Performing Department
(N/A)
Non Technical Summary
Improving livestock feed efficiency (FE) is essential to sustainably feed the world. Our long-term goal is to understand tissue and whole-animal level nutrient use efficiency to improve FE in dairy cows. Critical gaps remain in understanding coordinated nutrient use across the body, the relative role of differing adipose tissue depots in metabolism and body energy change, and the impact of maternal FE on in utero development. The objectives here are to determine if nutrient metabolism is coordinated across tissues to contribute to whole-body FE, evaluate the fatty acid composition and relative metabolic activity of adipose tissue depots, and examine if genomic and metabolic differences that lead to high FE influence fetal development. Quantification of key genes and proteins in liver, muscle, subcutaneous and visceral adipose, and lymph nodes will allow for determining relative tissue metabolism in high vs. low FE cows. RNA sequencing of fetal liver and muscle will determine pathways influenced through fetal programming. We will execute these aims through a collaboration with USDA AGIL and a comprehensive tissue collection during slaughter of 80 phenotypically characterized second lactation dairy cows. This once-in-a-career sample collection opportunity will allow for analysis that would otherwise be economically and ethically prohibitive; however, as the timeline for slaughter is not flexible, it presents an urgency and time-sensitivity. Our goals align closely with the Animal Nutrition, Growth and Lactation priority, within the Animal Health and Production Program. Focus on the contribution of nutrient use efficiency will serve to further improve dairy cattle efficiency and sustainability.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Experiments designed to answer the most pressing scientific questions must balance scientific appropriateness, ethicality, feasibility, and expense. As a result, we often face limitations to breadth or depth of analysis, studies are frequently underpowered, or we must use proxy measures or tissue subsamples. Rarely do we have the opportunity to access sufficient samples (quantity and replication) to allow us to address critical gaps in our understanding of nutrient use efficiency in dairy cattle that would otherwise be not only cost prohibitive, but ethically prohibitive. This proposal focuses on leveraging a unique, once-in-a-career opportunity, closure of a research dairy, to conduct a thorough examination of tissue-level metabolism in phenotypically characterized cows through an unfortunate but necessary culling of 80 dairy cows from the USDA Beltsville Agricultural Research Center (BARC) dairy and is being overseen by researchers of the Animal Genomics and Improvement Laboratory (AGIL). This situation provides a once-in-a-career opportunity for scientists to partner together to fully examine every tissue in phenotypically characterized dairy cows and their developing fetus in a comprehensive ethical manner to answer questions about whole-body nutrient use efficiency and coordination to yield a range of feed efficiency (FE) metrics and methane emissions. This affords us the ability to conduct an extensive tissue collection from 50 of the cows selected (highest and lowest FE) for scientists from USDA AGIL and the University of Wisconsin-Madison to partner and address a range of research questions. Collection of maternal and fetal tissues to this extent has rarely been done [1], and the animal expense alone would preclude the objectives presented herein from being completed without this timely collaboration. Slaughter of cows will begin in August 2023 and continue for 18 months. Dr. Ransom Baldwin, VI and colleagues at USDA AGIL will be addressing the role of gastrointestinal and service tissues as a component of energetic efficiency and how growth and differentiation of these tissues is regulated at the genomic level. USDA AGIL funds are available to address gastrointestinal objectives and to collect additional tissues. With this proposal, we are seeking funds to analyze the additionally collected tissues to answer novel objectives that would not be possible without additional funding. Given the lifespan of the tissues for the desired analysis, this proposal is unique, collaborative, and of a time-sensitive nature. The proposed objectives here will be executed in conjunction with serial slaughter of cows at the USDA BARC facility, and the research questions being answered herein are unique to those being addressed by AGIL research scientists and represent an attempt to maximize knowledge gained.Our long-term goal is to understand nutrient use efficiency at the tissue and whole-animal level to improve FE and reduce methane emissions in dairy cows. Although aspects