Progress 12/08/15 to 09/30/17
Outputs
Target Audience:Dairy producers. This audience was targeted because ultimately dairy producers will apply research results to their herds. Dairy producers were targeted through formal and informal classroom instruction and Extension/outreach activities. Personnel involved in livestock industry. Specifically, we targeted professionals who work for dairy nutrition consulting companies and feed supplement companies. These professionals are most likely to benefit from knowledge generated by this project. Research community. Specifically, we targeted animal scientist. An understanding of our research results will help other researchers develop hypotheses that will advance their own research programs utilizing the method outlined in this project. We targeted these individuals through a peer-reviewed publication and presentations at scientific meetings. Changes/Problems:Goal 4. Association of blood biomarkers of inflammation with fecal RNA gene expression related to inflammation during a mild-diarrhea (100% complete). During the on-farm experiment related to goal #2, we observed a maximal (2.6 ± 0.3) increased in fecal scores during week 2 of life. The latter was associated with an increase in transcription of genes related to inflammation in fecal RNA around the same time. Because of this effect, we evaluate blood biomarkers of inflammation, and we were able to confirm such inflammatory effect detected in fecal RNA. We plan to publish these data along with data from goal #2 in a peer-reviewed journal (anticipated publication date of 2017). What opportunities for training and professional development has the project provided?Two graduate students and two visiting scholars were trained on this project. Fernanda Rosa, M.S. student, 12/08/2015-9/30/16. No graduate research assistantship was provided under this funding. Assisted with on-farm experiment execution, including feeding, collecting samples, isolating RNA, etc. Kali Linville, M.S. student, 7/15/16-5/15/17. No graduate research assistantship was provided under this funding. Assisted with data analysis and drafting of a final manuscript submitted for publication. Two visiting scholars (Sebastiano Busato, B.S. student and Cecilia Avaroma, B.S. Student) from Zamorano University (Honduras, C.A.), learned skills in animal husbandry and good practices during research experiments involving animals, learned basic lab techniques (e.g., pipetting) as well as more advanced techniques such as RNA isolation, cDNA synthesis, and RT-qPCR. How have the results been disseminated to communities of interest?Research community. We generate one proceeding paper and two abstracts for this project. The PI has presented these data to the research community (sponsored by International Ingredient Corporation). A graduate student delivered a webinar to undergraduate students from the Universidad de La Salle, Bogota, Colombia. Stakeholders. A graduate student received placed third in a poster competition organized by the gamma sigma delta (GSD). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal 1: Validate in calves the method for isolation of RNA from exfoliated GI tract epithelial cell (100% complete). We have collected the fecal samples and successfully extracted sufficient total RNA for gene expression analysis. However, we observed significant variation in terms of total RNA yield per gram of fecal sample. However, the purity of RNA as measured by the 260/280 ratio was of consistently high across fecal samples. Additionally, the RNA quality as measured by the RIN factor was variable across fecal samples, however RIN factor was not associated with an impairment in the PCR efficiency. Goal 2: Evaluate the transcriptomics changes during the first 5 weeks of age in exfoliated GI tract epithelial cells in newborn dairy calves (100% complete). The on-farm experimental portion of the project has been conducted successfully and fecal and blood samples have been collected as well as performance parameters (e.g., body weight, starter grain intake, rectal temperature). The method to isolate RNA from fecal samples was satisfactory on the basis of RNA isolation, for which we obtained an average yield of 205.7 ng of RNA/mg of fecal sample. Similarly, the purity of RNA as measured by the 260/280 ratio wavelength in the spectrophotometer was also satisfactory with an average of 1.98. In terms of quality of RNA obtained by RIN factor in fecal samples, the results were variable as previously observed in goal #1, we observed an average RIN factor of 3.4 with a range from 1.8 to 7.4. Obtaining good quality of RNA from fecal samples it quite challenging, and will require additional research to improve this parameter. Despite the low RIN factor we successfully performed cDNA synthesis and RT-qPCR from fecal RNA. For this analysis, we evaluate genes related to inflammation and cell membrane. Additionally, we evaluated 7 internal control genes or reference genes in order to obtain a suitable gene expression normalization across fecal samples. The gene expression data has been normalized with these internal control genes, and the statistical analysis performed. We plan to publish these data along with data from goal #4 in a peer-reviewed journal (anticipated publication date of 2017). Goal 3: Evaluate the transcriptomic profile of exfoliated GI tract epithelial cells vs. small intestine epithelial cells (40% complete). The original idea for goal #3 was to isolate RNA from fecal samples and postmortem sections of the small intestine (i.e., duodenum, jejunum, ileum). Then, perform RNA-Seq analysis in these samples with the aim to establish correlations at a transcriptomic level between sections of the small intestine and the RNA isolated from fecal samples. Unfortunately, due to the inherently variable RIN factor observed in RNA isolated from fecal samples using the method outlined in this project we deemed unfeasible to acquire a good quality RNA-seq data to establish significant correlations between fecal RNA and small intestine. In the light of these changes, we have added goal #4 in the Changes/Problems section.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
F. T. Rosa, S. Busato, F. C. Avaroma, M. Bionaz, and J. S. Osorio. 2016. A non-invasive technique to evaluate transcriptional changes in the GI tract of neonatal dairy calves undergoing a mild diarrhea. Symposium on Gut Health in Production of Food Animals. St. Louis, MO.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
F. T. Rosa, S. Busato, F. C. Avaroma, K. Linville, E. Trevisi, M. Bionaz, and J. S. Osorio. 2017. Physiological adaptations in the GI tract detected by a fecal RNA method and blood inflammatory biomarkers in neonatal dairy calves undergoing a mild diarrhea. Midwest-ADSA-ASAS. Omaha, NE.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
F. Rosa, S. Busato, F. C. Avaroma, E. Trevisi, M. Bionaz, and J. S. Osorio. 2017. Transcriptional changes in the gut of neonatal dairy calves undergoing a mild diarrhea revealed by a non-invasive technique. J. Dairy Sci. Vol. 100, Suppl. 2.
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