Four female specimens were sacrificed each time. The ovaries www.selleckchem.com/products/VX-770.html were dissected for histological analysis, and a blood sample from the caudal vein was obtained for hormone determinations. 2.3. Estimation of the Gonadosomatic IndexThe weights of the specimens and the ovaries were determined using digital balances sensitive to 1g (Bonzo) and 10mg (Sartorius), respectively. The gonadosomatic index (GSI) was calculated as a percentage of the gonadal weight (Wg) over the total body weight (Wt) using the following formula: GSI = ((Wg/Wt) �� 100). 2.4. Histological Examinations of Ovaries and Oocyte GrowthThe ovaries were fixed in a solution made up of absolute ethanol (850mL), 40% formaldehyde (100mL), and glacial acetic acid (50mL) [12].
Gilson fixative (1:1:1 absolute ethanol: glacial acetic acid: chloroform and sublimate mercuric chloride to saturation) [13] was used for the fixation of oocytes with large vitelli. Ovary sections were cut to a 5��m thickness on a microtome, extended and adhered to a slide, and then they were stained with hematoxylin and eosin stain as well as Mason’s trichrome stain. The Bromage and Cumaranatunga criteria [14] were used to classify the different stages of oogenesis. The oocytes’ growth rates were determined by diameter measurements made on histological slides using a stereoscopic magnifier fit with a micrometer-graduated eyepiece. Five larger diameter oocytes from three slides of each female were chosen for this measurement.2.5. Plasma Level Determinations of Sex SteroidsTo determine the sex steroid profiles, blood samples were obtained in heparinized tubes and kept for 12 hours at 8�C12��C.
They were then centrifuged at 3,000rpm for 5min to obtain the plasma fraction, which was frozen at ?80��C until hormone determination. Plasma concentration levels were analyzed for estradiol-17�� (E2) and vitellogenin (V). The level of E2 was determined using radioimmunoanalysis (RIA) (BioSource Europe, S.A.), in which a fixed quantity of steroid labeled with 125I competes against the steroid present in the sample for the limited number of antibodies immobilized on the polystyrene tube wall. A standard curve was plotted, and the steroid concentration of the sample was determined by interpolation of the concentration from this standard curve. The minimum detectable quantity of E2 was 0.05ng/mL with less than 1% cross-reactivity to other hormones. Vitellogenin levels were determined by an indirect enzyme-linked inmunosorbent assay (ELISA) Entinostat method where a known ��capture�� antibody was first attached to the bottom of the well, the sample was added, and then a secondary (labeled) antibody was added to bind to the sample antigen.