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INVESTIGATOR: Proudfoot, K. L.

PERFORMING INSTITUTION:
OHIO STATE UNIVERSITY - VET MED
1900 COFFEY ROAD, 127L VMAB
COLUMBUS, OHIO 43210

LIVING UP TO HER POTENTIAL: INCREASING DAIRY COW PRODUCTIVITY AND WELFARE

NON-TECHNICAL SUMMARY: To remain sustainable, the U.S. dairy industry must adopt practices that ensure the efficiency and welfare of their animals. Dairy cows have a high potential for producing milk, yet many cows are unable to cope with intensive housing and succumb to disease, significantly reducing both productivity and welfare. Sensitive indicators of how animals cope with their environments are critically needed. High lying time is used as an indicator of good welfare, but is also associated with lameness, making it problematic as a direct measurement of welfare. A promising new method for measuring an animal's ability to cope is to determine the amount of sleep they are able to obtain. In humans, substantial epidemiological and experimental research has determined that sleep is critical to health, as even modest declines can increase the risk of illness and mortality. No research has determined if sleep loss reduces health and productivity in cattle. Thus, the objectives of the proposed project are to determine the effect of modest sleep loss on immune function, metabolism and milk production. As this is a novel area of research and a 'proof-of-concept' approach, the project fits with the Exploratory Research Program Priority. Results will provide the foundation for large-scale studies aimed at determining the relationship between sleep and disease in cattle, as well as the effect of common management practices on sleep loss. This line of research can lead to progressive changes that will increase animal efficiency and welfare, ultimately ensuring the sustainability of the dairy industry.

OBJECTIVES: The main goalof this research project is to determine the effect of sleep loss on the immunity of dairy cattle. There is promising evidence from humans and lab animals that even modest sleep deprivation can be detrimental to the immune system, but this will be the first project to our knowledge that will specifically measure the effect of sleep loss in a farm animal species. The overall goal of this line of research is to develop metrics for measuring the welfare of food animals, as well asmanagement and housing practices that improve both animal welfare and productivity. Lying time is often used to determine whether a cow is coping well with their environment, but this measurement is problematic as it also increases with painful conditions such as lameness. Measuring sleep as the quality of rest (rather than merely the quantity), will give us critical insight into the ability of food animals to cope with their environment. Ultimately, this research will be used to determine the effect of common management practices (e.g., milking at night, keeping lights on all night, lying surface type, stocking density) on the ability of lactating dairy cows to recieve sufficient rest as an indicator of how well they are coping with their environment.

