Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target audiences: University faculty members, scientific community, graduate students, reproductive technologies company scientists Products: Journal articles, book chapters Changes/Problems: We have altered Specific Objectives 2B and 2C because we were unable to identify allelic specific markers for IGF2R in the DNA of the tissues we had available to us for this project. Therefore, we altered Specific Objectices 2B and 2C to assess the methylation status of the DMR 2 region of IGF2R. We were successful in pursuing this objective and were able to identify the incidence of hypomethylation at DMR2 in specific fetal and placental tissues depending on the stage of gestation. We also added additional research objectives focused on assessing global gene expression in Day 15 bovine conceptuses that resulted from the transfer of blastocysts from in vivo vs. in vitro embryo productions systems. These conceptuses were also cross-classified by size to enable us to also assess mRNA transcriptomes from short (less developed) vs. long (more developed) conceptuses on Day 15 of gestation. What opportunities for training and professional development has the project provided? Two PhD students have been trained in association with this project over this past year. How have the results been disseminated to communities of interest? Yes in the form of book chapters and refereed journal publications. What do you plan to do during the next reporting period to accomplish the goals? Complete the dissertation of the second PhD student and publish papers from both of the dissertations have been produced as a result of this project.
Impacts
What was accomplished under these goals?
We have successfully completed a PCR-based characterization of the bovine AIRN ncRNA sequence. Using a primer walking strategy we have identified strong candidate sequences for both the promoter and the termination (poly-A tail) regions of this ncRNA. We have also examined IGF2R mRNA and AIRN ncRNA expression at Day 222, 70 and 15 of gestation in bovine fetuses and conceptuses resulting from embryos produced in vivo and in vitro. At Day 222, in pregnancies from in vitro produced embryos, IGF2R was significantly decreased in fetal liver; AIRN ncRNA was altered in cotlyedonary tissue (placenta); at Day 70 no differences were found in IGF2R in fetal liver or placenta but AIRN ncRNA was elevated in placenta; at Day 15 IGF2R expression was decreased in conceptuses. We have also examined patterns of methylation within the differentially methylated region 2 (DMR2) of IGF2R in both fetal liver DNA and cotyledonary (placental) DNA from pregnancies at Day 222, 70 and 15 of gestation. Overall, DNA is hypomethylated in this region in fetal liver at Day 222 but not at Day 70 for pregnancies resulting from the transfer of in vitro produced embryos. Hypomethylation is also evident at Day 15 in conceptuses from in vitro production. We have also examined gene expression in bovine conceptuses at Day 15 of gestation from blastocyst-stage embryos produced in either in vivo or in vitro embryo production systems that were of varying embryo quality grades (Grade 1 vs. Grade 3). Using bovine Affymatrix microarrays, we have identified differentially expressed mRNAs present in conceptuses resulting from blastocysts of these different embryo quality grades at the time of transfer. Furthermore, we cross-classified these conceptuses by length and completed gene expression analyses comparing short vs. long conceptuses resulting from the transfer of in vivo or in vitro produced embryos.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Farin CE, Barnwell CV, Farmer WT. 2014. Abnormal Offspring Syndrome. In: Bovine Reproduction. Hopper RM, editor. John Wiley & Sons, Inc:Ames, Iowa. pp.620-638.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Hicks JE, Petters RM, Sommer JR, Farin CE. 2014. Nr4A1 mRNA regulation of FSH-induced resumption in bovine cumulus oocyte complexes cultured in vitro. JBRA Assisted Reproduction 18(3):69-75.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2014
Citation:
Barnwell, CV. 2014. Early embryonic survival and conceptus elongation of bovine embryos produced in vivo or in vitro. North Carolina State University, PhD Dissertation.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Target audiences: University faculty members, scientific community, graduate students,reproductive technologies company scientists Changes/Problems: We originally intended to develop a model to examine allelic expression of IGF2R mRNA and AIRN ncRNA expression to better understand regulation of these two RNAs; however, we were unsuccessful in achieving this objective. Therefore, we modified this objective to pursue studies evaluating potentialfactors that affect regulation of the expression of these RNAs instead. Our focus has been on assessment of methylation status in specific regions of the IGF2R gene known in other speciesto have a role in regulation of expression. What opportunities for training and professional development has the project provided? Two PhD students are being trained under this project as well as 3 Master's students. We have also involved 2 undergraduate students for intensive experiences (honors project activities)in association with completion with data collection for this project. We also regularly involve 2-5 other undergraduate students as assistants for our on-farm data collection activities. How have the results been disseminated to communities of interest? Abstracts have been presented at regional, national and international scientific meetings; research seminars have been presented at our home university; initial publications have been presented in peer-reviewed journals What do you plan to do during the next reporting period to accomplish the goals? We plan to complete our analyses of the microarray data on conceptus development and the methylation data from the early, mid and late gestation pregnancies; additional publications will be prepared and submitted.
