation of strong complexes between protein and high molecular weight gallotannins. These complexes may have simultaneously protected these gallotannins from hydrolysis and allowed tannic acid to reduce digestive efficiency. Apparent protein and energy digestibilities of quebracho-containing diets were reduced, indicating protein complexing by these nonhydrolyzable tannins. Consistent with the hypothesis that hydrolyzable tannins are more likely to be broken down and absorbed internally than condensed tannins, only the 8% tannic acid diet tended to increase glucuronide excretion. / Master of Science

M. S.

Ninety 12 month old female rats were used to determine a protein requirement for tissue maintenance. Diets contained 0.84 to 4.69 percent protein and were fed for 28 days. Liver composition, carcass composition and serum protein levels were determined, and a protein requirement level was also predicted. Rats fed 0.84 and 1.86 percent protein had significantly lower serum protein values than a baseline group of rats sacrificed at the beginning of the study. Rats fed the two highest levels of dietary protein had serum protein values which were significantly higher than the baseline group and that of rats fed 2.81 percent protein was not significantly different from that of the baseline group. Rats fed 0.84, 1.86 and 2.81 percent protein had excessive liver fat, indicative of protein deficiency. No group was found to have liver nitrogen values comparable to that of the baseline group. All rats except those fed 0.84 percent protein had significantly more carcass fat than the baseline group. Total carcass nitrogen of rats fed 2.81, 3.77 and 4.69 percent protein were not significantly different from the baseline group indicating a general state of nitrogen equilibrium in these animals. Using linear regression, a protein requirement of 4.07 percent of the diet was predicted. Comparison of this value with other literature values was difficult due to differences in experimental design. It was suggested that in further research of this kind a longer study should be conducted and a concurrent baseline group should be used. / Master of Science

The amino acid requirement for milk protein synthesis was investigated in the rat mammary cells in tissue culture. Studies involved: 1) the amino acid reduction from a positive control (3X) back to a negative control (1X) for each of the essential amino acids that were present in the 1X-MEM (Omission Study) [X refers to the amino acid concentration of Eagle's minimal essential medium (MEM)], 2) the addition experiments which resulted in an inverse relation to those of the Omission studies, and 3) a three-dimensional central composite experiment with three variables (X₁ = lysine; X₂ = methionine, valine and arginine; X₃ = isoleucine, tryptophan, threonine, phenylalanine and histidine), embracing 15 treatment combinations. In addition, supplemental studies were conducted which determined: 1) the effect of varied labeled amino acid precursors (¹⁴C-U-lysine, phenylalanine and leucine) on the measured synthesis of milk protein, 2) the effect of varying cell density (6 x 10⁶ to 6 x 10⁷) on protein synthesis, and 3) the response to graded amino acid complement (1X through 5X) on milk protein synthesis. Supplemental studies indicated that: 1) synthesis of β-lactoglobulin and β-casein in response to the labeled amino acids were significant (P<.05), 2) cell numbers between 6 x 10⁶ to 6 x 10⁷ did not significantly (P>.05) alter the rates of the milk protein synthesis, and 3) the response of amino acid complement addition to the 1X-MEM was linear (P<.01) for β-lactoglobulin and β-casein synthesis. Data analysis from Omission and Addition experiments revealed that: 1) the first limiting amino acid was lysine, 2) the second limiting group of amino acids were methionine, valine and arginine, and 3) the least limiting amino acids were tyrosine, leucine and glutamine. The response surface study determined that the optimal combination of essential amino acids for the maximum milk protein synthesis were: 1) lysine = 15X, 2) methionine, valine and arginine = 4.5X, and 3) isoleucine, tryptophan, threonine, phenylalanine and histidine = 1. 5X elevation over that present in the 1X-MEM, and 4) leucine, tyrosine, cystine and glutamine = 1X. On the basis of these findings, it was possible to construct the optimal essential amino acid complement unique to the rat mammary cells in tissue culture for the maximum milk protein synthesis. / Ph. D.