APPROACH: To determine the effect of sleep deprivation on immune competence and milk yield in dairy cows, a within-cow experiment will be conducted at the University of Tennessee's East Tennessee Research and Education Center (Walland, TN).Power calculations, animals and housing. Based on prior results from Irwin et al. (2006), we expect an approximate two to three fold increase in pro-inflammatory cytokine gene expression. If we assume the more conservative increase (2 fold) with variation of 1.25, 12 cows per treatment group would be required to detect significance at the 0.05 level (two-tailed test) with >85% power. Cows from mid-lactation (90 to 150 days in milk) will be used to minimize interference from confounding factors such as immune dysfunction and degree of negative energy balance post-calving and maximize the likelihood of detecting significant effects of sleep. Cows will be housed in a free-stall pen with access to one lying stall and headlock per cow and will be milked twice a day at 0730 and 1700.Experimental design and treatments. All cows will encounter two 24 hour baseline phase each followed by a 24 hour treatment phase. Two treatments will be allocated to each cow: 1) sleep deprivation (S) and, 2) sleep and lying deprivation (SL). To induce sleep deprivation without affecting lying time (S), cows will be monitored by an observer every 10 minutes for 24 hours and will be gently stroked to waken them if their eyes close (a common technique used in rodent models of sleep; Vecsey et al., 2009). To restrict both sleep and lying time (SL), free-stalls will be blocked off using chains and a grid using 2 x 4s will be laid out in the alleys to ensure cows cannot lie down. As we will have access to 2 EEGs, we will run two cows through each baseline/treatment phase at a time. To start, all pairs will be exposed to their first baseline/treatment phase, taking 12 days (6 pairs at 48 hours each). This schedule will then be repeated for each pair with their second baseline/treatment phase (the order of treatments will alternated by pair). This design will ensure that the carry-over effects between treatments are minimal, as there will be at least 10 days between treatments for each pair.Measuring sleep and lying time. At 0900 on the day of the baseline phase, cows will be fitted with an EEG specially designed to record sleep in cattle (see Ternman et al., 2012 for a description) to provide a 'gold standard' of sleep duration. Each cow will also be fitted with a cost-effective and non-invasive sleep monitor that is in the process of being validated (see related work of investigators). To record lying time, cows will be fitted with IceTag™ accelerometers (IceRobotics Ltd., Roslin, Scotland, UK) on one of the hind legs. At the end of each baseline and treatment period, data from EEG and IceTag™ will be downloaded and stored on a computer.Measuring immune and metabolic function. To examine the effects of sleep loss on immune function and metabolism, whole blood will be collected and assessed for white blood cell counts, differential counts, inflammatory and metabolic molecules in sera, and ability of leukocytes to produce inflammatory cytokines. To account for changes in the diurnal pattern of immunity from baseline to treatment phases, blood samples will be taken every 6 hours during each phaseand will be assessed for TNF-a, IL-1, non-esterified fatty acids (NEFAs), and glucose using ELISAs. To measure the functional activity of peripheral blood leukocytes, blood will be stimulated with LPS and the generation of TNF-a, IL-1, and IL-6 mRNA via RT-qPCR will be measured. Briefly, whole blood will be incubated with LPS (0 or 5 µg/ml) for 4 hours at 37C (Rontved, et al., 2005). Total RNA will be isolated from cultures using the PaxGene RNA isolation system (Qiagen) and DNase treated to remove contaminating DNA. RNA quantity and quality will be assessed using a NanoDrop spectrophotometer and an Experion analyzer (BioRad), respectively. The cDNA will be synthesized using gene specific primers by RT-PCR as described by Aitken et al. (2009). Expression of targeted genes relative to two housekeeping genes will be analyzed by qRT-PCR using gene specific primers as described with modifications.Measuring milk production. Milk weights will be automatically collected during AM and PM milkings during the baseline phase, treatment phase and the 2 milkings post-treatment using a BouMatic system. Milk samples will be collected from each milking for component analysis. Component analysis will be completed in-house, at the Tennessee's Dairy Herd Improvement Laboratory (Knoxville, TN) without any extra cost.Data analysis. Data collected from the EEG and accelerometers will be used to verify that our treatments affected sleep and/or lying deprivation. To ensure this was the case, we will use a paired t-test to verify differences between baseline periods and both treatment periods. Data from both baseline phases will be averaged to create one value per cow and time period. A repeated measures ANOVA will be used to evaluate the fixed effects of treatment (baseline, S and SL), time, and the interaction of these two variables on leukocyte, cytokine, NEFA, and glucose concentrations in sera, cytokine gene expression following LPS stimulation, milk yield and components.

PROGRESS: 2015/09 TO 2017/08
Target Audience:During our final year the project results were shared with scientists, veterinary students,and producers through teaching efforts,scientific presentations, and Extension efforts. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the last year the training for the project has focused on one graduate student (Jessie Kull). Jessie was trained to 1) download and summarize the lying behavior data from the dataloggers, 2)run ELISAassays for evaluatingindicators of metabolic health (non-esterfied fatty acids and glucose), 3) runRT-qPCRfor evaluatingindicators of immunity (TNF-α, IL-1 and IL-6 mRNA expression and functional activity of blood leukocytes), and 4) use a statistical program (SAS) to summarized and analyze the data using repeated measures and mixed models. In addition, the student was able to gain extensive oral speaking opportunities through the 4 scientific presentations completed during the year. How have the results been disseminated to communities of interest?Yes, the results have been disseminated to 1) a scientific audience through 4 scientific presentations over the last year, 2) veterinary students through Dr. Proudfoot's (PI) Introduction to Animal Welfare course taught in Spring, 2017, and 3) producers through at least 5 Extension efforts made by both Dr. Proudfoot (PI) and Dr. Krawczel (co-PI) throughout the year. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2015/09 TO 2017/08
What was accomplished under these goals? Under this reporting periodwe finished all of the data analysis for the project. This includes the behavioral measurements (lying behavior), milk production, indicators of metabolic health (non-esterfied fatty acids and glucose), and indicators of immunity (TNF-α, IL-1 and IL-6 mRNA expression and functional activity of blood leukocytes). Once these data were complete, they were summarized into abstracts to be presented at 4 scientific meetings in 2017 by one graduate student (Jessie Kull): 1) the North American Regional Meeting for the International Society for Applied Ethology, 2) the Annual Meeting of the American Dairy Science Association, 3) the International Meeting for the International Society for Applied Ethology, and 4) theAnnual Conference of Research Workers in Animal Diseases. The data is currently being summarized into at least 3 manuscripts to be submitted to peer-reviewed scientific journals.

