Progress 05/01/17 to 04/30/21
Outputs
Target Audience:The target audience for this project is anyone concerned with or working toward increasing food production to feed the growing world population. Specific target audiences include other scientists in academia and industry working with poultry and the poultry companies. Changes/Problems:All research was halted due the COVID-19 pandemic. All laboratoires were closed from March to June of 2020. Social distancing requirements from June, 2020, to January, 2021, restricted our ability to complete some of the research. What opportunities for training and professional development has the project provided?Two postdoctoral researchers, two laboratory technicians, three graduate students, two international visiting graduate students, and five undergraduate students participated in this project. They gained training and experience in experimental design, molecular and cellular techniques such as total RNA isolation, reverse transcription, quantitative PCR, cell culture, cell transfection, and luciferase reporter assays, and data analysis. How have the results been disseminated to communities of interest?Results from this project have been presented at three national poultry science meetings. Abstracts for these presentations were published. Results were also submitted for publication. Additional manuscripts based on our results will be submitted for publication in the coming year. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Specific Aim 1: Characterize effects of premature somatotroph induction resulting from in ovo CORT injection on the growth performance of broiler chickens. The experiment required to evaluate growth performance and physiological effects of early GH induction for Specific Aims 1 and 2 was completed in year one. Standard growth performance parameters [body weight (BW), body weight gain (BWG), average daily gain (ADG), and feed conversion ratio (FCR)] were determined for each diet phase, as well as across the whole trial. At termination, a subset of birds from each pen were processed to determine carcass yield, meat yield, and abdominal fat. In ovo CORT injection on embryonic day 11 did not influence final BW, BWG, or ADG gain in either males or females. However, birds hatched from CORT-injected eggs tended to have improved feed efficiency as indicated by a reduced cumulative FCR between day of hatch and post-hatch day 42 (Non-injected, FCR=1.65; Saline-injected, FCR=1.64; CORT-injected, FCR=1.61; P=0.1070). On post-hatch day 43, birds were processed and carcass yields were determined. Injection with CORT significantly increased collective meat yield (Pectoralis major and minor, wing, thigh, and drumstick) by 1.2% (P=0.0485) and numerically reduced abdominal fat pad by 0.2% (P=0.1083) in males. These results suggest that premature induction of pituitary GH expression could have lasting effects on efficiency of feed utilization and nutrient partitioning, particularly in male broilers. Specific Aim 2: Determine the effect of in ovo CORT injection on metabolic indicators, hormone levels, and gene expression in broiler chickens. During the longitudinal trial where post-hatch growth performance was assessed, samples were collected to evaluate effects of in ovo CORT injection on metabolic indicators, hormone levels, and gene expression in neuroendocrine and somatic tissues. Plasma, hypothalami, and anterior pituitary glands were shipped to UMD for analysis of metabolites and hormones in circulation, as well as mRNA expression within somatotropic, corticotropic, and thyrotropic neuroendocrine axes. Reverse transcription-quantitative PCR analysis of somatotropic, corticotropic, and thyrotropic mRNA expression in liver and breast muscle was completed at UGA during years 2 through 4, and additional plasma was analyzed at UGA to determine levels of circulating insulin-like growth factor 1 (IGF1), CORT, thyroxine (T4), and triiodothyronine (T3) during years 2 and 3. Pituitary GH mRNA levels were increased by CORT administration on e11, however no effects of embryonic CORT treatment on pituitary mRNA levels for GH, GHRH-R, or SSTR2 or in hypothalamic mRNA levels for GHRH or SRIF during posthatch growth were found. No significant effects of in ovo CORT injection on post-hatch mRNA expression in liver or muscle were detected (P>0.05). However, developmental effects were observed for most genes in all tissues. In liver, only expression of insulin-like growth factor 2 (IGF2), IGF-binding protein (IGFBP) 3, and IGFBP7, did not change between hatch and D42 (P>0.05). The remainder of the genes examined either increased [GH receptor (GHR), thyroid hormone receptors (THR) A and B, glucocorticoid receptor (NR3C1), IGF receptor 1 (IGFR1), IGF1, IGFBP4, and IGF-acid labile subunit (IGFALS)] or decreased steadily (IGFBP1 and IGFBP5) or transiently (IGFBP2) between these ages (P≤0.05). IGFBP1 and IGFALS were not detected in breast muscle, and levels of mRNA for all other genes were affected by age. In contrast to liver, the majority of genes decreased steadily or transiently