Protocol for spawning. As females began to swell, they were observed daily and 

 occasionally picked up to determine if there were signs of eggs in the lower portion of the 

 oviduct. Fish were handled deliberately and gently to minimize stress. From the onset of 

 rapid swelling, it was usually only several days until females had freely flowing eggs. 



Many females needed only to be removed from the water and tipped vertically for the 

 weight of the fish to expel eggs from oviduct; other fish required only gently abdominal 

 pressure to start the free flow of eggs. From each of several successive 30-mL aliquots, 10 mL 

 of eggs were removed for the determination of percent floating eggs. A second 10-mL egg 

 aliquot was placed in a damp 400-mL beaker, two drops of sperm stripped within 30 seconds 

 from a male were added and rapidly swirled for 1 to 15 seconds. Approximately 400 mL of 

 sea water were then added to the beaker. A pooled aliquot was made up of the remaining 

 10 mL in each of the fourth through final egg aliquots. When egg volumes were small, 

 sometimes a portion of the third aliquot would be included in the pooled aliquot. Fertilized 

 egg aliquots were washed free of sperm approximately 20 min after fertilization by a 

 complete exchange of sea water. Usually only one male that met a simple sperm motility 

 test was necessary for the spawning tests as males contribute very little to the variability of 

 fertilization success. 



Measures of early reproductive success. Several measures of early reproductive success 

 were determined to facilitate the detection of effects at specific developmental stages. 

 Viable hatch is the proportion of spawned eggs that produce viable larvae. Hatching 

 success is the proportion of spawned eggs that hatch. Fertilization success is the proportion 

 of spawned eggs that are fertilized. In addition to these commonly used measures of 

 survival, it is important to recognize that often large numbers of spawned eggs sink. 

 Floating eggs contain the potentially viable gametes, since sinking eggs are usually not 

 fertilized and do not develop normally. Thus, where 



N = total number of eggs spawned, 



V = the number of eggs that float (viable eggs), 



F = the number of fertilized eggs, 



H = the number of eggs that hatched, and 



L = the total number of normal larvae, 



the following measures of survival through early life history stages were determined: 



(1) percent floating eggs = (V/N) *100 



(2) percent fertilization success = (F/V) *100 



(3) percent embryological success = (H/F) *100 



(4) percent normal larvae = (L/H) *100; 



in which the sources of variability for these measures in more than 100 spawnings have been 

 assessed and a standard protocol adopted for spawning and evaluation of developmental 

 success (Spies et al., 1985). 



Percent floating eggs was determined volumetrically in a calibrated centrifuge tube 

 approximately 10 min after spawning. Fertilization success was measured at the 4-8 

 blastomere stage after eggs had been held at 11-12°C for 3 to 4 h following fertilization in 

 the first incubation chamber, a 400-mL beaker. Eggs were scored in five categories: (1) 

 fertilized eggs with even cleavages, (2) fertilized eggs with uneven cleavages, (3) 

 unfertilized eggs with cortical reaction, (4) unfertilized eggs without a cortical reaction, and 

 (5) ill-formed opaque eggs or highly disorganized zygotes. Fertilized eggs were considered 

 to be those in categories 1 and 2. Eggs in category 2 never comprised more than 5 percent of 

 the total. Embryological success and hatching success were determined 80 to 100 hours after 

 fertilization based on the number of fertilized eggs that began incubation in a second 



23 



