Team KERMIT  Research Proposal Amy Chen Christina Gonzalez David Hu Richa Kalsi Tanya Kapoor HaeMin Park Sam Park Alex Pro...
<ul><li>Approximately 1.6 billion lbs of poultry litter (chicken manure mixed with bedding) is generated annually in the D...
<ul><li>Main component of PLAH: estradiol </li></ul><ul><li>Other components: estrone </li></ul><ul><li>Endocrine-disrupti...
It has been shown that at 200ng/L, feminization effects are visible.  <ul><li>Focuses on effects of estradiol only </li></...
What has not done yet been done… <ul><li>Use environmentally relevant concentrations and allow for natural biodegradation ...
Will exposure to environmentally relevant concentrations of PLAH in poultry litter negatively affect the sexual developmen...
<ul><li>Model organism for EDC research </li></ul><ul><li>Skin is semi-permeable </li></ul><ul><li>Growth cycles sensitive...
<ul><li>Initial Concentrations Setup: </li></ul><ul><li>Send poultry litter subsamples to University of Buffalo for gas ch...
<ul><li>Xenopus laevis  (African clawed frogs) tadpoles at 5 DPF </li></ul><ul><li>Testing conditions: </li></ul><ul><ul><...
5 DPF: expose to poultry litter 17 DPF: sex differentiation sensitive stage 55-75 DPF: Metamorphosis occurs.  120 DPF: Sac...
Sex Differentiation Determination <ul><li>Histology phase: determining sex ratios. </li></ul><ul><li>Stain gonads with hem...
Statistical Analysis Shapiro Wilk’s Test for Normality Bartlett’s Test for Normality Steel’s Many-One Rank Test (non param...
<ul><li>Threats to the ecosystem  </li></ul><ul><ul><li>destruction of frog populations </li></ul></ul><ul><li>Groundwater...
<ul><li>Lab space secured </li></ul><ul><li>IACUC Proposal submitted and approved  </li></ul><ul><li>Grants applications s...
Fall 2011 <ul><li>Begin research </li></ul><ul><ul><li>Feed frogs daily </li></ul></ul><ul><ul><li>Periodic water changes ...
Item Quantity Unit Cost Overall Cost 5-Gallon Aquaria 20 Donated $0.00 Histology Materials n/a n/a $500.00 X. Laevis  Tadp...
References <ul><li>Harrison, P. T. C., Holmes, P., & Humfrey, C. D. N. (1997). Reproductive health in humans and wildlife:...
 
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Sophomore Proposal Presentation 03172011

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  • Is 200 right???
  • PL from chicken house in eastern shore area,
  • $40 for containers, $60 for preservative, add on thingy for gloves, antibacterial soaps Histology materials:
  • Sophomore Proposal Presentation 03172011

    1. 1. Team KERMIT Research Proposal Amy Chen Christina Gonzalez David Hu Richa Kalsi Tanya Kapoor HaeMin Park Sam Park Alex Proctor Alec Ridgway Andrew Taverner Nick Vujcic Mentor: Steve Turley Librarian: Jim Miller
    2. 2. <ul><li>Approximately 1.6 billion lbs of poultry litter (chicken manure mixed with bedding) is generated annually in the Delmarva area </li></ul><ul><li>Large amounts are used as fertilizer for agricultural lands as a substitute for commercial fertilizers </li></ul><ul><li>Excessive application to agricultural fields often results in runoff of poultry litter associated hormones (PLAH) </li></ul>
    3. 3. <ul><li>Main component of PLAH: estradiol </li></ul><ul><li>Other components: estrone </li></ul><ul><li>Endocrine-disrupting compound (EDC) </li></ul><ul><li>Amphibians in the Delmarva area are being found to display intersex characteristics including malformed gonads. </li></ul><ul><ul><li>Feminization of male fish and frogs </li></ul></ul><ul><ul><li>Mixed ovarian and testicular tissues in gonads </li></ul></ul>Intersex tissue: oocytes mixed with seminiferous tubules.
