Fourteen days post fertilization (dpf) swim-up fry stage Oreochromis mossambicus juveniles were obtained from the Welgevallen experimental farm, University of Stellenbosch, South Africa. The juveniles used for each experiment were derived from a single breeding hatch of a single adult breeding pair (one female and male) tilapias for each experiment. Fish were kept in 10 litre stock tanks (30 x 22 x 16 cm) filled with tap water. The water was constantly aerated and filtered through activated charcoal. Fish were maintained in water of similar quality for all exposure experiments, but without activated charcoal filters. Fish were maintained at a temperature of 27 °C (± 1 °C) with a light regime of 14:10 (light:dark) throughout the duration of all experiments. Fish were fed daily with crushed commercial tilapia pellets (AquaNutro, South Africa). Water was replaced every 5 days.
7.3.2. Test chemicals and exposure design
All chemicals in this study were from either Sigma Chemical Company (St Louis, MO, USA) or Roche (South Africa) unless otherwise stated. All solvents and chemicals used were of analytical grade. Diethylstilbestrol (DES) was used for all estrogen exposures and was supplied by Sigma Chemical Company (St Louis, MO, USA). DES stock solutions were prepared to a concentration of 1mg/ml using either analytical grade ethanol or dimethyl sulfoxide (DMSO) as a solvent. The final solvent concentration never exceeded 0.01 % (v/v) according to OECD recommendations (OECD, 2004). Estrogen exposures were performed under semi static conditions. Exposures were performed in 1 litre glass bottles filled with 500 ml of water. Half the volume of the test solution was exchanged every five days. Prior to experiments, all glass containers were washed with Neptune Liquid salt (Bell Products, www.bellchemicals.com). Before all experiments, fish tanks and exposure systems were washed with Hibitane (5 % antiseptic) washing agent soap, followed by a thorough rinse with water that has been charcoal filtered. Tanks were then stripped with 100 % methanol followed by a rinse with charcoal filtered water. Tanks were filled with the required amount of water and aerated at 27 °C for 48 hours prior to putting fish into it.
Juvenile tilapias were transferred to the 1 liter exposure tanks. DES or vehicle control was added to the water and the fish were then exposed for 5 days. At the end of the exposure period the fishes were anaesthetized with MS-222 (100 mg/l water containing 200 mg NaHCO3). The fish were then used for further analytical procedures. The exposures were
done with groups of fish at various days post fertilization. The age of fish used for this study ranged between 15 to 70 days post fertilization.
7.3.3. Fish weight and length measurement
Fish were photographed using a Leica EZ4D microscope and measurements taken (to the nearest µm) using Leica Version 2.4.0 image analysis software (Leica Microsystems (Switzerland) Limited). The mass of juvenile fish was determined with a bench top precision scale (Ohaus Precision Scale) to nearest 0.01g.
7.3.4. Protein extraction and quantification
All the protein extraction procedures were carried out on ice. Protein extraction buffer (saline (0.9 % (w/v) NaCl in distilled water) and 0.01 % (v/v) PMSF) was added to the juvenile fish at a weight to volume ratio of 1g per 10 ml extraction buffer. Sample were then sonicated (Omni-Ruptor 400; Omni International INC.) at 40 % power. Samples were sonicated for 15 seconds in total, 5 second bursts at a time, followed by 1 min incubation on ice. Cell rests were removed by centrifugation at 12000 x g for 10 minutes at 4 ˚C. Cell pellets were discarded and the supernatants were aliquoted and stored at -80 ˚C until further use. Protein concentrations of the samples were measured according to the method of Bradford, (1976) using biovine serum albumin (BSA) as a standard protein (Sigma).
7.3.5. Vitellogenin quantification
The VTG content of the whole body homogenate preparations was determined using a competition ELISA. An ELISA system similar to the one described in chapter 6 was used to quantify VTG in juvenile fish. In this ELISA an in-house VTG antibody was used to detect VTG. Results and specifications regarding this ELISA system have been submitted elsewhere for publication (Journal of Immuno Assay and Immunochemistry). Nunc-Immuno Maxisorp plates (Nalge Nunc, Denmark) were used for all ELISA assays. Plates were coated overnight at 4°C with 50 µl per well of 1/2000 diluted anti-VTG antiserum in saline. At the end of the incubation period the antiserum was decanted and the plate was washed four times with saline. Following the wash procedure, the remaining adsorption sites were blocked by dispensing 0.2 ml of block solution (2 % v/v human serum albumin [HSA] in saline) per well. The plate was then incubated for one hour at room temperature. The plate was washed as before where after samples or purified VTG standards (50 µl) as well as 50 µl biotinylated- VTG were added to each well. Plates were then incubated for three hours at room
temperature. The plate was washed using the same procedure as earlier described. Avidin horse radish peroxidase (AV–HRP) was diluted 1/2000 with saline containing 1 % (w/v) HSA and dispensed at 50 µl per well. Plates were incubated for one hour at room temperature where after it was decanted and washed eight times with saline. BM Blue POD soluble substrate were heated to 37°C and dispensed at 50 µl per well. Plates were incubated at room temperature for twenty minutes followed by addition of 50 µl per well of stop solution (0.5 M H2SO4). The optic density was lastly determined at 450 nm. A standard curve was drawn from the VTG standards. The VTG concentration of juvenile tilapia homogenates were calculated using the standard curve.
7.3.6. 17β-estradiol quantification
Estradiol was quantified using a commercial ELISA kit (catalogue number RE52041, IBL, Germany) using the manufacturer’s instruction manual. In brief: microtitre plate strips precoated with rabbit anti-estradiol was removed from the strip holder and fixed firmly in the ELISA plate. All assays were done in duplicate. Samples and standards were transferred to the wells (25 µl/well). Estradiol – horseradish peroxidase conjugate was added to all the wells (200 µl/well). The solutions were mixed by gently tapping the plate, after which it was incubated for 120 minutes at room temperature. At the end of the incubation period the solutions in the wells were decanted. The wells were then washed three times with 300 µl/well of wash solution. Substrate was then dispensed at 100 µl per well after which the plate was incubated for 15 minutes at room temperature. The reaction was stropped by addition of stop solution (100 µl/well). The absorbance was determined at 450 nm using a plate reader. A standard curve was drawn using the reading obtained for the standards and the concentration of the samples was read off this curve.
7.3.7. Statistical analysis
Variation in body mass, length, total protein concentration, VTG concentration during development, as well as fold induction of VTG following exposure to DES and 17β-estradiol was assed by using single factor ANOVAs and pairwise multiple comparison procedure (Student-Newman-Keuls Method). A Pairwise multiple comparison procedure (Tukey’s HSD) was used to indicate significant different groups (P < 0.05).
e d d bc d b bc ac ac a D C C C B AB AB A A y = 8.1321e0.0863x R2 = 0.8801 y = 8.3745e0.0841x R2 = 0.9188 0 5 10 15 20 25 30 15 20 25 30 35 40 45 50 55 70
Days Post Fertilization
L en g th ( m m ) Control DES Exposed Expon. (Control) Expon. (DES Exposed)
7.4. Results