REGULATORY ROLES OF VOLATILE FATTY ACIDS IN CATTLE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1005238
• Project Start Date: Jan 1, 2015
• Project End Date: Dec 31, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061

Performing Department
Animal Poultry Sciences

Non Technical Summary
There were 1,550,000 cattle and calves in Virginia on January 1, 2010, and this number ranked 20th among all U.S. states (most recent data, from http://www.nass.usda.gov/Data_and_Statistics). The beef and dairy industries were the second and third largest agricultural commodities of Virginia, respectively in 2011; they together generated more than $800 million cash revenues (most recent data, from http://www.vdacs.virginia.gov/index.shtml). For Virginia beef and dairy industries to continue to be competitive on the national and international markets, production efficiency of cattle in Virginia must be improved. This requires a better understanding of cattle biology through research. Volatile fatty acids (VFA) are short-chain fatty acids produced by microbial fermentation in the gastrointestinal tract. The predominant forms of VFA are acetate, propionate, and butyrate. In ruminants such as cattle, the microbial fermentation occurs mainly in the rumen, the first of the four stomachs in those animals. The VFA produced in the rumen are the major source of energy for ruminants. Besides serving as substrates for energy production, VFA also function as regulatory molecules. It has been long known that VFA stimulate growth and functional maturation of the rumen epithelium in young ruminants and stimulate insulin and glucagon secretion. Recent studies in laboratory animals and humans suggest that VFA may mediate the effects of gut microbiota on energy intake, storage, obesity, and immunity. The mechanisms underlying the regulatory effects of VFA are poorly understood. In this project, we will study the mechanisms by which VFA stimulate rumen development and adipocyte differentiation in cattle. Rumen development and adipocyte differentiation are biological processes directly relevant to the productivity of cattle. A better understanding of how VFA regulate these processes could lead to the development of novel management strategies to control rumen development and adipocyte differentiation and thereby to improve productivity and production efficiency in cattle.

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
305	3399	1020	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3399 - Beef cattle, general/other;

Field Of Science
1020 - Physiology;

Keywords

cattle

rumen

preadipocytes

Goals / Objectives
Objective 1: Determine the effects of intraruminal infusion of VFA on cell proliferation in the rumen wall in pre-ruminant calves and determine if the proliferating cells express GPR41 and GPR43.Objective 2: Determine the effects of intraruminal infusion of VFA on blood concentrations of IGF-I, insulin, ghrelin, peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) and local mRNA expression of these hormones in pre-ruminant calves.Objective 3: Determine if VFA have direct effects on proliferation and differentiation of bovine preadipocytes in vitro and the potential involvement of GPR41 and GPR43 in these effects.

Project Methods
Objectives 1 and 2. Seven male 1-week-old Holstein calves will be infused with 100 ml of VFA mix consisting of 65 nM acetate, 25 mM propionate, and 10 mM butyrate through a rumen catheter once a day for 4 weeks. Seven similar calves will be infused with 100 ml of saline as controls. Calves of both groups will be fed milk replacer at 15% of body weight throughout the experiment. The rumen catheter will be inserted as described (Lane and Jesse, 1997). Blood samples will be taken weekly from each calf via jugular venipuncture. Weekly body weight will be recorded from each calf too. At the end of the four weeks infusion, calves will be euthanized through captive bolt stunning and subsequent exsanguination. Rumen, abomasum, colon, and pancreas tissue samples will be collected for histological, immunohistochemical, mRNA expression, and/or protein expression analyses. In histological analysis, length and density of rumen papilla and thickness of different layers of rumen wall will be measured to assess the level of rumen development. In immunohistochemical analysis, adjacent tissue sections of rumen will be stained with antibody for the Ki-67 antigen, a nuclear protein marker for proliferating cells, and antibodies for GPR43 and 41. mRNA and protein expression of IGF-I, ghrelin, peptide YY, insulin, proglucagon, GPR41 and GPR43 in rumen, abomasum, colon and pancreas will be measured by quantitative RT-PCR and western blotting, respectively. Serum concentrations of IGF-I, insulin, glucagon, ghrelin, peptide YY and GLP-1 will be measured using the commercially available radioimmunoassay or enzyme-linked immunosorbent assay kits.Objective 3.We will derive and culture preadipocytes from bovine subcutaneous adipose tissue as described (Lengi and Corl, 2010). Adipose tissue will be collected from crossbred Angus steers slaughtered at local slaughterhouse or the on-campus meat lab. The effects of acetate, propionate, and butyrate on the proliferation and differentiation of bovine preadipocytes will be assessed at different concentrations (0, 0.01, 0.1, 1, and 10 mM). Cell proliferation rate will be assayed as we previously described (Zhou et al., 2008). Differentiation of preadipocytes will be assessed by Oil Red O staining and by quantifying mRNA expression of lipogenic markers including PPARg,C/EBPα, FABP4, and leptin. If acetate, propionate, and butyrate have effects on proliferation or differentiation of bovine preadipocytes, we will determine if these effects are mediated by GPR41 and GPR43 through siRNA knockdown. Knockdown efficiency will be validated by real-time PCR and western blotting analyses of GPR41 and GPR43 mRNA and protein expression, respectively.

