Performing Department
Veterinary and Biomedical Scie
Non Technical Summary
The period around calving is a challenging time for dairy cows as cows experience challenging, though normal, physiological events such as inflammation and stress. Overweight cows, first calving cows, and cows that experience a difficult calving may experience a higher inflammatory response during this period. This exacerbated inflammatory response has been associated with a higher risk of cows developing diseases, underperforming, and ultimately, being removed from the herd. Because of this, these groups of animals should be considered high-priority groups for proactive disease prevention therapies. Considering that 40% of cows may be overweight around calving, 33% of the US dairy cows may be first lactation cows, and >15% of dairy cattle may experience calving issues in US dairy operations, this is not only an important animal welfare concern that requires immediate attention, but also represents great economic losses for dairy farmers. This impairs farm profitability, ultimately threatening the nation's food sustainability. Blanket treatments (which means treating all animals regardless of animal's risk factors) with aspirin after calving has been proposed as a proactive management strategy to reduce inlfammation and alleviate discomfort after calving in dairy cattle.However, there is a lack of research investigating the effects of targeted (e.g., treating only high priority cow groups) aspirin treatment during the pre-partum period when the inflammatory process actually begins. Furthermore, the effects of aspirin on the rumen microbe populations, which could be one of the main reasons of the positive effects observed in cows treated with this product, have not been assessed yet. The specific objectives of this study are to assess the effects of pre-partum oral aspirin treatment 14 days from expected calving date on: 1) milk yield and milk quality, clinical disease events, and cow fertility, 2) systemic inflammation and metabolic stress, and 3) rumen microbial diversity and metagenomics, in high-priority cow groups during the peri-parturient period. We hypothesize that this pre-partum therapeutic treatment may modulate early pro-inflammatory responses, and positively alter the rumen microbial composition, decreasing the likelihood of diseases and improving performance in post-partum cows. The proposed therapy will be applicable/easy-to-implement (adjust to farm logistics), and cost-effective (less labor involved, less animals treated). If this treatment is effective, it would greatly improve animal welfare by enhancing health of high-priority cow groups and decreasing unnecessary animal treatments (handling stress). Furthermore, the proposed therapy would optimize farm profitability by decreasing cow disease losses (e.g., treatment costs, loss in cow performance) and the costs of implementing disease prevention treatments (selective treatment vs blanket treatment) in dairy operations.
Animal Health Component
80%
Research Effort Categories
Basic
(N/A)
Applied
80%
Developmental
20%
Goals / Objectives
The major goal of the study isto improve animal welfare and farm profitability in dairy operations through developing an applicable and cost-effective disease prevention therapy. Blanket disease prevention therapies have been proposed to help cows cope with common physiological challenges during the post-partum period, such as high systemic inflammation, in order for them to maintain a strong health and perform properly. However, recent research has shown that in some groups of cows, such as over-conditioned cows (OVCOND), primiparous cows (PRIM) and cows that experience a difficult calving, known as dystocia (DYS), these physiological challenges may be exacerbated, suggesting that these high-priority groups of animals may benefit from disease prevention therapies the most, while other animals may not need it at all. Considering that around 40% of cows may be over-conditioned at dry off, and >15% of dairy cattle may experience dystocia in US dairy operations, developing selective disease prevention treatment strategies for these groups of animals is key to improve health in dairy farms.Anti-inflammatory treatment after calving has been proposed, and studied, as a proactive management strategy to reduce inflammation and alleviate discomfort after calving in dairy cattle. One of the most common family of drugs used for this purpose are non-steroidal anti-inflammatory drugs (NSAIDs). Several treatment approaches (e.g., number of doses, type of administration) using a variety of drugs from this family (e.g., carprofen, meloxicam) have been studied; however, results have been inconsistent. Notably, one of the promising NSAID drugs is acetylsalicylic acid (ASA) that has been shown to have consistently improved cow performance after calving (e.g., increased milk yield, improved fertility) regardless of the treatment approach used. As mentioned above, cows with higher inflammation at calving are at a higher risk of developing diseases during the first weeks after calving. Therefore, modulating the inflammatory response around calving could have the potential of decreasing diseases during the early lactation period. However, studies that have assessed variables related to cow health have shown inconsistent results on modulating inflammation or decreasing disease risk, suggesting that perhaps the treatment approach utilized (e.g., timing of treatment) may not be optimal. Although proposed ASA treatment approach features varied in the number of treatment administrations, drug use and dose, and route of administration, among others; administering treatment prior to calving has not yet been studied. To our knowledge there are no ASA studies focused on developing