Thirty-six mature, nongestating, crossbred ewes were randomly allotted to three diets: basal (6 ewes); 50% basal and 50% broiler litter (24 ewes), and 50% basal and 50% broiler litter with the addition of 25 ppm molybdenum (Mo) and 5 g sulfate/kg feed. The 24 litter fed ewes were allotted to withdrawal times of 0-, 30-, 60- and 120-d. The control (basal) diet contained and the deepstacked broiler litter contained 8 and 257 ppm Cu, dry basis, respectively. The ewes were fed the diets for 140-d at which time the control, the six litter-fed ewes with 0-d withdrawal and the Mo and SO₄ supplemented ewes were slaughtered. Liver samples were obtained from these ewes by biopsy initially and at 40-, 80-, and 120-d of the trial. At slaughter liver, kidney, muscle and duodenum samples were taken. Liver Cu content was increased (P<.01) by feeding broiler litter (404 vs 1543 ppm, dry basis). Liver Cu (962 ppm) accumulation and serum Cu were depressed (P<.05) by supplementing litter containing diets with Mo and SO₄. Zinc levels of the liver and Zn and Cu levels of the kidney and muscle were not affected by additional Mo and SO₄. Duodenum Cu and Zn levels reflected the dietary metal content. During the withdrawal period liver and duodenum Cu levels were not lowered (P>.05). A linear increase (P<.001) in SGOT activity was observed with time throughout the withdrawal period, suggesting possible liver damage. Serum Cu decreased at 30-d and increased thereafter (quadratic effect, P<.05). This observation coupled with increases in muscle and kidney Cu (P<.05) indicated an apparent redistribution of Cu stores. Metallothionein (MT) increased 12-fold above control values in the litter-fed ewes (P<.05) and tended to decrease during the withdrawal period. Therefore, it appears that supplementing Mo and SO₄ to a diet containing high Cu broiler litter may reduce the accumulation of hepatic Cu. Liver Cu stores will not be dissipated upon withdrawal of the high Cu litter from the diet. / Master of Science

Supplementation of the diet with low concentrations of antibiotics stimulates the growth of poultry by affecting the intestinal flora. The bacterial flora of the small intestine of turkey poults was extensively analyzed in an attempt to correlate changes in populations with growth response. Lactobacillus species comprised almost 100% of the duodenal flora of two-week-old poults but there was no difference in species associated with antibiotic (zinc bacitracin, 55 ppm) treatment. The ileal flora also was predominantly lactobacilli (average 75% of the flora). The most common lactobacilli from the turkey intestinal tract were several previously undescribed Lactobacillus species followed by L. acidophilus, L. salivarius subsp. salivarius, L. fermentum, and L. plantarum. Antibiotic treatment resulted in a shift in the proportions of several of the unnamed Lactobacillus sp. Preliminary feed trials using two strains of lactobacilli that belonged to species that increased in numbers with antibiotic treatment did not stimulate growth when one-day-old birds were colonized with the strains. A probable explanation for the increase in growth is the effect of antibiotic treatment on the multiplication of bacteria in the small intestine. As the digesta move from the gizzard to lower ileum an average 16-fold increase in bacteria occurs in untreated birds. In antibiotic-treated birds the increase was only 2-fold. This inhibition of growth is not due strictly to cell lysis because there are no significant differences in microscopic counts, but the viable counts do decrease. As a corollary there is significantly less lactic acid in the lower ileum of antibiotic-fed birds. Antibiotics did not affect total microscopic or viable counts in the crop or ceca. The above experiments were all done with zinc bacitracin; however, the inhibition of bacterial multiplication was also observed with procaine penicillin. The conclusion from my data is that zinc bacitracin, and probably procaine penicillin, stimulate the growth of turkey poults by a general suppression of the small intestinal flora rather than by an effect on any individual bacterial species. / Ph. D.

A set of experiments were conducted to evaluate the antibody response, serum vitamin E level, cortisol concentration and performance of pigs weaned at three ages (21, 28 or 35 d) and fed diet containing either 11 or 220 IU/kg diet recommended level of vitamin E. Supplemental dietary vitamin E (220 IU) increased the concentration of serum vitamin E but, did not affect performance, cortisol concentrations or the antibody response. As weaning age increased, weekly performance increased linearly as did cortisol levels. Animals weaned at 35 d age had the largest primary antibody response, but this difference was not observed for the secondary response. A second set of experiments evaluated effects of four dietary vitamin E levels (11, 110, 220 and 550 IU/kg feed) on the humoral and cell-mediated immune response and performance of 4 wk old weanling pigs housed at one of two nursery temperatures (19 or 30°C). Performance was greater for pigs housed at 19°C compared with pigs housed at 30°C, but mitogen stimulation indices of white blood cells, plasma cortisol levels, and antibody titers were similar. Serum and liver vitamin E levels linearly increased with increasing dietary vitamin E level, but performance, cortisol, antibody levels and mitogen induced stimulation indexes were not affected by supplemental vitamin E levels. In the third experiment, sows fed a NRC level of vitamin E demonstrated little fluctuation in serum vitamin E concentration during a 5 wk lactation period. There was a high concentration of vitamin E in colostrum, followed by a sharp decrease in milk vitamin E concentration after the first week of lactation and remained constant for the next four weeks. Piglet serum was initially low in vitamin Eat 1 d of age but increased with time peaking about week 3. These experiments suggest that supplementing dietary vitamin E above the levels recommended by the NRC to baby pigs (which nursed sows fed NRC recommended levels of vitamin E) will not influence cell-mediated or humeral immune response, performance parameters or cortisol levels when pigs were weaned at various ages or exposed to environmental temperature changes. / Doctor of Philosophy