Progress 01/01/15 to 12/31/19

Outputs
Target Audience:The target audiences of this project include animal science professionals and students in the areas of ruminantphysiology, nutrition, and growth biology. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided the opportunities for two graduate students to learn how to isolate cells, culture cells, transform cells, and analyze gene expression in cells by real-time PCR and RNA-sequencing. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During this final reporting period, we conducted experiments to establish a bovine rumen epithelial cell line. We dissociated the rumen epithelium collected from a steer at slaughter into individual cells through trypsin digestion. We transformed the isolated rumen epithelial cells with SV40 large T antigen. Through puromycin selection and limiting dilution, we generated six single-cell clones. We selected two morphologically different clones for further characterization. Based on RNA-sequencing, one clone appeared to be a type of rumen epithelial cells because it had high expression of many keratin (KRT) genes including KRT7, KRT8, KRT18, and KRT80, and cell adhesion genes including CAD1, CAD2, and CLDN1, which are markers of epithelial cells. The other clone was considered to be endothelial cells because many markers of endothelial cells including PECAM1, KDR, and NOS3 were expressed at high levels in this clone. Both clones will be valuable resources for studying the physiology of bovine rumen epithelial cells and endothelial cells.

Publications

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

Outputs
Target Audience:Ruminant physiologists; students; nutritionists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided a visiting graduate student the opportunity to learn about cell culture and RNA-seq analysis. How have the results been disseminated to communities of interest?The information was shared within the lab group and partnering laboratories within the Department of Animal Science at Virginia Tech, Blacksburg. What do you plan to do during the next reporting period to accomplish the goals?Conduct functional experiments using the cell clones as models

Impacts
What was accomplished under these goals? As mentioned in our last report, we had generated several clones of cells from bovine rumen and colon epithelium. During this reporting period, we analyzed the mRNA transcriptomes in two of the rumen-derived cell clones via RNA-seq. The RNA-seq analyses revealed that one clone was abundant with mRNAs that are typically expressed at high levels in endothelial cells, including CD34, PECAM1, and NOS3, while the other clone expressed mRNAs that are characteristic of epithelial cells, including KRT7, KRT8, and EGFR. These gene expression patterns suggest that the two clones were likely derived from a rumen endothelial cell and a rumen epithelial cell, respectively. These cell clones could be valuable in vitro models to study rumen cell biology.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Fellow scientists, resesearchers, and agriculture industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided a visiting graduate student the opportunity to learn about isolating and cloning the intestinal cells from cattle. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Perform additional mRNA and protein expression analyses to characterize and identify the cell clones.