Impacts
What was accomplished under these goals?
We have successfully completed a PCR-based characterization of the bovine AIRN ncRNA sequence. Using a primer walking strategy we have identified strong candidate sequences for both the promoter and the termination (poly-A tail) regions of this ncRNA. We have also examined IGF2R mRNA and AIRN ncRNA expression at Day 222, 70 and 15 of gestation in bovine fetuses and conceptuses resulting from embryos produced in vivo and in vitro. At Day 222, in pregnancies from in vitro produced embryos, IGF2R was significantly decreased in fetal liver; AIRN ncRNA was altered in cotlyedonary tissue (placenta); at Day 70 no differences were found in IGF2R in fetal liver or placenta but AIRN ncRNA was elevated in placenta; at Day 15 IGF2R expression was decreased in conceptuses. We have also examined patterns of methylation in fetal liver DNA and cotyledonary (placental) DNA from pregnancies at Day 222, 70 and 15 of gestation for sites within the differentially methylated region of the IGF2R gene. Overall, DNA appears to be hypomethylated in this region in fetal liver at Day 222 but not at Day 70 for pregnancies resulting from the transfer of in vitro produced embryos. Hypomethylationalso appears to beevident at Day 15 in conceptuses from in vitro production. We have examined gene expression in bovine conceptuses at Day 15 of gestation from blastocyst-stage embryos produced in either in vivo or in vitro embryo production systems that were of varying embryo quality grades (Grade 1 vs. Grade 3). Using bovine Affymatrix microarrays, we have identified differentially expressedmRNAs present in conceptuses resulting from blastocysts ofthese different embryo quality grades at the time of transfer.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Farmer WT, Farin PW, Piedrahita JA, Bischoff SR, Farin CE. 2013. Expression of antisense of insulin-like growth factor-2 receptor RNA non-coding (AIRN) during early gestation in cattle. Anim Reprod Sci 138 (1-2): 64-73; DOI: http://dx.doi.org/10.1016/j.anireprosci.2013.01.009.
Bowdridge EC, Knox WB, Whisnant CS, Farin CE. 2013. NCSynch: a protocol for ovulation synchronization and timed artificial insemination in goats. Small Rum Res 110:42-45.
Farin CE, Barnwell CV, Farmer WT. (2014). Abnormal Offspring Syndrome. In: Bovine Reproduction. Hopper RM, editor. John Wiley & Sons, Inc. (in press).
