1a. Objectives (from AD-416):
Objective 1. Identify proteins and their mechanism of action critical to the growth of the newborn pig by comparison of littermates with similar birth weights but with divergent growth rates. 1.A. Develop immunochemical methods and utilize an integrated proteomic and molecular approach to investigate the functional role of alpha-1 acid glycoprotein which is elevated in slow growing (SG) newborn piglets. 1.B. Identify additional plasma biomarkers for the early identification of SG piglets. Objective 2. Develop model(s) utilizing functional genomics to identify critical control points for economically important tissues of piglets impacted by growth rate. 2.A. Identify physiological differences between metabolically important tissues of SG and fast growing piglets at weaning by transcriptomic technology. 2.B. Perform a comparative analysis of runt (induced intrauterine growth retardation) and SG pigs to identify physiological similarities and differences between economically important tissues by transcriptomic technology. Objective 3. Develop industry applicable markers or screens to identify the slow growing piglet and develop strategies (nutritional/immunological) to promote more consistent and rapid growth. 3.A. Develop a strategy that can be readily adapted to large scale commercial environment to reduce growth variability in the pre- and post weaned piglet. 3.B. Identify and implement a treatment to bolster the growth performance of those pigs identified at postnatal day 1 as potential SG piglets.
1b. Approach (from AD-416):
Variability in growth rate between littermates within a production group leads to inherent inefficiencies in production of lean quality pork. This research will identify physiological mechanisms that contribute to piglet growth rate with the overarching goal of identifying markers predictive of growth rate to treat piglets of normal birth weight that, when left untreated, will exhibit slow growth. The research addresses four issues: 1) identification of plasma markers predictive for growth performance; 2) mechanisms of action for these markers and their relationship to growth rate; 3) identification of key physiological mechanisms modulating preweaning growth in critical metabolic organs; and 4) application of these markers as screens for use by industry and for use in an intervention strategy to improve performance of underachieving piglets. Plasma proteins will be identified that differ in concentration between slow and fast growing littermates and enzyme-linked immunosorbant assays (ELISA) for these proteins will be developed to identify the best predictors of growth rate. Functional analyses utilizing in vitro models will evaluate the mechanisms of action for these growth-related marker proteins in metabolically important tissues. Comparative genomic/proteomic analysis will be performed to elucidate mechanisms modulating preweaning growth in skeletal muscle, small intestine, and liver. Pathway analysis will examine relationships between the growth-related marker proteins and these physiological mechanisms. Lastly, additional swine populations will be surveyed with the ELISAs to complete development of the growth-related marker screen. Intervention strategies will then be developed to improve the growth rate of piglets predicted to underachieve.
3. Progress Report:
For Objective 1, two separate antisera for alpha 1 acid glycoprotein (AGP) were prepared in rabbit and goat for the development of a sensitive immunochemical assay to quantify the protein in sera of newborn piglets. The antisera were characterized by standard protein chemistry; however, due to high glycosylation and unique binding properties of AGP, assay development has not been successfully achieved and alternate strategies for using these antibody preparations are currently being developed. The function of AGP is currently being evaluated with piglet adipose (fat) tissue explants. These experiments have demonstrated that AGP is a potent inhibitor of fat synthesis by neonatal adipose tissue, reducing the expression of genes in the fat synthesis pathway by up to 80%. This suggests that the high levels of serum AGP in the fetal and neonatal pig may contribute to the limited accumulation of adipose tissue energy stores in the neonatal pig which reduces piglet viability. In addition, antisera for two related proteins, fetuin A and B, have been prepared and the specificity of these antisera is initially being evaluated. The goal will be to develop immunological assays to determine whether the fetuins can also be used as a biomarker for early growth impairment. For Objective 2, efforts focused on pinpointing physiological changes occurring in distinct models of piglet growth (normal birth weight with slow preweaning growth or intrauterine growth retarded – IUGR) via gene expression analyses to differentiate similar/dissimilar biological mechanisms associated with compromised growth. Surgical methods were utilized to increase the incidence of IUGR in a litter and generate animals for postnatal studies; animals are in the last trimester of pregnancy. Studies on physiological changes in placenta and fetal organs crucial for postnatal adaptation were performed via analysis of gene expression. The expression of the key rate limiting enzyme regulating estrogen production (aromatase) was decreased in the placenta of the IUGR fetus in late pregnancy, as was the level of estrogen. Also, aromatase expression was localized to a specific cell and region of the placenta. In lung, factors associated with epithelial cell survival were altered early in gestation; while in late gestation, molecules enhancing macrophage migration to the lung, a normal developmental phenomenon in pigs, had increased expression with IUGR. For liver, a master-regulator of gene expression and a factor modulating a key developmental growth factor were found increased with IUGR. For Objective 3, we have developed a reliable method to collect blood during the industry standard tail docking procedure which occurs within 24 hours following birth. Blood from every piglet in 25 healthy litters have been collected to date and the plasma obtained from these samples will be used to determine the concentration of AGP (a biomarker for growth) and for metabolomic studies. Pre-weaning growth performance has been monitored for all of these piglets so that correlation analysis can be performed between animal growth and AGP concentration or with data derived from the metabolomics studies.
1. Lower levels of a putative regulator of organ growth in the placenta coincides with diminished growth of the pig fetus. The selection for increased litter size in swine has resulted in a greater incidence of growth retarded (GR) fetuses with subsequent low birth weight piglets that have a greater susceptibility to disease, high mortality rate and poor meat quality in surviving animals; these characteristics contribute towards the approximate $1.6 billion loss of preweaned piglets and impact meat production for the swine industry. Coincident with retarded fetal growth is a smaller placenta, the organ that provides nutrients and produces hormones required for proper fetal organ development. However, information on functional changes in the placenta associated with a GR fetus is limited. Scientists in the Agricultural Research Service, Beltsville, Maryland examined the expression of genes regulating the production of estrogen, a hormone crucial for fetal organ development, as well as the level of estrogen in the placenta at four significant stages of fetal development. Findings showed for the first time that the protein regulating the production of estrogen (aromatase) was expressed in specific regions of the placenta, but more importantly, the level of aromatase and estrogen were decreased in the placenta of GR fetuses late in gestation when organ growth is the most rapid. The importance of estrogen for the development of key tissues (brain, liver, lung and muscle) and knowledge of factors estrogen controls, may provide a roadmap to further define a biological role for estrogen in the development of the GR phenotype, and more importantly, pinpoint potential ways to intervene and reduce the incidence of low birth weight.
2. Identification of a major blood protein that inhibits fat accumulation in neonatal pigs. Growth in the young pig is characterized by fast rates of muscle protein accumulation and very low rates of fat accretion, despite the consumption of a high fat diet. The identification of biological or genetic markers for growth is critical for the breeding of lean, efficient and fast growing pigs. Scientists in the Agricultural Research Service, Beltsville, Maryland identified a growth related marker (alpha 1 acid glycoprotein, AGP) that is elevated in poorly growing neonatal piglets but that also reduces the expression of genes associated with lipid synthesis by up to 80%. These data indicate that AGP may contribute to limiting the rate of fat accumulation in the neonatal pig. This inhibition of fat growth by AGP could be problematic as can result in development of inadequate energy stores that can result in poor growth performance or poor neonatal survival, thus reducing profitability for the swine producer and industry.Ramsay, T.G., Stoll, M.J., Caperna, T.J., Conde-Aguilera, J.A. 2013. Tumor necrosis a regulation of adipokine gene expression in neonatal adipose tissue. Veterinary Research Communications. 37(1):1-10.