of this are being examined, to date a comprehensive examination of whole-body influences on FE and methane emissions has not been conducted; thus, this situation presents an opportunity to fill a critical gap that is typically unattainable due to costs associated with slaughtering and collecting multiple tissues from multiparous lactating dairy cows of divergent FE. The objectives in this proposal will allow us to examine the contribution of the liver, muscle, adipose, and lymph nodes to multi-lactation, whole-animal FE and methane emissions phenotypes in conjunction with complimentary examination of gut physiology and microbiome analysis by USDA AGIL collaborators. Additionally, a subset of cows will be pregnant at the time of slaughter (150 days in milk, DIM), allowing us to examine the role of maternal FE on fetal tissue development. The central hypothesis of this proposal is that tissue efficiency and nutrient use influences whole-body FE and methane emissions, and that maternal FE status influences nutrient availability to, and programming of, tissues in the developing fetus.To test this, we propose three Specific Aims:Specific Aim #1: Determine if shifts in nutrient metabolism are coordinated across tissues to contribute to whole-body FE. Based on our current data, we hypothesize that gene expression and protein abundance related to fatty acid and amino acid oxidation and immune response in liver, muscle, adipose, and lymph nodes contribute to FE divergence and individual animal variance.Specific Aim #2: Evaluate the fatty acid composition and relative metabolic activity of adipose tissue depots. Of fatty acids that differ between cows of divergent FE, some are likely mobilized from internal adipose depots, consistent with varying metabolic activity of different adipose depots. We will determine if internal adipose tissue depots contribute to high vs. low FE.Specific Aim #3: Examine if the genomic and metabolic differences that lead to high vs. low FE influence fetal development. In utero exposure to nutrients presents a powerful mechanism to impact offspring growth rate and body composition from birth to maturity. We will determine the influence of maternal FE status on fetal weight and pathway regulation.
Project Methods
In order to complete the proposed aims, approximately 80 cows have been, or will be, phenotyped in the first and second lactation. This phenotyping includes routine growth data prior to the first lactation and milk yield and composition during both lactations. Additionally, RFI and methane emissions will be phenotyped over a 42-d period and used to identify the 50 cows with the most extreme RFI (25 highRFI, 25 lowRFI). During the second lactation, these cows will complete an additional 42-day RFI and methane emission phenotype characterization and be slated for comprehensive tissue collection at slaughter at 150 DIM. Detailed methods for animal handling, RFI and methane emission determination, tissue collection, and post-collection analysis are described in the General Methodology section below.Slaughter and tissue collection from lowRFI and highRFI cows. Cows determined to be lowRFI and highRFI will be scheduled for slaughter at 150 DIM (± 2 days) in the USDA AGIL facility. Given the intensity of tissue collection, only 1 or 2 cows will be slaughtered per day. Whole liver, adipose, muscle, and lymph node collections will be performed on all mid-lactation dairy cows and any present fetuses. All tissues will be weighed prior to division for downstream analysis. Liver tissue for both cows and fetal calves will be collected for protein abundance, triglyceride content, and fatty acid profile analysis. An additional liver tissue sample will be preserved in TRIzol for RNAseq (calves only) and gene expression analysis. Individual adipose tissue depots (subcutaneous, omental, renal, and mesenchymal) will all be individually weighed prior to division for downstream analysis in cows. All adipose tissue depots from cows will be collected for protein abundance and fatty acid composition. Muscle tissue from cows and fetuses will be collected for protein abundance and triglyceride content. An additional liver tissue sample will be preserved in TRIzol for RNAseq (calves only) and gene expression analysis. Lymph nodes from cows will be collected and a sample will be preserved in TRIzol for gene expression analysis.Specific Aim #1: MethodsDuring slaughter of the high and low FE cows, whole body live weight, carcass weight, and organ weights will be collected by USDA AGIL staff. Samples of gastrointestinal tissues, liver, semitendinosus muscle, adipose depots (Aim #2), and lymph nodes, will be collected as described below in the General Methodology section. Examination of gastrointestinal and service tissues, brain, mammary gland, kidney, and