DEVELOPING NOVEL ADDITIVES FOR THE DAIRY CATTLE AND FORAGE INDUSTRY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016261
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF MAINE
(N/A)
ORONO,ME 04469

Performing Department
Animal and Veterinary Sciences

Non Technical Summary
In the U.S., it is calculated that $3 and 1.2 billion are lost each year during hay and silage production, respectively. Spoilage does not only cause extensive dry matter (DM) losses in hay and silage production (up to 40% of DM), but also causes a significant decline in nutritive value and is a major source of pathogens that compromise animal performance and food safety. Hay is the third most valuable crop ($17 billion and 34 million/y, respectively) and the second and first in terms of harvested acres (54 million and 135 thousand acres) in the U.S. and Maine, respectively. Thus, it is imperative to develop technologies that can reduce hay and silage spoilage and reduce its negative impact. Another big challenge dairy farmers must face is mastitis, the most prevalent disease affecting lactating dairy cattle with an estimated cost over $2 billion/year in the U.S. alone. Considering that milk production is the second most valuable agricultural commodity in Maine ($126.6 million/year to Maine), decreasing the risk of mastitis and increasing awareness about its proper management are crucial for Maine's economy. Currently, environmental microbes (EM) are recognized as the major cause of mastitis in lactating dairy cattle and are the focus of the industry efforts to decrease its incidence.The purpose of this proposal is to develop novel compatible additives that can improve hay and silage nutritive value and production efficiency by preventing the negative effects of spoilage on forage quality and dry matter losses. Also, we seek to develop novel conditioners for dairy cattle organic beddings that can reduce the environmental pathogen load and help mitigate mastitis incidence. Current technologies used for these issues have limited action and have inconsistent results. We will evaluate a set of low-cost antimicrobial compounds against current technologies to prevent the spoilage of alfalfa 1) hay and 2) silage using DM losses, aerobic stability, nutritive value, and microbial counts as primary indicators of success. The same set of antimicrobial compounds will be used to 3) reduce pathogen loads in organic-based beddings for livestock, using well-establish antibacterial tests for major dairy cattle environmental pathogens. The results obtained will help to improve the competitiveness of Maine's livestock industry and expand our understanding of the impact of microbial communities in the food animal industry.

Animal Health Component

50%

Research Effort Categories

Basic

(N/A)

Applied

50%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	1640	1010	75%
311	3499	1100	25%

Knowledge Area
302 - Nutrient Utilization in Animals; 311 - Animal Diseases;

Subject Of Investigation
3499 - Dairy cattle, general/other; 1640 - Alfalfa;

Field Of Science
1010 - Nutrition and metabolism; 1100 - Bacteriology;

Keywords

spoilage

hay

silage

pathogens

beddings

mastitis

Goals / Objectives
My long-term goal is to develop novel technologies that solve key issues in livestock production, with an emphasis on additive development to prevent conserved forage spoilage. The overall objective of this proposal is to strategically evaluate additives that: 1) can improve hay and silage nutritive value and production efficiency by preventing spoilage; 2) can reduce pathogen loads in dairy cattle organic beddings; 3) can cause shifts in the rumen microbial community related to increase dairy cattle productivity. Technical lignins, mostly Kraft lignin and lignosulfonates, have antimicrobial properties against bacteria and fungi (Phillip et al., 2000; Baurhoo et al., 2008; Dong et al., 2011) and limited effects on ruminant performance except for a decrease in rumen proteolysis that has been used to improve the efficiency of protein use in ruminants (Wright et al., 2005; Wang et al., 2009).The following supporting objectives are proposed examples to address the overall objective:1) To compare an established hay preservative (e.g. propionic acid) and optimized novel additives effects (e.g. technical lignins) on forage quality preservation measures using lab-scaled hay bales.2) To determine the relative differences in antibacterial activity against pathogens causing environmental mastitis in a collection of technical lignins.3) To evaluate a selected technical lignin effects on the load of pathogens in dairy cattle organic beddings in vitro.4) To determine the effects of a collection of technical lignins on the proteolysis and dry matter (DM) losses of legume silages.5) To compare the effects of two Trichoderma reesei enzyme preparations in the taxonomic profile, OUT richness, and OUT shifts of planktonic, weakly, and tightly feed-adhered ruminal bacteria of lactating cattle.My programmatic objective will be to evaluate different technologies that 1) can reduce hay and silage spoilage; 2) reduce pathogen loads in organic-based beddings for livestock; and 3) relate gut microbiome community shifts to changes in animal performance. The results obtained will help to improve the competitiveness of Maine's livestock industry and expand our understanding of the impact of microbial communities in the food animal industry.

