CAMP DEPLETION IN FATTY LIVER OF PERIPARTURIENT DAIRY COW

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1020969
• Grant No.: 2020-67016-31343
• Cumulative Award Amt.: $200,000.00
• Proposal No.: 2018-06820
• Project Start Date: May 1, 2020
• Project End Date: Apr 30, 2023
• Grant Year: 2020
• Program Code: [A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals

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

Performing Department
Animal and Veterinary Sciences

Non Technical Summary
Fatty liver disease affects half of all dairy cattle that have recently given birth. Despite research into the causes and symptoms of fatty liver disease in dairy cattle, incidence rates remain high (costing farmers in excess of $60 million USD annually) indicating that on-farm management strategies are not enough to prevent fatty liver disease. Further understanding of the biochemical mechanisms that lead to the development of fatty liver is needed.Rodent research has indicated that a key coordinator of metabolism in the liver is dysregulated in cases of fatty liver. Because control of utilization of nutrients is critical to the survival of an organism, these processes are conserved throughout evolution. We, therefore, expect dairy cattle to have a similar dysfunction in cases of fatty liver.The overarching hypothesis for this project is, therefore, that this key enzyme (phosphodiesterase 4B (PDE4B)) is not regulated correctly in the liver of cattle who are affected by fatty liver disease after calving. To test this hypothesis, we will utilize over-conditioned (obese) dairy cattle and dairy cattle of normal body weight. We will collect tissue samples from healthy live animals and live animals that are experiencing fatty liver disease. Using these tissues, we will determine which of these key coordinators of energy usage in the liver are dysregulated. Second, based on the findings from the on-farm animal experiment, we will utilize the model of fatty liver disease developed in our laboratory that takes advantage of tissue collected from the slaughterhouse. By using our laboratory disease model, we will be able to test already commercially available treatments for efficacy in altering the activity of these enzymes and "fixing" the dysregulation. The ultimate goal of this research is preventing the development of fatty liver disease. Because commercially available drugs exist, we anticipate that upon identification of the dysregulated enzyme, we can ultimately develop an economical treatment to be used on the farm for prevention of fatty liver disease, thereby improving animal welfare and sustainability for dairy farmers.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3410	1010	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

fatty liver

periparturient

transition period

Goals / Objectives
Specific aim 1: Comparison of the hepatic lipid metabolism in the ideal-conditioned versus the over-conditioned periparturient dairy cow (PDC). We will purchase ideal-conditioned (body condition score (BCS) 3.0-3.25) and over-conditioned (BCS 3.75 or greater) dairy cows from a local dairy farm at the start of the dry period (approximately 60 days before expected calving) and then examine key hepatic differences in phosphodiesterase 4B (PDE4B) and protein kinase A(PKA) signaling and associated lipid metabolism in the healthy cow compared with the cow with fatty liver disease (FLD).Specific aim 2: Mechanistic exploration by using primary Holstein hepatic cell culture. By using our established in vitro model of bovine FLD, we will use overexpression and agonists/antagonists to probe the mediatory role of PDE4B-mediated cyclic adenosine monophosphate(cAMP) depletion and PKA inhibition in TG accumulation in cultured bovine hepatic cells.

