canned 5 from each group. The 

 other 5 in each group were frozen 

 in 10° F circulating brine for 20 hr, 

 then thawed in running seawater 

 (3 to 4 hr), sampled, and canned. 



B. 60° F study. 



Proceeded as in "A" except held fish in 

 60° F seawater for 6 hr. 



C. 78° F study. 



Proceeded as in "A" except held fish in 



78° F seawater for 6 hr. 



Note: Experiments B and C were 

 performed on the same day so 

 that the controls for these 

 groups are the same. 



D. 78° F study for 9 hr. 



Proceeded as in "A" but held fish in 

 78° F seawater for 9 hr, rather than 

 6 hr. 



E. The freezing of stressed and rested con- 



trols proceeded as in 1 and 2 of "A" 

 except that they were frozen in 10° F 

 brine for 20 hr before sampling and 

 canning. 



F. Live fish. 



1. 5 fish were placed alive in 0° F brine 



for 20 hr. 



2. The fish were then thawed, sampled 



and canned. 



Sampling 



The muscle sampling procedure has been 

 described by Crawford and Finch (1968). 

 Briefly, a 100-g wedge was removed from the 

 dorsal side of the fish. This wedge was bound- 

 ed by the first and fourth ray and just above 

 the lateral line. This wedge was immediately 

 frozen in liquid nitrogen and later packed in 

 dry ice and shipped to the BCF laboratory on 

 Terminal Island, Calif., and later the Univer- 

 sity of California in Berkeley for analyses. 



Chemical Analyses 



Four muscle wedges from each group of five 

 were used as follows by the BCF Laboratory. 

 The wedges were cut in half with one-half be- 

 ing returned to — 90° C storage for reference. 

 Twenty grams of the other half were ground 

 cold with 50 ml of 0.6 N HCIO4 and then neutral- 

 ized to pH 6.8 with 5N KOH. The potassium 



perchlorate precipate was allowed to settle and 

 was removed from the extract in the cold. The 

 extract was distributed into several vials and 

 frozen and stored at —90° C. In this manner, 

 no extract was thawed more than once when 

 analysis was carried out. 



Fructose (free) and fructose phosphate were 

 estimated on the neutralized extract by the 

 method of Roe (1934) and glucose phosphate 

 by the method of Morris (1948), after separa- 

 tion on Dowex 1x8 (chloride) resin and elu- 

 tion with 2N HCl (Spinelli et al, 1964). The 

 free sugars (glucose, fructose, and ribose) are 

 not retained on the column and are collected 

 as the extract is put over the column to remove 

 the phosphorylated sugars, glucose phosphate, 

 and fructose phosphate (nucleotides are also 

 held on the column). Glucose phosphate and 

 fructose phosphate were estimated by mixing 

 5 ml of the HCl wash in a 19-mm test tube with 

 10 ml of 0.2% Anthrone in 95% H2SO4; ab- 

 sorbance was read 10 min later at 620 mix. 



Fructose phosphate was determined on the 

 same HCl wash from the column by mixing 

 a 1-ml aliquot in a 19-mm test tube with 1 ml 

 of 0.1% resorcinol in 95% absolute alcohol and 

 3 ml of 30 % HCl. The test tube was heated for 

 8 min in an 80° C water bath, cooled and read 

 at 490 m/Li. This result was subtracted from the 

 glucose-fructose phosphate estimation, leaving 

 an estimation of glucose phosphate. Free fruc- 

 tose was determined by the Roe method. Ri- 

 bose was determined by the method of Mejbaum 

 (1939) after adjusting the extract to pH 11 and 

 passing it over Dowex 1x8 columns to remove 

 all ribose containing nucleotides, nucleosides, 

 and phosphorylated sugars. A 3-ml aliquot of 

 the resin treated extract was mixed in a glass 

 stoppered test tube with 1% resorcinol in 0.1% 

 FeCls in concentrated HCl. The tubes were 

 heated in boiling water for 30 min and cooled, 

 and the absorbance was read at 670 mfi. 



Free glucose, pyruvate, and lactate were de- 

 termined enzymatically with commercial kits 

 purchased from Calbiochem of Los Angeles, 

 Calif. Total i-educing sugars were estimated 

 by the previously described method of Morris. 



Hypoxanthine (Hx) was determined on the 

 neutralized extract by the xanthine oxidase 

 method of Spinelli et al. (1964). That is, the 

 shift in optical density at 290 mfi was deter- 

 mined on a known amount of Hx treated with 



14 



