McDermott et al: Annual fecundity, batch fecundity, and oocyte atresia of Pleurogrammus monopteryglus 



21 



Table 1 



Number of fish collected by year, month, and National 

 Marine Fisheries Service (NMFS) statistical area. The 

 number of samples with postovulatory follicles, i.e., in 

 spawning or postspawning condition are shown in paren- 

 theses. 



NMFS statistical areas 



Year Month 



541 542 



543 



620 Total 



1993 October 



1994 June 

 July 

 August 



Total 



57 



57 



4(4) 



49 



53 



5(5) 



34(11) 



10(9) 



44 



9 



57 



83 



10 



159 



with hematoxilin (Sigma Aldrich, St. Louis, MI) and eo- 

 sin counterstain (Sigma Aldrich, St. Louis, MI) (H&E). 

 Maturity stages were determined by using categories 

 developed by McDermott and Lowe (1997). Ovaries of 

 prespawning females were defined by the lack of hy- 

 drated oocytes in June and the absence of postovulatory 

 follicles once hydrated oocytes were present in July. 

 Presence of atresia was determined according to clas- 

 sifications developed by Hunter and Macewitz (1985). 



For the estimation of annual fecundity, only females 

 whose ovaries had not shown any signs of releasing 

 oocytes (i.e., all hydrated oocytes were within their fol- 

 licles and ovaries contained no postovulatory follicles) 

 were used. Different stages of postovulatory follicles 

 can be distinguished in fish that have spawned multiple 

 batches (McDermott and Lowe, 1997). These differ- 

 ent stages indicate that postovulatory follicles persist 

 in ovaries longer than the time period between batch 

 spawning events, minimizing the possibility of incor- 

 rectly classifying a partly spent fish as a prespawning 

 fish. Prespawning and partly spent specimens were 

 compared to examine determinate spawning. 



Gravimetric method 



The gravimetric method was used to count and measure 

 oocytes in weighed samples of Atka mackerel ovarian 

 tissue (Hunter et al., 1985). In our fecundity analy- 

 sis, small cross-sections were excised from preserved 

 Atka mackerel ovaries. The amount of tissue removed 

 was dependent on the size of eggs present, typically 

 ranging from 0.3 g to 2.5 g. The target sample size 

 was approximately 1000 eggs. The cross-section was 

 divided into wedges in order to ensure a representative 

 sample of ovarian wall tissue in relation to the tissue 

 containing eggs. Ovarian wall tissue weight represents 

 a substantial part of total ovary weight and needed 

 to be represented proportionally in the subsamples. 

 The samples were weighed to the nearest 0.001 g and 

 allowed to sit in water for several hours before han- 



dling to reduce exposure to formalin. Ovary tissue was 

 teased apart manually under a dissecting microscope 

 and spread evenly in a 50*7^ solution of glycerol in a dish 

 over a grid partitioned by 1-cm squares. The dissecting 

 microscope was equipped with a Javelin Ultrachip CCTV 

 video camera (model JE-7442, Javelin Electronics, Los 

 Angeles, CA), connected to a computer equipped with 

 BioScan Optimas 4.0 scanning software (Bioscan Inc., 

 Edmonds, WA). 



All oocyte stages used in our study were previously 

 described by McDermott and Lowe (1997). It was pos- 

 sible to distinguish the cortical alveoli oocyte stage 

 (stage 3) from the more advanced oocyte stages (stage 4 

 [oil droplet stage] and stage 5 [vitellogenic stage]) when 

 using the dissecting microscope and camera image to 

 examine whole eggs. Oocytes in stage 4 and above 

 showed a distinct ring around the nucleus, which was 

 attributed to the oil and yolk droplets accumulating 

 in the oocyte. Stage-3 oocytes did not show a distinct 

 ring around the nucleus. However, it was not possible 

 to distinguish oil-droplet oocytes (stage 4) from vitel- 

 logenic oocytes or more advanced stages (stage 5+) 

 when examining whole eggs. Because oocyte devel- 

 opment is usually attributed to oil-droplet and yolk 

 accumulation, it was initially assumed that oocytes 

 below stage 4 would not be spawned in the current 

 spawning event and therefore would serve as reserve 

 oocytes to be spawned in later spawning seasons. To 

 determine oocyte-size composition, all oocytes stage 4 

 and above were measured along a vertical axis in each 

 1-cm square of a grid until 250 eggs had been mea- 

 sured. All eggs in the sample determined to be oocyte 

 stage 4 or above were then counted manually under the 

 dissecting microscope to arrive at an estimate of the 

 number of eggs present per gram of tissue that were 

 oocyte stage 4 and greater. 



Location of tissue samples within the ovary 



We examined six specimens to determine if location of 

 tissue samples within the ovary affected estimates of 

 fecundity. Two specimens had ovaries in the vitellogenic 

 stage, two had ovaries in the early hydration stage, and 

 two had ovaries in the spawning stage. We took three 

 tissue samples per ovary lobe (six per specimen). The 

 samples were taken from the anterior, center, and pos- 

 terior location within each lobe of the ovary. 



All eggs within the tissue sample that were oocyte 

 stage 4 and above were counted and the first 100 oo- 

 cytes were measured. Analysis of variance (ANOVA) 

 (S-plus, 7.0 for Windows, Insightful Corp., Seattle, WA) 

 (Venables and Ripley, 2002) was used for this examina- 

 tion with ovary lobe (left or right), position within the 

 ovary, and maturity stage as factors. This analysis was 

 carried out for the mean number of eggs per gram of 

 tissue and mean egg size as response variables. The 

 ANOVA indicated that the tissue sample could be taken 

 from either lobe of the ovaries at any location. All fur- 

 ther samples were therefore taken from a central loca- 

 tion within either one of the ovary lobes. 



