Hunter et al : Fecundity, spawning, and maturity of Microstomus pacificus 



105 



Histological methods 



All of the preserved ovaries, regardless of develop- 

 ment, had a piece removed for histological analysis. The 

 pieces were dehydrated and then embedded in Para- 

 plast. Subsequently histological sections were cut at 

 5-6 /im and stained with Harris hematoxylin followed 

 by eosin counterstain (H&E). Each ovary was classified 

 histologically in the manner developed for northern 

 anchovy Engraulis mordax by Hunter and Goldberg 

 (1980) and Hunter and Macewicz (1980, 1985ab), with 

 a few modifications appropriate for Dover sole ovarian 

 structure. In the ovary we identified the presence or 

 absence of the following: oocjrtes that have not begun 

 vitellogenesis; oocytes in the first vitellogenic stages 

 (0.15-0. 55mm diameter); advanced yolked oocytes 

 (0.47-1. 4mm diameter) noting any stages of nucleus 

 migration (precursor to hydration); hydrated oocytes; 

 two stages of postovulatory follicles; and the different 

 stages of atresia. The rate at which postovulatory 

 follicles are resorbed in Dover sole is unknown. Hence 

 no ages were assigned to postovulatory follicles. 



Histological classification 



We used histological analysis of the ovaries to assess 

 the accuracy of our gross anatomical classification into 

 active and inactive states, to define the optimal criteria 

 for distinguishing mature from immature females, and 

 to calculate various indices of spawning activity and 

 postspawning states. The dendrogram (Fig. 1) indicates 

 the histological characteristics used to classify ovaries 

 into active and inactive states. The dendrogram also 

 gives the frequency of the classes in each state for the 

 prespawning period (November-December) and for the 

 spawning season (January-May) using combined data 

 from California and Oregon. The data are also given 

 by cruise and region in Table 2. 



Females were classed as active when histological 

 analysis indicated that the ovary contained the suffi- 

 cient number of advanced yolked oocytes for one 

 spawning. Active females were then separated into 

 spawning and nonspawning classes using additional 

 histological criteria. Spawning females were those 

 which showed histological evidence of past spawning 



