Ovarian Follicle Morphometric Analysis

Fixed ovaries were processed for microscopy and subsequently the entire ovary was sectioned at 8 µm. Every 5^th ovarian section was stained with haematoxylin and eosin for morphometric analysis. In order to prevent multiple counts of the same follicle, only those follicles with a visible oocyte nucleus were included. Since oocyte nuclei measured between 20–30 µm in diameter, counting every 5th section of the ovary ensured a distance of 40 µm between analysed sections, preventing multiple counts of the same ovarian follicle. Follicle classification based on characteristics proposed by Hirshfield & Midgley [66] (link) was as follows: type 1: primordial follicle, one layer of flattened granulosa cells surrounding the oocyte; type 2: primary follicle, one to fewer than two complete layers of cuboidal granulosa cells; type 3: secondary follicle, an oocyte surrounded by greater than one layer of cuboidal granulosa cells, with no visible antrum; type 4: antral follicle, an oocyte surrounded by multiple layers of cuboidal granulosa cells and containing one or more antral spaces, cumulus oophorus and theca layer may also have been evident. Total volume of each section was calculated (area of the section x thickness of the section) and follicle counts for each animal were corrected for the total volume of ovarian tissue counted. All follicle counts were then expressed as number of follicles counted per mm³ of ovarian tissue counted.