Project Methods
Proposed methodology for the supporting objectives examples:Objective 1: To compare an established hay preservative (e.g. propionic acid) and optimized novel additives effects (e.g. technical lignins) on forage quality preservation measures using lab-scaled hay bales.Hay. Within an alfalfa field (5% bloom) located in Penobscot, ME, 0.12 hectares will be divided into 6 areas and all treatment combinations will be applied to biomass harvested separately (6 replicates).Treatments. A CRD with a 2 (hay moisture) × 2 (additives) × 3 (doses) factorial arrangement of treatments will be used to determine the effects of additives dosing under field conditions. An optimized previously selected technical lignin and propionic acid (positive control) will be examined at 0× (negative control), 1×, and 2× of the optimal antifungal dose found in previous experiments with 0.6 kg of DM laboratory-scale hay bales baled at 15 and 32% moisture according to Coblentz et al. (1998). Technical lignin will be applied in powder form as are many other powder/granular commercial additives used in hay production.Measures. Heating (Coblentz et al., 1998), nutritive value, in vitro ruminal digestibility (Romero et al., 2015), and fungal counts (Romero et al., 2017) will be determined for all treatment combinations at bailing and after 120 d of storage in insulated boxes in a room kept at 24°C (Coblentz et al., 1998).Objective 2: To determine the relative differences in antibacterial activity against pathogens causing environmental mastitis in a collection of technical lignins. Treatments. 1) Sodium and magnesium lignosulfonate (Sappi NA, ME), 2) washed Lignoboost Kraft lignin (NCSU, NC, USA), and 3) alkali Kraft Lignin (Sigma Aldrich, MO, USA) will be tested in vitro against strains of Streptococcus uberis, Staphylococcus hyicus, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, and Prototheca zopfii isolated from mastitic cows (ATCC, Manassas, VA) using the procedures described (Ma et al., 2016). Measures. Briefly, Mueller Hinton broth or agar will be used to determine the minimum inhibitory concentration (MIC), defined as the lowest concentration of treatments that prevents visible growth of the pathogens; and minimum microbicidal concentration (MMC), defined as the first treatment dilution that decreases 99.9% of the initial microbial concentration. Analysis will be conducted in triplicate.Objective 3: To evaluate a selected technical lignin effects on the load of pathogens in dairy cattle organic beddings in vitro. Treatments. A selected additive from Obj.2 will be tested at multiple doses (across the bacteria mentioned above) using the method described by Adhikari et al. (2013) with "green" hemlock sawdust. Briefly, 5 randomly obtained sawdust bedding samples will be autoclaved and placed in sterile bags, treated with the conditioners, and then inoculated with bacteria or controls and incubated at 26°C with shaking for 48h. Measures. Afterwards, samples will be serially diluted and plated on TSA agar for colony counting (Ma et al., 2016). This experiment will have a complete randomized design with a 4 (conditioner dose) × 4 (bacteria) factorial arrangement of treatments replicated 5 times. The model will include the fixed effects of conditioners, bacteria, and their interaction and the data will be analyzed using the GLM procedure of SAS v. 9.4.Objective 4: To determine the effects of a collection of technical lignins on the proteolysis and dry matter (DM) losses of legume silages.Forage. An established stand of alfalfa located in Penobscot, ME will be harvested and chopped from 6 different areas in a field (0.04 ha total area needed).Treatments. A CRD with a 9 (additives) × 2 (moisture concentration) factorial arrangement of treatments will be used to determine the effects of additives below (72%) and at the recommended moisture concentration (58%) for alfalfa ensiling. Additives evaluated will be (1) LignoBoost Kraft lignin (NCSU, Raleigh, NC), (2) alkali Kraft lignin (Sigma-Aldrich, St. Louis, MO), (3) sodium lignosulfonate (Sappi North