2008 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 addresses National Program 101 action plan components 1(b) and 2(a & b) to identify the biology/genes governing fetal growth and early postnatal vitality, and to pinpoint biomarkers of development competence and intervention targets. Fetal growth retardation (runting) has been associated with retarded placental development and the impedance of placental blood flow. Surgical methods limiting placental development were tested for their ability to induce runting in swine. Our most efficient method involving aggressive uterine artery ligation (novel in swine) yielded runts in 75% of the animals at day 50 of gestation; growth retarded organs were collected for ensuing genomic screens to identify biomarkers. Studies focused on estrogen modulation of trophoblast (1o placental cells) physiology using inhibitory RNAs (siRNA) for key estrogen regulators (STAR and aromatase) identified specific siRNA that down-regulate aromatase mRNA. In vitro culture techniques were also developed to enhance estrogen detection in siRNA assays that suppress the expression of estrogen regulators. Coordinated responses to hormones alter gene expression and promote distinct ligand/receptor interactions that regulate cellular metabolic functions. To examine the control of insulin and glucagon signaling on liver protein expression, we developed an in vitro, serum-free model using porcine hepatocytes isolated from growing piglets. A proteomic approach (two dimensional electrophoresis, comparative software and mass spectrometry) was used to identify/quantify proteins differentially regulated by each hormone. The platform will enable studies to determine how specific stressors control liver inflammatory processes and energy metabolism. Chromatography techniques were developed to remove high abundance proteins and improve analyses of low expressed proteins; thus enhance biomarker discovery in neonatal pig serum. A new mass spectrometer has allowed the development of techniques to enhance protein identity and the discovery of secondary modifications that may alter protein function/activity. Pig adipose tissue undergoes its most rapid development during preweaning and the tissue is a source of numerous growth factors/cytokines. The establishment of adipose cytokine (adipokine) expression and an increased sensitivity of adipose to stressors during the preweaning period is likely critical for normal development and animal viability. Quantification of cytokine mRNA expression in neonatal pig subcutaneous and kidney adipose tissue postnatal days 1-21 was determined by real-time PCR (RT-PCR). Data generated enabled characterization of overall expression and highlighted cytokines potentially relevant to the growth of pig adipose. With this information, the impact of uterine stress (runting) on adipose tissue cytokine expression was examined. Data from RT-PCR clearly demonstrated that the physiological stress of runting impacts cytokine mRNA expression in adipose tissue. Preliminary in vitro data suggest glucocorticoids (stress hormones) rather than growth hormone or insulin may have a dominant effect on regulation of cytokine expression in neonatal adipose tissue. 4.Accomplishments 1. “Analysis of protein oxidation to monitor stress in newborn pigs.” In an effort to determine sources of stress in baby pigs, methods were developed to evaluate biomarkers associated with protein oxidative stress and damage. At birth, significant amounts of oxidized proteins were identified in piglet serum. We then determined that the standard iron dextran treatment used to prevent iron deficiency anemia in all newborns administered on the first day following birth, is associated with a two-fold increase in oxidized and/or damaged proteins. Enhanced levels of oxidation were observed in major serum proteins including, albumin, transferrin, alpha fetoprotein, immunoglobulins, and fetuin. Postponement of iron treatment until the third day of life resulted in an attenuation of protein oxidation. These results indicate that it may be possible to reduce the impact of this source of stress in newborn pigs by adjusting the timing of treatment with iron. This research contributes to the following National Program 101 research goals:. 1)Component I, Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources - problem (b); Identify functional genes and their interactions, and. 2)Component II, Enhancing Animal Adaptation, Well-Being and Efficiency in Diverse Production Systems - problem (a); Enhance animal well-being and reduce stress in Livestock production systems. 2. “Impact of neonatal runting on cytokine expression in swine adipose tissue.” Neonatal mortality in swine results in billion dollar losses to the swine industry. Neonatal piglet survival may be dependent upon maintenance/changes in normal adipose tissue cytokine production to aid in resistance to infection/disease. Analysis of the cytokine production profiles of adipose tissue in runt piglets demonstrated significant differences from their normal weight littermates indicating that specific adipose derived cytokines may have key roles in the stressed neonatal pig. These data indicate that adipose derived cytokines may serve as biomarkers for stress susceptibility for future use in genetic selection for enhanced survivability. This research aligns with the following National Program 101 research goals:. 