Progress 09/01/09 to 08/31/12
Outputs
OUTPUTS: Outputs: Eight experiments were conducted and analyzed to 1) Characterize fibrolytic activities and protein concentrations of 18 enzymes from 5 companies under ruminal conditions; 2) Examine how activities of the enzymes differed with pH and temperature changes; 3) Screen 18 enzymes for their effects on measures of rumen fermentation and nutritive value of bermudagrass haylage; 4) Examine effects of the application rate of the 5 most promising enzymes on measures of fiber digestion, rumen fermentation and nutritive value of bermudagrass haylage; 5) Examine effects of application of the best enzymes at the optimal dose on preingestive fiber hydrolysis and release of sugars and phenolic acids; 6) Examine effects of addition of 5 cofactors to the best 5 enzymes on fiber hydrolysis, release of sugars and fiber digestibility of bermudagrass haylage; 7) Evaluate regression relationships between enzyme activity and measures of fiber digestibility, nutritive value and rumen function; 8)Determine effects of enzyme addition to diets of dairy cows on their performance 9) Examine effects of enzyme application to ruminant diets on in vivo digestibility. Result dissemination: Four presentations were made at the 2011 Joint meeting of the of the American Society of Animal Science (ASAS), the American Dairy Science Association the American (ADSA) and the Poultry Science Association of America in 2011. In addition, two presentations were made at the 2012 XVI International Silage Conference in Finland. Two more presentations were made at the 2012 Joint meeting of the ADSA-AMPA-ASAS-CSAS-WSASAS. Each of these presentations was summarized into an abstract which was distributed to the approximately 3000 delegates who attended the ASAS ADSA meetings or the approximately 250 who attended the International silage conference in Finland. In addition, one presentation was made in July 2010 in Austria and another in February 2011 in Alberta Canada at the Research Coordination meetings of the International Atomic Energy Agency / FAO project titled Use of enzymes, direct-fed microbials and yeast to improve the efficiency of utilizing fibrous feeds in developing countries and reduce greenhouse gas emissions from livestock. The PI was invited to serve as a consultant on the project. The PI has been invited to present a keynote presentation summarizing findings from the project at the Ruminant nutrition symposium of the 2013 Joint meeting of ASAS and ADSA. Four manuscripts are in preparation from the project. PARTICIPANTS: Many of the experiments formed the basis of Juan Romero's Ph.D. thesis. Two post doctoral research associates, Kathy Arriola and Oscar Queiroz worked on the enzyme characterization and lactation performance trial respectively. Miguel Zarate, an MS student assisted with several of the in vitro enzyme evaluation tests. Dr. Elide Valencia conducted the ram digestibility trial as part of the subcontract to the University of Puerto rico. Drs. Charles Staples and Claudio Gonzalez provided advice on technical aspects and the project was led by the PI, Dr. Adegbola Adesogan. TARGET AUDIENCES: Target audiences include students and faculty at Universities, salesmen and technical services representatives working for nutrition companies, nutrition consultants and dairy producers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Results:Experiment 1 characterized the protein concentration and endoglucanase, esterase, exoglucanase, xylanase and beta-glucosidase activities of 18 enzymes sourced from Trichoderma longibrachiatum, Bacillus subtilis, Aspergillus niger, and Humicola spp. at pH 6 and 39oC. Considerable differences in these measures of enzyme activity were evident. Experiment 2 showed that xylanase and endoglucanase activities of the enzymes were optimal at 50oC for 83% and 78% of the enzymes and optimal at pH 4 -5 for 61 and 77% of the enzymes, respectively. This