America, Boston, MA), (4) magnesium lignosulfonate (Sappi North America, Boston, MA), plus (5) Lactobacillus plantarum (Volac, Orwell, UK), and (6) Control (untreated). A dose of 1% (w/v) of substrate will be evaluated, except for treatment 5 that will be applied at 105 cfu/g fresh forage. Technical lignins will be applied in powder form as many other powder/granular commercial additives used in silage production.Ensiling. Chopped alfalfa will be ensiled using 0.09 mm nylon-polyethylene embossed vacuum bags (0.3 kg, fresh basis) according to Romero et al. (2017).Measures. Nutritive value, ammonia-N, organic acids, alcohols, yeast, and mold counts will be determined according to Romero et al. (2017). Rumen in vitro digestibility will be assessed for the treatment with the least ammonia-N and propionic acid at both moisture concentrations as outlined by Romero et al. (2015).All work will be conducted at University of Maine Orono in the Animal Nutrition Lab, Animal Health Lab, and the Mycology Lab. All equipment needed is available and functional at these locations and preliminary tests have been already conducted for most of the objectives.Objective 5: To compare the effects of two Trichoderma reesei enzyme preparations in the taxonomic profile, OTU richness, and OTU shifts of planktonic, weakly, and tightly feed-adhered ruminal bacteria of lactating cattle.All animal components were done and published at University of Florida (Romero et al., 2016). UMaine will do all the bioinformatics on the microbial community data.Treatments. A factorial combination of 3 additives (control, CON; fibrolytic enzyme mixture, MIX; and high-xylanase fibrolytic enzyme, XYL) × 3 ruminal fractions (Ruminal planktonic, LIQ; weakly, ASO; and tightly, SOL, feed-adhered ruminal bacteria). Enzyme MIX was sprayed at 3.4 and XYL at 1 mL/kg of DM.Diet. Animals were fed a diet consisting of 35.1% corn silage, 9.9% bermudagrass silage, and 5.0% alfalfa-orchardgrass hay.Animals and Design. 3 ruminally-cannulated lactating Holstein cows were randomly assigned to treatments. A 3 × 3 Latin square design with 23-d periods including 18 d of adaptation, 3 d for in situ degradation, 1 d to rest the rumen, and 1 d for rumen contents sampling was used. The model contained the effect of additives, fractions, and their interaction and random effects of cow and period.Measures. Rumen samples were taken 3 h after morning feeding from several rumen locations. Bacterial pellets were extracted from the different rumen fractions according to Larue et al. (2005). DNA extraction was done using the Power Soil DNA kit (MO BIO, CA). The 16S rRNA gene (V1-V3 regions) was amplified with 27F-519R primers and normalized libraries were sequenced on a MiSeq platform v3 Reagent Kit 2 × 300 bp pair ends (600 cycles). Raw fastq files will be paired, trimmed, quality filtered (EE < 1%) and analyzed using USEARCH v7 (Edgar, 2010). Quality-filtered reads analyzed using QIIME 1.9.1. OTUs assigned using UCLUST (97% pairwise identity) and the Greengenes 13.8 (16S) database.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience may include producers, ruminant nutritionists, food safety researchers, dairy cattle managers and health professionals. Changes/Problems:Due to COVID we got behind out timeline, next year it will be more clear if there are any analysis that would not get finalized due to delays. What opportunities for training and professional development has the project provided?This project has allowed the training of two master students in the animal program. Both experienced presenting their results in national and international meetings. Also, this project has provided hands-on lab experience and training to six undergraduate students to further their careers in animal agriculture. How have the results been disseminated to communities of interest?2020. American Dairy Science Association Conference. West Pal, FL (Remote). What do you plan to do during the next reporting period to accomplish the goals?We are already working to complete Goal 3 and 5, there is a new grad student working in those objectives.

