RELATIONSHIPS BETWEEN LEAKY GUT, HEAT STRESS, AND MILK PRODUCTION IN DAIRY COWS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1021838
• Grant No.: 2020-67015-30830
• Cumulative Award Amt.: $495,000.00
• Proposal No.: 2019-06005
• Project Start Date: Apr 1, 2020
• Project End Date: Mar 31, 2025
• Grant Year: 2020
• Program Code: [A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals

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

Performing Department
Dairy Science

Non Technical Summary
Heat stress (HS) affects US dairy herds of all sizes and in every region of the country costing US agriculture $1.7 billion annually. In dairy cows, HS reduces milk production efficiency by affecting milk yield and milk composition. Much of the loss in milk production is due to reduced feed intake. However, reduced feed intake only explains about 50% of the observed decrease in milk production suggesting that HS itself affects milk production. We hypothesize that HS causes leaky gut in high-producing dairy cows resulting in immune system activation thereby partitioning nutrients away from milk production. Furthering the explanation of decreased milk production during HS, we hypothesize that amino acids (AA) supplied by the diet and body stores do not keep pace with demand for milk production and the immune response to leaky gut. Using a pair-feeding model to adjust for feed intake, high-producing dairy cows will be used in experiments to address two Specific Aims: 1) Determine how HS affects gut integrity, immune system function, and whole body AA utilization. 2) Demonstrate that supplementation of specific AA can amend negative effects of HS and improve whole body AA efficiency by balancing the effects of body AA release, gut integrity and function, immune system requirements, and milk production requirements. This project will improve US agriculture through the enhancement of AA utilization for milk production in high-producing dairy cattle under HS conditions, aligning well with the Animal Nutrition, Growth and Lactation (A1231) program area.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3410	1020	20%
302	3410	1010	60%
302	3410	1030	10%
302	3410	1040	10%

Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1010 - Nutrition and metabolism; 1020 - Physiology;

Keywords

dairy cow

heat-stress

milk

Goals / Objectives
Using a pair-feeding model to adjust for feed intake, high-producing dairy cows will be used in experiments to address two Specific Aims: 1) Determine how HS affects gut integrity, immune system function, and whole body AA utilization. 2) Demonstrate that supplementation of specific AA can amend negative effects of HS and improve whole body AA efficiency by balancing the effects of body AA release, gut integrity and function, immune system requirements, and milk production requirements.