other tissues will be completed by USDA AGIL staff and although analyses are outside of the scope of this proposal, findings will be considered collaboratively. The primary focus of this aim is on understanding pathways related to nutrient metabolism that support differences in FE between cows. PD White's current USDA grant focuses on understanding hepatic and muscle mitochondrial efficiency and protein turnover as contributors to FE and predicting RFI with blood metabolite concentrations in a broad population. Initial examination of these pathways by RNAseq have highlighted amino acid and fatty acid oxidation, inflammatory pathways, and cell cycling as potential pathways that differ between high vs. low FE cows. It is important to note that the work proposed here does not overlap with the current USDA grant; rather, this proposed aim will complement those studies to better understand metabolic differences between high and low FE animals.Together with RNAseq data, the current aim will examine rate limiting enzymes involved in amino acid and fatty acid oxidation, TCA cycle oxidation, and gluconeogenesis (liver only) in muscle and liver tissue. Specifically, we will examine genes considered rate limiting or indicators of complete oxidation via the TCA cycle, incomplete oxidation via ketogenesis, fatty acid β-oxidation, and propionate oxidation. In follow up to RNAseq analysis, we will quantify hepatic cell cycling genes and in muscle we will examine LPIN1, fatty acid binding protein (FABP), stearoyl-CoA 9-desaturase (SCD), and hexokinase 2 (HK2). Given the role in orchestrating many of these pathways, protein abundance of tissue specific isoforms of peroxisome proliferator-activated receptors (PPAR), sterol regulatory element-binding proteins (SREBP), and carbohydrate-responsive element-binding protein (ChREBP) will be quantified. Novel findings from our RNAseq analysis indicated the basal immune response differs between high and low FE. Although the activated immune system is now known to be energetically demanding [42-44], this is the first indication that there may be basal differences in immune response in high and low FE cows. To examine this, we will quantify gene expression for major histocompatibility complexes-Class I and II (BoLA-NC1, -NC3, -NC4, and -DRA), activation of innate immune cells (CD14, CD163), T lymphocyte activation (CD4, CD8B, CD80, and LAG3), natural killer cell activation (NCR1), and T lymphocyte suppression (CD274 and CTLA4).Specific Aim #2: MethodsAt slaughter, adipose tissue will be collected from visceral (mesenteric, omental, and perirenal) and subcutaneous (tail head) depots, weighed and subsampled for subsequent analysis (General Methodology). Analysis of fatty acid composition, and protein abundance will be interrogated by adipose depot, high and low FE, and the interaction.Fatty acid composition. Fatty acid composition will be determined by acid methylation and gas chromatography at UW. PD White's laboratory is well equipped and experienced in doing FA profile analysis. Briefly, FA will be methylated using 5% methanolic HCl and chloroform in a 70?C water bath for 2 h followed by neutralization with K2CO3. Chloroform will be removed, and samples evaporated under nitrogen to a volume of 100 μL to be analyzed by gas chromatography (GC). Fatty acid profiles will be determined on a PerkinElmer Clarus 680 GC (PerkinElmer, Norwalk, CT) using column specifications and temperature program described previously [55,56]. Peaks will be identified and integrated based on commercially available individual FAME mixtures: FIMFAME-5, -6, and -8 (4210, 2009, 2012; Matreya Inc., Pleasant Gap, PA) and analyzed using the TotalChrom Workstation V6.3.2 software (PerkinElmer).Indicators of Metabolic Activity. Protein abundance of acute phase proteins and lipases (will be quantified by Western Blot).Specific Aim #3: MethodsThe fetus will be collected from cows pregnant at the time of slaughter. Based on the past several years, USDA AGIL has a 50% conception rate at first service (~80 DIM) in multiparous cows; therefore, we anticipate 25 of the 50 cows to be pregnant (78 to 82 d) at the time of slaughter. During tissue collection, the whole fetus and fetal liver will be weighed and recorded. Muscle and liver tissue samples will be preserved in Trizol (General Methodology) for RNAseq analysis.RNA Sequencing. Liver and muscle tissue RNA will be isolated as described previously and in the General Methodology. We have been successful in past experiments achieving total RNA absorbance of 260/280 nm between 2.11 and 2.15 for liver and between 1.85 and 2.16 for muscle and RNA integrity numbers (Bioanalyzer 2100; Agilent Technologies, Santa Clara, CA) greater than 7 (mean ± SD; Liver = 7.2 ± 0.8; Muscle = 7.4 ± 0.6) for downstream RNAseq analysis.