between hatch and D42 (P≤0.05) in this tissue. Levels of GH-R, THRA, THRB, NR3C1, IGF1R, IGFBP2, IGFBP5, and IGFBP7 consistently decreased in expression throughout development, while IGF2 and IGFBP4 mRNA transiently decreased. Only IGF1 and IGFBP3 increased steadily or transiently in breast muscle after hatch, respectively (P≤0.05). Interestingly, the transient up or downregulation of select genes observed in both tissues occurred on D14, which suggests that dynamic metabolic changes are likely occurring around this age in broilers. Though circulating levels of IGF1, CORT, and T3 were not influenced by in ovo CORT injection on e11 (P>0.05), effects of age on each hormone were observed. Plasma IGF1 increased steadily between D0 and D28 and remained elevated on D42 (P≤0.05). Levels of CORT and T3 in circulation displayed an opposite pattern to IGF1, with both decreasing between D14 and D42 (P≤0.05). An age-by-treatment interaction was observed for T4, and levels of this hormone on D42 were marginally though significantly higher in male broilers hatched from eggs injected with CORT on D42 as compared to those hatched from eggs that were not injected or injected with saline (Non-injected, 10.9 ng/mL; Saline-injected, 8.7 ng/mL; CORT-injected, 11.7 ng/mL; P≤0.5).This apparent elevation of T4 as birds approach market weight might contribute to the observed increase in meat yield observed during processing by influencing nutrient utilization towards increased muscle accretion. Specific Aim 3: Identify key intracellular signaling mechanisms within the somatotropic axis of chickens Experiments under Specific Aim 3 were conducted during years 1 - 4. Reporter plasmids necessary to screen signaling pathways known to be activated by GH in mammals were amplified and purified for transfection. These included transcriptional reporters with the following response elements to assess signaling by JAK/STAT1 [Interferon-γ (IFN-γ) Activation Site (GAS)], JAK/STAT3 [Sis-inducible Element (SIE)], JAK/STAT5 [STAT5 Response Element (STAT5)], PKA/CREB [Cyclic AMP Response Element (CRE)], ERK/SRF [Serum Response Element (SRE)], JNK/AP-1 [AP-1 Response Element (AP1)], p38MAPK/ATF4 [Activating Transcription Factor 4 Response Element (ATF4)], PI3K/Myc [Myc Response Element (MYC)], and PI3K/FOXO [Forkhead Box Response Element (FOXO1)]. Preliminary experiments optimizing plasmid transfection and reporter analysis were completed in LMH cells using positive control agonists that included IFN-γ (JAK/STAT1), interleukin-6 (JAK/STAT3), interleukin-3 (JAK/STAT5), forskolin (PKA/CREB), epidermal growth factor (ERK/SRF), U46619 (JNK/AP-1), SB203580 (p38MAPK/ATF4), and insulin or PI3-K activating peptide (PI3K/Myc and PI3K/FOXO). Following optimization, dose response and time-course experiments were conducted using agonists in LMH cells and activation of each pathway was demonstrated. When similar dose-response and time-course experiments were conducted using either pituitary-derived or recombinant chicken GH, none of these pathways were activated. An identical set of preliminary experiments to validate these assays was conducted in a DF-1 chicken fibroblast cell line, and results demonstrated that signaling of each pathway was regulated by the positive-control agonists as expected. However, when transfected DF-1 cells were treated with pituitary-derived or recombinant chicken GH, pathway activation could not be detected. Expression of chicken GH-R in LMH cells and DF-1 cells was confirmed at the mRNA level, and cloning and sequencing of GH-R from each cell line verified that it did not contain any mutations. As a result of a lack of an ability to detect GH signaling activation, further experiments using select pathway inhibitors were not completed as proposed.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Vaccaro L, Porter TE, Ellestad LE. Effects of genetic selection on components of the thyrotropic and corticotropic axes in the modern broiler. Domest Anim Endocrinol
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
1. Sparling B, Payne J, Vaccaro L, Porter TE, Ellestad LE (2018) Ontogenic changes in the Cobb broiler following early-embryonic induction of growth hormone. Poultry Science Association Annual Meeting, July 23 26, San Antonio, TX
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
2. Vaccaro L, Sinpru P, Brady K, Chaiseha Y, Porter TE, Ellestad LE (2018) The effect of genetic selection on gene expression in the developing somatotropic axis of broilers. Poultry Science Association Annual Meeting, July 23 26, San Antonio, TX
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
3. Marshall BJ, McClure TA, Porter TE, and Ellestad LE (2019) Development of an in vitro system to evaluate intracellular signaling cascades regulated by somatotropic hormones in chickens. Poultry Science Association Annual Meeting, July 15 18, Montreal, Quebec
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
4. Vaccaro L and Ellestad LE (2019) Effects of genetic selection on endocrine mechanisms regulating growth and metabolism in the modern broiler. Poultry Science Association Annual Meeting, July 15 18, Montreal, Quebec