Impacts
What was accomplished under these goals? During this reporting period, we conducted experiments to isolate and clone enteroendocrine cells from the small intestine and colon of cattle so that we could test the effects of short-chain fatty acids on secretion of gastrointestinal hormones such as peptide YY and glucagon-like peptide 1 and their mRNA expression in an in vitro system. We have generated and expanded a dozen clones. We have selected two clones and analyzed them for expression of marker genes. Based on initial qPCR analyses, none of those two clones was derived from enteroendocrine cells. We are in the process of further characterizing and identifying them and the remaining clones.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Graduate students, animal scientists and biochemists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided a graduate student the opportunity to learn and practice cell culture and mRNA analysis techniques. How have the results been disseminated to communities of interest?Students and the PI discussed the data within the Animal and Poultry Sciences Department at Virginia Tech. What do you plan to do during the next reporting period to accomplish the goals?We will continue to conduct research on the regulatory roles of volatile fatty acids in cattle for the next reporting period.

Impacts
What was accomplished under these goals? In this past reporting period, we conducted a study to determine the effects of major short-chain fatty acids acetate, propionate, and butyrate on the expression of different myosin heavy chain genes in bovine myoblasts. We isolated satellite cells from four Angus or Angus crossbred steers. We induced myoblasts to differentiate and fuse into myotubes in the presence of three different concentrations of acetate (0.1, 0.5, and 2.5 mM), propionate (0.01, 0.05, and 0.25 mM), or butyrate (0.005, 0.025, and 0.1 mM), for 72 hours. We quantified the expression levels of MYH1, MYH2, MYH3, MYH4, MYH7, and MYH8 mRNAs by real-time PCR. We also stained the myoblasts with Giemsa and measured the fusion index (percentage of nuclei located in multinucleated myotubes). Our data showed that MYH mRNA expression or fusion index in bovine myoblasts was not affected by acetate, propionate, or butyrate at the three concentrations tested. These results do not support a direct effect of short-chain fatty acids on fiber differentiation or myoblast fusion in cattle.

Publications

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Rumen animal physiologists; animal scientists; dairy and beef farmers and associated industries. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided a graduate student the opportunity to learn about isolating and culturing bovine preadipocytes and satellite cells. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Continue withconducting additional research on volatile fatty acids in bovine.

Impacts
What was accomplished under these goals? In the past reporting period, we conducted experiments to determine the effects of volatile fatty acids on differentiation of bovine preadipocytes into adipocytes and of bovine satellite cells into myotubes. We isolated preadipocytes from subcutaneous fat and satellite cells from skeletal muscle of Angus crossbred steers at slaughter. We differentiated these into adipocytes and myotubes in the presence of different concentrations of acetate, butyrate and propionate and assessed the differentiation status of cells by quantitative PCR of mRNA expression of adipogenic (PPARG, LEP, FABP4) and myogenic (MYH3, MYOG, CKM, MB) markers. Data indicated that acetate, propionate, or butyrate had no effect of differentiation of bovine preadipocytes into adipocytes, but that these short chain fatty acids, in particular, butyrate, inhibited differentiation of bovine satellite cells into myotubes. We are in the process of repeating these experiments.

Publications

Progress 01/01/15 to 09/30/15

Outputs
Target Audience:Ruminant animal physiologists, and animal scientists, veterinarians, dairy and beef farmers and associated industries. Changes/Problems:In the past reporting period, we attempted to conduct experiments to determine the effect of intra-ruminal infusion of butyrate on cell proliferation in the rumen wall in pre-ruminant calves (Objective 1). However, we have made little progress toward this objective for two reasons. One was that we had difficulty in getting newborn calves in 2015. The second reason was that newborn calves we got were often unhealthy to sustain the proposed experiments. What opportunities for training and professional development has the project provided?The project provided twograduate students the opportunity to learn about how to feed and care fornewborn calves and to work with veterinarians and dairy farmers. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Conduct experiments under objective 3.

Impacts
What was accomplished under these goals? In the past reporting period, we attempted to conduct experiments to determine the effect of intra-ruminal infusion of butyrate on cell proliferation in the rumen wall in pre-ruminant calves (Objective 1). However, we have made little progress toward this objective for two reasons. One was that we had difficulty in getting newborn calves in 2015. The second reason was that newborn calves we got were often unhealthy to sustain the proposed experiments.

Publications