PHEROMONAL STIMULATION AND METABOLITE INHIBITION OF 

 OVULATION IN THE ZEBRAFISH, BRACHYDANIO RERIO' 



Lo-CHAi Chen and Robert L. Martinich' 



ABSTRACT 



Female zebrafish, Brachydanio rerio, would not ovulate in the absence of males in waters previously 

 inhabited by the fish. Chemical presence of males and fresh, dechlorinated tap water each induced 

 ovulation in about half of the trials. Application of the two factors in combination gave 100% ovulation. 

 These results suggest that in the zebrafish a pheromone released by the males stimulates ovulation and 

 that metabolites produced by the fish repress ovulation. It is postulated that metabolites restrict 

 spawning of the fish to the rainy season and that the pheromone functions in synchronizing reproduc- 

 tive readiness between sexes or in conserving courtship energy expenditure. 



In a study concerning egg size, incubation period, 

 and growth in the zebrafish, Brachydanio rerio 

 (Hamilton-Buchanan), we encountered the 

 problem of having to strip eggs from the females 

 to synchronize the fertilization of the eggs ar- 

 tificially. We tried the method described by Hart 

 and Messina (1972) without successes. We have 

 regularly been able to induce natural spawning by 

 introducing ripe individuals of both sexes from 

 their holding tanks at 27°C together into a bowl of 

 fresh, dechlorinated tap water at 21 °C. Some of 

 the changes associated with this introduction, such 

 as the chemical presence of the males 

 (pheromone), the physical presence of the males 

 (visual, auditory, tactile, and lateral line), fresh 

 tap water (absence of accumulated metabolites), 

 and temperature shock (from 27° to 21°C), may be 

 capable of inducing ovulation. 



The roles of these factors in controlling 

 reproduction in fishes have been well documented. 

 Some of the examples are: 



Pheromones — Aronson 1945; Tavolga 1956; 

 Amouriq 1965; Gandolfi 1969; Rossi 1969; and 

 Chien 1973. 



Visual-Aronson 1945, 1965; Tavolga 1956; Rossi 

 1969; and Chien 1973. 



Auditory-Tavolga 1956; Brawn 1961; Gray and 

 Winn 1961; and Myrberg and Spires 1972. 



Tactile-Egami and Nambu 1961. 



Metabolites— Swingle 1956; and Greene 1966. 



'The data in this paper were extracted from the master's thesis 

 of R. L. Martinich. 



-Department of Zoology, San Diego State University, San 

 Diego, CA 92182. 



Temperature— Harrington 1959; Aronson 1965; 

 and de Vlaming 1972a, b. 

 Much of the information in the literature, 

 however, does not clearly distinguish between 

 gonad development, ovulation, and spawning. The 

 present study was undertaken to single out such 

 ovulation-inducing factors. 



MATERIALS AND METHODS 



Female zebrafish were kept at 27.0 ±. 1.0°C in 

 40- or 60-liter aerated aquaria subdivided into 

 three or four compartments by perforated plastic 

 dividers, one female per comparment to enable 

 identification. Male zebrafish were isolated in 

 aerated 20-liter aquaria at room temperature (21.0 

 ±. 1.0°C). No visual contact was permitted 

 between sexes. All individuals were subjected to 

 12 h of light per day and were generously fed 

 "Tetramin"' in the morning and frozen brine 

 shrimp in the evening. To ensure fertility, each 

 fish was initially permitted to spawn naturally. 

 This was done by introducing the fish into fresh, 

 dechlorinated tap water at room temperature in a 

 20-cm finger bowl with another individual of the 

 opposite sex. A total of 27 fertile females and 19 

 fertile males were used. 



Eight experiments were designed to test the 

 relative contribution of each factor individually, 

 and in combination, on ovulation in the zebrafish 

 (Table 1). In experiments 1 to 7, a female was 

 transferred from the holding compartment into 



Manuscript accepted February 1975. 

 FISHERY BULLETIN: VOL" 73, NO. 4, 1975. 



'Reference to trade names does not imply endorsement by the 

 National Marine Fisheries Service, NOAA. 



889 



