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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
1. Establish benchmark transcriptome profiles induced by uterine stress during gestation that are predictive of altered physiological mechanisms in key organs critical to the piglet immune and metabolic stress responses. 2. Identify critical gene(s), gene products, and their mechanism of action in the stress response of piglets associated with morbidity, growth rate, and body composition. 2.A. Identify key secretory proteins that are regulated by stress in the preweaning pig. 2.B. Identify key secretory proteins produced by adipose tissue that are regulated by stress in the preweaning pig. 3. Develop comprehensive in vitro models to analyze the mechanistic role of select, important developmental or metabolic factors in mediating the organism’s response to stresses via functional genomic approaches. 3.A. Determine the physiological mechanisms of key stress-regulatory proteins that regulate nutrient partitioning in the liver and adipose tissue. 3.B. Characterize the role of trophectoderm-derived estrogen as a modulator of “programmed” set points that determines the development potential of the peri-implantation embryo. 4. Define the repertoire of biomarkers that may be predictive of neonatal growth potential by using models of induced metabolic stress.

1b.Approach (from AD-416)
High preweaning mortality or impaired stress-related growth of live-born piglets continue to be major problems that negatively impact commercial swine production. Piglets exhibiting decreased vitality are at greater risk of morbidity or death and a decreased growth rate extending the farrow-to-market time, which results in increased producer costs. Considering embryo development as a continuum, it is plausible that abnormal piglet development and loss through adulthood is a consequence of aberrant embryonic/uterine development. This research will identify physiological mechanisms modulating piglet stress responses to pinpoint targets for interventions to improve “at risk” piglet survival. The research addresses three elemental issues.
1)elucidation of the relationship between developmental perturbations and etiology of abnormal postnatal stress responses,.
2)paucity of predictive screening tools for “at risk” neonates, and.
3)lack of interventions to ameliorate postnatal development of “at risk” piglets. The impact of the uterine milieu on alterations of key physiological mechanisms that modulate stress response in metabolic or immune organs will be evaluated by comparative transcriptomic analysis between induced intrauterine growth retardation (IUGR), i.e. runting, and control concepti. To identify postnatal stress responses that are disrupted and persist ex utero as a consequence of in utero growth retardation or parturition complications and detect compensatory mechanisms, the gene expression of key metabolic and immune tissues from growth retarded piglets, (induced IUGR or spontaneous IUGR) and piglets exhibiting decreased vitality will be assessed by in-depth proteomic or transcriptomic analyses throughout the preweaning period. Functional analyses utilizing in vitro model systems and technologies, such as RNAi, will evaluate the mechanistic role of specific stress-related factors/pathways that are identified in metabolically important tissues. The relevance of putative stress-related factors/pathways will be assessed in vivo, employing distinct models of induced metabolic stress. The knowledge acquired will enable.
1)the discovery of new biomarkers indicative of metabolic or immune stress response,.
2)the identification physiological mechanisms/factors that can be targeted to develop new improved interventions that decrease mortality and days to market of “at risk” piglets and.
3)the establishment of public “systems biology” database for specific gestational and environmental stresses.

