ENRICHING BRANCHED-CHAIN FATTY ACIDS IN CELLULAR LIPIDS OF RUMEN BACTERIA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1016999
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF VERMONT
(N/A)
BURLINGTON,VT 05405

Performing Department
Animal Science

Non Technical Summary
Branched-chain fatty acids (BCFA) are unique bioactive compounds in bovine milk fat that are principally derived from rumen bacteria. The biosynthetic pathway of BCFA is well established in some selected bacteria taxa, but little is known concerning the collective biosynthesis of BCFA in rumen bacteria and, particularly, in the context of the rumen ecosystem. The purpose of this project is to i) assess the conversion of various BCFA substrates, branched-chain amino acids, and their respective downstream metabolites, to BCFA by rumen bacteria, and ii) resolve a better understanding of how these substrates/intermediates affect the type and relative levels of BCFA produced (and transferred to milk) as a result of the bacterial community structure. Using complementary in vitro and in vivo approaches, this project will expand our knowledge of rumen BCFA-generating bacteria and elucidate strategies to manipulate and enhance the concentration of bioactive BCFA in milk fat. We expect to identify both BCFA precursors and synthesis intermediates that elicit bacterial species-specific responses, synthesis, and incorporation of BCFA in their membranes, ultimately translating to secretion into the milk.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
308	3410	1010	100%

Knowledge Area
308 - Improved Animal Products (Before Harvest);

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

rumen microbiota

dairy cow

fatty acids

milk

Goals / Objectives
Rumen bacteria and its metabolites play a pivotal role in the dairy cow's milk composition, specifically fatty acids. Our long-term goal is to develop a fundamental understanding of these interactions and dynamics to provide the scientific basis for strategies to enhance health attributes of bovine milk fat. Branched-chain fatty acids (BCFA) are a unique group of fatty acids synthesized by specific rumen bacteria and may be one of the most important bioactive lipids in milk and dairy products. Yet, it is not known which substrates are relevant to or preferred by rumen bacteria and in what proportions they are needed to increase the yield of bacterial membrane BCFA translating into an increased milk BCFA content. Critically examining the rumen bacterial population and metabolites (BCFA of bacterial lipids) will provide the framework for the development of innovative feeding strategies designed to enhance the content of BCFA in milk/dairy products. Because there is a need to characterize quantitative relationships between dietary substrates of BCFA, the rumen microbiota community structure and their membrane BCFA contributing to the fatty acids in milk, the overarching objective in this Hatch grant application is to i) assess the conversion of branched-chain amino acids and their respective down-stream metabolites (i.e., branched-chain a-keto acids and branched short-chain carboxylic acids) to BCFA by rumen bacteria, and ii) provide a better understanding of how these precursors/primers affect the type and amount of BCFA produced as a result of the bacterial community structure. Our central hypothesis is that the carefully gauged supply of BCFA precursors and, particularly, down-stream metabolites, will generate bacterial BCFA with a distinct profile and relative proportions that are associated with a specific bacteria community signature. Using an in vitro continuous culture technique followed by a subsequent in vivo trial in dairy cows, we will test the contribution of different BCFA substrates and corresponding intermediates in the biosynthesis of BCFA by rumen bacteria. Our specific aims are to:Specific Aim 1: Determine type, concentration, and ratios of BCFA in rumen bacterial cells maintained in a continuous culture fed either a) branched-chain amino acids (BCFA substrate), b) branched-chain a-keto acids (BCFA intermediate), or c) branched short-chain carboxylic acids (BCFA intermediate).Specific Aim 2: Analyze the composition of rumen bacterial communities maintained in a continuous culture fed either a) branched-chain amino acids (BCFA substrate), b) branched-chain a-keto acids, or c) branched short-chain carboxylic acids and elucidate their relationship with the type and concentration of BCFA synthesized.Specific Aim 3: Develop a feasible feeding strategy (in vivo trial in dairy cows) based on the outcomes in Specific Aims 1 and 2 and verify efficacy.

