On the proper rearing density in domesticated zebrafish to avoid unwanted masculinization. Links with the stress response

Abstract

The zebrafish (Danio rerio) has become a well-established experimental model in many research fields but the loss of the primary sex-determining region during the process of domestication renders laboratory strains of zebrafish susceptible to the effects of environmental factors on sex ratios. Further, an essential husbandry aspect -the optimal rearing density to avoid stress-induced masculinization -is not known. We carried out two experiments: the first focusing on the effects of density on survival, growth and sex ratio by rearing zebrafish at different initial densities (9, 19, 37 and 74 fish per litre) for 3 months (6-90 days post-fertilization, dpf ), and the second focusing on the effects of cortisol during the sex differentiation period (15-45 dpf) for zebrafish reared at low density. The results showed an increase in the number of males in groups subjected to the two highest initial rearing densities; we also observed a reduction of survival and growth in a density-dependent manner. Furthermore, zebrafish treated with cortisol during the sex differentiation period showed a complete masculinization of the population; treatment with the cortisol synthesis inhibitor metyrapone negated the effects of exogenous cortisol. Our results indicate that the process of sex differentiation in domesticated zebrafish can be perturbed by elevated stocking density and that this effect is likely to be mediated by an increase in cortisol through the stress response. However, the underlying mechanism needs further study.

Response to “The importance of controlling genetic variation – remarks on ‘Appropriate rearing density in domesticated zebrafish to avoid masculinization: links with the stress response’”

“…If the data for all five families are combined, differences are significant (P=0.0014). These data, along with the data presented in Ribas et al (2017a), clearly illustrate that at a rearing density of 40 fish l −1 or higher, masculinization occurs in zebrafish. Although most families tend to increase the number of males in response to elevated density, some show statistical significance and some do not.…”

“…1C of Ribas et al, 2017b). Furthermore, in Ribas et al (2017a) we clearly stated that ongoing experiments in our laboratory confirm the masculinizing effects of elevated densities. We advance the sex ratio results of these experiments below, which are part of a larger study on other aspects of the effects of rearing density.…”

“…Although some sources (e.g. FishBase) claim higher values, the absolute fecundity of zebrafish females is typically around 300 eggs (Ribas and Piferrer, 2014 Ribas et al (2017a). In that study, pairs 2, 3 and 4 showed an increase in the number of males, while pair 1 showed the opposite effect but, again, the masculinizing effect of elevated density was seen when the sex ratios obtained at each density were compared with the sex ratios of the lowest density, as also stated.…”

“…PLoS ONE 7, e34397. Ribas, L., Valdivieso, A., Dıáz, N. and Piferrer, F. (2017 Response to "The importance of controlling genetic variationremarks on 'Appropriate rearing density in domesticated zebrafish to avoid masculinization: links with the stress response'" We appreciate the comment by Delomas and Dabrowski to the Ribas et al (2017a) paper and want to mention that we were very much aware of the possible effect of genetic differences in the sex ratio response to increasing rearing density from the beginning. This is why, in each one of the four replications of experiment 1, we avoided using pooled eggs and sperm from different progenitors, and instead used eggs and sperm derived from a single dam and sire, respectively.…”

Ovarian transcriptomic signatures of zebrafish females resistant to different environmental perturbations

“…In contrast, in the European sea bass, no effects were observed when fish were confined during the early stages of development (Saillant et al, ). In zebrafish ( Danio rerio ), elevated density resulted in male‐biased sex ratios (Ribas, Valdivieso, Díaz, & Piferrer, ) with interfamily variation (Liew et al, ; Ribas, Valdivieso, Díaz, & Piferrer, ) due to genotype–environment interactions.…”