3.Progress Report
The research aims to identify in growth-retarded piglets, alterations of physiological mechanisms/factors in vital metabolic tissue that modulate environmental stress responses during gestation and preweaning to, ascertain biomarkers for early screening and, intervention targets to ameliorate adverse responses. Crucial to the study, particularly during gestation, was the establishment of an inducible animal model of runting that is reproducible. Retarded fetal growth (runting) in the pig is often a result of uterine crowding and impeded placental development. The yield of runts in 75% of gilts at gestational day 50 with the initial surgical method was improved by a more robust method of surgically-induced uterine crowding that has yielded runts in 100% of the gilts. Fetuses were generated for gestational day 37 and 50; liver, lung, placenta, and muscle tissues were collected. Global and candidate gene expression analyses between control and runt were initiated in liver and in placenta, respectively, and differentially expressed mRNAs were found that warrant further investigation. In vitro experiments to evaluate the regulation of cytokine expression in neonatal swine adipose tissue revealed significant, rapid increases in the production of two cytokines in the absence of hormonal or metabolic stimulation when the tissue was incubated in standard medium. Experiments to determine the cause of the elevated cytokine expression under basal media conditions identified bovine serum albumin as the source. Bovine serum albumin is routinely used in media for adipose tissue in vitro studies because it prevents the tissue’s adhesion to culture vessels and absorbs fatty acids released by adipose tissue that would compromise the tissue’s metabolic and paracrine responses. The source and method of albumin preparation were found to influence responses by these cytokines in adipose tissue. An albumin source (low in endotoxin (< 0.1 ng/mg) and organic solvent purified) that elicited a minimal cytokine response was identified and will be used in the future to examine the metabolic effect of cytokines on adipose tissue and the regulation of adipokine expression. New techniques were used to further characterize protein oxidation in serum from fetal and neonatal pigs. A strategy was developed to label the oxidized proteins with biotin which were then separated by two dimensional gel electrophoresis and the modified proteins were visualized with a specific fluorescent stain. The same gels were next stained with a colorimetric total protein stain and both oxidized and unmodified proteins from the same gel could then be isolated and identified by mass spectrometry. Samples from fetal and neonatal pigs were quantified, analyzed by these methods. Identifications of specific proteins by mass spectrometry are currently being conducted. Additionally, a preferable non-invasive method of body composition analyses, Quantitative Magnetic Resonance (QMR), has been validated for is applicability to evaluate piglet growth in longitudinal studies. Data is currently being acquired to establish body composition curves in normal and growth retarded piglets.

1. Metabolic stress induces cytokine expression by adipose tissue in vitro. Cytokine release is known to be altered by stress in a variety of tissues, whether psychological, physiological or metabolic; however the role of stress in the expression of adipose derived cytokines is unknown. The present study was designed to examine cytokine expression by neonatal porcine adipose tissue in response to metabolic stress. Metabolic stress was induced by methyl-ß-cyclodextrin (MCD), which interferes with cell signaling mechanisms by reducing intermembrane fluidity. The expression of tumor necrosis factor A was elevated by more than twenty fold within one hour of exposure to MCD relative to control medium while interleukin 6 mRNA abundance was increased by more than a hundred fold within 8 hours of exposure to MCD relative to the response to the control medium. These data clearly demonstrate that metabolic stress can induce the expression of cytokines in adipose tissue of neonatal swine. Since stress in the neonatal pig contributes to high mortality rates, the present data suggest that cytokine production by the adipose tissue may have some role in neonatal survival.

2. Establishment of technology to improve growth characterization during preweaning. Important for accurate changes in body composition over time is the availability of technology that enables measurements without the administration of sedatives or anesthesia, as these can affect growth. A comparative study between dual-energy X-ray absorptiometry (DEXA), an established technology requiring animal sedation, and QMR, not requiring sedation, demonstrated that both technologies exhibited similar precision and accuracy. The establishment of QMR for body composition analysis during the preweaning period in the piglet provides a very useful tool to measure changes in growth characteristics with minimal influence due to the omission of antagonistic sedatives/anesthesia.

3. Serum protein biomarkers in normal and runt piglets. To investigate biomarkers associated with poor growth performance in piglets, stress related proteins and oxidative protein modifications were investigated in serum proteins at 1, 7, and 21 days of age. Newborn runt piglets had significantly less total protein per unit volume in serum compared to their one day old normal littermates. An examination of stress related proteins demonstrated that the acute phase or heat shock protein, alpha-1-acid glycoprotein was markedly elevated in the serum of runts at birth whereas the acute phase protein, haptoglobin, was similar in runts and control littermates. By 7 and 21 days, the level of neither acute phase protein was different between control and runt piglets. Quantitative analyses of protein carbonyls, which are good indicators of oxidative stress, were similar in all runt and normal pigs at 1, 7, and 21 days of age. These data indicate that specific protein biomarkers associated with impaired growth (total protein and alpha-1-acid glycoprotein) are present at birth but may be an unreliable predictive tool if evaluated at 1 week of age or beyond. In addition, it does not appear that runts are more sensitive to oxidative stress than their normal littermates.