Project Methods
In vitro continuous culture (Specific Aims 1 and 2) To address our Specific aims 1 and 2, three studies will be performed using in vitro continuous culture fermenters. Each study will be conducted in a 4 x 4 Latin square design, including four 11-day periods testing three treatments and a control. Using 'bioreactors' (i.e., continuous culture fermenters, FerMac 320 modular bioreactors), in each study (Specific Aim 1 and 2), three different BCFA precursors/primers will be evaluated. In experiment 1, branched-chain amino acids (valine, leucine, and isoleucine) will be tested (Specific aims 1a and 2a). Bioreactors will be randomly assigned to one treatment for the duration of the 11-day experiment and will receive either 1) a control treatment consisting only of the standard total mixed ration (TMR) diet, 2) a TMR diet supplemented to include 200% of the valine content of the control diet, 3) a TMR diet supplemented to include 200% of the leucine content of the control diet, or a TMR diet supplemented to include 200% of the isoleucine content of the control diet. We chose the particular levels of branched-chain amino acids as a proof of concept. The amount of amino acid supplementation will be determined based on the amino acid content of the TMR at the time of the experiment. The amino acid treatments will be mixed with TMR and added (fed) twice daily at two equal portions. The length of each experiment will be 11 days comprising of one rumen fluid collection day (day 0), a 7-day adaptation (days 1 to 7), and a 3-day sampling period (day 8 to 10). This 4 x 4 Latin square design will be repeated for two further experiments. in experiment 2, three branched-chain a-keto acids (a-ketovalerate, a-ketoisocaproate, and a-keto-b-methyl-valerate) will be assessed (Specific aims 1b and 2b); and in experiment 3, three branched short-chain carboxylic acids (isovalerate, 2-methylbutyrate, and isobutyrate) will be examined (Specific aims 1c and 2c). Samples will be analyzed for microbial DNA extraction (bacterial density and community structure), volatile fatty acids, and bacterial cell membrane fatty acids. The PROC MIXED procedure in SAS 9.4 will be used to analyze data using a repeated measures ANOVA. The statistical model will include the fixed effect of treatment and the random effect of bioreactor. Data will be adjusted for multiple comparisons using Bonferroni's method. Significance will be declared at P≤0.05. Correlation matrices and principal component analyses will be created in RStudio.In vivo trial in dairy cows (Specific Aim 3) This experiment will be performed using the four lactating fistulated dairy cattle and will address Specific Aim 3. Of the 9 treatments tested in Specific aims 1 and 2 (branched-chain amino acids valine, leucine, and isoleucine; branched-chain a-keto acids a-ketovalerate, a-ketoisocaproate, and a-keto-b-methyl-valerate; and branched short-chain carboxylic acids isovalerate, 2-methylbutyrate, and isobutyrate), three will be selected based on their impact on the rumen microbiota and BCFA profile observed in experiments 1-3. These treatments will be utilized in a 4 x 4 Latin square design, where cows will each be randomly assigned to one treatment for a 21-day period, and treatments will be rotated for each of the subsequent three periods to achieve a balanced 4 x 4 design. Cows will be fed ad libitum, and individual feed intakes will be recorded on day 0 and days 15-21 of each period. On day 0, 17, 19, and 21 of each period, rumen fluid samples will be collected four times daily via the fistula and will be stored for rumen digesta analysis. Milk yield will be recorded daily and milk samples will be collected at both the AM and PM milking on day 0, 16, 18, and 20 of each period and stored for analysis. Since this trial will also employ a 4 x 4 Latin square design, statistical analyses are performed as described above

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

Outputs
Target Audience: Target audiences include academic members, dairy farmers, and dairy industry partners. This project supports a graduate student at the University of Vermont who works directly on the research. Furthermore, two undergraduate students are involved in this research providing them with hands-on experience in the field and laboratory and a deeper understanding of the scientific process. We submitted several abstracts to UVM's Student Research Conference (this conference showcases undergraduate and graduate student's research to fellow students, faculty, and the public) and the ADSA Annual Meeting where preliminary results are presented to inform the scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The second in vitro trial investigating the effect of isoleucine supplementation on 1) the content and composition of branched-chain fatty acids in rumen microbial cell membranes and 2) the composition of rumen microbial communities is currently being conducted. Our goal is to complete the trial and lab analyses, conduct statistical analyses, and draft and submit abstracts and papers. How have the results been disseminated to communities of interest?Two abstracts were submitted and accepted for presentation at UVM's annual Student Research Conference. At the conference, two students (an undergraduate and a graduate student) presented their results to their peers (undergraduate and graduate students), UVM faculty, and the participating community. Another abstract has been submitted for presentation at the annual ASDA meeting. Lastly, one paper has been published and a manuscript is currently under review. What do you plan to do during the next reporting period to accomplish the goals?The second in vitro trial investigating the effect of isoleucine supplementation on 1) the content and composition of branched-chain fatty acids in rumen microbial cell membranes and 2) the composition of rumen microbial communities is currently being conducted. Our goal is to complete the trial and lab analyses, conduct statistical analyses, and draft and submit abstracts and papers.

