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
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.
It has been shown that at 200ng/L, feminization effects are visible.
Focuses on effects of estradiol only
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
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:
Send poultry litter subsamples to University of Buffalo for gas chromatography- mass spectroscopy analysis
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
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
Observe external malformations
Feed floating frog food
Observe external malformations
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 Normality Steel’s Many-One Rank Test (non parametric test) Dunnett’s Test (Parametric test) Normal Non-normal Homogenous variance Heterogenous variance
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