Project Methods
Specific Aim 1 Methods Overview. Work for this Aim will center on one in vivo experiment designed to test the following hypotheses, phrased as questions: 1a) Does HS directly cause leaky gut and activate the immune system in lactating cows? 1b) Is the supply of AA from the diet and from endogenous proteolysis significantly altered by HS, leading to either inadequate or of an improper balance to support demands for both milk production and an immune response? 1c) To what extent does HS alter enterocyte fuel substrate use and mitochondrial function? Sixteen healthy lactating Holstein cows will be used in a pair-feeding experiment, as described by Gao et al. (2017). Cows will be randomly allocated to one of two treatments with two periods per treatment. After 14 d of acclimation (period 1), cows on the heat stress treatment (HS) will be exposed to cyclical heat stress for 4 d whereas cows on the pair-fed thermal neutral treatment (PFTN) will not be exposed to HS and intake will be matched to HS. A common diet will be fed to all cows twice daily; intake will be measured. Cow vital signs will be recorded 3x/d throughout the experiment. To assess gut integrity, each cow will be orally drenched with inert markers: chromium-EDTA, D-mannitol, and sucralose on a single day per period. Hourly subsamples of total volume of urine collected within 24 h of dosing will be used to assess percent recovery of each marker. On d 7 of period 1, two jugular catheters will be placed in each cow; catheters will be maintained until the end of the experiment and used for labelled AA infusion (below) and blood sampling for measurement of acute phase proteins. Histology and immunohistochemistry will be used to assess intestinal integrity at the end of period 2. Cows will be milked twice daily throughout both periods; milk yield and composition will be measured at each milking. Amino acid entry from the diet (EAA), de novo synthesis (non-essential AA), release from whole body protein, incorporation into body protein, and clearance will be assessed over a 24 h period at the end of both periods. We will use the basic methodology of Estes et al. (2018), wherein each cow will receive a constant infusion of 0.5 g of a 13C-AA mixture. Blood samples will be assessed for AA enrichment (Estes et al., 2018). A 3-pool model (mirrored for isotope movement) will be fitted to the resulting data to derive rates of whole body AA incorporation into and release from protein, AA entry rates (absorption for EAA and absorption plus synthesis for NEAA), and AA irreversible loss rates. At the end of period 2, all cows will be slaughtered. To quantify HS effects on skeletal muscle metabolism, semitendinosus muscle biopsies (excisional or surgical) will be obtained from all cows on the final day of each period and analyzed according to methods of Xie et al. (2016). Jejunal mucosal scrapings from all HS and PFTN animals will be obtained at slaughter. These enterocyte-rich scrapings will be used in substrate oxidation and metabolic flexibility assays (Zhao et al., 2018, Frisard et al., 2010, Yang et al., 2014, Tarpey et al., 2017).Evaluation, Specific Aim 1:Aim 1 will use a randomized complete block design with 16 cows allotted to one of two treatments (HS or PFTN) and one of four (1, 2, 3, 4) groups. Each cow will experience 2 periods. In period 1, no treatments will be assigned and therefore data for period 1 will be considered baseline data. Data pertaining to period 2 will be adjusted with the baseline data for period 1. Cow within treatment and group will be considered the random term used to test for differences. Data will be reported as least square means and considered significant if P ≤ 0.05.Expected Outputs, Specific Aim 1:Compared to PFTN, HS will exhibit changes to vital signs (e.g, increased respiration, increased rectal temperature). Also, relative to our original questions, the following results are expected:1a) Does HS directly cause leaky gut and activate the immune system in lactating cows?We expect increased urine excretion of all 3 orally dosed inert markers in HS compared to PFTN.Visual evidence for reduced gut integrity in HS compared to PFTN by histological assessment.Increased circulating blood leukocytes and blood acute phase proteins in HS compared to PFTN, indicative of an inflammatory immune response.Increased irreversible loss of EAA during HS due to immune requirements.1b) Is the supply of AA from the diet and from endogenous proteolysis either inadequate or of an improper balance to support demand for both milk production and an immune response in HS cows?Despite similar dry matter intake, HS cows are expected to show ~50% further reduction in milk yield compared to PFTN.Altered blood plasma free AA concentrations and net supply in HS compared to PFTNThe same or reduced rates of absorbed AA due to gut integrity problems, and increased rates of irreversible loss of one or more AA.Increased whole body proteolysis.1c) To what extent does extent HS alter enterocyte fuel substrate use and mitochondrial function?Enterocytes exposed to HS are expected to switch their major oxidative fuel source away from the preferred substrate glutamine, perhaps to glucose.The molecular basis for apparently altered fuel substrate metabolism in HS will be revealed through our mitochondrial assays.Specific Aim 2 Methods Overview. Work for this Aim will center on one in vivo experiment designed to test the following hypothesis, phrased as a question:2) Can increasing the AA status of HS cows be a way to support milk production, obtain less protein mobilization, improve gut integrity, and avoid of immune system activation?The animal experiment for this Aim will be similar to the methods reported for Aim 1. Notable updates here include:Doubling number of total cows used (32 used in Aim 2; 16 used in Aim 1) to account for 4 treatments:PFTN-0; pair-fed thermoneutral, minus EAA+Gln (n = 8).PFTN-AA; pair-fed thermoneutral, plus EAA+Gln (n = 8).HS-0; heat stress, minus EAA+Gln (n = 8).HS-AA; heat stress, plus EAA+Gln (n = 8).No stable isotope infusions.No slaughter in this experiment.No assays of enterocyte fuel substrate preference or mitochondrial function in this experiment.The crystalline AA in the treatment mixture will contain a blend of all EAA and the NEAA Gln (included for its purported role in intestinal function; Harmon, 1986; Kessel et al., 2007; Oba et al., 2004; Okine et al., 1995; El-Kadi et al., 2009; Zuhl et al., 2015). The supplemented amount of each EAA will be determined based on Aim 1 results using the model of the effects of individual AA and energy supply on milk protein production developed by co-investigator Hanigan as described in Aim 1. Cows assigned to AA treatments will be infused continuously via a jugular catheter with a sterile AA mixture designed to correct the apparent imbalance or deficiency in EAA supply caused by the HS and the Gln. The same blood, feces, and urine sampling scheme used in Aim 1 will be repeated here.Expected Outputs, Specific Aim 2:Compared to PFTN, HS will exhibit changes to vital signs (e.g, increased respiration, increased rectal temperature). Also, going along with our originally phrased question for this Aim, we expect the following:Lower evidence of leaky gut and immune system activation in HS+AA when compared to HS-0.Plasma AA concentrations for the HS animals that more closely match the PFTN-0.Improved AA utilization and improved N balance.Higher milk yield, milk lactose, and milk protein in HS+AA when compared to HS-0, that is similar to PFTN-0.Improved N balance, maintained gut integrity and function (and thus no inflammatory immune response), and improved milk yield and protein yield in HS-AA compared to HS-0.Evaluation, Specific Aim 2:Data analysis will be as in Aim 1, with statistical models updated to include the fixed effect of AA supplementation.