indicates that full the hydrolytic potential of most of the enzymes was unlikely to be realized in the rumen. Experiment 3 identified 5 of the 18 enzymes that showed the greatest potential to improve the neutral detergent fiber (NDF) digestibility of bermudagrass haylage when applied at rates recommended by the enzyme supplier. Enzymes sourced from Trichoderma longibrachiatum had the greatest response. Experiment 4 showed that increasing the enzyme application rate resulted in nonlinear increases in dry matter (DM) and NDF digestibility of bermudagrass haylage. At the optimum application rate, NDF digestibility was increased by 2.6 to 5.7% relative to the untreated control, but the optimum rate was enzyme-specific. Experiment 5 showed that on average, enzyme application increased preingestive hydrolysis of NDF, p-coumaric acid, ferulic acid, and water-soluble carbohydrates by 5, 10, 30 and 62%, respectively. Furthermore, increasing the enzyme application rate resulted in non-linear enhancements of these responses. In Experiment 6, addition of cofactors to specific enzymes numerically reduced the NDF concentration of bermudagrass haylage, but increased water-soluble carbohydrate (WSC) release significantly (P<0.01). In Experiment 7, fibrolytic enzyme activities and protein content explained 64, 72, 99 and 93% of variation in NDF, hemicellulose, WSC (%), and FER. The greatest (P < 0.01) contributors to accuracy (R2) of predicting NDF, WSC, and FER were endoglucanase activity (EN; 0.34) and beta glucosidase (0.15), EN (0.65) and protein content (0.14), and EN (0.17) and xylanase activity (0.71), respectively. Enzyme activity and protein concentration explained 56 and 81% of the variability in digestibility of DM and NDF and the greatest contributions to these predictions were EN and XY, which accounted for 0.19 and 0.11 of the variability in DMD and 0.31 and 0.13 of the variability in NDF digestibility, respectively. Experiment 8 showed that application of a mixture of the two of the most promising fibrolytic enzymes to bermudagrass silage did not improve milk production by lactating dairy cows. Experiment 8 showed that application of one of the most promising enzymes to old world bluestem hay had no effects on the intake or digestibility of the hay in rams.
Publications
- Arriola, K. G., J. J. Romero Gomez, and A. T. Adesogan. 2011. Effects of pH and temperature on fibrolytic enzyme activities of various commercial exogenous enzyme preparations. Proc. ASAS-ADSA-PSA Annual meeting, 2011. New Orleans, LA.
- Romero, J.J., K.G. Arriola, M.A. Zarate, A.T. Adesogan. 2011. Screening exogenous fibrolytic enzyme products for improved in vitro ruminal fiber digestibility of bermudagrass. Proc. ASAS-ADSA-PSA Annual meeting, 2011. New Orleans, LA.
- Romero, J.J., K.G. Arriola, M.A. Zarate, A.T. Adesogan. 2011. Effect of rate of application of various exogenous fibrolytic enzyme products on in vitro ruminal fiber digestibility of bermudagrass. Proc. ASAS-ADSA-PSA Annual meeting, 2011. New Orleans, LA.
- Romero, J.J., K.G. Arriola, M.A. Zarate, A.T. Adesogan. 2011 Relationships between exogenous fibrolytic enzyme product activities and in vitro ruminal digestibility of bermudagrass. Proc. ASAS-ADSA-PSA Annual meeting, 2011. New Orleans, LA
- Queiroz, O. C. M., A.T. Adesogan, J. L. P. Daniel, J. J. Romero, J. H. Shin, C.R. Staples, and J. E.P. Santos. 2011. Effects of adding fibrolytic enzymes to diets containing bermudagrass silage harvested at two maturity stages on the performance of lactating Holstein cattle. Proc. ASAS-ADSA-PSA Annual meeting, 2011. New Orleans, LA.
- Romero, J.J., K.G. Arriola, M.A. Zarate, C.R. Staples, C.F. Gonzalez, W. Vermerris and A.T. Adesogan. 2012. Screening exogenous fibrolytic enzyme products for improved in vitro ruminal fiber digestibility of bermudagrass haylage. Proceedings of the XVI International Silage Conference, Hameenlinna, Finalnd, July 2-4, 2012.