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Specific Objective 1: Assessment of IGF2R mRNA and AIRN RNA expression in bovine conceptuses during early gestation from in vivo- and in vitro-produced pregnancies. This experiment is ongoing. Conceptuses were collected from recipient females between on Day 15 of gestation. Recovered conceputses were assessed for the presence of an embryonic disc, their length determined, biopsied for determination of conceptus sex and remaining tissue was snap-frozen for subsequent RNA extraction. Additional fetal liver and cotyledonary tissues from Day 222 of gestation from fetuses exhibiting abnormal offspring syndrome that had been collected previously were also obtained. These tissues were subjected to RNA extraction, cDNA synthesis and real-time RT-PCR to assess IGF2R mRNA, AIRN RNA and H2AZ (housekeeper) RNA levels. Differences were found for expression of IGF2R mRNA in fetal liver but not cotyledons. In contrast, differences were found for expression of AIRN RNA in cotyledons but not in fetal liver. In addition, conceptuses collected on Day 15 of gestation from in vivo-produced blastocysts were found to vary significantly in length and were classes as either short or long and assessed for gene expression by microarray analysis. Differences in expression of classes of genes associated with immune function and cytoskeletal development were identified. We are currently collecting additional Day 15 conceptuses following the transfer of in vitro produced blastocysts for identification of short vs. long lengths and assessment of gene expression by microarray analysis. Specific Objective 2: Assessment of allelic expression of bovine IGF2R and AIRN during conceptus and fetal development. Objective 2A: Characterization of bovine AIRN RNA sequence. This subobjective has been completed and the bovine AIRN RNA has been characterized. A publication is currently in preparation. Objective 2B: Identification of allelic markers for bovine IGF2R and AIRN gene expression. Objective 2C: Determine the effect of in vitro embryo production on the patterns of allelic expression of bovine IGF2R and AIRN during conceputs and fetal development. Objectives 2B and 2C have been altered and combined. Rather than assess allelic expression, we are planning to use the Day 222 and Day 15 fetal, placental and conceptus tissues obtained in SO1 to determine if the differences in mRNA and RNA expression observed between tissues from fetuses following the transfer of in vivo vs. in vitro produced blastocysts differ in their levels of methylation at specific loci within the differentially methylated region of the IGF2R gene. These data will provide insights into the role of embryo production method to alter gene expression and DNA methylation patterns in association with the occurrence of abnormal offspring syndrome. PARTICIPANTS: Charlotte E. Farin, BS, MS, PhD, Professor, Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621; Peter W. Farin, BS, MS, DVM, PhD, Associate Professor, Department of Population Health and Pathobiology, North Carolina State University, Raleigh, NC 27606 (Deceased August 2011). William T. Farmer, BS, MS, Doctoral Graduate Student, Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621; Callie Barnwell, BS, Doctoral Graduate Student, Department of Population Health and Pathobiology, North Carolina State University, Raleigh, NC 27606 TARGET AUDIENCES: Livestock producers, livestock scientists, basic research scientists, graduate and undergraduate students PROJECT MODIFICATIONS: Altered Specific Objective 2 to examine methylation differences in regions known to be associated with control of expression of IGF2R and AIRN genes rather than location of differences in allelic sequences between maternal and paternal alleles. Change made due to information obtained as a result of pursuing Specific Objective 1.
Impacts
Production or manipulation of embryos in vitro during the first week following fertilization alters the normal pattern of development of conceptuses and fetuses throughout gestation. Understanding mechanisms underlying the occurrence this phenomenon, known as abnormal offspring syndrome, will facilitate identification of in vitro methods that promote normal development during gestation.
Publications
- Jackson LR, Farin CE, Whisnant CS. 2012. Tumor necrosis factor alpha inhibits bovine embryo development through a prostaglandin mediated mechanism. J Anim Sci Biotech 3:7-10.
- Bing J, Pratt-Phillips S, Farin CE. 2012. Effect of supplemental online resources in distance education format on undergraduate animal science laboratory instruction. NACTA J 56(1):67-72.
- Curry Jr. KW, Wilson EB, Flowers JL, Farin CE. 2012. Scientific basis vs. contextualized teaching and learning: the effect on the achievement of postsecondary students. J Agricultual Education 53(1):57-66.
- Barnwell CV, Farin PW, Ashwell CM, Farmer WT, Farin CE. 2012. Global mRNA expression in short vs long day 15 conceputses derived from grade 1 in vivo produced embryos. 45th Annual Meeting Soc for the Study of Reprod, State College PA. (abstr #192).
- Bing JQ, Pratt S, Gillen L-A, Farin CE. 2011. Undergraduate performance in a domestic animal laboratory taught via distance education. J Anim Sci 89:297-301. Epub 2010 Sept 10.