Impacts
What was accomplished under these goals? Milk and dairy products represent one of the most important agricultural commodities in regard to human nutrition. The marketplace for milk and dairy products is currently becoming increasingly more competitive and volatile. Greater consumer awareness of the relationship between diet and health is fueling a niche market for foods that promote optimal health and wellness as well as reducing the risk of chronic disease. Such a niche market can be rewarding to small and medium-sized producers to better compete in the marketplace. This presents opportunities to the dairy producers, processors, and manufacturers in Vermont to develop and market value-added milk/dairy products, thereby diversifying their revenue streams away from the volatility of commodity milk markets. About one-quarter of all solid fat intake in the American diet comes from milk and dairy products. Milk fat contains a unique variety of bioactive lipids that can support human health. One group of these bioactive lipids in bovine milk are branched-chain fatty acids. The biosynthetic pathway of branched-chain fatty acids is well established in some selected bacteria taxa, but little is known concerning the collective biosynthesis of branched-chain fatty acids in rumen microbes and, particularly, in the context of the rumen ecosystem. Our long-term goal is to develop a commercially viable value-added product enriched in bioactive components, such as branched-chain fatty acids, that places milk/dairy products in a more favorable context to consumers (i.e., health benefits) and thereby enhances the profitability of dairy producers through knowledge that will either reduce production costs and/or enable producers to capitalize on quality premiums for their milk. Because there is a need to characterize quantitative relationships between dietary substrates of branched-chain fatty acids, the rumen microbiota community structure and their membrane branched-chain fatty acids ultimately contributing to the fatty acids in milk, the goal of this project is to i) assess the conversion of branched-chain amino acids and their respective down-stream products to branched-chain fatty acids by rumen microbes, and ii) provide a better understanding of how these precursors/primers affect the type and amount of branched-chain fatty acids produced as a result of the bacteria community structure. Our specific aims are to: Specific aim 1: Determine the type, concentration, and ratios of branched-chain fatty acids in rumen microbial cells maintained in a continuous culture fed dietary substrates of branched-chain fatty acids. Specific aim 2: Analyze the composition of rumen microbial communities maintained in a continuous culture fed dietary substrates of branched-chain fatty acids. Using an in vitro technique, we quantified the relationship between the dietary substrate leucine and the membrane composition of leucine's down-stream metabolites, iso-15:0 and iso-17:0 BCFA, in rumen bacteria and protozoa. Dual-flow continuous culture fermenters were used in a repeated design with 4 periods of 10 d each to evaluate the dose-response of dietary leucine. Treatments were 1) a control (CON) comprising of 100% leucine, 2) CON + 150% leucine, 3) CON + 200% leucine, and 4) CON + 300% leucine. On d 8-10, bacteria and protozoa were isolated from each fermenter, and their lipid membranes and DNA were analyzed for fatty acids and community structure, respectively. Specific aim 1: The total content of branched-chain fatty acids in bacterial cell membranes was close to 5% of the total fatty acid identified. Except for the 300% group, leucine did not influence the content or concentration of iso-15:0 in bacterial cell membranes across all treatment groups. However, the content and concentration of iso-17:0 increased with both the 200% and 300% supplementation. Rumen bacterial cell membranes had the largest concentrations of individual BCFA with the 300% leucine supplementation, including iso-13:0, anteiso-14:0, iso-15:0, iso-17:0, and anteiso-17:0, compared to 150% and 200% leucine inclusion. In protozoal cell membranes, the content of total BCFA was less than 3% of total fatty acids identified. Yet, the supplementation of leucine caused more pronounced shifts in the branched-chain fatty acid profile in protozoal cell membranes compared to that of bacteria. Notably, the content of iso-15:0 increased approximately 2-fold with 200% and 300% supplementation in protozoal cell membranes. Similarly, the content of iso-17:0 increased 1.5-fold with 200% and 300% supplementation. Our data confirm that the metabolism of branched-chain fatty acids can be influenced in the rumen environment. Moreover, the results indicate that dietary leucine may be used as a tool to increase the proportions of iso-15:0 and iso-17:0 in rumen microbial cell membranes. Specific aim 2: Rumen microbial community structure was analyzed via shotgun sequencing. Leucine treatment did not impact the top 15 most abundant taxa - likely due to the high variation within each treatment group. There were only a few minor significant changes between individual leucine treatment groups.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Taormina VM, Unger AL, Schiksnis MR, Torres-Gonzalez M, Kraft J. Branched-chain fatty acids-an underexplored class of dairy-derived fatty acids. Nutrients. 2020 Sep 20;12(9):2875. doi: 10.3390/nu12092875.
• Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Schiksnis MR, Burke J, Baker LM, Greenwood SL, Kraft J. Technical Note: Modification of a filtration system to increase protozoa retention in continuous culture fermenters. Journal of Dairy Science
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Schiksnis MR, Burke J, Kraft J. Enriching branched-chain fatty acids in cellular lipids of rumen bacteria and protozoa. UVM 2020 Annual Student Research Conference
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Burke J, Schiksnis MR, Kraft J. Improvement of a filter system for in vitro continuous culture fermenters to maximize protozoa retention. UVM 2020 Annual Student Research Conference
• Type: Conference Papers and Presentations Status: Submitted Year Published: 2021 Citation: Schiksnis MR, Greenwood SL, Kraft J. In vitro supplementation of leucine impacts the composition of branched-chain fatty acids in rumen bacterial and protozoal cell membranes. ADSA Conference 2021.
• Type: Websites Status: Other Year Published: 2019 Citation: https://www.uvm.edu/uvmnews/dayinthelife2019