Progress 04/01/20 to 03/31/25

Outputs
Target Audience:The target audience focused on for the project duration included: scientific peers - whom we communicated with through manuscripts, professional society meeting abstracts, and scientific conferences. members of Virginia's dairy industry - whom we communicated with through magazine articles written for lay audience. graduate students interested in dairy science - who had experiential learning opportunities linked to the project. undergraduate students interested in dairy science -who had experiential learning opportunities linked to the project. Changes/Problems:Changes/Problems Objective 2aimed to demonstrate that supplementation of specific amino acids (AA) could mitigate the negative effects of heat stress (HS) and improve whole-body AA efficiency by balancing AA release from body tissues, gut integrity and function, immune system demands, and milk production requirements. Two primary challenges impacted our ability to fully complete this objective and ultimately resulted in returned budget funds: Unexpected findings from Objective 1:Contrary to our initial hypothesis, no changes in branched-chain amino acid (BCAA) flux were observed during short-term HS. This suggests that protein turnover may not be significantly altered under these conditions, which limited the rationale for proceeding with AA supplementation trials as originally designed. COVID-19-related supply chain disruptions:Delays in acquiring essential medical-grade supplies for AA infusion work slowed progress and affected the rate of award expenditure. Despite these challenges, we adapted our approach to maximize the value of the project. Notably, we leveraged archived samples from the 16 cows used in Objective 1 to pursue additional lines of inquiry. This work was conducted without additional use of project funds but still acknowledged USDA support. These efforts yielded new knowledge and resulted inthree peer-reviewed publications, which are included in the project products. Highlights of these additional outcomes are summarized below: Metabolic Flexibility during Heat Stress. We investigated the ability of mammary, muscle, and liver tissues to switch between carbohydrate and lipid energy sources--a concept known as metabolic flexibility (Met Flex). Tissue samples collected on the final day of Period 2 revealed that: • HS reduced metabolic flexibility inskeletal muscle, but not inmammary or livertissue. • This reduction may contribute to decreased milk yield during HS and warrants further investigation. Mammary Tissue Structure and Function during Heat Stress Histology, protein abundance, and gene expression analyses were performed on mammary tissue samples. Key findings include: A 4.3 kg/day reduction in milk yield after 4 days of HS was primarily linked to impairedprotein synthesisandcell survivalpathways. The decline in milk yield was associated more withreduced cellular activitythan with changes in cell number. These results highlight the complex impact of HS on mammary gland function and suggest new targets for intervention. Mammary Transcriptome during Heat Stress Transcriptomic analysis of mammary tissue revealed: • 54 genes upregulatedand74 genes downregulatedunder HS conditions. • HS activated stress-response genes while suppressing those involved inDNA repairandmitochondrial function. • These molecular disruptions may underlie the observed 4.35 kg/day milk loss and provide a foundation for future mechanistic studies. In summary, while Objective 2 could not be completed as originally planned, the project adapted effectively to unforeseen challenges. The additional research conducted using archived samples significantly expanded our understanding of the physiological and molecular responses to HS in dairy cows and contributed valuable new knowledge to the field. What opportunities for training and professional development has the project provided?The project team provided hands-on training to both undergraduate (8 over the life of the grant)and 2 PhDstudents, led by team members with advanced professional expertise. Training activities encompassed: Animal Handling and Husbandry: Instruction in safe handling of research animals and standard dairy husbandry practices, including milking procedures, safe cow handling, and feeding protocols. Research-Specific Technical Skills: Training in specialized research techniques such as jugular catheter placement and maintenance, urinary catheter placement and maintenance, and biological sample collection and processing. In addition to technical training, the project offered professional development opportunities aimed at enhancing participants' communication skills. These included experience in both written and oral dissemination of research findings, contributing to the development of scientific communication competencies. How have the results been disseminated to communities of interest?Overal project results have been disseminated through the publishing of 5 peer-reviewed journal articles, 2 PhD dissertations, and a handful of conference abstracts, as reported in the "Products" and "Other Products" section of this report. