Stereological comparison of oocyte recruitment and batch fecundity estimates from paraffin and resin sections using spawning albacore (Thunnus alalunga) ovaries as a case study

Traditional histological protocols in marine fish reproductive laboratories using paraffin as the embedding medium are now increasingly being replaced with protocols using resin instead. These procedures entail different degrees of tissue shrinkage complicating direct comparisons of measurement results across laboratories or articles. In this work we selected ovaries of spawning Mediterranean albacore (Thunnus alalunga) as the subject of our study to address the issue of structural changes, by contrasting values on oocyte recruitment and final batch fecundity given from the same tissue samples in both paraffin and resin. A modern stereological method, the oocyte packing density (OPD) theory, was used supported by initial studies on ovarian tissue sampling and measurement design. Examples of differences in the volume fraction of oocyte stages, free space and connective tissue were found between the embedding media. Mean oocyte diameters were smaller in paraffin than in resin with differences ranging between 0.5% in primary growth and 24.3% in hydration (HYD) stage oocytes. Fresh oocyte measurements showed that oocytes shrank as a consequence of the embedding process, reaching the maximal degree of shrinkage for oocytes in the HYD stage (45.8% in paraffin and 26.5% in resin). In order to assess the effect of oocyte shrinkage on the OPD result, and thereby on relative batch fecundity (Fr), oocyte diameters corrected and uncorrected for shrinkage, were used for estimations. Statistical significant differences were found (P < 0.05) between these two approaches in both embedding media. The average Fr was numerically smaller in paraffin compared to resin (86 ± 61 vs. 106 ± 54 oocytes per gram of body mass (mean ± SD)). For both embedding media statistical significant differences (P < 0.05) were seen between Fr results based on either oocytes in the germinal vesicle migration stage or HYD stage. As a valuable adjunct, the present use of the OPD theory made it possible to document that the oocyte recruitment of spawning ovaries of Mediterranean albacore followed the typical pattern of an asynchronous oocyte development and indeterminate fecundity.