chamber, a 400-mL beaker provided with a screen-covered port that allowed sea water to 

 flow through but retained the eggs. In most cases, between 150 and 200 eggs were used to 

 determine embryological success. This second chamber was held at the same temperature as 

 the first, but was provided with flow-through seawater at the rate of 2 to 3 mL per min. 



A major source of variability in egg survival is related to the sequence of eggs in 

 spawning: eggs spawned (= stripped) first do not develop as well as those spawned last and 

 a regular progression of egg quality in the early part of a spawning can be seen as aliquots 

 are taken during the course of a stripping. This phenomenon is a function of egg position 

 within the ovarian lumen after ovulation and may be related to the time between ovulation 

 and stripping. Although we cannot precisely control this source of variability due to 

 differences between females, we have incorporated two procedures into our protocol that 

 minimize its effect. Firstly, the first two to three aliquots (39 mL each) of spawned eggs are 

 not used in estimating reproductive success. Secondly, the second and subsequent spawnings of 

 each female are done at 48-h intervals to standardize as much as possible the time between 

 ovulation and stripping. Since the sources of between-spawn variability are unknown, there 

 was no basis for evaluating or controlling its effect on the outcome of these experiments, we 

 therefore simply averaged the reproductive success measures for all spawnings of each 

 female. Males contribute less than 1 percent to the variability in fertilization success 

 provided they have motile sperm, therefore the main effects of contaminants on egg survival 

 could best be determined by evaluating the spawning females. With these features in the 

 protocol between-female variance for the reproductive success measures from 123 spawnings of 

 43 females was as follows: 45.3 percent for floating eggs, 48.2 percent for fertilization 

 success, 35.7 percent embryological success, and 16.1 percent for percent normal larvae (Spies 

 and Rice, 1988). We therefore concluded that substantial differences in reproductive success 

 measures could be detected between females with the following procedure: 



1. Females that were noticeably hydra ted were watched closely and stripped once 

 they had freely flowing eggs. Subsequent spawnings were carried out at 48-h intervals. 



2. Eggs were collected in a series of 30-mL aliquots. The number of aliquots varied 

 between two and eight, with five to six being the usual number. 



3. From each aliquot two 10-mL portions of eggs were removed to evaluate reproductive 

 success. The remainder of eggs (excluding those from aliquots 1 to 3, or 1 to 2) were combined 

 and 10 mL of eggs taken to produce a pooled aliquot which was independently evaluated for 

 the reproductive success measures. 



4. To evaluate percent floating eggs in an aliquot, 10 mL of eggs were placed in a 

 graduated, glass 40-mL centrifuge tube, 30 mL of seawater added, the contents stirred, and 

 after standing for 15 to 30 min, the volume ratios of floating and sinking eggs (V:N) 

 determined. 



5. A male which had been determined to have sperm that remained motile for 2 min 

 after stripping was used to fertilize the eggs. 



6. To evaluate fertilization success in an aliquot, 10 mL of eggs were placed in a wetted 

 glass 400-mL beaker, 2 drops of sperm added, the contents vigorously stirred, 300 mL of 

 seawater added, and the beaker placed in a seawater table for 20 to 30 min. Floating eggs 

 were then transferred to clean seawater in a second 400-mL beaker with a piece of nytex 

 screen. Fertilization success (F:V) ratio was determined at the 4 to 8 blastomere stage after 

 3 to 4 hat 11 to 13°C. 



7. To determine embryological success and percent normal larvae, 150 to 200 eggs that 

 had been evaluated for fertilization success were transferred to a 500-mL glass beaker 

 provided with a 2 to 3 mL/min flow of seawater and a screened outflow to retain the eggs. 



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