Abstract:

Evaluation of fecundity, fertility, hatch success and larval survival to determine reproductive success. Reproductive steroid and protein concentrations in adult pompano after reproduction and exposure to chemically enhanced water accommodated fraction (CEWAF) of south Louisiana crude oil.

Suggested Citation:

DL Wetzel, TA Sherwood, NR Rhody, MJ Resley, RL Medvecky. 2019. CEWAF-exposed Florida pompano reproduction: Spawn and Hatch & Reproductive Function Assays. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7FF3QTH

Purpose:

Controlled oil exposure study to evaluate the effects of crude oil on fecundity, fertility, hatch success and larval survival, and steroid and protein expression for assessment of normal or aberrant reproduction system function.

Data Parameters and Units:

Sample Key worksheet: Collected date (mm/dd/yyyy), Organism Species, Treatment, Gender, Spawning Tank, Comment. Samples 6071, 1C20, 124B, 112F, 4C73, 5550E, 6650 died so they were not sampled for plasma. Fish 112F and 5550E were not spawned, dissected, or sampled. Exposure Tank Description and Spawning Tank Description worksheets: Tank ID, Start and End Date (mm/dd/yyyy), Treatment, Number of Fish. CEWAF Prep worksheet: Start and End Dates (mm/dd/yyyy), Carboy, Oil (g), Corexit (g), Volume Water (L), Treatment, Amt Stock Soln (L), and Amt Seawater (L). Reproductive Function worksheet: Spawn #, Field ID, Treatment, Gender, Spawning Tank, MML, E2 (Estradiol Rep 1, Rep 2, and average), 11-kt (11-keto testosterone , concentration pg/ml, %B/Bo (%bound/maximum bound-ratio of absorbance of particular sample or standard well to that of maximum binding well) for two dates (pg/ml), Vtg (vitellogenin Rep 1, Rep 2, and average for two dates (ng/ml). Spawn and Hatch worksheet: Treatment, Date (mm/dd/yyyy), Tank, Total # eggs, fish wt (g), Fecundity (#eggs/fish wt), # Fertilized Eggs, % Fertilized Eggs, % Hatch, % 1 day survival, % 3 day survival, Average % hatch, Average % 1 day survival, Average % 3 day survival, Fecundity (#eggs/g). Growth worksheet: Exposure Tank, Field ID, Measurement Date (mm/dd/yyyy), Weight (g), Fork Length (cm), Jun-Oct Weight Change (g), and Jun-Oct Length Change (cm).

Methods:

Sample Collection: plasma For VTG assay, 4 mL whole blood were collected into a lithiuum heparin blood collection tube preserved with 25TIU/ml of aprotinin. For the remaining assays, 4 mL whole blood were collected into a sodium citrate or EDTA blood collection tube according to kit manufacturer's protocol. Tubes were gently inverted 5 times and centrifuged at 2000 x g for 10 minutes between 30- 60 minutes from collection time. Supernatant was removed and placed in a clean cryovial preserved with 10TIU/ml of aprotinin. Samples were stored in -80degC freezer. When samples were removed and thawed for the first time they were aliquotted out to prevent numerous freeze/thaw cycles. Sample Processing: Samples were assayed according to manufacturer's protocol; Fish Estradiol ELISA (abx054981) by Abbexa, 11-keto Testosterone ELISA (Item #582751) by Cayman Chemical, Rainbow Trout Vitellogenin ELISA (Prod. #V01004402) by BioSense Laboratories.

Instruments:

DS2 Automated ELISA instrument by Dynex Technologies

Error Analysis:

%CV for the standard curve less than 25% is acceptable, less than 15% for sample replicates.

Individual Files: