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Team KERMIT Research Proposal
Amy ChenChristina Gonzalez
David HuRicha Kalsi
Tanya KapoorHaeMin Park
Sam ParkAlex ProctorAlec Ridgway
Andrew TavernerNick Vujcic
Mentor: Steve TurleyLibrarian: Jim Miller
• Approximately 1.6 billion lbs of poultry litter (chicken manure mixed with bedding) is generated annually in the Delmarva area
• Large amounts are used as fertilizer for agricultural lands as a substitute for commercial fertilizers
• Excessive application to agricultural fields often results in runoff of poultry litter associated hormones (PLAH)
• Main component of PLAH: estradiol• Other components: estrone• Endocrine-disrupting compound (EDC)
•Amphibians in the Delmarva area are being found to display intersex characteristics including malformed gonads.
• Feminization of male fish and frogs • Mixed ovarian and testicular tissues in gonads
Intersex tissue: oocytes mixed with seminiferous tubules.
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
It has been shown that at 200ng/L, feminization effects are visible.
• Focuses on effects of estradiol only
What has not done yet been done…What has not done yet been done…
• Use environmentally relevant concentrations and allow for natural biodegradation
• Observing synergistic effects among the various chemicals in poultry litter—Most studies look at the effects of estradiol alone
• Directly connect poultry litter to frog deformations
Will exposure to environmentally relevant concentrations of PLAH in poultry litter negatively affect the sexual development of Xenopus laevis?
• Model organism for EDC research• Skin is semi-permeable• Growth cycles sensitive to hormonal
control• Easy to induce reproduction
– Large number of offspring• Entire life cycle is aquatic so
exposure can be maximized
X. Laevis tadpole
X. Laevis adults; female on left and male on right
Initial Concentrations Setup:1.Send poultry litter subsamples to University of Buffalo for gas chromatography- mass spectroscopy analysis
• Estradiol• Estrone• Estriol• Testosterone
2.Dilute to desired concentrations based on estradiol concentration data
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
•Xenopus laevis (African clawed frogs) tadpoles at 5 DPF•Testing conditions:
• 23 ± 2 ⁰C• 16:8 hrs of light: dark• Vented to decrease ammonia concentrations• Grown in 5 gal. aquaria
0 ng/L estradiol 15 ng/L 60 ng/L 300 ng/L
25 tadpoles 25 tadpoles 25 tadpoles 25 tadpoles
X 5 replicates
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)
• Feed floating tadpole food
• Monitor survival• Observe external malformations• Monitor behavior
• Feed floating frog food
• Monitor survival • Observe external malformations• Monitor behavior
• Monitor time to metamorphosis • Measure wet weight and snout-vent length
Sex Differentiation Determination
Histology phase: determining sex ratios.• Stain gonads with hematoxylin and eosin solution
– Ovarian and testicular tissues will stain selectively• Observe gonadal abnormalities (as below)
Statistical Analysis
Shapiro Wilk’s Test for Normality
Bartlett’s Test for NormalitySteel’s Many-One Rank Test
(non parametric test)
Dunnett’s Test (Parametric test)
Normal Non-normal
Homogenous variance
Heterogenous variance
• Threats to the ecosystem – destruction of frog populations
• Groundwater contamination – can lead to eventual human intake of
EDCs
• Increased EDCs in environment linked to – Higher breast cancer rates, – Higher prostate cancer rates– Higher testicular cancer rates, – Decreased sperm counts……in HUMANS.
• Lab space secured
• IACUC Proposal submitted and approved
• Grants applications submitted:•ACCIAC•Sea Grant
• Grant application in Progress:•HHMI Grant
• Pilot Study has been started
• Tadpole/frog care training has been scheduled
Fall 2011• Begin research
• Feed frogs daily• Periodic water changes• Sacrifice frogs
• Present at Colloquium
• Complete thesis• Prepare & practice for thesis defense
Fall 2012
Spring 2012• Begin analyses of gonadal tissues• Complete a poster of our work• Present at Undergraduate Research Day• Begin writing thesis
Spring 2013*
• Defend thesis• Revise thesis for publication
*Provided world does not end in 2012
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
References• 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
• 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
• 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.
• 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.
• 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.
• 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/