International Journal of Aquaculture, 2025, Vol.15, No.4, 184-196 http://www.aquapublisher.com/index.php/ija 192 treatment at 37 °C. Similarly, juvenile fish with zebrafish genotype of female can all develop into "pseudo-male" individuals after treatment at 37 °C. These high temperature-induced maleization effects are related to the increased cortisol levels in the body and the inhibition of aromatase activity. In addition to temperature, breeding density and social structure are also key factors. Groupers may delay sexual maturation and sexual transition under high-density feeding conditions, as crowded environments usually increase fish body stress levels (elevated cortisol) and alter their behavioral ratings, which may inhibit the formation of dominant individuals. Many hermaphrodite fish are more likely to undergo sexual reversal or earlier in solitary or low-density conditions. This is because social stimulus is reduced and individuals do not need to maintain female identity to integrate into the group structure, so they tend to be more male (Wu and Chang, 2017). The relationship between nutritional status and gender is also worth mentioning. Full nutrition and rapid growth often enables the fish to reach the physique required for sexual maturity, which may promote gender switching (in hermaphrodite fish, large individuals are usually able to play the role of males). Some studies have pointed out that sexual reversal will only occur when the weight of a red spot grouper reaches a certain threshold, otherwise small females tend to maintain females to preferentially increase their body shape. Therefore, in breeding practice, improving the nutrition of the bait and managing stocking density well, so that some individuals can grow rapidly, which is expected to shorten the time required for their sexual reversal, which is beneficial for breeding plans that require male fish to be used as seeds. 6.2 Experiment on induction of gender conversion by exogenous hormone treatment Artificial application of exogenous hormones is a classic way to change the gender of fish, and has been tried on many species such as grouper. For hermaphrodite fish, exogenous hormones are mainly used to accelerate or synchronize their gender conversion process, so that they can change in the direction expected by humans. The most commonly used method is to feed or inject androgens to induce maleization of female fish. Literature reports that semen tissue can be observed in orange grouper less than one instar after MT feeding for more than 8 weeks, although sexual reversal is temporary at this time (requiring continuous hormone administration). Once the medication is stopped, the unstable semen structure may partially degenerate or even restore ovarian function (Figure 3) (Soyano et al., 2022). On the other hand, the application of estrogen (such as estradiol E2) can be used to inhibit maleization and maintain the female state. In commercial seedling cultivation, there have been attempts to prevent the juvenile fish from maleization by short-term soaking estrogen to obtain a whole female population. However, since grouper is usually female first and then male, the whole female group has little significance for production, but the whole male group has attracted more attention. In addition to sexual steroids, gonadotropins (such as human chorionic gonadotropins hCG, carp luteinizing hormone, etc.) are also used to try to trigger sexual reversal. For example, injection of large amounts of hCG in mature female fish can indirectly increase endogenous androgen levels, and as a result, partial reversal was observed on oblique band grouper (Peng et al., 2020). Furthermore, the application of corticosteroid hormones provides another idea. In practical applications, MT is still the most effective and commonly used inducer. To ensure food safety, hormonal feed is rarely used directly in commercial cultivation, but MT treatment is still a convenient means to establish a whole-male or whole-female experimental group in scientific research. It is also worth noting that different species and developmental stages respond to hormones differently, and the dose and treatment time need to be adjusted. Therefore, for grouper, it should be based on its own physiological optimization plan. Some studies compared the effects of MT oral injection and intramuscular injection on grouper, and found that oral injection is mild but takes longer, the injection starts quickly but the dose is not easy to control and has a risk of trauma. 6.3 Interaction between environmental factors and gene expression Environmental factors affect gene expression through endocrine pathways, and changes in gene expression will in turn shape the phenotype. This interaction is particularly complex in the process of gender determination. Taking temperature as an example, high temperature not only directly affects the activity and gene expression of aromatase in the gonad, but also inhibits the expression of genes such as Foxl2 and cyp19a1a through cortisol-mediated pathways, thus making gender differentiation biased towards males. In addition, high temperatures may alter the activity of DNA methylases and histone modifier enzymes in the gonad, resulting in changes in the apparent modification status of certain key genes (Wang et al., 2020). Looking at social factors, the
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