Performing Department
(N/A)
Non Technical Summary
Poor colostrum consumption by neonatal gilts has been linked to poor growth and reproductive competency at puberty, which can lead to premature culling. Colostrum is rich in immunoglobulins to support the immune system, but also has bioactive factors like growth factors, that may help initiate pathways needed for nutrient metabolism. Previous studies in our lab, have linked increased colostrum consumption to better reproductive competency at puberty, and in turn, when piglets did not receive colostrum there was an accumulation of very long chain fatty acids in the vagina of puberty age gilts. The peroxisome is a cellular organelle that is responsible for metabolizing very long chain fatty acids, and further studies have identified proteome signatures that demonstrated peroxisomal dysregulation. However, the peroxisome is not the only cellular organelle responsible for fat metabolism in various tissues nor is the vagina a primary site for fat metabolism in the body. Therefore, we hypothesized that poor fat metabolism in neonatal pigs due to inadequate colostrum consumption is not localized to the vagina and is likely systemic, and the mitochondria, another cellular organelle necessary for fat metabolism, may be experiencing dysregulation like the peroxisome.The investigate this hypothesis, 60 female neonatal piglets will be identified at birth and birth body weight (BBW) will be immediately obtained. Piglets only within 1.3 to 1.7 kg will be used and piglets will be allocated to 1 of 6 treatments: colostrum fed at 20% of BBW (COL20; n=10), colostrum fed at 10% of BBW (COL10; n=10), formula fed at 20% of BBW (PF-20; n=10), formula fed at 10% of BBW (PF-10; n=10), piglets that will receive ad libitum colostrum from the sow (SOS; n=10), and piglets that will be immediately euthanized at birth (Zero-hour; n=10). Piglets will not be allowed to suckle from the sow, unless allocated to the SOS group. Animals allocated to either COL or PF will be fed their allotted amount of colostrum or formula every 2 hours over a 24 h period. After receiving their treatments for 24 hours, piglets will be euthanized and the liver tissue will be collected. The liver tissue will be incubated in stable isotopes to track carbon movement from fats, glucose, and other precursors. Another piece of liver tissue will be collected to evaluate all of the genes changed due to colostrum consumption. We will match these datasets to create a global molecular picture of the piglet liver.From these data, we will be able to identify potential bottlenecks, impairments, or road-blocks in metabolism when piglets are not fed adequate amounts of colostrum. We will also be able to identify molecular targets for future studies to develop intervention strategies to alleviate the negative impacts of poor colostrum consumption. We will also be able to give recommendations on the level of colostrum piglets should consume to support growth and reproductive competency at puberty.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overall goal of this project is to determine the effect of high versus low colostrum intake on the activation of metabolic pathways and the flow of carbons from fatty acids and other energy substrates in the liver of neonatal pigs. The following objectives will be accomplished to meet this goal:1. Quantify metabolic flux of long chain fatty acids, glucose, and lactate in the liver of neonatal gilts fed different levels of colostrum or nutrient matched formula.2. Analyze the effect of different levels of colostrum or nutrient matched formula on hepatic transcriptome to determine metabolic pathways activated by colostrum feeding.3. Correlate changes in metabolic flux measures with gene expression counts to identify molecular targets of colostrum and the impact of nutrient matched formula.In addition to the research objectives of this proposal, a menotring plan has been put in place to help Dr. Beckett advance. The mentoring plan is below:1. Grant Identification and Preparation Counseling2.Prepare and Develop Manuscripts for Publication3.Develop Teaching and Instruction Skills4.Participate in Diversity, Equity, and Inclusion (DEI) Initiatives5.Mentor Undergraduate Students
Project Methods
Neonatal gilt piglets will be identified at birth and birth body weight (BBW) will be immediately obtained. Piglets only within 1.3 to 1.7 kg will be used and randomly allocated to 1 of 6 treatments: colostrum fed at 20% of BBW (COL20; n=10), colostrum fed at 10% of BBW (COL10; n=10), formula fed at 20% of BBW (PF-20; n=10), formula fed at 10% of BBW (PF-10; n=10), piglets that will receive ad libitum colostrum fromsow (SOS; n=10), and piglets that will be immediately euthanized at birth (Zero-hour; n=10).After 24 hours of receiving their respective treatments, a blood sample will be collected from the piglets and the immunocrit protocol by Vallet et al., (2013) will be used to evaluate passive transfer of immunity. Immediately after the blood sample, piglets will be euthanized using CO2 inhalation, and the left lateral lobe of the liver will be excised. Liver slices weighing approximately 30 mg will be immediately placed in vials for parallel flux incubations containing 1 mM [U13C] palmitic acid, 1 mM [U13C] lactate, or 1 mM [U13C] glucose for 2 hours to achieve isotopic steady state. After incubation, the samples will be blotted dry and a wet weight will be obtained to later standardize the flux data. The flux samples will be extracted and derivatized for gas chromatography mass spectrometry (GC-MS) analysis using the protocol by Kennedy and Allen (2019). MIDA will be performed, and isotopic enrichment of intermediates will be determined.Another piece of liver tissue (~ 50 mg) will be transferred to a cryovial containing 500 μL of Trizol and immediately frozen in liquid nitrogen to preserve RNA for RNA-sequencing.SAS 9.4 will be used to analyze the effect of colostrum versus formula feeding and the level of colostrum and formula on flux. RNA-sequencing measurements as identified by read count matrices will be used as input into EBseq software (Leng et al. 2013), which will determine differentially expressed genes between treatments. For biological interpretation of data, differentially expressed genes will be used for gene ontology and pathway enrichment analysis using DAVID (Huang et al. 2007; Huang et al. 2009) and GSEA analysis using GenePattern (Reich et al, 2006; Subramanian et al, 2005). Qiagen's Ingenuity Pathway Analysis will be performed as complementary analysis (e.g. identifying upstream regulatory factors).Efforts: These data will be published in multiple different manuscripts to be deseminated to the academic community. Data and publicationswill be used for extension communictions.Undergraduate students will be involved in sample and data acquisition.Evaluation: Confirmation of tissues enriched with label will be one measure of success of this proposal. Having a robust data set between flux and transcriptome data will be a measurement of success of this proposal. Advancement in Dr. Beckett's career will be another measurement of success of this proposal.