J. K. Ha

RELATIVE CONTRIBUTIONS OF BACTERIA, PROTOZOA, AND FUNGI TO IN VITRO DEGRADATION OF ORCHARD GRASS CELL WALLS AND THEIR INTERACTIONS

ABSTRACT To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.

Comparative studies of microbial populations in the rumen, duodenum, ileum and faeces of lactating dairy cows.

Aims:  Understanding factors that influence the composition of microbial populations of the digestive system of dairy cattle will be key in regulating these populations to improve animal performance. Although rumen microbes are well studied, little is known of the dynamics and role of microbial populations in the small intestine of cows. Comparisons of fingerprints of microbial populations were used to investigate the effects of gastrointestinal (GI) segment and animal on community structure.

Starch Source Evaluation in Calf Starter: II. Ruminal Parameters, Rumen Development, Nutrient Digestibilities, and Nitrogen Utilization in Holstein Calves

Ruminal parameters, rumen development, nutrient digestibilities, and N utilization were estimated in Holstein calves fed starch from different sources. Ground corn, ground barley, ground wheat, and crimped oats were used to formulate 4 isostarch (25% of starter dry matter) pelleted diets. These diets were randomly allocated to calves (16 calves per treatment, 8 female and 8 male) and fed ad libitum along with mixed grass hay throughout the experiment. Ruminal contents and blood were sampled at d 35, 50, and 70 of age to estimate ruminal parameters and plasma beta-hydroxybutyrate, respectively. At d 70, twenty-four male calves (6/treatment) were randomly selected, euthanized, and forestomach weight, papillae length (PL), papillae width (PW), rumen wall thickness (RWT), and papillae concentration were measured. At d 63, twenty-four female calves (6/treatment) were randomly selected and moved to metabolism stalls to estimate total tract apparent nutrient digestibilities and N utilization. Female calves were given 2 wk for adaptation to experimental facilities and then total collections of feces and urine were made from d 77 to 84 of age. Ruminal pH at d 35 of age was higher in calves fed corn and oat diets than in those fed barley and wheat diets. Ruminal pH at d 50 and 70 of age was the lowest in calves on barley diets followed by those on oat and wheat diets and then by those on the corn diet. Ruminal total volatile fatty acid concentrations at d 35 of age were greatest in calves fed corn or wheat diets followed by those fed barley and oat diets. Calves on corn and wheat diets maintained greater ruminal volatile fatty acids concentrations at d 50 and 70 of age. Ruminal ammonia, acetate, propionate, butyrate, and blood beta-hydroxybutyrate concentrations were also greater in calves on the corn and wheat diets. Full and empty weights of forestomach, PL, PW, RWT, and papillae concentrations were greater in calves on corn and wheat diets. Daily average intake of nutrients (dry matter, crude protein, neutral detergent fiber, starch, Ca, and P) was greater in calves fed corn and wheat diets than in those fed barley and oat diets. Starch source did not influence the total tract apparent digestibilities of nutrients in calves. Daily N retention (g/d) was greatest on the corn diet followed by the wheat diet and then the barley and oat diets. In conclusion, calves on a corn diet have greater ruminal capacity to accommodate feed bulk. More physically and metabolically functional rumens in calves on corn and wheat diets probably resulted in greater feed consumption and N retention.

Effect of silica on the colonization of rice straw by ruminal bacteria

The relationship between silica distribution and microbial colonization in the cuticle of rice straw was examined using scanning electron microscopy and energy dispersive X-ray microanalysis (EDX). Rice straw stems cut into 1 -cm lengths were either treated with 40 g NH3 kg−1 dry matter (DM) or left untreated. Microbial colonization was examined using cultures of Fibrobacter succinogenes S85 grown on rice straw as the sole source of carbohydrate. The nylon bag technique was also used to study the colonization of rice straw by mixed ruminal microorganisms and to determine in sacco dry matter disappearance (ISDMD). Examination by EDX revealed the outer (abaxial) surface of the leaf sheath to be virtually covered by silica, with high levels of accumulation within distinctly arranged opaline bodies (phytoliths). Silica was distributed diffusely over most of the inner (adaxial) surface of the leaf sheath, but phytoliths were absent and some regions were devoid of silica. Ammonia treatment dramatically increased the ISDMD of rice straw, but did not alter the content or distribution of silica on either surface of the leaf sheath. Trichomes contained no silica and were major sites of invasion and colonization in the intact cuticle of rice straw. Cuticular damage and loss were more prevalent in treated than in untreated rice straw, and underlying tissues were found to be devoid of silica and extensively colonized. The improved digestibility of ammonia-treated rice straw may be partially due to a weakening of the adhesion between the cuticle and underlying tissues. Detachment of the cuticle removes this barrier to digestion and exposes underlying silica-free tissues to bacterial colonization.

The stress of weaning influences serum levels of acute-phase proteins, iron-binding proteins, inflammatory cytokines, cortisol, and leukocyte subsets in Holstein calves.