targeted pre-partum treatment approaches for the high-priority cow groups mentioned above.Efforts to elucidate the mechanisms involved on the observed benefits of ASA treatment have been focused on identifying specific effects of ASA treatment on cow metabolic stress and systemic inflammation variables, such as haptoglobin (HP) and β-hydroxybutyrate (BHB) concentrations, without being able to accurately explain the relationship between ASA treatment and cow health and performance. There is existing evidence that ewes fed willow trees, containing salicin, which is metabolized into ASA during absorption, not only showed similar animal performance benefits observed in dairy cattle treated with ASA, but also altered rumen aspects, such as rumen microbial profiles. Since most of the studies that showed positive results of ASA treatment administered this product orally, it remains to be determined if the possible mechanism of action of ASA is via modulating the rumen microbiome, metabolites and fermentation. To our knowledge till date, there is a lack of research reports investigating the direct effect of ASA on ruminal function.The proposed study will address these critical knowledge gaps and anticipate unravelling novel information on the mechanism of action of ASA in reducing inflammation during the pre-partum period by investigating the link between cow physiological parameters, inflammatory responses and the rumen microbiome and metabolites. By using this integrated approach in highly disease predisposed groups, we anticipate identifying factors that predispose to stress and cause inflammation that will eventually lead to developing targeted therapeutic applications. We hypothesize that early therapeutic treatment with ASA 14 d prior to calving date may modulate early pro-inflammatory responses, preventing the onset of an exacerbated inflammatory response around calving, and alter the rumen microbial composition and their metabolites, which may improve adaptation to dietary management changes during this period; thus, decreasing the likelihood of cows developing diseases and improving cow performance. This project brings together an interdisciplinary team of experts from Penn State University and University of Pennsylvania to fill these critical knowledge gaps and help in better serving the dairy industry. Drs. Barragan and Hovingh have extensive experience on the assessment of metabolic stress and systemic inflammation and Drs. Pitta and Bender, have been working on USDA-funded grants to investigate the role rumen microbiome and ruminal function in improving health, well-being, and productivity of dairy cattle.The long-term goal of this study is to uncover the underlying physiological mechanisms involved with improved performance observed in cows treated with ASA after calving and develop an applicable and targetable ASA treatment regimen aimed at preventing diseases and improving performance in high-priority cow groups. The specific objectives are to assess the effects of pre-partum oral ASA treatment 14±3 days from predicted calving date on:1) milk yield, milk components and milk quality (somatic cell counts) during the first 60 days in milk, number of clinical disease events during the first 60 days in milk, and cow fertility (i.e., days in milk [DIM] to conception, numbers of service to conceive, abortion rate) in high-priority cow groups (i.e., OVCOND, PRIM and DYS) during the peri-parturient period; 2) systemic inflammation (HP, cytokines [IL-6, IL-1β, TNF-α]) and metabolic stress (NEFA, BHB) in high-priority cow groups (i.e., OVCOND, PRIM and DYS) during the peri-parturient period; and 3) rumen microbial diversity and metagenomics in high-priority cow groups (i.e., OVCOND, PRIM and DYS) during the peri-parturient period.
Project Methods
Objective 1:To be able to enroll the required number of cows within the proposed time frame and with the financial resources available, this randomized block experimental study will be performed in a large dairy farm (2,000 milking cows) located in Central Pennsylvania. Mr. John Proskine is the herd manager of this farm and has a long-standing collaboration relationship with the study team. He will collaborate with the team to arrange the logistic of performing the research component in this farm. This operation has the facilities and technology needed for successfully running the field component of the study and data collection, such as ability to record daily milk yield from calibrated meters, daily rumination, electronic records, and headlocks in pre- and post-partum cow pens. Assuming a power of 80% and at least a difference of 5% in abortion rate between treatment groups, with adequate statistical significance (alpha=0.05), and considering an estimated experimental unit (cow) attrition rate of 20%, a sample size of 300 cows per group (total of 600 animals; Image 2) will be required (Sample Size Calculator GPower 3.1).Pre-partum cows will be sampled (i.e., blood sample) right after moved into the close-up pen, which is performed 24±3 d from the cow predicted calving date (pcd) in this farm. One week after animals are moved to this pen, cows will be blocked by BCS category (low≤3 pts.; optimal=3.25-3.5; high≥3.75) and parity (PRIM, multiparous [MULT]), and randomly allocated to one of two treatment groups: 1) ASA (n = 300), or 2) PLC (n = 300). Cows in the ASA group will receive one oral treatment with 4 ASA boluses (480 grain/bolus; equivalent to 125 g/d; drug dose used in Barragan et al., 2020c and 2021 that showed improved milk yield and reproductive performance in treated cows) one week after cows are moved into the close-up pen (14±3 days from pcd); while cows in the PLC group will receive one oral treatment with gelatin capsules (4 capsules; Torpac Inc., Fairfield, NJ) filled