Project Methods
Specific aim 1: Comparison of the hepatic lipid metabolism in the ideal-conditioned versus the over-conditioned PDC:By using ideal-conditioned (3.0-3.25 body condition score (BCS), 5 point scale) and over-conditioned (3.75+ BCS, 5 point scale) multiparous dairy cows, we will examine the signaling pathways related to hepatic lipid absorption in addition to hepatic lipid mobilization described above as they relate to hepatic cAMP concentration and associated downstream PKA activation in biopsied liver that has been flash frozen with liquid nitrogen.Twenty pregnant multiparous Holstein dairy cattle will be enrolled (i.e., purchased from regional dairy farms) into the experiment at the beginning of the dry period (60 days prior to the expected date of calving). Cows entering their second lactation or greater will be assigned to groups based upon their BCS at time of dry-off and enrollment. Groups will be balanced based upon milk yield from the prior lactation. Group one will be composed of cows that are ideal-conditioned, and group two will be composed of cows that are over-conditioned. Data will be analyzed as a mixed model including the fixed effects of cow, treatment, and group. Twenty animals were chosen to provide statistical power to detect differences of < 3% liver TG content with a P < 0.05 at a 90% confidence interval based upon previously published data. Both groups will be fed a common total mixed ration (TMR) that provides 100% of National Research Council (NRC) protein and energy requirements for dry cows. Dry cows will be fed their respective dietsad libitum. The cattle will be fed individually in tie-stalls, and individual feed intake will be recorded. Fourteen days prior to expected calving, all cattle will be moved to pens to provide comfort for calving and switched to bunk-fed rations that meet 100% of NRC protein and energy requirements. After calving, cows will be returned to tie-stalls and switched to a typical corn/soy/hay NRC ration for high-producing dairy cattle and fed individually and intake will be recorded.Liver samples (~300 mg liver) will be obtained by puncture biopsy under local anesthesia at 14 days prior to predicted date of parturition and at 14 days in milk (DIM) in all cows 4 ± 1/2 h after feeding and analyzed for total triglyceride (TG). To monitor energy balance effects on lipid mobilization, adipose samples will be collected on d -14 and d +14 from the pericaudal (tail head) region on the same days as liver biopsy for determination of total TG content and activities of HSL and other TG-mobilizing enzymes including ATGL and MGL. Liver samples will be analyzed for protein concentrations of PDE4B/Phospho-PDE4B, PKA/phospho-PKA, AMPK/phospho-AMPKa subunit, and CREB/phospho-CREB by western blotting to determine activation of these phosphoproteins. Markers of inflammation and apoptosis also will be analyzed by western blotting and IHC. The mRNA concentrations of PGC-1α, IL-1B TFEB, PEPCK, and PC will be determined by quantitative real-time PCR. ATP/AMP/cAMP weight ratios will be analyzed by high-performance liquid chromatography (HPLC) by using a previously established method.To monitor metabolic changes throughout the experiment, serial jugular blood samples will be collected on d -14, +2, +14, +28, and +35 relative to parturition. Blood samples will be collected at the same time of the day relative to feeding and milking. Blood will be collected into tubes containing anticoagulant, placed on ice to retard the degradation of hormones and metabolites, and centrifuged to separate the plasma from the whole blood. Individual aliquots of plasma will be stored at -20°C for subsequent analyses of glucose, NEFA, beta-hydroxybutyrate, TG, cholesterol, insulin, and glucagon by using appropriate commercially available kits. Circulating blood inflammatory cytokine TNFα concentration also will be quantified by western blotting. We expect to identify key differences in mediators of hepatic TG accumulation related to cAMP depletion, activity of PDE4B, and PKA activity in cattle that have a PP period and those that do not upon completion of specific aim 1.Specific aim 2: Mechanistic exploration by using primary Holstein hepatic cell culture utilizing ourin vitromodel of bovine FLD: Numerous studies have focused on the mechanisms of development of FLD (AFLD and NAFLD) in rodent models and in humans; the physiology and biochemistry of FLD in the ruminant animal is, however, less understood. A fewin vitroculture methods for bovine hepatic cells have been presented, but they are complicated in a number of ways because they involve collagenase perfusion (which tends to isolate epithelial cells before reaching parenchymal cells)and/or complex culture mediaoften supplemented with exogenous growth factors or steroids. Furthermore, these studies typically utilize male calvesor female calvesthat likely do not represent the physiology of the adult cow. Abattoir-derived liver from adult animals is readily available and has been utilized for primary cell culture in a number of species such as equine, porcine, and bovine. Because of the lack of available established cell lines or establishedsimpleprimary cell culture methodologies and with the need for further mechanistic studies in mind, we developed anin vitromodel of bovine FLDfrom abattoir-derived liver from adult cattle making us uniquely qualified to investigate mechanisms of disease developmentBy using our establishedin vitromethod of primary bovine FLD, we will examine the influence of TG accumulation with a combination of TNFα and fatty acid concentration on the observed differences in the markers of apoptosis, inflammation, and hepatic TG metabolism from specific aim 1. Palmitate and TNFa were chosen as the treatment because palmitate, along with oleate, are the fatty acids that increase the most in the liver of cattle with FLD, palmitate alone is most effective in promoting fatty acid accumulation in anin vitromodel of hepatic steatosis, and daily TNFa injections promote hepatic TG accumulation in dairy cattle. Finally, we chose to use a co-culture (mixed) method instead of a monoculture because co-culture allows for cell-to-cell communication and has the advantage of being a more faithful representation of FLDin vivo.The same methodologies listed in detail under specific aim 1 will be used to analyze those specific mRNAs and proteins. By using mixed primary cell-culture, we will over-express PDE4B by using an appropriate transfection kit/method or by using electroporation if no suitable kit/method is available. Transfection efficiency will be confirmed in each experiment, and, through work with various suppliers, we will identify an optimal method for transfecting primary bovine hepatic cells. Furthermore, we will downregulate PDE4B with an antagonist (e.g., rolipram, anti-PDE4B siRNA) and examine the associated changes in PKA and AMPK signaling pathways (specifically the proteins and mRNAs listed in specific aim 1) by western blotting, IHC, and qPCR. ATP/AMP/cAMP weight ratios will be determined by using HPLC. We also will use glucagon to stimulate the production of cAMP, thereby activating PKA to rescue the depletion of cAMP by overexpression of PDE4B. Finally, if modulation of AMPK activation becomes necessary, commercially available agonists (e.g., AICA riboprotein (AICAR), metformin) will be used to specifically activate the AMPK signaling pathway. A critical endpoint for these describedin vitrostudies will be the accumulation of lipid and TG, which will be quantified by oil red O staining and by commercially available TG quantification kits, respectively. Markers of inflammation (NF-κB P65) and apoptosis (caspase-3/7) will be assessed by western blotting and IHC, and key proteins and mRNAs (specific aim 1) involved in lipogenesis and lipolysis and fatty acid oxidation will be measured by western blotting and qPCR.