Impacts
What was accomplished under these goals? Goal 4: We evaluated the effects of untreated (0%), NaL and MgL applied independently at 0.5, 1, and 1.5 (% w/w, fresh basis) and INO (Pediococcus pentosaceus and Lactobacillus plantarum; 5 and 4 log cfu/fresh alfalfa g, on high moisture alfalfa (Medicago sativa L.) silage nutrient preservation. Data were analyzed as a randomized complete block design (RCBD; 5 blocks) and linear and quadratic polynomial contrasts were used to determine dose rate effects for NaL and MgL and orthogonal contrasts for INO effects. At opening (d 229), both MgL and INO increased DM loss (~13.7 vs 11.3% of DM) due to a lower production of lactic acid (~7.55 and 7.83 vs 9.23% of DM, respectively) which resulted in a higher pH relative to untreated (~4.41 and 4.46 vs 4.33; respectively). The high acidification in untreated resulted in additives not reducing further the proteolysis that occurred relative to untreated, measured as NH3-N (~11% of N). Overall, all additives tested failed to improve the preservation of high moisture alfalfa silage nutrients.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020. Leon-Tinoco A., B. Costa, S. Almeida, D. Reyes, S. Rivera, M. Killerby, R. Hollandsworth, Z.X. Ma, B. Perkins, S. L. Annis, C. Wu, C. Knight, A. Lichtenwalner, D. Skonberg, M. Stokes, and J.J. Romero. Effect of lignosulfonates on the dry matter loss, nutritional composition, and microbial counts of high moisture alfalfa silage. Presented at ADSA Annual Meeting, West Palm, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020. Killerby, M., R. White, D. C. Reyes, A.Y. Leon-Tinoco, S. Rivera, H. Paz, J. A. Jendza, and J. J. Romero. Meta-analysis of the effects of preservatives on hay spoilage I: Chemical treatments. Presented at ADSA Annual Meeting, West Palm, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020. Killerby M., S.T.R. Almeida, R. Hollandsworth, B. C. Guimaraes, A. Leon-Tinoco, Z. Ma, D. Coffin, B. Perkins, S. Annis, C. Knight, C. Wu, J. Bolton, and J.J. Romero. Effect of chemical and biological preservatives on the dry matter loss, nutritional composition, microbial counts, and aerobic stability of ensiled wet brewers grain. Presented at ADSA Annual Meeting, West Palm, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020. Hollandsworth, R., M. Killerby, S.T.R. Almeida, Z.X. Ma, A.Y. Leon-Tinoco, B.C. Guimaraes, and J.J. Romero. Effect of chemical and biological preservatives on the dry matter loss, nutritional composition, microbial counts, and heating of aerobically exposed wet brewers grain silage. Presented at ADSA Annual Meeting, West Palm, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020. A. Leon-Tinoco, S. L. Annis, S.T.R. Almeida, B. C. Guimaraes, R. Hollandsworth, M. Killerby, C. Wu, R. Kersbergen, A. Lichtenwalner, B. Perkins, C. Knight, D. Skonberg, Z.X. Ma, and J.J. Romero. An optimized lignosulfonate-based product matched propionic acid preservation effects on high-moisture alfalfa hay. Presented at ADSA Annual Meeting, West Palm, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020. A. Leon-Tinoco, S. L. Annis, S.T.R. Almeida, B. Guimaraes, R. Hollandsworth, A. Poulin, K. Dean, M. Killerby, C. Wu, A. Lichtenwalner, B. Perkins, D. Skonberg, Z.X. Ma, K.C. Jeong, R. Causey, and J.J. Romero. Assessing the antifungal activity of various sources of sodium lignosulfonate and chitosan against fungi isolated from spoiled hay. Presented at ADSA Annual Meeting, West Palm, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 2020. Killerby, M., R. White, D. C. Reyes, A.Y. Leon-Tinoco, S. Rivera, H. Paz, J. A. Jendza, and J. J. Romero. Meta-analysis of the effects of preservatives on hay spoilage II: Biological treatments. Presented at ADSA Annual Meeting, West Palm, FL.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:This research seeks to develop novel additives to reduce aerobic spoilage in wet baled hays and at silo opening and will benefit dairy and beef farmers. Accordingly, the target audience may include producers, forage professionals, ruminant nutritionists, food safety researchers, and enzymologists. Forages represent at least 50% of the diet in cattle. By reducing forage spoilage the efficiency of nutrient production increases, emissions decrease, and food safety of the animal food chain increases. I expect this research to benefit cattle and forage producers in Maine and across the nation. Changes/Problems:Funding to hire more research staff What opportunities for training and professional development has the project provided?This project has allowed the training of two master students in the animal program. Both experienced presenting their results in national and international meetings. My first student has already submitted an original research article as a first author to J Dairy Sci, which is under review. Also, this project has provided hands-on lab experience and training to three undergraduate students to futher their careers in animal agriculture. How have the results been disseminated to communities of interest?Presentations: 2019. American Dairy Science Association Conference. Cincinnati, Ohio. 2019. University of Maine Student Symposium. Bangor, Maine. 2019. Animal Health and Antimicrobial Research Field Day. Gainesville, Florida. Publications: Journal of Dairy Science What do you plan to do during the next reporting period to accomplish the goals?We are currently collaborating with industry partners to set a series of experiments that will transition our results to the field so optimized sodium lignosulfonate can be used as a hay preservative. Furthermore, we are workig on a meta-analysis on hay preservatives so the impact of current hay preservatives can be fully understood and compared against our proposed novel hay preservative.