Studies were conducted to determine the effects of feeding lasalocid and monensin upon mineral status of ruminants, changes in digestive and renal physiology which bring about altered mineral status, and the effects of dietary K upon the actions of ionophores. Two 84-d grazing trials were conducted with steers fed no ionophore, 200 mg lasalocid, or 150 mg monensin. Monensin tended to increase gain of steers, but lasalocid had no effect. Feeding supplemental lasalocid and monensin altered Ca, P, Mg, Na, K, Cu, Fe and Zn status of grazing steers, but effects were not consistent. Twenty-four crossbred steers were individually fed in a 147-d finishing trial to study a possible K x monensin interaction. Steers were fed diets ad libitum containing .4% or 2.3% K, with or without 23 ppm monensin, dry basis. Feeding monensin with low K increased serum Na, Mg, inorganic P, Cu and Zn, but monensin fed with high K had no effect or decreased these serum minerals. Fifteen wethers, fitted with abomasal and ileal cannulae, were fed a basal diet alone or supplemented with 23 ppm lasalocid or monensin; Apparent absorption of Mg, K and Fe increased when ionophores were fed. Magnesium flow through the small and large intestines was decreased by lasalocid and monensin. Effects of lasalocid and monensin differed for metabolism, digestive tract flow and (or) partial absorption of Ca, P, Cu, Fe and Zn. Twelve ewe lambs were used in a renal clearance experiment. Lambs were fed a basal diet with no ionophore, 23 ppm lasalocid, or 23 ppm monensin, dry basis. Ionophores were fed starting on d 1 of the experimental period. Serial collections of blood and urine were made during d 1 and d 5 of the experimental period. Monensin reduced urine flow rate at d 1 and d 5. Feeding monensin lowered serum clearance and urinary excretion rates of Na and K at d 5. Serum clearance and urinary excretion rates of Ca were lower in sheep fed ionophores at d 1. Feeding monensin decreased serum clearance and urinary excretion rates of Cu at d 1. At d 5, urinary excretion rates of Zn were greater when lasalocid was fed, and lower when monensin was fed, compared to feeding no ionophore. These studies indicate that dietary lasalocid and monensin can alter mineral status of ruminants, although their effects may differ. The effects of ionophores may be dependent upon dietary K levels. / Ph. D.

Purified brush border and basolateral membranes were isolated from homogenized intestinal enterocytes of Holstein steers by divalent cation precipitation followed by differential and sucrose density gradient centrifugation. Membrane marker enzymes were used to determine the effectiveness of the fractionation procedure. Alkaline phosphatase and sodium-potassium adenosine triphosphatase served as the marker enzymes for the brush border and basolateral membranes, respectively. The brush border fraction was enriched 5.1-fold over the cellular homogenate. Purification of 10.1-fold over cellular homogenate was obtained for the basolateral membrane fraction. Electronmicrographs and osmotic response data were used to confirm the vesicular nature of the membrane preparations. Brush border membrane vesicles from bovine jejunal and ileal tissue were used to evaluate lysine (LYS) and methionine (MET) transport. Total transport of LYS and MET was divided into mediated and diffusion components. Mediated uptake was further divided into sodium-dependent (Na⁺) and sodium independent (Na⁻) systems. Total LYS and MET uptake by ileal tissue tended to be higher than jejunal tissue at all concentrations evaluated but differences were significant (P<.O5) at 2.5 and 7.5 mM for LYS and 5, 12.5 and 15 mM for MET. The greater capacity of ileal tissue appeared to be due to the Na⁺ component of LYS uptake and the diffusion component of MET uptake. Methionine transporters had lower affinities and higher capacities than the corresponding LYS transporters in both ileal and jejunal tissue. Methionine transport was greater (P<.O5) than LYS transport in both ileal and jejunal tissue when initial amino acid concentration was 7.5 mM. When initial amino acid concentration was 1.25 mM, MET uptake was greater (P<.13) than LYS uptake in jejunal but not ileal tissue. The relative contribution of mediated and diffusion uptake systems to total MET and LYS uptake was found to be dependent of substrate concentration. / Ph. D.