THE IMPACT OF PREPARTAL LIVER GLUTATHIONE ON MILK PERFORMANCE PARAMETERS AND WELFARE OF PERIPARTAL DAIRY COWS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028416
• Grant No.: 2022-67015-37085
• Cumulative Award Amt.: $300,000.00
• Proposal No.: 2021-07102
• Project Start Date: Jun 1, 2022
• Project End Date: May 31, 2024
• Grant Year: 2022
• 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
The US dairy industry recognizes that animal health plays an essential role in feed efficiency and milk quality, as evidenced by the wealth of research conducted on this topic in the last two decades. A major challenge in dairy cow health is the transition from late pregnancy to lactation. This stressful period is characterized by substantial metabolic and physiological changes due to energy demands for fetal growth and lactation. This transition is often correlated with a significant increase in health disorders occurring within the first weeks after birth. These health disorders can severely reduce milk production, reproductive parameters, and cause welfare issues that can lead to death or removal of cows from the herd. These health disorders can have a tremendous economic impact on dairy farms and subsequently make the dairy industry at large less environmentally sustainable.Oxidative stress is a biological condition common to all multicellular organisms, and in humans, it has been ascribed as an underlying factor for severaldiseases, including Parkinson's, Alzheimer's, and cancer, among others. Oxidative stress is an imbalance between oxidants, also called reactive oxygen species, and antioxidants such as glutathione. Dairy cows are commonly exposed to the highest levels of oxidative stress during the transition from pregnancy to lactation. Therefore, the aim of this study is to understand the crucial role glutathione plays during the transition period of dairy cows by comparing cows with high glutathione against low-glutathione cows.To reach this goal, we will determine glutathione concentrations in liver biopsies samples from dairy cows taken before calving. We will use this data to perform a retrospective analysis based on cows with high versus low liver glutathione levels. Additionally, we will perform contemporary gene expression and western blot analyses to give us a clearer picture of the transcriptional and metabolic status of enzymes related to glutathione synthesis and storage in the liver of transition dairy cows. We will also collect blood samples to monitor concentrations of glutathione, biomarkers of oxidative stress, liver function, inflammation, as well as hormones such as insulin and cortisol. This information will give us a broader understanding of how adequate reserves of liver glutathione may impact the animal at a systemic level. In addition, we will correlate performance parameters such as milk yield, feed intake, and body condition score in transition cows with high and low liver glutathione.Collectively, data from this studywill allow us to delineate the relationships between glutathione precursors and glutathione storage in the liver of dairy cows under stress conditions. Understanding the mechanisms that regulate glutathione storage in transition dairy cows will enable us to devise nutritional and management strategies for dairy farmers and dairy consultantsto prevent oxidative stress and thereby improve animal health and performance.

Animal Health Component

60%

Research Effort Categories

Basic

30%

Applied

60%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3410	1010	34%
305	3410	1020	33%
311	3410	1090	33%

Knowledge Area
302 - Nutrient Utilization in Animals; 311 - Animal Diseases; 305 - Animal Physiological Processes;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1090 - Immunology; 1010 - Nutrition and metabolism; 1020 - Physiology;

Keywords

oxidative stress

rnaseq

gene expression

glutathione

transition dairy cows

Goals / Objectives
Goal: To define the mechanisms that control glutathione storage or accumulation in the liver of dairy cows during prepartum and assess its effect on performance parameters and welfare postpartum.Objectives: In Holstein dairy cows, we will analyze liver glutathione at various time points during the transition period from late pregnancy to lactation to determine what are the major control points or rate-limiting factors regulating prepartum liver glutathione storage via RNA-seq, RT-qPCR, and western blot methods. In mouse liver models, we will characterize the effects of precursors of glutathione such as cysteine and methionine as well as hormones and metabolites on glutathione storage.

