I 



E. Casillas 



L. Smith 



B. G. D'Aoust 



Effects of Stress 



on Salmonid 



Blood Clotting Mechanisms 



ABSTRACT 



Enhancement of blood clotting functions is a possible factor 

 which could kill fish following stress resulting from exposure to 

 supersaturation. During examination of various hematological 

 parameters in rainbow trout which were subjected to stress from 

 exercise, blood coagulation times were found to decrease to 45% 

 of the original pre-stress values within a half hour after the 

 termination of the stress period. Thrombocyte counts were 

 found to increase three- to fourfold in the same period. Hemato- 

 crits and blood plasma glucose also rose significantly with 

 respect to the stress applied. Red blood cell and white blood 

 cell counts, however, did not increase in response to the stresser. 

 The degree of the responses observed were compared between 

 members of a wild trout population from Chester Morse, Wash- 

 ington, and hatchery-reared Donaldson-strain rainbow trout. 

 The wild strain showed a more rapid return to pre-stress condi- 

 tions than the hatchery-reared trout. Based on very preliminary 

 experiments, the responses of the clotting mechanisms in Pacific 

 salmon are similar and probably change even more rapidly than 

 in rainbow trout. This variability in the blood coagulation rate 

 is proposed as a mechanism to avoid disseminated intravascular 

 coagulation (D.I.C.) in the poorly perfused muscles of fish. 

 Experiments in this area are continuing. 



Most of the bioassays regarding gas bubble disease 

 have reported that the incipient lethal dose ol 

 supersaturation for salmonids kept in shallow water 

 is in the vicinity of 120% saturation for exposures of 

 more than 24 hr (Meekin and Turner, 1974). Work 

 reported by Beyer, D'Aoust, and Smith (this confer- 

 ence) indicated that 6 cm fish come to equilibrium 

 with the gas content of its environment in less than 

 2 hr. Based on these results, maximum lethality 

 should occur soon after 2 hr of exposure to any 

 supersaturation level, which however, is only the 

 case at high level supersaturation. It therefore seems 

 reasonable to propose that some other mecha- 

 nism(s) in addition to or instead of simple bubble 

 formation operate to cause mortality in fish during 

 long-term exposure to low levels of supersaturation. 

 One such factor — blood clots — will be presented in 

 this report as a possible contributing factor in 

 making low-level supersatu rations eventually lethal. 



METHODS 



Rainbow trout from the University of Washing- 

 ton hatchery (Donaldson strain) and from Lake 



Chester Morse (an undisturbed watershed 30 miles 

 east of Seattle) were used in all of the experiments. 

 A typical trout from the hatchery was about 700 g 

 in weight, 12 to 14 months of age, and reared on a 

 commercial diet of Oregon Moist Pellets®. The trout 

 from Lake Chester Morse were of similar size and 

 weight, but of unknown age and diet. They were 

 captured by hook and line and transported to our 

 university laboratory in oxygenated tanks. They 

 were kept in the lab in running lake water for at 

 least 2 weeks before being used in experiments. 



To perform an experiment, a plastic tube was 

 inserted into the dorsal aorta and anchored in the 

 nasal cartilages using the method of Smith and Bell 

 (1964). MS-222 anesthetic, but no anticoagulants, 

 was used during the surgery, and then a recovery 

 period of 48 hr was allowed before beginning an 

 experiment. Stress was produced by placing a hook 

 in the caudal peduncle and producing vigorous 

 activity by the fish for 2 min, after which the fish 

 exhibited strong signs of fatigue. One blood sample 

 was taken before the stress period and additional 

 samples were taken for up to 5 hr afterwards. Blood 

 samples were replaced with an equal volume of 

 physiological saline, with the total blood volume 

 removed by sampling being less than 5% of the 

 fish's total blood volume. Blood samples used to 

 determine clotting time were collected into non- 

 heparinized capillary tubes, while those samples 

 used for other analyses were collected into hepa- 

 rinized tubes. Analyses included determination of 

 red and white cell counts, thrombocyte count, 

 hematocrit, and plasma glucose concentration. 



Control fish were treated exactly the same as 

 the experimental fish except for the omission of the 

 period of stress. 



RESULTS 



The decrease in blood clotting times for the 

 stressed fish are shown in Fig. 1 and are nearly 

 the same in both strains of fish, except that the 



Casillas and Smith: University of Washington, Seattle, Washing- 

 ton; and D'Aoust: Virginia Mason Research Center, Seattle, 

 Washington. 



93 