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
5. Vaccaro L and Ellestad LE (2019) The effect of genetic selection on gene expression regulating growth and metabolism in broiler muscle tissue during embryonic and post-hatch development. International Poultry Scientific Forum, February 11-12, Atlanta, GA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
6. Vaccaro L, Ellestad LE (2020) Differential expression of insulin-like growth factor (IGF) binding proteins indicates potential differences in their endocrine or paracrine regulation of IGF signaling. International Poultry Scientific Forum, January 27-28, Atlanta, GA
- Type:
Journal Articles
Status:
Other
Year Published:
2022
Citation:
1. Vaccaro L, Porter TE, Ellestad LE. The effect of genetic selection on somatotropic gene expression in modern broilers: a potential role for insulin-like binding proteins in regulating broiler growth & body composition. J Endocrinol., in preparation
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Progress 05/01/19 to 04/30/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The COVID-19 shutdown has substantially delayed our progress. What opportunities for training and professional development has the project provided?Two postdoctoral researchers, one laboratory technician, two graduate students, and two undergraduate students assisted with this project during the past year. Theygained experience in experimental design, molecular and cellular techniques such as total RNA isolation, reverse transcription, quantitative PCR, cell culture, cell transfection, and luciferase reporter assays, and data analysis. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will identify intracellular signaling pathways activated by GH in primary hepatocytes and/or an avian myoblast cell line and confirm pathway activation by GHRH in primary cultures of chicken anterior pituitary cells.
Impacts
What was accomplished under these goals?
Objective 1 has been completed. Substantial analysis was conducted related to Objective 2. Plasma levels of T3, T4, IGF-I, and GH were measured. RNA was extracted from the hypothalamus, pituitary, liver, and breastmuscle samples collected from birds from the performance trial conducted for Objective 1. Results were analyzed. Administration of corticosterone on embryonic day 11 resulted in premature induction of GH and a subsequent increasein the ratio of lean mucle to adipose at market weight on day 42 after hatch. However, no differences in circulating hormone levels were found at 2, 4, or 6 weeks of age. Though the anticipated longitudinal changes in gene expression over time were observed in all tissues studied, there were no effects of CORT injection on these levels. Pituitary GH mRNA levels were increased by CORT administrationon embryonic day 11, however no effects of embryonic CORT treatment on pituitary mRNA levels for GH, GHRH-R, or SSTR2 or in hypothalamic mRNA levels for GHRH or SST during posthatch growth were found.Assays evaluating circulating levels of IGF-1, CORT, T3, and T4 in all six replicates were also completed. Similar to the gene expression data, while the plasma hormones changed over time as expected, this was not impacted by CORT injection on embryonic day 11. Experiments were conducted related to Objective 3. Transfection and analysis protocols for LMH cells were optimized, and time-course and dose response experiments with known agonists of each signaling pathway as well as GH have been completed. Results indicate that none of the tested pathways appear to be activated by GH in LMH or DF-1 cells in the absence or presence of overexpressed growth hormone receptor. Similar experiments using primary hepatocytes isolated from D3 liver and/or an avian myoblast cell line are underway. Primary cultures of pituitary cells were found to respond to GHRH treatment through multiple pathways.
Publications
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Progress 05/01/18 to 04/30/19
Outputs
Target Audience:Research results were presented to scientists attending the annual meeting of the Poultry Science Association. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral researcher, one laboratory technician, two graduate students, and three undergraduate students assisted with this project during the past year. They gained experience in experimental design, molecular and cellular techniques such as total RNA isolation, reverse transcription, quantitative PCR, cell culture, cell transfection, and luciferase reporter assays, and data analysis. How have the results been disseminated to communities of interest?One abstract was presented at the annual meeting of the Poultry Science Association. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will complete RNA extraction from remaining liver and muscle samples and assess gene expression related to hormonal regulation of growth in these tissues. Intracellular signaling pathways activated by GHRH, SST, and GH will be determined.
Impacts
What was accomplished under these goals?