Project Methods
Welfare of dairy cows can impact feed efficiency and milk quality. A major challenge in maintaining adequate cow health in dairy farms is the period when cows transition from late pregnancy to lactation. This transitional period is challenging because cows are exposed to higher levels of oxidative stress, resulting in oxidative damage at the cellular level to lipids, DNA, proteins, and other macromolecules. Oxidative stress has been identified as the underlying factor to dysfunctional host immune and inflammatory response in transition dairy cows.Antioxidants such as glutathione (GSH) are well known to be effective in mitigating oxidative stress. Glutathione synthesis is affected by the energy density of the diet and the hormonal balance in the cow. Glutathione is primarily stored in the liver, and such reserves may help minimize adverse effects during periods of excessive oxidative stress (such as the transition period of dairy cows). Therefore, this research project seeks to understand the impact of previously accumulated antioxidants such as glutathione (GSH) on health and performance parameters during periods of high oxidative stress. Additionally, we expect to reveal essential molecular mechanisms driving the synthesis and accumulation of GSH in the liver of transition dairy cows. The information gathered in this project has the potential to provide nutritional recommendations to enhance the antioxidant capacity of transition dairy cows.Because of the importance of GSH as an antioxidant, liver GSH will be our primary target. Liver biopsies will be performed on pregnant multiparous Holstein dairy cows at 10 days before the expected calving date, and GSH levels will be measured using established colorimetric methods. Cows will then be grouped into high GSH and low GSH categories. This classification will allow us to understand how the level of liver GSH before calving can affect the oxidative status, health, and performance postpartum. In the same animals, subsequent liver biopsies will be performed at 7 and 21 days postpartum. All cows in this study will be offered the same standard husbandry conditions. Cows enrolled in this project will be housed in a naturally ventilated barn with bedded packs during the dry period. Three days prior to expected calving, they will be moved to individual maternity pens bedded with straw until parturition. After parturition, cows will be housed in a naturally ventilated barn with freestalls that are rubber mattresses bedded with straw and milked 2 times daily. Throughout the experiment, cows will be fed using a Calan gate feeding system to measure individual daily intakes and regulate the amount of feed offered to each animal. From -21 d to calving, cows will receive a basal close-up diet once a day [1.39 Mcal of net energy lactation (NEL)/kg and 12.3% crude protein]. After calving, all cows will receive the same basal lactation diet once a day (1.60 Mcal of NEL/kg and 15.6% crude protein).To understand the importance of prepartal liver GSH, a comparison between the high and low GSH groups will be performed across performance parameters, including milk yield, milk components, feed intake, energy balance, feed efficiency, and BCS. The ability of liver GSH accumulation prepartum to mitigate postpartal oxidative stress and overall improve animal welfare is a major outcome to be confirmed in this research. Therefore, blood samples will be collected at -21, -10, 1, 7, 14, and 21 +/- 2 days relative to calving to evaluate blood biomarkers of oxidative stress, metabolism, inflammation, and liver function. Among the oxidative stress markers, the reactive oxygen metabolites (ROM) and oxidative radical absorbance capacity (ORAC) will be measured. The combination of these markers will provide an idea of the oxidative status, that is, the balance between oxidants (i.e., ROM) and antioxidants (i.e., ORAC). Additional markers of inflammation (i.e., haptoglobin) and liver function (i.e., albumin) will provide a complete picture of the animal welfare status of dairy cows calving under a high and low GSH condition.Understanding what controls the prepartal accumulation of GSH in the liver is a core goal of this research. This will be evaluated by measuring main hormones responsible for the metabolic adaptations dairy cows undergo from pregnancy to lactation. We will measure insulin, growth hormone, insulin-like hormone, and leptin in the same blood samples described above. These hormones are associated with energy balance and body fat reserves. Thus, these data might help us understand how the energy balance, influenced by the diet, can potentially affect liver GSH storage prepartum.The liver biopsy samples collected above will also be utilized to assess the molecular mechanisms regulating GSH synthesis and storage in liver. Because GSH is a tripeptide composed of glutamate, cysteine, and glycine, relevant metabolic pathways to these amino acids will be targeted, including methionine cycle, transsulfuration pathway, one-carbon metabolism, and the gamma-glutamyl cycle. These targeted metabolic pathways will be evaluated via contemporary methods, including real-time qPCR, western blot, and colorimetric and fluorometric assays. Then, to provide a more comprehensive view of the effect of high and low GSH liver storage, high-throughput analysis via RNA-seq and complementary bioinformatics will be conducted. RNA-seq will be performed as paired-end reads for an estimated yield of 25-20M reads/sample. Trimmed reads will be aligned to the Bos taurus genome using genome-guided alignment software. Final gene expression data will be used to perform a gene enrichment analysis to understand which metabolic and physiological pathways are activated or inhibited when cows present a higher accumulation of GSH in the liver before parturition. In the same way, after calving, this gene enrichment analysis will allow us to dissect what alterations occur at the molecular level when high GSH vs low GSH cows face oxidative stress.The study proposed here will shed light on the potential benefits of increasing liver GSH storage prior to calving on animal health and welfare. In addition, this project is expected to reveal key regulatory mechanisms and nutrients that enhance prepartal liver GSH in dairy cows, which could be translated into nutritional and management recommendations at the farm level to improve the antioxidant capacity of cows in early lactation. For instance, there is evidence that increasing dietary Met supply during prepartum increases the accumulation of GSH in liver, and other factors such as energy density in the diet or vitamins may play a role in liver GSH accumulation.