- Farmer WT, Farin PW, Farin CE. 2011. Characterization of the bovine antisense to IGF2R (bAIRN) non-coding RNA. Reprod Fertil Dev 23(1):182 (abstr. #140).
- Barnwell CV, Whisnant CS, Farin CE, Alexander JE, Farin PW. 2011. Recipient serum progesterone concentration and early conceptus development in bovine embryos produced in vivo or in vitro. Reprod Fertil Dev 23(1):152 (abstr. #80).
- Bowdridge EC, Knox WB, Whisnant CS, Farin CE. 2011. NCSynch: a protocol for ovulation synchronization and timed artificial insemination in goats. Proc Am Soc for Anim Sci Annual Meetings, July 10-14, New Orleans LA (abstr).
- Whitley NC, Farin CE, Knox WB, Townsend L, Horton JR, Moulton K. 2011. Comparison of two ovulation synchronization methods for timed artificial insemination in goats. Proc Am Soc for Anim Sci Annual Meetings, July 10-14, New Orleans LA (abstr).
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Specific Objective 1: Assessment of IGF2R mRNA and AIRN RNA expression in bovine conceptuses during early gestation from in vivo- and in vitro-produced pregnancies. This experiment is ongoing and conceptuses are currently being collected from recipient females between Days 13-18 of gestation. Recovered conceputses are being assessed for the presence of an embryonic disc, their length determined, biopsied for determination of conceptus sex and remaining tissue is being snap-frozen for subsequent RNA extraction. Specific Objective 2: Assessment of allelic expression of bovine IGF2R and AIRN during conceptus and fetal development. Objective 2A: Characterization of bovine AIRN RNA sequence. This objective is currently being pursued and primers encompassing the first 30 Kb have been designed and products produced for sequence verification. Additional primers are being designed to map the remainder of this non-coding RNA. Objective 2B: Identification of allelic markers for bovine IGF2R and AIRN gene expression. Objective 2C: Determine the effect of in vitro embryo production on the patterns of allelic expression of bovine IGF2R and AIRN during conceputs and fetal development. Objectives 2B and 2C were not yet started during the first year of this project. PARTICIPANTS: Charlotte E. Farin, BS, MS, PhD, Professor, Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621; Peter W. Farin, BS, MS, DVM, PhD, Associate Professor, Department of Population Health and Pathobiology, North Carolina State University, Raleigh, NC 27606; J. Eric Alexander, BS, Research Specialist, Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621; William T. Farmer, BS, MS, Doctoral Graduate Student, Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621; Callie Barnwell, BS, Doctoral Graduate Student, Department of Population Health and Pathobiology, North Carolina State University, Raleigh, NC 27606 Elizabeth C. Compton, BS, MS, Graduate Student (completed), Department of Animal Science, North Carolina State University, Raleigh, NC 27695-7621; TARGET AUDIENCES: Livestock producers, livestock scientists, basic research scientists, graduate and undergraduate students PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Production or manipulation of embryos in vitro during the first week following fertilization alters the normal pattern of development of conceptuses and fetuses throughout gestation. Understanding mechanisms underlying the occurrence this phenomenon, known as abnormal offspring syndrome, will facilitate identification of in vitro methods that promote normal development during gestation.
Publications
- Farin CE, Farmer WT, Farin PW. 2010. Pregnancy recognition and abnormal offspring syndrome. Reprod Fertil Dev 22:75-87.
- Barnwell CV, Farin PW. 2010. Embryo evaluation and pregnancy outcomes following embryo transfer in cattle. Clinical Theriogenology 2:264-274.
- Farin CE, Alexander JE, Farin PW. 2010. Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro. Theriogenology 74:1288-1295.
- Wrench N, Pinto CRF, Klinefelter GR, Dix DJ, Flowers WL, Farin CE. 2010. Effect of season on fresh and cryopreserved stallion semen. Anim Reprod Sci 119:219-227.
- Bing J, Pratt S, Gillen LA, Farin CE. 2010. Undergraduate performance in a domestic animal laboratory taught via distance education. J Anim Sci [Sept 10; epub ahead of print].
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