Cooper et al.: Fecundity of Sebastolobus alascanus and Sebastolobus altivelis 



17 



Stereological fecundity estimation 



The majority of oocytes within an ovary were found to 

 be at the same developmental stage; however develop- 

 ment was not completely synchronous. Some ovaries 

 containing stage-5 and -6 oocytes (late vitellogenesis 

 to migratory nucleus) also contained a few stage-4 

 oocytes, which although unsuitable for fecundity esti- 

 mation with the gravimetric method, could be used 

 with the stereological method described by Emerson et 

 al. (1990). Fecundity was estimated from ten of these 

 samples by using the stereological method to complete 

 the shortspine thornyhead collection from Alaska. 



Fecundity was estimated per unit of volume and 

 then multiplied by the volume of both ovaries. The 

 formula used to estimate fecundity per unit of vol- 



" PV?' 



where N = the number of oocytes per unit of 

 volume; 

 k = an oocyte size correction coefficient; 

 /3 = an oocyte shape correction coefficient; 

 N„ = the average number of vitellogenic 



oocytes per unit of area; and 

 V = the average fractional volume of vitel- 

 logenic oocytes per unit of area. 



The method for estimating the parameter k is given in 

 Emerson et al. (1990) and the parameter k was estimated 

 for six shortspine thornyhead samples. The resulting k 

 values had a small range (1.0088-1.022), and a small 

 standard deviation (0.0066), and a mean k value of 1.017 

 was used for all samples as a result, ft was calculated 

 by using the method given in Weibel and Gomez (1962). 

 The ft parameter was calculated from one shortspine 

 thornyhead sample (53 oocytes) to be 1.565. 



Exact volume of sample ovaries was impossible to 

 determine because portions of the ovaries had already 

 been removed for histological study (Pearson and 

 Gunderson, 2003). Volume was estimated by dividing 

 whole ovary weight by an average density of 1.052 g/ 

 mL. This was the average density from six samples 

 (SD = 0.0297) estimated by water displacement in a 

 graduated cylinder. 



Values for N a and V, were estimated by using a sim- 

 plified Weibel grid for particulate structures (Weibel 

 et al., 1966) instead of a Weibel multipurpose grid. A 

 square containing 13 rows of 13 points was created 

 and printed out on a clear acetate sheet. This overlay 

 was taped to the front of a monitor. A video camera 

 mounted to a stereomicroscope sent the image of the 

 histology section to the computer monitor. The num- 

 ber of vitellogenic oocytes per grid and the number of 

 points falling on vitellogenic oocytes were recorded and 

 used to estimate N a and V,, respectively. The Weibel 

 grid was used at 25x magnification, and 50x magnifica- 



NV 



V 



0.5 cm 



Figure 1 



Partial cross section of shortspine thornyhead rockfish 

 {Sebastolobus alascanus) ovary showing bands of vitel- 

 logenic (V) and nonvitellogenic (NV) oocytes. 



tion was used to help distinguish borderline vitellogenic 

 oocytes. 



A sampling grid was placed under the ovary histologi- 

 cal section. The corner of the Weibel grid was aligned 

 with corners of the sampling grid in order to systemati- 

 cally sample the ovary cross section. Two histological 

 sections were sampled per ovary. 



The number of Weibel grid counts per ovary depended 

 on the size of the ovary cross section. An average of 

 55.9 (range: 29-103) Weibel grid counts were taken per 

 ovary. This number was greater than the average num- 

 ber of Weibel grid counts used by Emerson et al. (1990), 

 but the extra counts were made because shortspine 

 thornyhead vitellogenic oocytes develop on peduncles 

 (Erickson and Pikitch, 1993; Pearson and Gunderson 

 2003) and are distributed in a band around the central 

 part of the ovary (Fig. 1). Because the vitellogenic oo- 

 cytes are not uniformly distributed, the Weibel grid was 

 applied systematically at more points across the entire 

 ovary, and the counts were averaged. Because the whole 

 cross section could not be systematically sampled and 

 averaged, cross sections of larger fish were not used for 

 stereological estimates. 



Statistical methods 



Length-fecundity relationships were estimated by using 

 the following equation: 



Fee = al b , 