Progress 05/01/20 to 04/30/23

Outputs
Target Audience: Nothing Reported Changes/Problems:This project experienced serveral setbacks (in addition to those that were covid-related) : 1. The graduate student who had been working on this project for her masters thesis left the university for health reasons and has been unable to continue her work. 2. The PI of this project is leaving the university. For these reasons, we have been unable to publish the results of this work. What opportunities for training and professional development has the project provided? Nothing Reported 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? We observed nearly a 100% incidence rate of fatty liver disease in dairy cows prior to calving regardless of treatment with over nutrition or optimal nutrition. It has been previously shown that the incidence rate of fatty liver disease in 2000 was about 50% and that overfeeding in the way that we did would induce fatty liver disease. Both of those results were not repeated here, nor have they been repeated in other research projects reviewed by the PI (as an ad hoc manuscript reviewer for Journal of Dairy Science and Nature Scientific reports). We believe that the reason for this is that the physiology of the dairy cow has changed over the last 20 years because of genetic selection for production without consideration for health of the animal. This observation was unexpected and will lead us own novel research pathways. We followed several metabolic markers over time prior to calving and after calving in blood. We also collected adipose and liver tissue prior to calving and after calving. We will be able to describe the metabolic physiology of the cow over time as it relates to gene expression and expression of proteins over time as it relates to the metabolism of the transition dairy cow. We explored novel biochemical pathways, and these results will lead us down novel research pathways. We anticipate publications related to the development of in vitro disease modeling in cows. (Choudhary et al., 2021; Testroet et al., 2022)

Publications

Progress 05/01/21 to 04/30/22

Outputs
Target Audience: Nothing Reported Changes/Problems:This project experienced several setbacks (described in the final report) and our access to information about progress and accomplishments is unfortunately limited. What opportunities for training and professional development has the project provided? Nothing Reported 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? Please refer to our final report.

