132 



Fishery Bulletin 105(1) 





LNl?"(!- 'm 



Figure 1 



(A) Microphotograph of a postovulatory follicle (POF) situated at the epithelium of an ovarian 

 lamella (L) in the Iberian sardine Sardina pilchardus; (B) the same POF magnified, indicating 

 the perimeter of its cross-sectional area (XSA) and its diameter (D). Scale bar = 0.1 mm. 



estimates of the spawning fraction and biomass when 

 spatial variation in ambient water temperature is large 

 within a survey area. This dependence can be assessed 

 under laboratory conditions through inspection of speci- 

 mens spawning under different temperature regimes 

 (Fitzhugh and Hettler, 1995). Alternatively, the effect 

 of temperature on POF degeneration can be studied in 

 the wild by the examination of individuals caught at 

 variable environmental conditions (Ganias et al., 2003; 

 Roumillat and Brouwer, 2004). 



Because of the above unresolved issues of the POF 

 method (that can be species specific), the spawning 

 fraction remains the most poorly estimated DEPM pa- 

 rameter (Hunter and Lo, 1997; Stratoudakis et al., 

 2006). The main objective of our study was to devise 

 a method whereby one can test indirectly for potential 

 sources of bias in the attribution of stage and age to 

 the POFs of the field-collected sardine S. pilchardus. 

 We used the size of POFs (cross-sectional area and dia- 

 meter) as an index of POF age and, together with other 

 histomorphological characteristics (follicle shape, state 

 of the granulosa layer), we refined existing criteria 

 for determining the stage and age of POFs in sardine. 

 Then, we modeled the size of POFs as a function of POF 

 age, ambient temperature, type of preservative, and 

 type of embedding material. The analysis allowed us 

 to test whether our staging criteria were valid and to 

 examine whether temperature and laboratory process- 

 ing introduced bias in the process. 



Materials and methods 



Adult sardines were collected off Portugal and the Gulf 

 of Cadiz in 1997, 1999, and 2005 within the framework 

 of DEPM surveys for the estimation of the spawning 

 biomass of the Atlanto-Iberian sardine stock (ICES, 



2004). Sampling was conducted during peak spawning 

 months for sardine (January-March), either on board the 

 RV Noruega or from the commercial purse-seine fleet. 

 During the surveys, sea surface temperature (SST) was 

 recorded on an extensive grid representing hydrographic 

 casts that covered the whole sampling area during the 

 ichthyoplankton surveys (ICES, 2004). 



Fish gonads were immediately removed after capture 

 and placed in jars either with AFA (65 parts by volume 

 of 50% alcohol, 32 parts formalin, and 13 parts glacial 

 acetic acid) solution [1997, 1999] or with 4% formalin 

 [2005]). In the laboratory, ovaries were embedded ei- 

 ther in paraffin (1997, 2005) or in resin (1999), and 

 histological sections (paraffin: 5 f<m; resin: 3 ^m) were 

 stained with hematoxylin and eosin. Histological slides 

 were examined and scored for maturity, atresia, and 

 presence of POFs. When POFs were detected, slides 

 were scanned in detail to locate the largest follicle 

 situated along the epithelium of the lamellae. These 

 POFs were photographed with a digital camera at- 

 tached to the microscope at various magnifications so 

 that the whole follicle would fit into the microphoto- 

 graph (Fig. lA). The cross-sectional area of the whole 

 POF (all samples) and the distance between the two 

 extremes of the follicle on the lamella (POF diameter, 

 paraffin samples) were measured with UTHSCSA im- 

 age analysis software (Univ. Texas Health Science 

 Center, San Antonio, Texas) (Fig. IB). Differences in 

 the dimensional (size, shape) and fine histological (de- 

 terioration of the granulosa layer) characteristics were 

 used, together with existing descriptions for Sardina 

 populations (Atlantic: Perez et al., 1992; Mediterra- 

 nean: Ganias et al., 2003), to describe the pattern of 

 POF resorption. 



The evolution in the shape of POFs was assessed 

 through the allometric relationship between diameter 

 (D) and cross-sectional area (XSA): 