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

Outputs
Target Audience:Target audiences include academic members, dairy farmers, and dairy industry partners. This project supports a graduate student at the University of Vermont who has been working directly on the research. Furthermore, one Animal Science undergraduate student has been directly involved in this research providing her with hands-on experience in the field and laboratory and a deeper understanding of the scientific process. A subproject of this project will serve as the basis of her Honors College Thesis to be completed by May 2020. Changes/Problems:A major part of the first year was spent troubleshooting the fermenters (continuous culture setup), specifically the filter system which is essential to retaining protozoa and equilibrating the inflow/outflow of the rumen fluid. Specifically, during the first trial, we noticed that the protozoa were not retained in the fermenter setup. The filter proved to be a major obstacle. While we used a filter system where the details and specs were published, the assembly and application of the (multistage) filter were neither straight forward nor useful. Hence, our team spent months to establish and optimize the filter system for the continuous culture setup. The issue with the filter system was eventually resolved through the use of the novel technology of 3D printing. Importantly, the exertion of the 3D-printed filter work will lead to a Technical Note considered to be published in the Journal of Dairy Sciences. Unexpectantly and beyond our control, we lost an entire trial due to an unscheduled power outage in the building that occurred near the end of the trial. Therefore, as a result of the above-mentioned difficulties, our research progress was significantly slowed down at the beginning of the project. However, lately, we have completed the first major experiment and are now back on track despite these initial setbacks. What opportunities for training and professional development has the project provided?A graduate student (MS candidate) and an Honors College undergraduate student have been trained and mentored in research methods; both students have been conducting independent research projects based on this project. How have the results been disseminated to communities of interest?Two abstracts have been submitted to the UVM Student Research Conference. Preliminary results will be presented (one poster, one oral presentation) in April at the UVM Student Research Conference. What do you plan to do during the next reporting period to accomplish the goals?1) Complete all analyses pertaining to the first major experiment (supplementation of leucine and its metabolic intermediate a-ketoisocaproate). 2) Prepare/submit 2 manuscripts for publication in peer-reviewed journals (1 original research, 1 technical note). 3) Conduct the second major experiment (continuous and batch culture) to determine the conversion of valine and its respective down-stream metabolite (i.e., α-keto acid) by rumen microbes (specific aims 1 and 2).

Impacts
What was accomplished under these goals? Preliminary studies, as well as continuous culture and batch culture trials, have been conducted (specific aims 1 and 2). Assays and analyses at the laboratory are being performed and data are being analyzed. Preliminary research/studies developed a protein-deficient diet to exemplify the effects of leucine developed a specific filter system for the continuous culture system to retain the protozoa populations Main experiments 1) 6 continuous culture trials were performed (dose-response study of leucine supplementation). Collections/analyses: daily rumen fluid effluent for rumen digesta composition, bacteria and protozoa enumeration, microbial community analysis (metagenomic approach), bacteria and protozoa membrane fatty acids composition, and volatile fatty acids. 2) 4 batch culture trials were performed (2 treatments: unlabeled and isotope-labeled leucine and a-ketoisocaproate) to trace isotopes (C13) leucine and a-ketoisocaproate through 48-hour trials. Collections: supernatants at 0, 3, 6, 9, 12, 24, 48 hrs post-inoculation, GC/MS analyses are currently being performed to determine how leucine and its intermediate a-ketoisocaproate are being metabolized by rumen microbes

Publications