In sexually reproducing species, the quality of gametes (eggs and sperm) is essential for reproductive success. High-quality gametes can enhance fertilization rates, embryo survival, and offspring fitness, ultimately leading to the production of high-quality offspring. This principle is common across vertebrates, including fish (Valdebenito et al., 2015), birds (Assersohn et al., 2021), reptiles (Friesen et al., 2020), and mammals (Leoni et al., 2007). In animals undergoing in vitro fertilization (such as many fish species), gamete quality becomes especially important because parental control over the gametes is limited after release. Therefore, the viability of eggs and sperm is a key indicator in predicting reproductive potential, and it influences the developmental quality and early survival of the offspring (Ochokwu et al., 2015; Valdebenito et al., 2015). The quality of fish eggs and sperm is a crucial reproductive factor that affects the normal development of embryos, and differences in quality have significant adaptive consequences for reproductive success. For instance, in evolutionary ecology, gamete quality can influence sexual selection and mate choice strategies (Kekäläinen and Evans, 2018; Kelly and Jennions, 2011). In aquaculture practice, these differences are directly linked to the efficiency of aquatic breeding and the survival rates of offspring (Bobe and Labbé, 2010). In the realm of population conservation, gamete quality serves as a crucial foundation for assessing habitat quality and protecting endangered species (Viveiros and Godinho, 2009; Yaripour et al., 2020). Many studies have shown that the quality of both male and female gametes is determined by several factors, including environment, age, nutrition, stress, husbandry practices, etc. (Bobe and Labbé, 2010), often ignoring the impact of inter-individual differences in behavior. As is widely known, inter-individual differences in behavior, commonly referred to as personality traits (Gosling, 2001), are consistent over time and across contexts. Personality influences important life activities of species such as resource acquisition, health, survival, and reproduction, and ultimately their fitness (Sgoifo et al., 2005). A meta-analysis on the fitness consequences of personality indicates that bolder individuals tend to have increased reproductive success in various animal species (Smith and Blumstein, 2008). For example, bolder and more aggressive male zebrafish (Danio rerio) can fertilize more eggs (Ariyomo and Watt, 2012). Bolder black-legged kittiwake pairs (Rissa tridactyla) show higher chick survival compared to shyer pairs (Collins et al., 2019). Bolder male eastern chipmunks (Tamias striatus) produce more offspring (Patterson and Schulte-Hostedde, 2011). Bolder young female gray seals (Halichoerus grypus) give birth to heavier pups than their shyer counterparts (Bubac et al., 2018).

In the field of animal personality, the bold-shy continuum is one of the most widely studied axes of behavioral variation, which refers to the propensity of an animal to take a risk in new environments (Coleman and Wilson, 1998; Toms et al., 2010). Bold individuals take more risks in unfamiliar environments (Smith and Blumstein, 2010; Wilson et al., 1994), respond to stress more actively (Rupia et al., 2016), forage more successfully (Bell and Stamps, 2004; Sundström et al., 2004), are more preferred in mate choice (Godin and Dugatkin, 1996), and have higher reproductive success than shy individuals (Collins et al., 2019; Patterson and Schulte-Hostedde, 2011). To date, the link between personality and reproductive success has been extensively studied in various animal taxa, including mammals (Martin-Wintle et al., 2017; Masilkova et al., 2022), birds (Both et al., 2005), fish (Ariyomo and Watt, 2012), and crustaceans (Bridger et al., 2015). Most of the research results on fish provide useful information on the effect of personality on reproduction by measuring reproductive performance indicators, mainly quantitative traits such as number of eggs, sperm quality, number of fertilized eggs, and volume of yolk sac (Ariyomo and Watt, 2012; Gasparini et al., 2019; Torres et al., 2023; Vargas et al., 2018). It is important to note, however, that the reproductive success of fish under given environmental conditions depends on a variety of complex networks of physiological processes, including multiple neuroendocrine systems (Yaron and Sivan, 2011; Zohar et al., 2010). Yet, the mechanistic pathways linking behavioral phenotypes to reproductive fitness—particularly the potential mediating role of these physiological systems—remain poorly understood. Therefore, this study examines the differences in the hypothalamic-pituitary-gonadal (HPG) axis between bold and shy individuals, along with their implications for gamete quality, which can influence reproductive success.

Gonadal development and maturation, as well as reproductive performance in teleost fishes, are complexly regulated by the neuroendocrine HPG axis. When fish gonads are stimulated by gonadotropins (GtH), glycoprotein hormones that regulate gonadal development and maturation, sex hormones are secreted to promote gonadal development and ultimately affect the reproductive outcomes (Schulz et al., 2001). Specifically, gonadotropin-releasing hormone (GnRH) secreted by the hypothalamus binds to its receptor (GnRHR), stimulates the pituitary to synthesize and release GtH, namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH), thereby regulating the secretion of testosterone (T) and 17β-estradiol (E2) by the gonad to control gonadal maturation and spawning in fish (Elakkanai et al., 2015). As is well known, FSH is believed to control vitellogenesis and spermatogenesis in fish, while LH regulates oocyte maturation/ovulation and sperm production (Levavi-Sivan et al., 2010), both of which affect gamete quality. E2 in females can promote oogonial proliferation and vitellogenesis (Yaron and Sivan, 2011), and T in males can promote spermatogonial multiplication and spermatocyte formation (Billard et al., 1982), while the formation of fertilized ovum and normal embryo development are controlled by sperm (such as motility and volume) and egg quality (including yolk composition) (Mahalingam and Santhanam, 2023). Sex hormones, an important category of steroid hormones, are synthesized beginning with the transfer of cholesterol, which is a rate-limiting process mediated by the steroidogenic acute regulatory protein (StAR) on the outer mitochondrial membrane. Cholesterol is converted to sex hormones by a series of steroidogenic enzymes. The genes encoding these enzymes include three key cytochrome P450 enzymes: cholesterol side-chain cleavage enzyme (cyp11a1), CYP17α hydroxylase/17,20 lyase (cyp17), and aromatase (cyp19a), as well as hydroxysteroid 17β-dehydrogenase (hsd17β) and hydroxysteroid 3β-dehydrogenase (hsd3β) (Tokarz et al., 2015). In addition, it has been reported in the literature that individual differences in aggressive behavior affect sex hormone levels in fish (Alcazar et al., 2016; da Silva et al., 2021; Elofsson et al., 2000). Therefore, we conjecture that the effect of boldness on fish reproductive success may be mediated by key molecular factors within the HPG axis; their biological roles, however, remain incompletely understood.

Zebrafish has the advantages of small size, rapid development, and high fecundity, and as a popular model organism, is often used in various behavioral paradigms. Established evidences have denoted that the boldness traits of zebrafish can be quantified effectively through the open field test (Bellot et al., 2022; Lucon-Xiccato et al., 2020). Additionally, as a highly social species, zebrafish exhibit significant individual differences in behaviors such as aggression and dominance-related interactions. These behavioral variations have been shown to significantly impact reproductive performance (Ariyomo and Watt, 2012; Paull et al., 2010). This is conducive to making zebrafish a valuable model for exploring how behavioral phenotypes influence reproductive physiological differences among individuals. Existing evidence shows that bold individuals have a higher reproductive success (Smith and Blumstein, 2008). Based on this, we assume that bold zebrafish possess advantages in reproductive physiology compared to shy individuals. Therefore, to investigate the physiological processes of reproduction in zebrafish with different behavioral traits, we used the open field test to distinguish between bold and shy adult zebrafish, and then measured body condition, sex steroid hormones, and analyzed gonad histology, as well as further detected the relative mRNA levels of HPG axis-related genes.