Publications

Progress 05/01/20 to 04/30/21

Outputs
Target Audience:1. University of Vermont - Reporting period 2020-2021 2. Eric D. Testroet 3. Accomplishents: Many of our research plans were delayed significantly by the COVID-19 pandemic. It is our hypothesis that dysregulation of PDE4b results in hepatic lipidosis and that ultimately modulation of PDE4b activity can lead to prevention of hepatic lipidosis in the periparturient cow. Currently we are in the process of finalizing an in vitro model of bovine hepatic lipidosis (manuscript in final revision to be submitted upon validation of hepatic cell function and gluconeogenic behavior in response to glucagon stimulation). We have isolated and cultured primary bovine hepatocytes and have completed functional validation (i.e., lipid accumulation, urea production, albumin production, LDH leakage, and cytotoxicity markers) but are completing the above further validation at the request of the associate editor. We have performed liver and adipose biopsies on 24 cattle that were meant to either experience hepatic lipidosis or not based on dietary treatment during the dry period. We had expected to examine key signaling pathways related to protein kinase A (PKA), AMP-activated protein kinase (AMPK), and phosphodiesterase 4b (PDE4B). We have analyzed key gene and protein expression data collected from these 24 cows and are in the process of interpreting these data (to be submitted for publication and form the M.S. thesis of Ms. Michelle LaCasse - defense scheduled for 12/2021). Unexpectedly, during our on-farm experiment, all cattle, regardless of treatment, developed fatty liver disease as characterized by percentage lipid in liver tissue on a wet weight basis. We have contacted authors of recently published manuscripts that have utilized similar methodologies and found that they have observed similar results (induction of fatty liver disease regardless of dietary treatment in the dry period). We are undergoing efforts to perform a limited meta-analysis of these data from combined studies. Our hypothesis is that genetic selection has altered the physiology of the "modern" dairy cow such that previously established experimental protocols to induce fatty liver disease are no longer effective (and in fact nearly all dairy cattle develop fatty liver disease in the periparturient period). In addition, we have received funding to characterize the bovine hepatic lipidosis model as a model for human NAFLD and NASH, and to examine use of a novel siRNA as a preventative for development of hepatic lipidosis in the transition period of dairy cattle. We have made little progress on this objective because of pandemic-related delays, but currently have RNA collected from cattle with fatty liver disease from our on-farm experiment, primary cells grown and induced to develop fatty liver disease in vitro, and we will be submitting these samples for RNA-seq and bioinformatic analyses and comparison to published sequencing data of humans with NAFLD and NASH. Following are funded projects, publications, and manuscripts in preparation: 1. 1. S. Shome, A. Testroet, J. Reecy, K. Amin, K. Conley, J. Reecy, R. Jernigan, D. Dobbs, M. Du, S. Clark, and D. C. Beitz, E. D. Testroet. 2021. Non-Coding RNA in Raw and Commercially Processed Milk and Putative Targets Related to Growth and Immune-Response. BMC Genomics. 22(749). 2. 2. S. Dankwa, U. Humagain, C. Yeoman, S. Clark, D. C. Beitz, S. Ishaq, and E. D. Testroet. 2021. Reduced-fat dried distillers grains with solubles does not negatively impact gut bacteria. Animal. 15(7)100281. In Revision Testroet, E. D., J. M. de Avila, S. Clark D. C. Beitz, and M. Du. 2021.The effect of palmitate and TNFα on abattoir-derived Holstein cow liver primary cell culture. In preparation to be re-submitted to J. Dairy Sci. In Preparation S. Choudhary, M. LaCasse, R. K. Choudhary, M. Rincon, D. C. Beitz, and E. D. Testroet. 2021. In vivo and in vitro expression of mitochondrial complex 1 inhibitor in bovine liver. In preparation to be submitted to J. Dairy Sci. Published Abstracts LaCasse, M., S. Choudhary, R. Choudhary. J. de Avila, D. C. Beitz, M. Du, and E. D. Testroet. A nonperfusion-based method of hepatic cell isolation and development of fatty liver disease model for dairy cattle. Poster. To be presented at the 2020 Experimental Biology Annual Meeting, San Diego, CA. S. Choudhary, R. Choudhary, LaCasse, M., J. de Avila, D. C. Beitz, M. Du, M. Rincon and E. D. Testroet. Expression of mitochondrial complex 1 inhibitor in bovine tissue, primary hepatic cells, and detection of its' transcript in conditioned media mimicking fatty liver disease. Poster. To be presented at the 2020 Experimental Biology Annual Meeting, San Diego, CA. Book Chapters S. Choudhary, M. LaCasse, D. C. Beitz, and E. D. Testroet. 