    4. 4. It has been shown that at 200ng/L, feminization effects are visible. <ul><li>Focuses on effects of estradiol only </li></ul>CONCENTRATION of Estradiol LOCATION 5-25 ng/L Pond that received runoff (general) 19 to 75 ng/L MD Eastern Shore streams/ rivers receiving agricultural runoff 20-2330 ng/L Runoff near litter pastures 38.7-196 ng/L Runoff from freshly manured field 245 ng /L Pond following a rain event after poultry litter application 730 ng/L Eluant from pile of poultry litter
    5. 5. What has not done yet been done… <ul><li>Use environmentally relevant concentrations and allow for natural biodegradation </li></ul><ul><li>Observing synergistic effects among the various chemicals in poultry litter—Most studies look at the effects of estradiol alone </li></ul><ul><li>Directly connect poultry litter to frog deformations </li></ul>
    6. 6. Will exposure to environmentally relevant concentrations of PLAH in poultry litter negatively affect the sexual development of Xenopus laevis ?
    7. 7. <ul><li>Model organism for EDC research </li></ul><ul><li>Skin is semi-permeable </li></ul><ul><li>Growth cycles sensitive to hormonal control </li></ul><ul><li>Easy to induce reproduction </li></ul><ul><ul><li>Large number of offspring </li></ul></ul><ul><li>Entire life cycle is aquatic so exposure can be maximized </li></ul>X. Laevis tadpole X. Laevis adults; female on left and male on right
    8. 8. <ul><li>Initial Concentrations Setup: </li></ul><ul><li>Send poultry litter subsamples to University of Buffalo for gas chromatography- mass spectroscopy analysis </li></ul><ul><ul><li>Estradiol </li></ul></ul><ul><ul><li>Estrone </li></ul></ul><ul><ul><li>Estriol </li></ul></ul><ul><ul><li>Testosterone </li></ul></ul><ul><li>Dilute to desired concentrations based on estradiol concentration data </li></ul>Days 1-30 Days 30-120 Send 2 homogenized, aqueous subsamples to setup initial concentrations Send 2 subsamples for analysis every week Send 2 subsamples for analysis biweekly
    9. 9. <ul><li>Xenopus laevis (African clawed frogs) tadpoles at 5 DPF </li></ul><ul><li>Testing conditions: </li></ul><ul><ul><li>23 ± 2 ⁰C </li></ul></ul><ul><ul><li>16:8 hrs of light: dark </li></ul></ul><ul><ul><li>Vented to decrease ammonia concentrations </li></ul></ul><ul><ul><li>Grown in 5 gal. aquaria </li></ul></ul>0 ng/L estradiol 15 ng/L 60 ng/L 300 ng/L 25 tadpoles 25 tadpoles 25 tadpoles 25 tadpoles X 5 replicates
    10. 10. 5 DPF: expose to poultry litter 17 DPF: sex differentiation sensitive stage 55-75 DPF: Metamorphosis occurs. 120 DPF: Sacrifice juvenile frogs with tricaine methanesulfonite (MS-222) <ul><li>Feed floating tadpole food </li></ul><ul><li>Monitor survival </li></ul><ul><li>Observe external malformations </li></ul><ul><li>Monitor behavior </li></ul><ul><li>Feed floating frog food </li></ul><ul><li>Monitor survival </li></ul><ul><li>Observe external malformations </li></ul><ul><li>Monitor behavior </li></ul><ul><li>Monitor time to metamorphosis </li></ul><ul><li>Measure wet weight and snout-vent length </li></ul>
    11. 11. Sex Differentiation Determination <ul><li>Histology phase: determining sex ratios. </li></ul><ul><li>Stain gonads with hematoxylin and eosin solution </li></ul><ul><ul><li>Ovarian and testicular tissues will stain selectively </li></ul></ul><ul><li>Observe gonadal abnormalities (as below) </li></ul>
    12. 12. Statistical Analysis Shapiro Wilk’s Test for Normality Bartlett’s Test for Normality Steel’s Many-One Rank Test (non parametric test) Dunnett’s Test (Parametric test) Normal Non-normal Homogenous variance Heterogenous variance