Progress 06/01/22 to 05/31/24

Outputs
Target Audience: Dairy farmers, nutritionists, dairy science students, animal nutrition companies, dairy scientists, and the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project allowed onegraduate student to carry out novel research, analyze data, and present findings at national and international conferences. In addition, two undergraduate students gained experience in lab analysis, including colorimetric assays, gene expression, and flow cytometry. How have the results been disseminated to communities of interest?During this project, 4 oral and poster presentations at 4 scientific meetings have provided the opportunity for hundreds of animal and dairy scientists, nutritionists, and veterinarians to learn from our findings. Additionally, we have presented two abstracts at local conferences with a more applied nutrition background. A popular press article was published in the Virginia Dairyman magazine where we highlighted the main findings from this research. Other avenues to dessiminate our findings were through several invited presentations at dairy nutrition and management conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In this final year of the project, we focused on completing lab analysis, interpretation, and reporting on our goals for this project. Four abstracts have been presented at national and international conferences and a manuscript from this work is undergoing final edits before journal submission. One of the main goals of this project was to further provide evidence of liver glutathione as a promoter of better postpartal lactation performance. We observed clear improvements in milk production in cows with high prepartal liver glutathione concentrations. This was further verified by a linear increase in energy-corrected (ECM) milk from cows with low to high prepartal liver GSH. Similarly to ECM, milk protein yield increased linearly from cows with low to high prepartal liver GSH. Regarding mechanisms that control liver GSH synthesis and storage, we observed a distinctive association between liver GSH concentration and energy metabolism. Overall, prepartal glucose and insulin were positively correlated with prepartal liver GSH. Similarly, prepartal energy balance and net energy intake were positively correlated with prepartal liver GSH. This evidence led us to believe there is an indispensable energy requirement for liver GSH accumulation beyond the three amino acids required to synthesize GSH. This is supported from a biochemical standpoint, where the two steps for GSH synthesis require ATP. Additionally, those correlations between energy-related parameters and liver GSH were translated in cows with high prepartal liver GSH having higher BCS before and after calving. Interestingly, this effect was not conducive to higher blood NEFA or BHB levels postpartum or increased ROM due to higher fatty acid beta-oxidation in the liver. Additionally, no effects on liver triglycerides were observed. This suggests that liver GSH may protect transition dairy cows against fatty liver or ketosis. At the molecular level, we observed a transcriptional regulation of key genes related to GSH metabolism, including GGT (gamma-glutamyl transpeptidase) and GCLC (gamma-glutamylcysteine synthetase), where GGT is in charge of catabolism of GSH, while GCLC is the rate-limiting step in GSH synthesis. Both of these genes were linearly upregulated across cows with low to high liver GSH prepartum. This suggests that mRNA transcription may respond to liver GSH accumulation, but liver GSH accumulation is not necessarily regulated at the gene level. Taken together, we first observed that prepartal liver GSH does improve lactation performances, and it may be partially regulated by energy status.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Souza, A. F., G. G. Begalli, M. H. Oliveira, R. C. B. Grazziotin, J. Halfen, E. Trevisi, and J. S. Osorio. 2023. The impact of prepartal liver glutathione on milk performance parameters and welfare of peripartal dairy cows. The First International Conference on Antioxidants: Sources, Methods, Health Benefits and Industrial Applications. May 10-12. Barcelona, Spain.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Souza, A. F., G. G. Begalli, M. H. Oliveira, R. C. B. Grazziotin, J. Halfen, E. Trevisi, and J. S. Osorio. 2023. The impact of prepartal liver glutathione on milk performance parameters and welfare of peripartal dairy cows. American Dairy Science Association Annual Meeting. June 25-28. Ottawa, Ontario, Canada.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Souza, A. F., G. G. Begalli, M. H. Oliveira, R. C. B. Grazziotin, J. Halfen, E. Trevisi, and J. S. Osorio. 2024. Prepartal liver glutathione improves liver function, and it may be regulated by prepartal energy status in transition dairy cows. American Dairy Science Association Annual Meeting. June 16-19. Palm Beach, Florida.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Souza, A. F., G. Begalli, M. H., Oliveira, R.C.B., Grazziotin, J. Halfen, E. Trevisi, and J. S. Osorio. 2024. Connecting prepartal liver GSH with postpartal performance parameters, blood biomarkers, and gene expression in transition dairy cows. International Symposium on Ruminant Physiology. August 26-29. Chicago, Illinois.

