Performing Department
Fisheries Animal & Veterinary Science
Non Technical Summary
Raising young dairy calves for replacement stock or for veal can be challenging for the producer. This is due in part to the susceptibility of calves to disease. For example, 5.8% of calves die due to causes un-related to predation with dairy operations experiencing a high rate of calf mortality due to digestive (30.6%) and respiratory issues (36.7%; USDA, APHIS 2011). This is problematic because every calf that dies represents lost genetic potential, lost income for the producer and reduced meat and milk production per operation. Overall, issues with calf health and mortality reduce operation sustainability and hinder advancement towards greater food security. If we can better understand the causes of calf mortality, then research can be conducted to develop effective intervention strategies and reduce animal loses. Therefore, understanding the effects of maternal programming on calf health and immunity will ultimately lead to reduced calf losses, increased operation sustainability and improved food animal production. This project will evaluate the effects of high maternal milk production and mastitis infection during pregnancy. Specifically blood will be collected from calves over time and blood biomarkers for inflammation and overall health will be evaluated. The metabollic status of the calves will also be evaluated. Tissue samples will also be utilized. The data yeilded from these analyses will help to better understand what factors contribute to calf morbidity and mortality. These data will in turn be used to develop effective intervention strategies to help improve calf health and livestock operation productivity as well as sustainability.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The research goals of the proposed projectare 1) identify the effects of high maternal milk production and maternal mastitis infection during gestation on passive transfer and blood biochemistry during the first 48 hrs post-partum in bull calves and 2) identify the long-term effects of high maternal milk production and maternal mastitis infection on bull calf health and metabolism.Other goals include helping develop the PIs independent research program in the area of calf health and maternal programming.
Project Methods
Objective 1. Using an 18-gauge needle, 10 mL of blood from the jugular vein will be collected prior to transport of calves from the farm, upon arrival at URI, as well as at 36 and 48h of age (Figure 2). Calf body weights and body measurements (crown-rump length, heart girth and height) will also be recorded at 24 h. Blood samples will be placed in heparinized or additive-free tubes and processed as described in Hoffman et al. (2016a). To evaluate the effects of maternal programming on passive transfer in calves, circulating IgG concentrations will be determined from calf serum samples using a commercially available kits (Abcam; Cambridge, MA). Circulating GGT, total protein and 17 other parameters will also be determined via blood biochemistry analyses at the Missouri State Veterinary Medical College Diagnostics Laboratory (Columbia, MO). Calf serum data will be analyzed in SAS using ANOVA with repeated measure. These assays and practices were utilized in the preliminary study and the PI has extensive experience with this form of sample collection/analyses which contributes the feasibility of this objective.Objective 2. Blood (20 mL per week; Figure 2) and measurements will be collected weekly using similar procedures as described in Objective 1. Samples will be sent to Missouri State Veterinary Medical Diagnostics Laboratory for blood biochemistry analyses to evaluate key blood biomarkers over time. An LPS challenge will be performed to evaluate the immune response of the calves at 2 weeks of age using methods described in Benjamin et al. (2016). Two weeks of age was selected as calves are vulnerable to both enteric and respiratory diseases at this time due to the reduction of maternal antibodies and the immaturity of the calves' immune system (Hulbert and Moisa, 2011). Briefly, catheters will be places and LPS (0.25 μg/kg body weight; Benjamin et al., 2016) will be injected. Samples will be collected at -30 min, 0 min and every hour for eight hours. Calf respiration rates and body temperatures will be collected at t 0 and t 8 hrs and 24 hrs post challenge. Serum and plasma samples collected will be used to determine circulating Haptoglobin, IL-6, IL-1B, and TNF-alpha via commercially available kits as described in Benjamin et al. (2016). These factors were chosen as they play a key role in mediating the immune/inflammatory response of the animal after