2021. Fatty liver disease and utility of stem cells in developing the disease model. Ch. 8 in Stem Cells in Veterinary Science. Vol. 1. R. K. Choudhary and S. Choudhary, ed. Springer Nature. ISBN - 13: 978-9811634635 Changes/Problems:We have experienced significant issues related to COVID-19 and laboratory shutdowns and research shutdowns. These difficulties are, however, not unique to our laboratory. In addition, we unexpectedly saw no treatment effect from cattle fed high-energy diets, which has prompted the question of if the disease estimates of fatty liver disease in the transition period are correct, or if the disease is potentially much more prevalent than previously thought. We are pursuing a potential and practical on farm treatment targetting down-regulation of the MCJ protein and our goals have shifted towards evaluation the effects of secondary metabolic disorders resulting from fatty liver disease in addition to fatty liver disease as described in the original proposal. What opportunities for training and professional development has the project provided?Both Ms. LaCasse and Drs. S. and R. Choudhary were able to publish conference papers and would have attended the conference if it were not cancelled. The results were still presented at the NCCC-210 MS meeting and at the virtual conference. Ms. LaCasse is working on completing her M.S. dissertation and has also taken a job working at the Minnesota Veterinary Diagnostic Laboratory. How have the results been disseminated to communities of interest?The results have/will be disseminated via conference papers, manuscript publications, multistate meetings, and extension articles as appropriate. Following are funded projects, publications, and manuscripts in preparation: S. Shome, A. Testroet, J. Reecy, K. Amin, K. Conley, J. Reecy, R. Jernigan, D. Dobbs, M. Du, S. Clark, and D. C. Beitz, E. D. Testroet. 2021. Non-Coding RNA in Raw and Commercially Processed Milk and Putative Targets Related to Growth and Immune-Response. BMC Genomics. 22(749). S. Dankwa, U. Humagain, C. Yeoman, S. Clark, D. C. Beitz, S. Ishaq, and E. D. Testroet. 2021. Reduced-fat dried distillers grains with solubles does not negatively impact gut bacteria. Animal. 15(7)100281. In Revision: Testroet, E. D., J. M. de Avila, S. Clark D. C. Beitz, and M. Du. 2021.The effect of palmitate and TNFα on abattoir-derived Holstein cow liver primary cell culture. In preparation to be re-submitted to J. Dairy Sci. In Preparation: S. Choudhary, M. LaCasse, R. K. Choudhary, M. Rincon, D. C. Beitz, and E. D. Testroet. 2021. In vivo and in vitro expression of mitochondrial complex 1 inhibitor in bovine liver. In preparation to be submitted to J. Dairy Sci. Published Abstracts: LaCasse, M., S. Choudhary, R. Choudhary. J. de Avila, D. C. Beitz, M. Du, and E. D. Testroet. A nonperfusion-based method of hepatic cell isolation and development of fatty liver disease model for dairy cattle. Poster. To be presented at the 2020 Experimental Biology Annual Meeting, San Diego, CA. S. Choudhary, R. Choudhary, LaCasse, M., J. de Avila, D. C. Beitz, M. Du, M. Rincon and E. D. Testroet. Expression of mitochondrial complex 1 inhibitor in bovine tissue, primary hepatic cells, and detection of its' transcript in conditioned media mimicking fatty liver disease. Poster. To be presented at the 2020 Experimental Biology Annual Meeting, San Diego, CA. Book Chapters: S. Choudhary, M. LaCasse, D. C. Beitz, and E. D. Testroet. 2021. Fatty liver disease and utility of stem cells in developing the disease model. Ch. 8 in Stem Cells in Veterinary Science. Vol. 1. R. K. Choudhary and S. Choudhary, ed. Springer Nature. ISBN - 13: 978-9811634635 What do you plan to do during the next reporting period to accomplish the goals?We intend to compile the results of the protein expression in cattle pre- and post-partum and examine the signalling pathways that were proposed in our seed grant. We have submitted the methodology to Journal of Dairy Science for publication, but have been asked for minor additional characterizations of our disease model. We intend to complete these and publish the methodology and to publish the newer direction this work has taken regarding expression of the MCJ protein in the liver of transition dairy cattle. The PD believes that this protein has potential for establishing both USDA and potentially NIH funding lines and is pursuing those opportunities in the upcoming year.