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Determine how heat stress (HS) affects gut integrity, immune system function, and whole-body amino acid (AA) utilization, in addition to animal feed intake and milk production characteristics in dairy cows. What was done: A controlled animal experiment was conducted using 16 multiparous Holstein cows (~100 days in milk), randomly assigned to one of two treatments: Pair-fed thermoneutral (PFTN, n = 8):Housed in thermoneutral conditions (temperature humidity index, THI = 64), feed intake matched to HS cows. Heat stress (HS, n = 8):Exposed to cyclical HS conditions (THI = 76-80). The study consisted of two 4-day periods: Period 1 (P1):Thermoneutral conditions with ad libitum intake. Period 2 (P2):Treatment-specific conditions as described above. Data collected included: Physiological and production parameters:Milk yield and composition, rectal temperature, respiration rate, and dry matter intake (DMI). Gut integrity:Assessed using oral administration of Cr-EDTA and sucralose with 24-hour urine collection. Immune activation:Measured via blood concentrations of lipopolysaccharide-binding protein (LBP). Amino acid metabolism:Branched-chain amino acid (BCAA) entry rates into blood were measured using a stable isotope infusion and a 4-pool model. Isotopic enrichment was assessed using a mass spectrometer coupled to a GC by a combustion oven. A 4-pool model was used to determine apparent post-absorptive entry rates for each BCAA. All other data were analyzed using the GLIMMIX procedure in SAS. P1 data were used as covariates when significant. What was learned: Feed intake and milk production:HS reduced voluntary DMI by 35%, which accounted for 66% of the observed reduction in milk yield. HS cows had lower yields (but not concentrations) of milk protein, fat, and other solids. Physiological responses:HS increased rectal temperature (38.4°C vs. 39.4°C) and respiration rate (40 vs. 71 breaths/min). Nitrogen metabolism:HS cows had elevated milk urea nitrogen (MUN) and a tendency for higher plasma urea nitrogen, indicating a shift in whole-body nitrogen metabolism. Glucose and insulin:HS cows had 7% lower plasma glucose and 71% higher insulin concentrations. Gut integrity and immune activation:No differences were observed in urinary recovery of Cr-EDTA or sucralose, nor in LBP concentrations, suggesting no compromise in gut integrity or systemic immune activation. Amino acid metabolism:No differences were detected in BCAA entry or flux rates, contrary to the hypothesis that HS would increase BCAA turnover. Impact: This study provides new insights into the physiological and metabolic responses of lactating dairy cows to moderate heat stress. Key findings include: Quantification of HS impact:HS directly accounted for 34% of the reduction in milk yield, independent of reduced feed intake. Refined understanding of nitrogen metabolism:Elevated MUN and plasma urea nitrogen suggest altered nitrogen handling under HS. Revised hypothesis on gut integrity:Contrary to expectations, HS did not compromise gut integrity or activate the immune system via gut-derived endotoxins. Unexpected stability in BCAA metabolism:No changes in BCAA flux suggest that protein turnover may not be significantly altered during short-term HS. These findings refine our understanding of the mechanisms by which HS affects dairy cow productivity and can inform future nutritional and management strategies to mitigate HS effects.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Ellett, M. D. 2024. The Influence of Heat Stress on Milk Yield, Gastrointestinal Permeability, and Nutrient Partitioning in Lactating Dairy Cattle. PhD Dissertation. Virginia Tech, Blacksburg, VA. https://vtechworks.lib.vt.edu/items/cfff9af3-60b0-44c3-9de4-95d76c480606
• Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: Perez-Hernandez. 2023. Local Regulation of Milk Synthesis Capacity in the Mammary Gland of Lactating Dairy Cows. PhD Dissertation. Virginia Tech, Blacksburg, VA. https://vtechworks.lib.vt.edu/items/b27e05cd-e26c-4789-9850-01714783e881
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: M.D. Ellett, R.P. Rhoads, M.D. Hanigan, B.A. Corl, G. Perez-Hernandez, C.L.M. Parsons, L.H. Baumgard, K.M. Daniels. 2024. Relationships between gastrointestinal permeability, heat stress, and milk production in lactating dairy cows. J. Dairy Sci., 107: 5190-5203.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: M.D. Ellett, K.M. Daniels, M.D. Hanigan, B.A. Corl, G. Perez-Hernandez, C.L.M. Parsons, J.A. Melvin, D.W. Fausnacht, R.P. McMillan, L.H. Baumgard, R.P. Rhoads. 2025. Tissue-specific responses to oxidative fuel source preference during heat stress in lactating dairy cows. JDS Communications. 6:160-164.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: G. Perez-Hernandez, M.D. Ellett, L.J. Banda, D. Dougherty, C.L.M. Parsons, A.J. Lengi, K.M. Daniels, B.A. Corl. 2024.Cyclical heat stress during lactation influences the microstructure of the bovine mammary gland. J. Dairy Sci. 107:8609-8628.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: G. Perez-Hernandez, M.D. Ellett, B. Pokhrel, C.L.M. Parsons, B.A. Corl, K.M. Daniels. 2025. Transcriptomic changes induced by controlled cyclical heat stress in the bovine mammary gland during lactation. JDS Communications.6:604-609.