1)Component I, Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources- problem (b); Identify functional genes and their interactions, and. 2)Component II, Enhancing Animal Adaptation, Well-Being and Efficiency in Diverse Production Systems- problem (a); Enhance animal well-being and reduce stress in Livestock production systems. 3. “Ontogeny of cytokine expression in developing swine adipose tissue.” Neonatal stress has a severe negative impact on neonatal survival, in part through reducing infection resistance. Cytokines mediate this resistance and adipose tissue has recently been identified as a rich source of cytokines. The first study to examine the ontogeny of cytokines in neonatal adipose tissue was performed between birth and 21 days (d21) of age in preweaning pigs. Analysis of cytokine gene expression within adipose tissue between d1 to d21 of age demonstrated that the cytokine expression profiles change during neonatal development and differ between sites of adipose tissue deposition (subcutaneous or intra-abdominal). Analysis of the cytokine distribution revealed that only a few adipose derived cytokines may serve as key markers for stress resistance/susceptibility for future use in selection of animals for improved health and well being. This research aligns with the following National Program 101 research goals:. 1)Component I, Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources - problem (b); Identify functional genes and their interactions, and. 2)Component II, Enhancing Animal Adaptation, Well-Being and Efficiency in Diverse Production Systems - problem (a); Enhance animal well-being and reduce stress in Livestock production systems. 4. “Gestational abnormalities in metabolic organs induced by uterine stress.” In the swine industry, low birth weight or runting is often a consequence of placental insufficiency and a prominent contributor to early neonatal mortality/morbidity and the culling of piglets. Chronic under nutrition of the fetus leads to the retarded growth of organs/tissues important for the adaptation to and defense against environmental stresses and thus, yields an animal with inferior economic traits. Surgical methods were established to generate a model of placental insufficiency that was reproducible, i.e. an increased incidence of runting, to identify physiological perturbations induced in the liver and lung at the time of placenta establishment (gestational day 50). Results from the aggressive uterine artery ligation method, novel in swine, suggested it to be our best method to date with 75% percent of the animals yielding statistically defined runt(s). Collected liver and lung tissues will be utilized for ensuing genomic studies to identify perturbations in the development and physiology of these organs that may pinpoint intervention targets to improve postnatal survival. This research aligns with the following National Program 101 research goals:. 1)Component I, Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources - problem (b); Identify functional genes and their interactions, and. 2)Component II, Enhancing Animal Adaptation, Well-Being and Efficiency in Diverse Production Systems - problem (b) Reducing Reproductive Losses. 6.Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings 1 Review Publications Blomberg, L., Schreier, L.L., Talbot, N.C. 2008. Expression Analysis of Pluripotency Factors in the Undifferentiated Porcine Inner Cell Mass and Epiblast During In Vitro Culture. Molecular Reproduction and Development. 75(3):450-63. Bee, G., Pursel, V.G., Mitchell, A.D., Maruyama, K., Wells, K.D., Solomon, M.B., Wall, R.J., Coleman, M.E., Schwartz, R.J. 2007. Carcass composition and skeletal muscle morphology of swine expressing an insulin-like growth factor-I transgene. Archives of Animal Breeding. 50(5):501-519. Mitchell, A.D., Scholz, A.M. 2008. Efficiency of energy and protein deposition in swine measured by dual energy X-ray absorptiometry (DXA). Archives of Animal Breeding. 51:159-171. Caperna, T.J., Shannon, A., Garrett, W.M. 2008. A gel-based reference map of the porcine hepatocyte proteome. Domestic Animal Endocrinology. 35(1):142-156. Miles, J.R., Blomberg, L.A., Krisher, R.L., Everts, R.E., Sonstegard, T.S., Van Tassell, C.P., Zuelke, K.A. 2008. Comparative transcriptome analysis of in vivo- and in vitro- produced porcine blastocysts by small amplified RNA-serial analysis of gene expression (SAR-SAGE). Molecular Reproduction and Development. 75:976-88. Blomberg, L., Hashizume, K., Viebahn, C. 2008. Blastocyst elongation, trophoblastic differentiation, and embryonic pattern formation. 2008. Reporduction. 135:181-95. Ramsay, T.G., Mitchell, A.D., Richards, M.P. 2007. Comparison of gene expression in lean contemporary and crossbred obese swine. Adipocytes 2(3/4):133-142. Ramsay, T.G., Mitchell, A.D., Richards, M.P. 2008. Uncoupling protein expression in skeletal muscle and adipose tissue in response to in vivo porcine somatotropin treatment. Domestic Animal Endorcinology. 35(2): 130-141. Elsasser, T.H., Caperna, T.J., Li, C.J., Kahl, S., Sartin, J.L. 2008. Critical control points in the impact of the proinflammatory immune response on growth and metabolism. Journal of Animal Science. 86(E. Suppl):E105-E 125. Ramsay, T.G., Mitchell, A.D. 2008. Impact of dietary protein content on uncoupling protein mRNA abundance in swine. Comparative Biochemistry and Physiology. 149:562-571.