Impacts
What was accomplished under these goals? Specific aim 1 has been completed and the results are being compiled by Ms. Michelle LaCasse for an M.S. dissertation. Specific aim 2 is still underway, but multiple laboratory shutdowns have inhibited the continuity of our cell culture system and caused issues. We are working on publishing this methodology and then we will examine the key findings from specific aim 1. It is our hypothesis that dysregulation of PDE4b results in hepatic lipidosis and that ultimately modulation of PDE4b activity can lead to prevention of hepatic lipidosis in the periparturient cow. Currently we are in the process of finalizing an in vitro model of bovine hepatic lipidosis (manuscript in final revision to be submitted upon validation of hepatic cell function and gluconeogenic behavior in response to glucagon stimulation). We have isolated and cultured primary bovine hepatocytes and have completed functional validation (i.e., lipid accumulation, urea production, albumin production, LDH leakage, and cytotoxicity markers) but are completing the above further validation at the request of the associate editor. We have performed liver and adipose biopsies on 24 cattle that were meant to either experience hepatic lipidosis or not based on dietary treatment during the dry period. We had expected to examine key signaling pathways related to protein kinase A (PKA), AMP-activated protein kinase (AMPK), and phosphodiesterase 4b (PDE4B). We have analyzed key gene and protein expression data collected from these 24 cows and are in the process of interpreting these data (to be submitted for publication and form the M.S. thesis of Ms. Michelle LaCasse - defense scheduled for 12/2021). Unexpectedly, during our on-farm experiment, all cattle, regardless of treatment, developed fatty liver disease as characterized by percentage lipid in liver tissue on a wet weight basis. We have contacted authors of recently published manuscripts that have utilized similar methodologies and found that they have observed similar results (induction of fatty liver disease regardless of dietary treatment in the dry period). We are undergoing efforts to perform a limited meta-analysis of these data from combined studies. Our hypothesis is that genetic selection has altered the physiology of the "modern" dairy cow such that previously established experimental protocols to induce fatty liver disease are no longer effective (and in fact nearly all dairy cattle develop fatty liver disease in the periparturient period). In addition, we have received funding to characterize the bovine hepatic lipidosis model as a model for human NAFLD and NASH, and to examine use of a novel siRNA as a preventative for development of hepatic lipidosis in the transition period of dairy cattle. We have made little progress on this objective because of pandemic-related delays, but currently have RNA collected from cattle with fatty liver disease from our on-farm experiment, primary cells grown and induced to develop fatty liver disease in vitro, and we will be submitting these samples for RNA-seq and bioinformatic analyses and comparison to published sequencing data of humans with NAFLD and NASH.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: LaCasse, M., S. Choudhary, R. Choudhary. J. de Avila, D. C. Beitz, M. Du, and E. D. Testroet. A nonperfusion-based method of hepatic cell isolation and development of fatty liver disease model for dairy cattle. Poster. 2020 Experimental Biology Annual Meeting, San Diego, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: S. Choudhary, R. Choudhary, LaCasse, M., J. de Avila, D. C. Beitz, M. Du, M. Rincon and E. D. Testroet. Expression of mitochondrial complex 1 inhibitor in bovine tissue, primary hepatic cells, and detection of its transcript in conditioned media mimicking fatty liver disease. Poster. 2020 Experimental Biology Annual Meeting, San Diego, CA.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: S. Choudhary, M. LaCasse, R. K. Choudhary, M. Rincon, D. C. Beitz, and E. D. Testroet. 2021. In vivo and in vitro expression of mitochondrial complex 1 inhibitor in bovine liver. In preparation to be submitted to J. Dairy Sci.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: Testroet, E. D., J. M. de Avila, S. Clark D. C. Beitz, and M. Du. 2021. The effect of palmitate and TNF? on abattoir-derived Holstein cow liver primary cell culture. In preparation to be re-submitted to J. Dairy Sci.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: S. Shome, A. Testroet, J. Reecy, K. Amin, K. Conley, J. Reecy, R. Jernigan, D. Dobbs, M. Du, S. Clark, and D. C. Beitz, E. D. Testroet. 2021. Non-Coding RNA in Raw and Commercially Processed Milk and Putative Targets Related to Growth and Immune-Response. BMC Genomics. 22(749).
• Type: Journal Articles Status: Published Year Published: 2021 Citation: S. Dankwa, U. Humagain, C. Yeoman, S. Clark, D. C. Beitz, S. Ishaq, and E. D. Testroet. 2021. Reduced-fat dried distillers grains with solubles does not negatively impact gut bacteria. Animal. 15(7)100281.
• Type: Book Chapters Status: Published Year Published: 2021 Citation: S. Choudhary, M. LaCasse, D. C. Beitz, and E. D. Testroet. 2021. Fatty liver disease and utility of stem cells in developing the disease model. Ch. 8 in Stem Cells in Veterinary Science. Vol. 1. R. K. Choudhary and S. Choudhary, ed. Springer Nature. ISBN - 13: 978-9811634635