Progress 04/01/23 to 03/31/24

Outputs
Target Audience:Peer scientists researching ruminant gut physiology was the main target audience for this reporting year. Changes/Problems:COVID-19-related supply chain disruptions slowed our overall progress during this reporting period. However, the results obtained for Specific Aim 1, focused on whole-body protein turnover, will provide valuable insights to guide the implementation of Aim 2. What opportunities for training and professional development has the project provided?Two PhD students continued to work on research for specific aim 1 and gained experience in written and verbal scientific communication. 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?Two PhD students will defend their PhD dissertations and make dissertations avilable online through Virginia Tech's electronic library system.They will draft and submit manuscripts of dissertation work to peer-reviewed journals for publication consideration. Team members may also attend conferences and disseminate research findings. Ongoing discussion between investigators regarding Specific Aim 2 will occur.

Impacts
What was accomplished under these goals? Two PhD students worked on parts of specific aim 1 and will incorporate findings into their respective PhD dissertations and peer-reviewed manuscripts.

Publications

Progress 04/01/22 to 03/31/23

Outputs
Target Audience:Peer scientists researching ruminant gut physiology was the main target audience for this reporting year. Changes/Problems:In this reporting period the team experienced lingering COVID-19 supply chain issues, slowing our overall progress,but no major deviations from our experimental timeline occured. What opportunities for training and professional development has the project provided?The PhD student, Ellett, presented data from this work (aim 1) at the 2022 annual meeting of the American Dairy Science Association. The PhD student associated with this grant (Ellett) passed his preliminary exams and advanced towards candidacy. How have the results been disseminated to communities of interest?Results-to-date were disseminated to research peers through submisison and presentation of scientific abstracts. What do you plan to do during the next reporting period to accomplish the goals?Manuscripts related to aim 1 are being drafted and will be submitted for peer review in late 2023. The experiment for aim 2 must still be conducted.