The purpose of our study was to investigate changes in immunological parameters induced by weaning stress (including milk restriction) in calves. Fifteen Holstein calves were subjected to weaning at 6 weeks of age. Blood samples were collected at -14, -7, -2, 1, 3, and 5 days post-weaning (DPW; 0 DPW = 42 days). Weaning caused significant (p < 0.01) increases in the neutrophil (NE):lymphocyte (LY) ratio at 5 DPW with a significant (p < 0.05) reduction of LYs. The concentration of acute-phase proteins (haptoglobin and serum amyloid A) also increased significantly (p < 0.05) at 3 and 5 DPW compared to -2 DPW. Levels of the iron-binding protein lactoferrin decreased significantly (p < 0.05) after weaning. Serum tumor necrosis factor-α and cortisol levels were elevated (p < 0.05) at 3 DPW, while those of serum interferon-γ decreased (p < 0.05) at 1 and 3 DPW compared to levels observed before weaning. Weaning significantly (p < 0.05) decreased the percentage of CD25+ T cells in the peripheral blood. In conclusion, weaning stress affected the NE:LY ratio along with the levels of acute phase proteins, lactoferrin, cortisol, and inflammatory cytokines in the peripheral blood of calves. Weaning stress may induce an acute phase response possibly through the elevation of cortisol production and modulation of inflammatory cytokines.

Comparative study on the aflatoxin B1 degradation ability of rumen fluid from Holstein steers and Korean native goats

The aflatoxin B1 degrading abilities of two different ruminants were compared in this study. One set of experiments evaluated the aflatoxin B1 degradation ability of different rumen fluid donors (steers vs. goats) as well as the rumen fluid filtration method (cheese cloth filtered vs. 0.45 µm Millipore) in a 2 × 2 factorial arrangement. Additional studies examined aflatoxin B1 degradation by collecting rumen fluid at different times (0, 3, 6, 9 and 12 h) after feeding. Cannulated Holstein steers (740 ± 10 kg bw) and Korean native goats (26 ± 3 kg bw) were fed a 60% timothy and 40% commercial diet with free access to water. Rumen fluid from Korean native goats demonstrated higher (p < 0.01) aflatoxin B1 degradability than Holstein steers. However, filtration method had no significant influence on degradability. In addition, aflatoxin degradation did not depend upon rumen fluid collection time after feeding, as no significant differences were observed. Finally, a comparison of two types of diet high in roughage found aflatoxin degradability in goats was higher with timothy hay opposed to rice straw, although individual variation existed. Thus, our findings showed the aflatoxin degradability is comparatively higher in goats compared to steers.

Characterization of Cellulolytic and Xylanolytic Enzymes of Bacillus licheniformis JK7 Isolated from the Rumen of a Native Korean Goat

A facultative bacterium producing cellulolytic and hemicellulolytic enzymes was isolated from the rumen of a native Korean goat. The bacterium was identified as a Bacillus licheniformis on the basis of biochemical and morphological characteristics and 16S rDNA sequences, and has been designated Bacillus licheniformis JK7. Endoglucanase activities were higher than those of β-glucosidase and xylanase at all temperatures. Xylanase had the lowest activity among the three enzymes examined. The optimum temperature for the enzymes of Bacillus licheniformis JK7 was 70°C for endoglucanase (0.75 U/ml) and 50°C for β-glucosidase and xylanase (0.63 U/ml, 0.44 U/ml, respectively). All three enzymes were stable at a temperature range of 20 to 50°C. At 50°C, endoglucanse, β-glucosidase, and xylanase had 90.29, 94.80, and 88.69% residual activity, respectively. The optimal pH for the three enzymes was 5.0, at which their activity was 1.46, 1.10, and 1.08 U/ml, respectively. The activity of all three enzymes was stable in the pH range of 3.0 to 6.0. Endoglucanase activity was increased 113% by K+, while K+, Zn+, and tween 20 enhanced β-glucosidase activity. Xylanase showed considerable activity even in presence of selected chemical additives, with the exception of Mn2+ and Cu2+. The broad range of optimum temperatures (20 to 40°C) and the stability under acidic pH (4 to 6) suggest that the cellulolytic enzymes of Bacillus licheniformis JK7 may be good candidates for use in the biofuel industry.