- Romero J. J., K. Arriola, Miguel Zarate, Charles Staples, Claudio Gonzalez, Wilfred Vermerris and Adegbola Adesogan. 2012. Effect of rate of application of various commercial exogenous fibrolytic enzymes on fiber hydrolysis and in vitro digestibility of bermudagrass haylage. Proceedings of the XVI International Silage Conference, Hameenlinna, Finalnd, July 2-4, 2012
- Romero, J.J., K.G. Arriola, M.A. Zarate, C.F. Gonzalez, W. Vermerris and A.T. Adesogan. 2012. Exogenous fibrolytic enzyme effects on preingestive fiber hydrolysis and release of sugars and phenolics from bermudagrass haylage. Proc. ASAS-ADSA-PSA Annual meeting, 2012. Phoenix, AZ.
- Romero, J.J. K.G. Arriola, M.A. Zarate, C.R. Staples, C.F. Gonzalez, W. Vermerris and A.T. Adesogan; 2012. Effect of rate of application of various commercial exogenous fibrolytic enzymes on preingestive fiber hydrolysis and release of sugars and phenolics from bermudagrass haylage. Proc. ASAS-ADSA-PSA Annual meeting, 2012. Phoenix, AZ.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Four experiments have been conducted. The first experiment screened 18 exogenous fibrolytic enzymes from 5 companies for a comprehensive array of fibrolytic activities and examined how cellulase and xylanase activities were influenced by the prevailing pH and temperature. The second experiment examined effects of applying these enzymes on the degradability of bermudagrass fiber and dry matter(DM) and on the composition of fermentation products. The third experiment examined the optimal application rate of the 12 most promising enzymes on fiber and DM degradadability of bermudagrass. The fourth experiment evaluated relationships for predicting fiber digestiblity of bermudagrass using individual fibrolytic enzyme activities or combinations of such activities. An ongoing study is examining the preingestive hydrolytic effects of the 5 most promising enzymes. The results of the experiments described above were presented as four papers at the Joint Meeting of the American Dairy Science Association and the American Society of Animal Science in New Orleans in 2011. One paper was presented in poster form and three were presented in oral form with power point. Each of these presentations was summarized into an abstract which was distributed to the approximately 3000 delegates who attended the meeting. In addition, the project contributed to the invitation extended to the PI to serve as a consultant on an International Atomic Energy Agency -UN Food and Agricultural Organization international project that aims to use fibrolytic enzymes to improve animal performance and mitigate greenhouse gas production by livestock. The results of the study mentioned above were presented by the PI at the first meeting of the project management committee consisting of consultants and research contract holders in Lethbridge, Canada in February, 2011. PARTICIPANTS: Individuals working on the project include the following: Juan Romero, Ph.d. student Kathy Arriola, Post doctoral researcher Adegbola Adesogan, PI Claudio Gonzalez, Co-PI. The project is subcontracted to the University of Puerto Rico under Dr. Elide Valencia's supervision. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report except a change in timelines due to reasons elucidated earlier that led to the no-cost extension.
Impacts
Some of the key findings of the study are as follows: 1. Fibrolytic enzyme activities of various commercially available products vary tremendously and fluctuate with temperature and pH. 2. The optimal temperature (50C) and pH (4.5) of most fibrolytic enzymes tested was beyond the normal ranges found in the rumen, indicating that the full potential of most enzymes was unlikely to be realized in ruminant animals. 3. The optimal application rate of the enzymes was enzyme specific. The above statements emphasize the importance of testing enzymes using a screening assay like ours before feeding them to animals. 4. Certain fibrolytic activities explained considerable amounts of the variability in bermudagrass fiber digestibility but others did not. In particular, accurate (r2 = 80%) multivariate regression equations relating enzyme activity to fiber digestibility were generated. 5. The most effective enzyme increased the digestibility of bermudagrass fiber by about 10 percentage units, which represents a significant improvement that will likely result in improved animal performance and reduced methane emissions when this enzyme is fed to cattle.
Publications
- K. G. Arriola*, J. J. Romero Gomez, and A. T. Adesogan. 2011. Effects of pH and temperature on fibrolytic enzyme activities of various commercial exogenous enzyme preparations. Abstract W119.
