Pieces of ovary were also kept in glutaraldehyde fixative Belinostat molecular weight to study subcellular alterations. Occurrence of estrus cycle The rats were observed for occurrence of estrus cycle every day in the morning between 9.00 AM and 10.00 AM by examination of cellular morphology of vagina by cotton swab smear technique.[11] The cotton wool tip was moistened slightly by dipping in saline. The rat was held around the thorax, ventral surface facing up. The tip of the swab stick was inserted carefully into the vagina to a depth of about 1 cm with a rotating action of swab and at an angle of 45�� to animal body. The tip was rolled gently onto a clean pre labelled glass slide and the smears were examined under light microscope.

Basing on the cell types, viz nucleated epithelial cells – Proestrus (PE), swollen cornified cells – Estrus (E), combination of nucleated epithelial cells, swollen cornified cells and leucocytes – Metestrus (ME), leucocytes-Diestrus (DE), each phase of estrus cycle was identified. The rats were examined for estrus cycle phase continuously for 3 consecutive cycles. The findings were tabulated as % of each estrus cycle phase continuously in 3 consecutive cycles. Biochemical analysis Antioxidant markers SOD was estimated by the method that involved inhibition of superoxide-dependent reduction of tetrazolium dye methyl thiazolyl tetrazolium (MTT) to its formazan.[12] GSH was estimated based on a reaction of reduced glutathione with 5-5ditiobis-2-nitrobenzoic acid (DTNB).[13] Peroxidation markers Malondialdehyde, the product of lipid peroxidation, was estimated by reaction with thiobarbituric acid as per the method prescribed by Balasubramanian et al.

[14] Protein carbonyls were estimated, based on the reaction of amino carbonyls with 2, 4-dinitrophenyl hydrazine to form hydrazones, which can be detected spectrophotometrically at 372 nm.[15] Sero-biochemical markers Total protein, ALT, BUN and creatinine were estimated in serum by using the standard diagnostic kits. Total Protein Total protein in the ovarian tissue was quantified as per Lowry et al.’s[16] method. Histology For light microscopy examination, the formalin fixed tissues were dehydrated through ascending grades of alcohol, cleared in three changes of xylene, and were embedded in paraffin. Serial sections, each of 4-micron thickness, were cut and stained with H and E as per standard protocols.

[17] For transmission electron microscopy (TEM), the glutaraldehyde-fixed tissues were used. Specimen preparation, staining and the observations Dacomitinib were done at the designated RUSKA Lab, Hyderabad. Statistical analysis The data were subjected to statistical analysis by applying one way ANOVA using statistical package for social sciences (SPSS) version 12.0. Differences between means were tested using Duncan’s multiple comparison tests and significance was set at P < 0.05.