PUBLICATIONS (not previously reported): 2015/09 TO 2017/08
1. Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kull, J. A., G. M. Pighetti, K. L. Proudfoot, J. M. Bewley, B. F. O?Hara, K. D. Donohue, P. D. Krawczel. 2017. Effects of acute lying and sleep deprivation on behaviour and milk production of lactating Holstein dairy cows. J. Dairy Sci. 100(E-Suppl. 2):138.
2. Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kull, J. A., G. M. Pighetti, K. L. Proudfoot, J. M. Bewley, B. F. O?Hara, K. D. Donohue, P. D. Krawczel. 2017. Effects of acute lying and sleep deprivation on behavior and productivity of Holstein dairy cows. Page 134 in Proceedings of the 51st International Congress of the International Society of Applied Ethology. Aarhus, Denmark.
3. Type: Conference Papers and Presentations Status: Under Review Year Published: 2017 Citation: Kull, J. A., G. M. Pighetti, K. L. Proudfoot, J. M. Bewley, B. F. O?Hara, K. D. Donohue, P. D. Krawczel. 2017. Effects of acute lying and sleep deprivation on metabolism and the immune response of Holstein dairy cows. 98th Annual Conference of Research Workers in Animal Diseases 2017. Chicago, USA.
4. Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kull, J. A., G. M. Pighetti, K. L. Proudfoot, J. M. Bewley, B. F. O?Hara, K. D. Donohue P. D. Krawczel. 2017. The effect of 24 hour lying and sleep deprivation on energy balance, behavior, and milk yield in dairy cows. Page 27 in Proc. of The 13th International Society of Applied Ethology North-American Regional Meeting. Ames, Iowa.

PROGRESS: 2015/09/01 TO 2016/08/31
Target Audience: Nothing Reported Changes/Problems:The IACUC process through the University of Tennessee (sub-award) delayed the project start date by 4 months (from Aug 31, 2015to Jan 1, 2016). This change caused a delay in the hiring of an MSc student to run the project at the University of Tennessee;the student was hired on Jan 1, 2016instead of August 31, 2015. In addition, this change delayed the data collection period of the project from October, 2015 to April, 2016. Due to this delay, we will be formally requesting a no-cost extension through the appropriate channels. What opportunities for training and professional development has the project provided?One graduate student was intensively trained to manage a large number of student volunteers, read EEG monitors, attach EEG electrodes to cattle, collect blood samples, follow herd biosecurity measures, collect milk samples, and provide ad libitum access to feed for individually housed cattle. In addition, 17 undergraduate student volunteers, and 3 graduate student volunteers were trained to read EEG monitors and adjust electrodes, follow herd biosecurity measures, collect milk samples, and provide ad libitum access to feed for individually housed cattle. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Over the next reporting period, we plan to finish data analysis including the immune measures from blood samples.We also plan to continue training the graduate student, including the creation oftwo abstracts to be presented attwo international conferences,complete at least one manuscript to submit to a high impact journal, and share the results of the project with other communities of interest, including scientists, veterinarians,dairy producers, and members ofindustry.

IMPACT: 2015/09/01 TO 2016/08/31
What was accomplished under these goals? All data collection for the research project was accomplished during this reporting period. Specifically, 12 mid-lactation cows were enrolled in the study conducted at the University of Tennessee. Each cowexperienced two 24 hour baseline phases followed by two treatment periods: 1) sleep and lying deprivation, and 2) sleep deprivation. Cows were affixed with an EEG to monitor sleep, and a 3D accelerometer to measure lying time. EEG and lying time data has beenanalyzed during the reporting period. In addtion, milk production data from experimental cows has been collected and analyzed.

PUBLICATIONS: 2015/09/01 TO 2016/08/31
No publications reported this period.