Progress 06/01/22 to 05/31/23

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One postdoc, two graduate students, and two visiting scholars were trained during this evaluation period. Jessica Halfen, Postdoctorate, entire reporting period. Provided support to this NIFA-funded research project at the farm and in the lab. Helped organize research materials and sampling activities. Helped to mentor and guide the graduate student in charge of this project as well as troubleshooting lab assays. Ana Souza Lima, Ph.D. student, entire reporting period. Responsible for conducting this transition cow research project. During this experiment, she learned basic experimental design, blood and milk sampling, surgical procedures to collect sample specimens, and molecular biology techniques, including real-time qPCR. She has already presented some of the results of this research project at The First International Conference on Antioxidants in Barcelona, Spain. Gustavo Begalli, Ph.D. student, entire reporting period. During this experiment, Gustavo learned basic experimental design, blood and milk sampling, surgical procedures to collect sample specimens, and molecular biology techniques, including flow cytometry. Rodrigo Grazziotin, visiting scholar, 09/28/22 - 03/28/23. Rodrigo assisted and provided support to the main graduate student in charge of this research experiment. He learned the basics of animal husbandry, experimental design, and sampling various biological fluids and tissues. Maria Helena Oliveira, visiting scholar, 09/28/22 - 01/15/23. Mariaassisted and provided support to the main graduate student in charge of this research experiment. He learned the basics of animal husbandry, experimental design, and sampling various biological fluids and tissues. 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?Our upcoming plans are twofold: research and dissemination. On the research front, we aim to complete benchwork analyses that encompass several key areas. These include profiling blood biomarkers related to metabolism, oxidative stress, inflammation, and liver function. We'll also conduct gene expression studies focusing on the GSH metabolism/cycle in prepartal liver samples, utilizing RT-qPCR techniques. Furthermore, we'll perform RNAseq analyses on prepartal liver samples from both HGSH and LGSH cow groups. As for dissemination, we're committed to sharing our findings far and wide. We'll be presenting our research at both national and international conferences, targeting a broad audience that includes dairy farmers, dairy nutritionists, and researchers in dairy-related fields. Beyond presentations, we plan to publish our results in two forms: a popular press article for wider public engagement and a peer-reviewed scientific paper for the academic community.

Impacts
What was accomplished under these goals? During this reporting period, significant progress was made toward the main objective of this project. We completed a transition cow experiment during this objective. Sixty-three multiparous Holstein dairy cows were enrolled at -21 d relative to calving and remained on trial until 30 days in milk (DIM). All cows received the same close-up diet from -21 DIM until calving (1.59 Mcal/kg of DM and 14.6% CP) and a lactation diet from calving until 30 DIM (1.82 Mcal/kg of DM and 18.4% CP). Before calving, cows were housed in a compost-bedded pack barn, and after calving, cows were moved to a free-stall barn. Throughout the trial, cows were fed once a day with an individual Calan gate feeding system. After calving, cows were milked twice a day at 0100 and 1200 h. Blood samples were collected at -21, -10, 2, 7, 14, and 21 d relative to parturition. These blood samples were used to analyze biomarkers of oxidative stress, metabolism, inflammation, and liver function. Liver biopsies were performed at -10, 7, and 21 d relative to calving. The liver biopsy at -10 d relative to calvingwas used to perform a retrospective analysis based on total glutathione (GSH) and assign cows to high GSH(HGSH; n = 14, 2.4 mM), medium-high GSH (MHGSH; n = 15, 1.63 mM), medium-low GSH (MLGSH; n = 16, 1.13 mM), and low GSH (LGSH; n = 15, 0.47 mM). Overall, liver GSH at -10 d relative to parturition ranged from 0.01 to 3.38 mM concentration. This prepartal liver GSH concentration across cows was skewed to the low end of the GSH concentration. This suggests that most cows normally calved with a low liver GSH concentration. The milk production was affected (P = 0.04) by prepartal liver GSH concentration, where there was an evident increase in milk production in HGSH cows compared to MHGSH and LGSH cows. Although milk production in HGSH cows was 2.5 kg/d greater than MLGSH, this effect did not reach statistical significance (P = 0.21). Additionally, we observed a strong effect on milk protein yield, where HGSH cows had a greater (P < 0.04) milk protein yield than the other groups from week 2 to 4 postpartum. There was an interesting effect on body condition score (BCS), where there was a noticeable increase in BCS in HGSH cows before calving. This higherBCS (P = 0.03) was maintained by HGSH after calving when compared to other groups. These effects observed in BCS were accompanied by a positive correlation between total GSH and energy balance prepartum (r = 0.35; P < 0.01) and a trend for a positive correlation between total GSH and net energy intake prepartum (r = 0.23; P 0.07). Among the health outcomes recorded during this trial, a trend (P = 0.07) in mastitis events was observed, where LGSH cows had more mastitis events than other groups.

Publications