Impacts
What was accomplished under these goals? Goal 1A: After we learned in our previous experiments that sodium lignosulfonates showed the most promise as hay preservatives across our collection of technical lignins, we screened five different sources of sodium lignosulfonate across 3 molds and one yeast to select the most promising sodium lignosulfonate (in terms of minimum inhibitory concentration and minimum fungicidal concentration) and identify the chemical characteristics that are related to antimicrobial activity. We had standard chitosan and chitosan nanoparticles as added treatments that could be developed for the organic hay producers since there are no current preservatives available for this type of producer. From the results, we also selected standard chitosan for latter tests. Goal 1B: From Goal 1A we compared a selected commercially available sodium lignosulfonate product and standard chitosan against propionic acid (positive control) in terms of hay preservation. Because of our previous efforts we were able to further decrease the dose of sodium lignosulfonate application from 2% to 0.5% w/w, fresh basis. The chitosan treatment failed to preserve hay. Goal 2: We screened a collection of technical lignins against 6 strains of pathogenic bacteria that causes mastitis in dairy cattle. We determined the minimum inhibitory concentrations and minimum bactericidal concentrations and selected the most promising technical lignin, also sodium lignosulfonate. These results will allow us to test this potential bedding conditioner with actual contaminated bed to assess its potential to prevent mastitis.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Reyes, D. C., S. L. Annis, A. Y. Leon-Tinoco, S. A. Rivera, H. M. Dubuc, L. B. Perkins, J. J. Perry, R. J. Kersbergen, C. Wu, C. W. Knight, M. S. Castillo, and J. J. Romero. 2019. Increased high-moisture alfalfa hay preservation, in vitro ruminal degradability, fermentability, and decreased proteolysis with the novel use of paper mill byproducts. Journal of Dairy Science 102:409.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Reyes, D. C., S. A. Rivera, Z. X. Ma, D. P. Marcinkowski, K. C. Jeong, and J. J. Romero. 2019. Mitigating environmental mastitis microbes with the novel use of paper mill byproducts. Journal of Dairy Science 102:182.
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Screening technical lignins for their potential as hay preservatives
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Developing animal feed preservatives from paper mill byproducts
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Microbial feed spoilage. Animal Health and Antimicrobial Research Field Day. Gainesville, Fl.