Specific Aim 1: Characterize effects of premature somatotroph induction resulting from in ovo CORT injection on the growth performance of broiler chickens. The experiment required to evaluate growth performance and physiological effects of early GH induction for Specific Aims 1 and 2 has been completed. Further evaluation of growth performance parameters confirmed that in ovo CORT injection on embryonic day 11 did not influence final body weight, body weight gain, or average daily gain in either males or females; however, birds hatched from CORT-injected eggs tended to have improved feed efficiency as indicated by a reduced cumulative feed conversion ratio (FCR) between day of hatch and post-hatch day 42 (Non-injected, FCR=1.65; Saline-injected, FCR=1.64; CORT-injected, FCR=1.61; P=0.1070). On post-hatch day 43, birds were processed and carcass yields were determined. Injection with CORT significantly increased collective meat yield (Pectoralis major and minor, wing, thigh, and drumstick) by 1.2% (P=0.0485) and numerically reduced abdominal fat pad by 0.2% (P=0.1083) in males. Specific Aim 2: Determine the effect of in ovo CORT injection on metabolic indicators, hormone levels, and gene expression in broiler chickens. Extraction of total RNA from hypothalamus, pituitary gland, and muscle and evaluation of mRNA expression for somatotropic, corticotropic, and thyrotropic hormones and hormone receptors has been completed. Preliminary results suggest that expression of pituitary GH was increased by CORT injection and mucle GH-R may be higher on post-hatch day 14 in males hatched from CORT-injected eggs, while levels of mRNA for IGFR1, IGF2, and several IGFBPs (2, 3, 5, and 7) appear reduced on post-hatch day 42 in muscle in this group. Extraction of total RNA from liver and evaluation of mRNA expression in these samples are underway and expected to be completed within the next 4 months. Specific Aim 3: Identify key intracellular signaling mechanisms within the somatotropic axis of chickens Preliminary experiments to optimize transfection and analysis protocols have been completed, and time-course and dose response experiments using known agonists of each signaling pathway are being conducted to establish positive controls for use in comparisons with GHRH, SST, and GH treatments.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Sparling B, Payne J, Vaccaro L, Porter TE, Ellestad LE (2018) Ontogenic Changes in the Cobb Broiler Following Early-Embryonic Induction of Growth Hormone. Program 2018 Annual Meeting of the Poultry Science Association.
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Progress 05/01/17 to 04/30/18
Outputs
Target Audience:This is the first year of this project. A presentation was given to scientists and students at the Poultry Science Association annual meeting in 2017 and two more will be given in 2018. Changes/Problems:We were provided poor quality eggs from the supplier. This resulted in low hatching rates. As a result, fewer pens were used in experiment 1 than called for in the design. We are analyzing the results, but we might have to repeat the experiment. What opportunities for training and professional development has the project provided?One postdoc, one technician and six graduate students assisted with this project in the past year. They gained experience in raising chickens, experimental design, tissue collection, and data analysis. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the coming year, plasma hormone and metabolite levels will be measured. RNA will be extracted from tissue samples collected, and levels of gene expression related to hormonal regulation of growth will be measured.
Impacts
What was accomplished under these goals?
A preliminary trial was conducted to determine the optimum age for egg injection to maximize growth hormone (GH) mRNA induction. Eggs were left uninjected or injected with saline or corticosterone (200 ng/egg) on embryonic day (e) 10 or e11, and pituitary glands were collected 6h and 12h post-injection on each age. While CORT injection increased pituitary GH mRNA on both ages, the response was greater and more consistent on e11 and this was the age chosen to use for the trial to evaluate growth performance. The experiment required to evaluate growth performance and physiological effects of early GH for Specific Aims 1 and 2 has been completed. We have evaluated growth parameters (body weight, body weight gain, average daily gain, feed intake, and feed conversion ratio) and determined that CORT injection did not appear to influence performance in this trial. At the conclusion of the study, on post-hatch day 43, birds were processed and carcass yields were determined. Injection with CORT increased collective meat yield breast (Pectoralis major and minor, wing, thigh, and drumstick) by 1.2% in males and 0.7% in females. All samples have been collected to evaluate effect of in ovo CORT injection on metabolic indicators, hormone levels, and gene expression in neuroendocrine and somatic tissues.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Vaccaro L, Sinpru P, Brady K, Chaiseha Y, Porter TE, Ellestad LE (2018) The effect of genetic selection on gene expression in the developing somatotropic axis of broilers. Program 2018 Annual Meeting of the Poultry Science Association.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Sparling B, Payne J, Vaccaro L, Porter TE, Ellestad LE (2018) Ontogenic Changes in the Cobb Broiler Following Early-Embryonic Induction of Growth Hormone. Program 2018 Annual Meeting of the Poultry Science Association.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Ellestad LE, Proszkowiec-Weglarz M, Porter TE (2017) Ets-1 is a target of MAPK signaling in the embryonic anterior pituitary gland during glucocorticoid initiation of pituitary growth hormone expression. Program 2017 Annual Meeting of the Poultry Science Association.
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