Effects of hydrolyzed yeast supplementation in calf starter on immune responses to vaccine challenge in neonatal calves

The effects of hydrolyzed yeast supplementation on growth performance, health and immune-physiological parameters in neonatal calves challenged with vaccine were investigated. Twelve Holstein calves were started in the experiment at 2 ± 1 days of age and were studied for 35 days. Calves were randomly assigned to each of two dietary treatments, a control (CON) and hydrolyzed yeast (HY) group. The calves in the HY group received control calf starter supplemented with 0.2% HY. All calves were given calf starter ad libitum for 5 weeks starting in week 1. Calves were also given whole milk according to a step-down milking protocol. In order to induce immune responses, all calves were challenged with Hog cholera and Erysipelothrix insidiossa live vaccines by intramuscular injection at 3 weeks of age. Growth performance and feed intake were not affected by dietary treatment throughout the experimental period, except that the HY group had significantly higher (P < 0.05) milk intake than did the CON group at 3 weeks of age. Calves in the HY group showed significantly better (P < 0.05) fecal and health scores at 3 weeks compared to those in the control group. After vaccine challenge, neutropenia, lymphophilia and thrombocytopenia were observed in the CON group, but calves in the HY group did not show significant changes of leukocytes. The average concentration of serum haptoglobin in the HY group was significantly higher (P < 0.05) at 1 and 3 days post-vaccine challenge (DPVC) than that of CON group. Feeding HY supplemented calf starter resulted in a higher (P < 0.05) relative amount of bacterial and viral - specific IgA than in the CON group at 5 DPVC. Although the percentage of CD4+ T cells was significantly (P < 0.05) higher in the HY group than in the CON group at -2 DPVC, significant differences between groups after vaccine challenge was not observed during the experimental period. These results suggest that 0.2% HY supplementation in calf starter can improve the health status and immune-related serum protein production and affect blood cell composition in neonatal calves after vaccine challenge.

Influence of equalizing the gross composition of milk replacer to that of whole milk on the performance of Holstein calves

This study compared the performance of female Holstein calves fed either whole milk (WM) or milk replacer (MR) having similar gross composition to WM. Calves (n = 20) were separated from their mothers within 2 h of birth, weighed, moved into individual pens, and fed colostrum (10% of their BW over 2 feedings daily) for the first 3 d. Calves were alternately assigned to either WM (n = 10) or MR (n = 10) and were fed using mobile plastic bottles. At each feeding, diluted MR was prepared in buckets by mixing 0.143 kg of MR powder (as-is basis) in 1 L of lukewarm water. Calves on both treatments were fed (1.8 L/feeding) for 4 times daily for the first 25 d of age. Feeding frequency was reduced to 3 times daily for next 5 d, then to 2 times daily for next 14 d, and then to once daily during the last 5 d of the preweaning period. Feed intake, growth, and health variables were monitored until calves were 70 d of age. Mean daily consumption of WM and MR was similar in both treatment groups (P = 0.74). Initial BW of calves fed either WM or MR was similar (42.1 +/- 3.3 vs. 41.9 +/- 2.9 kg, respectively; P = 0.83). Calves fed WM or MR daily consumed similar amounts of calf starter (1,019 vs. 1,056 g, respectively; P = 0.32), hay (121.5 vs. 126.3 g, respectively; P = 0.30), and water (5.2 vs. 5.0 kg, respectively; P = 0.54). At weaning (d 49) and postweaning (d 70), BW was greater (P = 0.03 and P = 0.02, respectively) in calves fed WM than in those fed MR. Mean DMI from both solid and liquid feeds was not affected by the treatments during the preweaning (P = 0.21) and postweaning (P = 0.16) periods. Body weight gain efficiency during preweaning and overall was improved (P = 0.001 and P = 0.002, respectively) in calves fed WM than in those fed MR. Health (days scoured, rectal temperature, respiratory score, and general appearance score) and serum chemistry variables (glucose, total protein, urea N, nonesterified fatty acids, and creatinine) in calves were not affected (P >or= 0.12 and P >or= 0.12, respectively) by the treatments. Even though gross composition of the MR and WM was similar, growth was greater in calves fed WM. Calves fed WM consumed similar amounts of DM and were heavier than those fed MR, probably because of better bioavailability (digestion and assimilation) of nutrients and availability of some unknown growth factors from WM.

Production of Endoglucanase, Beta-glucosidase and Xylanase by Bacillus licheniformis Grown on Minimal Nutrient Medium Containing Agriculture Residues

Bacillus licheniformis was grown in minimal nutrient medium containing 1% (w/v) of distillers dried grain with soluble (DDGS), palm kernel meal (PKM), wheat bran (WB) or copra meal (CM), and the enzyme activity of endoglucanase, β-glucosidase, xylanase and reducing sugars was measured to investigate a possibility of using cost-effective agricultural residues in producing cellulolytic and hemicellulolytic enzymes. The CM gave the highest endoglucanase activity of 0.68 units/mL among added substrates at 48 h. CM yielded the highest titres of 0.58 units/ml of β-glucosidase, compared to 0.33, 0.23, and 0.16 units/mL by PKM, WB, and DDGS, respectively, at 72 h. Xylanase production was the highest (0.34 units/mL) when CM was added. The supernatant from fermentation of CM had the highest reducing sugars than other additional substrates at all intervals (0.10, 0.12, 0.10, and 0.11 mg/mL respectively). It is concluded that Bacillus licheniformis is capable of producing multiple cellulo- and hemicellololytic enzymes for bioethanol production using cost-effective agricultural residues, especially CM, as a sole nutrient source.