CHEN and MARTINICH: OVULATION IN ZEBRAFISH 



chemically continuous with the experimental 

 chamber because of the water circulation. 



Visual stimuli from a male were provided in 

 experiment 5 by allowing the test female to be in 

 visual contact with a mature male in a separate, 

 chemically discontinuous all-glass aquarium. In 

 experiment 3, the perforations of the divider par- 

 titioning the male chamber and the experimental 

 chamber provided questionable visual stimuli 

 from the male. In all other experiments (1, 2, 4, 6, 

 and 7), visual stimuli from males were screened by 

 visually isolating the experimental chambers with 

 cardboard or black plastic sheet. 



Possible auditory and lateral line stimuli from 

 male were allowed in experiment 3 through the 

 perforated condition of the partition separating 

 the experimental chamber from the male chamber. 



In experiment 6a, the experimental chamber 

 was a simple 5-liter all-glass aquarium, whereas in 

 experiment 6b a current similar to that in 

 experiments 1, 2, 4, and 7 was provided by air lift- 

 ing and back-siphoning of water between the 

 experimental chamber and a vacated 5-liter 

 aquarium. 



Prior to each experiment, the glass aquaria and 

 hoses were scrubbed, soaked, and thoroughly 

 rinsed with tap water. Experiments testing the 

 pheromonal responses utilized a different set of 

 hoses from those used in experiments lacking the 

 male pheromones. Different nets were used for 

 netting males and females as a precaution against 

 contacting a test female with the slime of a 

 male. 



RESULTS 



The number of positive responses (trials eggs 

 obtained) and negative responses (trials no eggs 

 obtained) are given for each of the eight 

 experiments in Table 1. The results of each 

 experiment were compared with those of 

 experiment 8 and the chi-square value was cal- 

 culated. The percent of trials resulting in a posi- 

 tive response is also given for each experiment. 



It is apparent from the results that presence of 

 pheromone and absence of metabolites are the two 

 most influential factors stimulating ovulation in 

 the zebrafish. Experiments 1, 2, and 3 in which 

 pheromone was provided and metabolites were 

 absent invariably gave nearly 100''^ ovulation. 

 Experiments 4, 5, 6, and 7 in which either 

 pheromone was provided or metabolites were ab- 

 sent gave 40-50% ovulation. However, in 



experiment 8 in which pheromone was absent and 

 metabolites were present, no ovulations were ob- 

 served. The roles of metabolites and male 

 pheromone are further indicated by the fact that 

 females which initially responded negatively in 

 experiments lacking the male pheromone and/or 

 fresh tap water (experiments 4, 5, 6, and 7) gave 

 eggs in the second stripping in all cases upon being 

 presented with the missing factor(s). 



None of the other factors tested, including 

 temperature shock and auditory and/or lateral 

 line stimulations seem important in controlling 

 ovulation, as in no cases did their presence or ab- 

 sence significantly alter the results. The influences 

 of water movement between the experimental 

 chamber and the male chamber were insignificant, 

 as there is no difference between results of 

 experiments 6a and 6b. 



Successful stripping was recorded at all times 

 over the morning, afternoon, and early evening. 

 After the stripping, without exception, a pair 

 would commence natural spawning immediately 

 upon introduction regardless of the time of day. 



DISCUSSION 



In the absence of the male pheromone and the 

 presence of the metabolites, females consistently 

 failed to ovulate (experiment 8). Under similar 

 condition, Eaton and Farley (1974) also failed to 

 strip eggs from isolated females. Histological 

 studies of zebrafish ovaries by Hisaoka and Firlit 

 (1962) indicated that oocytes are not released from 

 the ovarian stroma into the central lumen and 

 oviducts (ovulation) until stimulated by males 

 during the breeding process. Eaton and Farley 

 (1974) were able to obtain ripe ova from isolated 

 females in the morning only after a brief (7 h) 

 introduction of a male into the female's tank on 

 the previous day. They suggested that the 

 vigorous chasing behavior exhibited by the male 

 toward the female might have provided the 

 stimulus, although no supporting data were 

 provided. 



The results of the present study clearly establish 

 that a male pheromone stimulates ovulation of the 

 female. Little is known about the nature of the 

 male pheromone. It appears not to be species 

 specific since circulation of water between 

 aquarium containing male Brachydanio al- 

 hoUneatuH and aquarium containing female B. 

 rerio elicited ovulation in the female in four out of 

 four trials. Intrageneric interspecific effec- 



891 