4. Perturbation of placental gene expression with growth retardation. Concomitant with the establishment of the placenta (placentation), beginning at about gestational day 18 and complete by gestational day 50, is the initiation of the development of metabolically important organs, liver, lung and muscle. At gestational day 37, the sites for endometrial gland secretion uptake in the placenta (areolae) are beginning to form and, by gestational day 50, the areolae are fully formed within the mid-placental region where the fetus is oriented; however, areolae are less developed in the elongating, less mature distal ends of the placenta. The expression of fifteen factors regulating placental growth, placental tissue differentiation, placental transport, or response to hypoxia (a state associated with poor placentation) was examined post-placentation (day 50) in the more and less mature placental tissue of runt and control fetuses. The mRNA for endothelial nitric oxide synthase (eNOS), an enzyme involved in the synthesis of an important nutrient transporter, nitric oxide, was significantly increased in whole placental tissue and the areolae of runts. Compared to gestational day 37, eNOS mRNA was significantly decreased by gestational day 50 in controls, however, in runts, the higher level of eNOS was maintained. Considering insufficient nutrition is an important contributor to the retarded growth of runts and their organs, this finding highlights a potential nutrient transporter in the placenta whose functional role should be studied further.

5. Impact of media composition on in vitro cytokine expression by adipose tissue explants. Recent studies have demonstrated that adipose tissue is a significant source of cytokines which can subsequently act to alter adipose tissue growth and metabolism. This may be of importance in the survival of the neonatal pig when adipose tissue is limited. The present study was performed to determine why in vitro experiments to assess these adipose tissue derived cytokines has been so difficult to interpret. The data from the present study indicate that a protein (albumin) commonly used in the medium to lubricate the tissue, and to absorb fatty acids released by the tissue, can acutely and dramatically alter the expression of a number of adipose derived cytokines. These data indicate that previous in vitro studies to examine regulation of cytokine production by porcine adipose tissue must be reexamined to enable an appropriate understanding of the production and secretion of cytokines by adipose tissue in swine.

6. Measurement of body composition of pigs during the neonatal growth period. Studies were conducted to assess the body composition of neonatal pigs during growth from birth to four weeks of age – a critical time period for survival of the neonate. Using Quantitative Magnetic Resonance (QMR) technology sequential measurements of body composition, that is total body fat, lean, and water content, were made on live piglets. Both fat and lean content increases linearly over time in piglets from birth to 4 kg and their content were more closely related to body weight than to age of the piglet. Differences in fat and lean content were not observed between normal birth weight and low-birth weight (non-runt) piglets. The information obtained from this study will help to establish a database that can be used to compare the growth composition of normal birth-weight pigs to low birth-weight pigs.

Review Publications
Mitchell, A. 2009. Effect of ractopamine on growth and body composition of pigs during compensatory growth. Animal. 3(1):173-180.

Degrelle, S.A., Blomberg, L., Garrett, W.M., Li, R.W., Talbot, N.C. 2009. Comparative Proteomic and Regulatory Network Analyses of the Elongating Pig Conceptus. Proteomics. 9(10):2678-94.

Mitchell, A.D., Scholz, A.M. 2009. Relationships among dual-energy X-ray absorptiometry (DXA), bioelectrical impedance (BIA), and ultrasound measurements of body composition of swine. Archives of Animal Breeding. 52(1):28-39.

Ramsay, T.G., Caperna, T.J. 2009. Ontogeny of adipokine expression in neonatal pig adipose tissue. Comparative Biochemistry and Physiology. 152:72-78.

Natarajan, S.S., Xu, C., Cregan, P.B., Caperna, T.J., Garrett, W.M., Luthria, D.L. 2008. Utility of proteomics techniques for assessing protein expression. Journal of Regulatory Toxicology and Pharmacology. 54:S32-S36.

Last Modified: 7/24/2014
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