FISHERY BULLETIN: VOL. 69, NO. 1 



expose the brain; the gland's cavity and stalk 

 are readily seen when the brain is lifted from 

 its cavity and the entire gland can be removed 

 with forceps. A direct approach is more appro- 

 priate to the speed with which spawning ac- 

 tivities proceed and 1 day of practice is suffi- 

 cient to enable one person to remove glands as 

 fast as fish are available without undue loss of 

 damaged or partial pituitaries. 



The glands are placed directly in chilled ace- 

 tone until spawning and gland-taking activities 

 are finished. The acetone is decanted and re- 

 placed 2 or 3 times over a period of 3 days until 

 all loose material is washed away. The final 

 wash of acetone is decanted, and the glands are 

 blotted gently on filter paper, placed in tightly 

 stoppered vials, and kept at — 10° C in a jar 

 of desiccant. Glands stored 1 or 2 years by 

 this method showed no detectable loss of activity. 



It was found that pituitaries were easier to 

 homogenize if they were not completely dried. 

 Just prior to preparing a stock solution, the 

 glands were removed from vials, air-dried for a 

 few minutes, and weighed. They were then 

 immediately placed in glass tissue grinders and 

 homogenized in sesame oil or saline. The re- 

 sulting brei was finely divided and was used 

 unfiltered without clogging 25- and 26-gauge 

 needles. Stock solutions (1-10 mg/ml) were 

 stored in rubber-capped serum bottles (5 ml) at 

 — 10° C. All injections were 1.0 to 0.5 ml im 

 and up to 1 ml ip. I used oil rather than saline 

 in most cases on the assumption that oil may 

 slow the rate of absorption and more evenly dis- 

 tribute the hormone. 



EVALUATING THE EFFECTS OF 

 HORMONE TREATMENT 



Three basic criteria — gonad index, spawning, 

 and fertilization of eggs — were chosen to test 

 the effects of gonadotropin injections. Devel- 

 opment of fertilized eggs to hatching is also a 

 useful test of the absolute success of the hormone 

 treatment used to obtain spawn, but hatching 

 success is also very sensitive to other factors, 

 e.g., salinity, temperature, dissolved oxygen, and 

 bacterial contamination. 



A commonly used measure of the effects of 



hormone treatment is the gonosomatic index 

 (GSI) which expresses wet gonad weight as a 

 percent of total wet body weight. The GSI is a 

 reasonable measure of the state of reproductive 

 maturity of a fish. Its measurement, of course, 

 requires that the fish be sacrificed. Histological 

 examination of gonads or measuring egg di- 

 ameters to assess the stage of maturation are 

 more elaborate approaches which were not used 

 after it was found that GSI accurately predicted 

 spawning readiness of the fish. Mclnerney and 

 Evans (1970) have shown a direct relationship 

 between GSI and the histological index in fe- 

 male threespine stickleback. Among more re- 

 cent approaches to this problem is Stevens' 

 (1966) development of a technique for removing 

 eggs from striped bass by means of a catheter. 

 He was able to determine the stage of the eggs 

 and predict the time of optimal ripeness with 

 a fair degree of accuracy. 



Samples of 4 or 5 fish were used to assess the 

 effects of hormone treatment. Controls of un- 

 injected or sham-injected groups of fish were 

 maintained where these were appropriate to 

 understanding the eflfects of hormone treatments 

 on GSI. The GSI of laboratory-held fish var- 

 ied, understandably, through time, and the ef- 

 fects which this had on the results obtained are 

 discussed where appropriate. The dates of most 

 experiments are given to help explain this var- 

 iation. 



Two fundamental processes in spawning, hy- 

 dration and ovulation, were evaluated separately 

 with respect to various hormone treatments. 

 Hydration was measured as an increase in total 

 body weight over a period of 1 to 2 days after 

 injection, a process distinct from gonad growth 

 which occurs over longer time periods. This 

 rapid weight gain is due mostly to water uptake 

 by the fi.=h and is reflected in much higher GSI 

 values, as most of the water appears to go into 

 the gonad. Externally, a hydrated fish is grossly 

 bloated, and, in some cases, the fish are listless 

 and remain motionless on the bottom of the 

 tank. Ovulation was assessed by attempting to 

 strip eggs from fish at various times after in- 

 jection. Eggs may be forced from nearly ripe 

 fish, but these eggs invariably are surrounded 



162 