with water one week after cows are moved into the close-up pen (14±3 days from pcd). The treatment approach mentioned above was developed considering farm logistics and treatment applicability, while matching cow physiological processes (onset of inflammatory response) as accurately as possible without losing treatment practicality. At the time of animal enrollment and treatment allocation, it will be uncertain if cows will experience DYS at calving. However, by blocking treatment allocation based on BCS and parity groups, researchers expect that DYS cows will be evenly distributed across blocking factors and treatment levels. Accounting for a 15% incidence of DYS, researchers are expecting to have 90 cows that will experience DYS at calving, which will be a sufficient sample size to test differences on performance (e.g., milk yield) and metabolic (e.g., HP concentration) parameters in this group of animals. Clinical disease events, milk yield and milk components, SCC for the first 60 DIM will be collected from the on-farm computer records (Dairy Comp 305, Valley Ag Software, Tulare, CA). Furthermore, on-farm records will be used to collect reproductive performance data (DIM to conception, numbers of service to conceive, abortion rate) of study animals up to 300 DIM. In addition, investigators will assess weekly body condition score (BSC), using a 5-point scale, of study animals from enrollment until 21 days after calving. An offload of computer farm records will be collected weekly, and information needed (e.g., clinical disease events) will be compiled in a Microsoft Excel® (2017) file for further analysis.Objective 2.A random subsample (n=200) of the original enrolled cows evenly distributed based on BCS category, parity and treatment will be randomly selected. Assuming a power of 80% and at least a difference of 19.57 µg/mL in HP concentration between treatment groups, with adequate statistical significance (alpha=0.05), and considering an estimated experimental unit (cow) attrition rate of 20%, a sample size of 100 cows per group (total of 200 animals) will be required (Sample Size Calculator GPower 3.1).Blood samples will be collected through coccygeal bleeding one week before treatment, the day of treatment, and once a week until 21 days after calving for assessment of circulating biomarkers of metabolic stress (i.e., NEFA, BHB) and systemic inflammation (i.e., HP, IL-6, IL-1β, TNF-α). All samples will be centrifuged within 2 h of collection and stored in a freezer at -20 ? C for further analysis. Determination of BHB will be performed using a NovaVet® electronic handheld device (Nova Biomedical Corporation, Waltham, MA) in the laboratory of the study team (Central Milk Testing Laboratory, University Park, 16802). The assessment of circulating concentration of HP, NEFA, and cytokines will be performed in the laboratory of the study PI using commercially available ELISA kits (Life Diagnostics, West Chester, PA) following manufacturer's instructions.Objective 3. A subsample (n=80) of the original enrolled cows evenly distributed based on BCS, parity and treatment will be randomly selected and sampled. Drs. Pitta and Bender sampled 5 PRIM and 5 MULT cows over transition period for 20 times obtaining 100 timepoints for each group of animals, which enabled them to identify differences between treatments. In the proposed study, the sample size would increase to 40 primiparous and 40 multiparous cows that will be sampled 5 times, which will provide enough samples to detect differences. Ruminal samples will be collected 24 h prior to treatment, within 28 h post-treatment followed by weekly sampling during the transition period. All rumen samples will be collected between 2-4 h post-feeding. Samples for rumen contents will be collected using oro-gastric tube as described previously.Microbial diversity: Rumen samples will be extracted for DNA using the "Repeated Bead Beating and Column" (RBB+C) purification method followed by extraction with a commercial kit. The extracted DNA will be amplified using polymerase chain reaction (PCR) for the 16S rRNA gene for bacteria and archaea using our established methods. For fungal diversity, procedures will be similar to the method described in Kumar et al. (2015) and that of protozoa as per the method of Tapio et al. (2016). The PCR products will be bead purified using Beckman Coulter Agencourt AMPure XP Beads (Beckman-Coulter, CA) as per our established methods. The 16S rRNA libraries will be prepared at ASMG Laboratory, University of Pennsylvania using our established pipelines, and sequenced using the Illumina MiSeq platform available at the PennCHOP Microbiome facility.Metagenomics: 1 μg of extracted DNA from each rumen sample will be prepared for whole genome shotgun sequencing using the Nextera DNA Library Prep Kit. The unamplified library (tight insert size of 250 bp for high-throughput sequencing from both ends by 2 × 150 bp) will be sequenced on an Illumina NextSeq500 instrument available at the Center for Host-Microbe Interactions, School of Veterinary Medicine, University of Pennsylvania.Bioinformatic and Statistical Analysis: Dr. Pitta's Agricultural Systems and Microbial Genomics (ASMG) laboratory is equipped with the required bioinformatic tools to perform analysis of large sequencing data sets retrieved from next-generation sequencing (NGS) platforms. We will process all samples for bacterial and archaeal diversity using the QIIME pipeline as described in our previous papers. Metagenomic sequencing data will be processed for quality control, sequence assembly, and annotations to assign taxonomic profiles (with the LCA algorithm) as well as to associate taxonomy with functions using the KEGG database as per our established methods (Pitta et al., 2016 a, b).