Stereological point-counting; an accurate method for assessing ovarian function in tilapia

Abstract

We discuss the use of stereology in fisheries research and by presenting novel data, along with comparisons to conventional methodology, further confirm the effectiveness of a point-counting method that estimates the relative distributions of oocyte developmental stages within an asynchronous ovary. Point-counting methodology was applied in three experiments involving the ovaries of Tilapia zillii (Gervais), a teleost that exhibits highly asynchronous ovarian development. Quantitative data were first obtained from conventional digestion of ovarian tissue with mercury-based Gilson's Fluid and compared to that derived from stereology. Analysis of covariance demonstrated no significant difference ( P≥0.05) between counts of discrete oocyte developmental stages determined by either method. However, counts of post-ovulatory follicles (POFs) using Gilson analysis were not possible, due to the highly irregular size and shape of these structures. A second experiment compared an immediately post-spawned ovary with an ovary 5 days after spawning, presenting comparative data determined by stereology and Gilson digestion; on this occasion, Gilson analysis significantly underestimated the proportion of atretic ooytes ( P<0.05). A further experiment forms a preliminary investigation of the effect of intra-muscular injections of human chorionic gonadotropin (hCG) upon early ovarian recrudescence and is presented as an example to demonstrate the highly informative nature of stereological analysis; injections of hCG on 3 days after spawning resulted in a significant decrease of stage 5 (early vitellogenic) but significant increase of stage 6 (late vitellogenic/maturing) oocytes by day 5. We conclude that point-counting stereology is a powerful technique for the quantitative analysis of ovarian development. Moreover, stereology avoids the prolonged use of dangerous mercury-based solutions and classifies oocytes into discrete developmental stages using a combination of criteria (biochemical, histological and size distribution) and not simply oocyte size.