    13. 13. <ul><li>Threats to the ecosystem </li></ul><ul><ul><li>destruction of frog populations </li></ul></ul><ul><li>Groundwater contamination </li></ul><ul><ul><li>can lead to eventual human intake of EDCs </li></ul></ul><ul><li>Increased EDCs in environment linked to </li></ul><ul><ul><li>Higher breast cancer rates, </li></ul></ul><ul><ul><li>Higher prostate cancer rates </li></ul></ul><ul><ul><li>Higher testicular cancer rates, </li></ul></ul><ul><ul><li>Decreased sperm counts </li></ul></ul><ul><ul><li>…… in HUMANS. </li></ul></ul>
    14. 14. <ul><li>Lab space secured </li></ul><ul><li>IACUC Proposal submitted and approved </li></ul><ul><li>Grants applications submitted: </li></ul><ul><ul><li>ACCIAC </li></ul></ul><ul><ul><li>Sea Grant </li></ul></ul><ul><ul><li>Grant application in Progress: </li></ul></ul><ul><ul><ul><li>HHMI Grant </li></ul></ul></ul><ul><ul><ul><li>Pilot Study has been started </li></ul></ul></ul><ul><ul><ul><li>Tadpole/frog care training has been scheduled </li></ul></ul></ul>
    15. 15. Fall 2011 <ul><li>Begin research </li></ul><ul><ul><li>Feed frogs daily </li></ul></ul><ul><ul><li>Periodic water changes </li></ul></ul><ul><ul><li>Sacrifice frogs </li></ul></ul><ul><li>Present at Colloquium </li></ul><ul><li>Complete thesis </li></ul><ul><li>Prepare & practice for thesis defense </li></ul>Fall 2012 Spring 2012 <ul><li>Begin analyses of gonadal tissues </li></ul><ul><li>Complete a poster of our work </li></ul><ul><li>Present at Undergraduate Research Day </li></ul><ul><li>Begin writing thesis </li></ul>Spring 2013 * <ul><li>Defend thesis </li></ul><ul><li>Revise thesis for publication </li></ul>*Provided world does not end in 2012
    16. 16. Item Quantity Unit Cost Overall Cost 5-Gallon Aquaria 20 Donated $0.00 Histology Materials n/a n/a $500.00 X. Laevis Tadpole and Adult Powdered Food n/a n/a $40.00 Tank Maintenance Items n/a n/a $210.00 Poultry Litter I pile n/a <$50.00 X. Laevis Specimens 400 Donated $0.00 pH/ DO2 1 $500 $500 Lighting 20 $10 $200 Lab Space 1 n/a $0.00 Poultry Litter Analyses 25 300 $7500.00 TOTALS ~$9000.00
    17. 17. References <ul><li>Harrison, P. T. C., Holmes, P., & Humfrey, C. D. N. (1997). Reproductive health in humans and wildlife: are adverse trends associated with environmental chemical exposure? Science of The Total Environment , 205 (2-3), 97-106. doi:doi: DOI: 10.1016/S0048-9697(97)00212-X   </li></ul><ul><li>Houlahan, J. E., Findlay, C. S., Schmidt, B. R., Meyer, A. H., & Kuzmin, S. L. (2000). Quantitative evidence for global amphibian population declines. Nature , 404 (6779), 752-755. doi: 10.1038/35008052   </li></ul><ul><li>Lutz, I., Kloas, W., Springer, T., Holden, L., Wolf, J., Krueger, H., & Hosmer, A. (2008). Development, standardization and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of <i>Xenopus laevis</i>. Analytical and Bioanalytical Chemistry , 390 (8), 2031-2048.   </li></ul><ul><li>Welshons, W. V., Thayer, K. A., Judy, B. M., Taylor, J. A., Curran, E. M., & Saal, F. S. V. (2003). Large Effects from Small Exposures. I. Mechanisms for Endocrine-Disrupting Chemicals with Estrogenic Activity. Environmental Health Perspectives , 111 (8), 994-1006.   </li></ul><ul><li>Wolf, J. C., Lutz, I., Kloas, W., Springer, T. A., Holden, L. R., Krueger, H. O., & Hosmer, A. J. (2010). Effects of 17 β- estradiol exposure on Xenopus laevis gonadal histopathology. Environmental Toxicology and Chemistry , 29 (5), 1091-1105. </li></ul><ul><li>Seth (2009). Intersex fish and how we’re causing it. City Renewed, http://www.cityrenewed.com/2009/04/intersex-fish-and-how-were-causing-it/ </li></ul>

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