- J.J. Romero*, K.G. Arriola, M.A. Zarate, A.T. Adesogan. 2011. Screening exogenous fibrolytic enzyme products for improved in vitro ruminal fiber digestibility of bermudagrass. Abstract 219.
- J.J. Romero*, K.G. Arriola, M.A. Zarate, A.T. Adesogan. 2011. Effect of rate of application of various exogenous fibrolytic enzyme products on in vitro ruminal fiber digestibility of bermudagrass. Abstract 220.
- J.J. Romero*, K.G. Arriola, M.A. Zarate, A.T. Adesogan. 2011. Relationships between exogenous fibrolytic enzyme product activities and in vitro ruminal digestibility of bermudagrass. Abstract 221.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Activities: Experiment 1. Enzyme characterization: Eighteen fibrolytic enzyme mixtures were obtained from different commercial companies and analyzed for protein concentration, endoglucanase, esterase, exoglucanase, xylanase and beta-glucosidase activities at pH 6 and 39C. Experiment 2. Determination of enzyme effects on ruminal digestion: Bermudagrass haylage was ground to pass a 1 mm screen with a Wiley mill and placed into incubation tubes in quadruplicate in each of two runs. Exactly 2 mL of a buffered enzyme solution was added. Each enzyme was diluted in the buffer and added at previously effective or manufacturer-recommended rates for other forages. Tubes were incubated at 20C for 24 h. Subsequently, 40 mL of anaerobic buffer and 2mL of reducing agent and 10 mL of rumen fluid were added and tubes were incubated at 39C for 24 h. Upon termination of the incubation, tubes were placed on ice to stop the fermentation and residues and fresh samples were analyzed for dry matter and neutral detergent fiber digestibility and chemical composition. The DM and NDF digestibility of the 18 enzymes were statistically compared using the Dunnet's test. The 12 enzymes with the greatest digestibility values were used in a similar, follow up experiment (Experiment 3) to confirm their efficacy and to identify the 6 most potent enzymes. The most effective application rate for increasing the digestion of the 6 enzymes selected in Experiment 3 were evaluated in Experiment 4. Application rates of 0, 0.5x, 1x, 2x, and 3x were tested in each of two runs, where 1x is the application rate used in Experiments 2 and 3. All enzyme activity and digestibility assays involved at least 3 replicates per sample. Services: Due to his work on this and other projects, the PI has been recruited as a consultant on a International Atomic Energy Agency and Food and Agricultural Organization international collaborative project that aims to train scientists from several developing countries on using enzymes and similar technologies to improve forage quality. The project has also resulted in development of techniques for enzyme characterization in our laboratory. Dissemination:Results of the project will be disseminated at the next annual meeting of the American Society of Animal Science. PARTICIPANTS: Dr. Kathy Arriola, a post doctoral researcher performed all the enzyme characterization tests on the project. Dr. Arriola and Juan Romero, a Ph.D. student conducted the in vitro digestibility experiments. Students trained on the techniques used in the project included Miguel Zarate, an M.S. student and Lucas Giordano Paranhos, a visiting scholar. This project involves collaboration with the University of Puerto Rico, Mayaguez. Dr. Elide Valencia, the Co-PI at that institution has been sent a list of the most promising enzymes for testing in his lab. Drs. Charles Staples and Claudio Gonzalez are Co-PIs who have played an advisory role in various stages of the project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Experiment 1 provided detailed characterization of the activities of the 18 enzymes. Experiment 2 indicated the 12 most promising enzymes for digesting bermudagrass. Further examination of these 12 enzymes in Experiment 3 identified the 6 most promising enzymes. In Experiment 4, the effects of application rate of the 6 most promising enzymes on digestibility of bermudagrass was examined. Preliminary results from the latter do not indicate significant differences between the application rates tested. This project has shown that enzymes with high cellulase and xylanase activities from different companies have varying effects on the digestibility of bermudagrass. Six promising candidates have been identified. Further tests will aim to determine if the potency of these enzymes can be increased by certain strategies, to elucidate modes of action, and to better understand enzyme characteristics necessary to optimize digestion of tropical grasses.
Publications
- No publications reported this period
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