exposure to LPS. Performing these assays in the lab is feasible as we have all the necessary equipment and the PI is highly experienced in performing these assays and training others to do so. An IV Glucose Tolerance Test (IVGTT) will be performed at 7 weeks of age to evaluate changes in glucose homeostasis as this can not only effect calf health but calf growth as well. Calves will be catheterized, and initialblood samples collected at 30 min and 15 min prior to and just before (t 0) the administration of an IV bolus of glucose (150 mg/kg body weight; Bossaert et al., 2009). Then blood will be sampled at 2, 5, 10, 15, 30, 60, and 120 min post glucose administration (Hoffman et al., 2016a). Blood glucose and insulin concentrations will be determined using assays from Alpco (Salem, NH) and Abcam respectively. Glucose tolerance tests have been utilized in several ruminant species to evaluate the effects of maternal programming on offspring metabolic health. Data will be analyzed in SAS with repeated measure. The PI has experience performing these IVGTT in sheep and these skills can be readily adapted to calves.Objective 3. Calves will be slaughtered at RI Beef and Veal at 10 weeks of age. Live weights and empty carcass weights will be recorded. Pancreas and liver tissue will be weighed. Tissue samples will be snap frozen for later RNA isolation and preserved in formalin for later histological analyses. We are focusing on the liver and the pancreas tissue as evaluating these tissues will provide information about the animals' metabolism and health. Specifically, both the liver and pancreas are vulnerable to the effects of maternal programming (Wu et al., 2006; Ford et al., 2009; Hoffman et al., 2017) and play a role in regulating the metabolism of the animal. Additionally, the liver performs other critical functions such as detoxification as well as production of various proteins needed for immune response (Robinson et al., 2016). Therefore, if the development of these organ has been compromised by maternal programming alterations to the animal's metabolism and immunity can occur. Formalin preserved liver and pancreas samples will be sent to Mass Histological Services (Cambridge, MA) and will be embedded in paraffin, sectioned and stained using Harris Hematoxylin and Eosin Y. Unstained slides will be processed in lab via deparaffinization and immunostaining. Specifically, changes in liver and pancreas cellular proliferation will be determined using Ki-67 primary antibody (Abcam). Insulin and glucagon staining will also be conducted for the pancreas tissue using primary antibodies from Dako (Santa Clara, CA) and Sigma-Aldrich (Carlsbad, CA), respectively. Nuclei were visualized using DAPI (Abcam, Cambridge, MA). Corresponding secondary antibodies (AlexaFlour; Thermofisher; Waltham, MA) will be used to visualize the desired targets. Sections will be imaged using a Nikon Upright Microscope System at URI-INBRE Centralized Core Facility. Quantification of cell number, hepatocyte size, islet number, islet size, cellular proliferation, insulin positive area and glucagon positive area will be performed using ImageJ. Data will be analyzed using Proc Mixed in SAS. Theses analyses can readily be performed in the PI's lab as the appropriate equipment is present and there is access to the microscope at our core facility. The lead PI has experience with immunostaining, tissue imaging and histological data analyses as well as training others in these practices.RNA will be isolated from pancreas and liver tissue using a commercially available kits (Qiagen, Germantown, MD). Library preparation will be performed at the Brown University Genomic Core and sent to GeneWiz (Boston, MA) for sequencing on a Hi-Seq 2500. Performing RNA-seq will provide valuable insight to changes in the transcriptome that were caused by maternal programming without being restricted by probes or primers. Paired-end reads will be trimmed and filtered using Sickle (Josh and Fass, 2011) and mapped to the Ovis aries reference annotation (Oar_V.4.1) using STAR aligner (Dobin et al., 2015). Differentially expressed genes will be determined using HTseq counts (Anders et al., 2015) and DESeq in R. A gene will beconsidered differentially expressed when q ≤ 0.05. Gene ontology and pathways analyses will be performed using PANTHER (Mi et al., 2013). Data analyses will be performed on the URI INBRE server that is available to PI. The PI has extensive experience with RNA isolation and samples preparation for sequencing as well as the corresponding analyses that are performed with the raw sequencing reads. The PI also has experience training students to perform RNA isolation and sample preparation.