Impacts
What was accomplished under these goals? In the previous reporting period, we used a pair-feeding model to adjust for feed intake in high-producing dairy cows in an experiment designed to (aim 1): Determine how HS affects gut integrity, immune system function, and whole body amino acid (AA) utilization. In this project period we continued to perform laboratory analyses on the samples collected from project cows. Statistical analyses were performed when laboratory analysis concluded. In this project year we presented urine marker data (2022 ADSA abstract, Ellett) that shows, under conditions of this experiment and contrary to our hypothesis, 4 days of heat stress did not directly impair gut integrity (i.e., cause "leaky gut") in lactating dairy cows. Our original thought was that heat stress would directly damage the gut, allowing gut microbes to gain access (i.e., "leak into") to the cow's tissues and trigger an immune response. Work in the upcoming year will assess immune cell infiltration in the gut tissue we collected, evaluate integrity of cell-to-cell junctions in the gut tissue we collected, and we will also quantify immune cell markers in the blood we collected. It is now anticipated that these measurements, once analyzed statistically, will support our new finding that 4 days of heat stress does not directly impar gut integrity in lactating dairy cows. Nonetheless, the heat-stressed cows in our experiment demonstrated a decrease in milk production that could not be explained by feed reduction alone (manuscript to be submitted in late 2023). This tells us that nutrient metabolism (our findings continue to point to amino acid metabolism), is affected in heat-stressed cows in ways that we do not yet fully understand. Our whole body amino acid utilization analyses have been long-delayed by COVID-19 supply chain issues but in the next reporting period, results will be available wherein we expect to identify which amino acids to supplement during heat stress in lactating dairy cows. These findings, in turn, will allow us to proceed with aim 2) Demonstrate that supplementation of specific amino acids can amend negative effects of heat stress and improve whole body amino acid efficiency by better balancing amino acid requirements.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: M. Ellett, M. Hanigan, C. Parsons, R.Rhoads, and K. Daniels. 2022. Heat stress and total-tract gastrointestinal permeability in lactating dairy cows. J. Dairy Sci. 105(Suppl. 1): 382. (Abstr.)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Perez-Hernandez, G., L. J. Banda, D. Dougherty, M. D. Ellett, A. J. Lengi, C. L. M. Parsons, K. M. Daniels, and B. A. Corl. 2023. Bovine mammary epithelial cell number and cell losses after a short period of heat stress during lactation. J. Dairy Sci. 106(Suppl. 1): 92. (Abstr.)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Parsons, C. L. M., L. J. Banda, M. D. Ellett, and K. M. Daniels. 2023. Impacts of acute in vitro heat stress on preruminant jejunal integrity. J. Dairy Sci. 106(Suppl. 1): 297. (Abstr.)

Progress 04/01/21 to 03/31/22

Outputs
Target Audience:Peer scientists researching ruminant gut physiology was the main target audience for this reporting year. Changes/Problems:COVID-19 supply chain issues slowed our overall progress during this reporting period but no major deviations from our experimental timeline occured. What opportunities for training and professional development has the project provided?Training activities included hand-on instruction of undergraduates and graduate studentsby project team members with advanced professional skills. Training included instruction on: safe research animal handling, dairy husbandry practices (e.g., milking cows, safe handling of cows, feeding cows), and specific research task training (e.g., jugular catheter placement and maintenance, urinarycatheter placement and maintenance, sample collection and processing). 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?Laboratory analyses for Specific Aim 1 will be completed by project team members and an at least one manuscript summarizing major findings will be submitted for peer review. Team members may also attend conferences and disseminate research findings. Discussions will commence regarding initial of experiments for Specific Aim 2.

Impacts
What was accomplished under these goals? Our project team conducted the in vivo animal experiment and began laboratory analysesfor Specific Aim 1 during this reporting peroid.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Ellett, M. D., C. L. M. Parsons, J. M. Hay, and K. M. Daniels. 2022. Persistence of sugars used for intestinal permeability measures in an in vitro rumen environment. J. Dairy Sci. Comm. 3: 245-249. https://doi.org/10.3168/jdsc.2021-0202

Progress 04/01/20 to 03/31/21

Outputs
Target Audience: Nothing Reported Changes/Problems:The COVID-19 pandemic severely limited research effforts in this reporting period but the project team met periodically to plan and purchase items needed to support Specific Aim 1 of the project. What opportunities for training and professional development has the project provided?The graduate student involved in the project received training on research project design and one-on-one support from the project director and technical professional associated with the project. 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?During the next reporting period, research activities for Specific Aim 1 should be underway. A manuscript summarizing the in vitro findings should be submitted for peer review.

Impacts
What was accomplished under these goals? The COVID-19 pandemic severely limited research effforts in this reporting period but the project team met periodically to plan and purchase items needed to support Specific Aim 1 of the project. In October to December of 2020 the team conducted an in vitro experiment to support Specific Aim 1.The objective was to evaluate persistence ofd-mannitol, sucralose, and lactulose in a closed in vitro rumen fermentation system over 48 h. The null hypothesis was that sugar concentration would not be affected by time.

Publications