Bone deformation is a multifactorial pathology of fish mainly associated with environmental, nutritional, and genetic factors (Iaria et al., 2021). More importantly, its incidence is considered a quality index (Boglione et al., 2013; Le Luyer et al., 2015; Lall and Kaushik, 2021). Environmental and nutritional factors related to management conditions, physicochemical characteristics, fish density, nutrients, and microelement composition within the diet (Boglione et al., 2013). At the same time, genetic factors such as the aberrant expression of genes participating in processes like bone tissue formation, remodeling, and repair can also affect different and relevant pathways (Krossøy et al., 2009; Boglione et al., 2013).

Many genes have been implicated in different stages of skeleton formation in salmonids, constituting a complex of highly regulated signaling pathways represented by families such as bone morphogenetic proteins (Bmps), waterproof (Wat), and neurogenic locus notch homolog protein (Notch) proteins (Day et al., 2005; Lin and Hankenson, 2011; Zhao et al., 2019). During osteogenesis, the expression of sonic hedgehog (Shh) genes involved in axial bone growth, the spine, and fins (Olivares and Rojas, 2013) and homeobox (Hox) genes related to skull and tooth growth regulate the activation of these signaling pathways (Theslelf, 1995; Liu et al., 2004; Lin and Hankenson, 2011).

BMPs represent one of the best-described pathways, with more than 30 isoforms reported. Bone morphogenetic protein 2 (BMP2), bone morphogenetic protein 4 (BMP4), bone morphogenetic protein 5 (BMP5), bone morphogenetic protein 6 (BMP6), bone morphogenetic protein 7 (BMP7), bone morphogenetic protein 8 (BMP8), bone morphogenetic protein 9 (BMP9), bone morphogenetic protein 11 (BMP11), bone morphogenetic protein 12 (BMP12), bone morphogenetic protein 13 (BMP13), and bone morphogenetic protein 14 (BMP14) have osteogenic activity (Dumic-Cule et al., 2018). However, one of the critical BMP markers corresponds to Bmp2/BMP2 since it allows bone regeneration by activating the expression of transcription factors such as SRY-box transcription factor 9 (Sox9), RUNX family transcription factor 2 (Runx2), and Osterix (Osx), leading to bone formation and development (Ducy et al., 1997; Kirkham and Cartmell, 2007; Matsubara et al., 2008; Lin and Hankenson, 2011).

Runx-2 has been a transcription factor considered an exclusive marker of mineralized tissues, playing a central role during osteogenesis (Kirkham and Cartmell, 2007). Its activation directly stimulates the transcription of genes such as osteocalcin, osteopontin, collagen I, collagenase 3 (matrix metalloproteinase 1), sialoprotein, and alkaline phosphatase (ALP), all related to extracellular matrix formation (Ducy et al., 1997; Franceschi and Xiao, 2003; Kirkham and Cartmell, 2007; Lin and Hankenson, 2011). Osterix, also known as transcription factor Sp7, is another transcription factor essential for osteoblast differentiation and bone construction (Nakashima et al., 2002; Kirkham and Cartmell, 2007). Its activation has been related to the formation, development, and maturation of bone tissue during embryogenesis, the formation of new bone and osteoblast differentiation (Komori et al., 1997; Nakashima et al., 2002; Kirkham and Cartmell, 2007; Matsubara et al., 2008; Zhou et al., 2010; Sinha and Xin Zhou, 2013).

Among the proteins involved in bone matrix formation, the most relevant are osteocalcin (OCN), collagen type I (COL-I), and alkaline phosphatase (ALP). Osteocalcin is a protein secreted by osteoblasts as part of the extracellular matrix and is the most abundant of the non-collagenous proteins (Koga et al., 2005; Fernández-Tresguerres et al., 2006; Perez-Amodio et al.; 2006; Hayman, 2008). Collagen type I is an extracellular matrix protein in bone tissue and represents the most important and abundant of the collagen proteins found in bone (Matsubara et al., 2008; Gistelinck et al., 2016). The expression of Col-I has been considered an early marker of osteoblast activity and plays a crucial role in osteoblast differentiation (Matsubara et al., 2008). According to research, rainbow trout have a subunit composition of 1 (I), 2 (I), and 3 (I), with 3 (I) being specific for bone (Saito et al., 1998; Saito et al., 2001).

Alkaline phosphatase, due to its wide distribution in the body and the many isoenzymes it presents in the blood, saliva, liver, and kidney, represents an excellent early clinical marker for multiple pathologies in fish (Congleton and Wagner, 2006; Matsubara et al., 2008). Osteoblasts produce the bone ALP fraction, a feature of phenotypic differentiation, proliferation, and migration (Matsubara et al., 2008). Plasma levels of ALP represent a marker of osteoblastic activity (Delmas et al., 2000; Kuo and Chen, 2017). Moreover, there was a direct relationship between the presence of ALP and the correct mineralization of bone tissue in vertebrates (Delmas et al., 2000; Fernández-Tresguerres et al., 2006; Kuo and Chen, 2017). Another phosphatase fraction in bone tissue is an acid phosphatase, a marker of tartrate-resistant acid phosphatase (TRAP) activity (Fernández-Tresguerres et al., 2006; Perez-Amodio et al., 2006; Hayman, 2008). Finally, calcium and phosphorus in both bone and plasma represent systemic regulation of bone formation and remodeling, as these minerals are bioavailability indicators (Beck et al., 2000; Sugiura et al., 2003; Pombinho et al., 2004). Therefore, we aimed to evaluate the changes in the expression of genes involved in formation and regeneration and their relationship with bone mineralization in rainbow trout with mandibular deformation.

Successful genetic modification initiatives have been implemented in various salmonid species to enhance the production of commercially significant characteristics (Gjedrem, 2000; Gjedrem, 2012). However, the potential adverse outcome of selective breeding has been the accumulation of inbreeding (Robertson, 1961). In fact, there is a lot of inbreeding among rainbow trout because of the selective breeding for desirable traits in small, isolated populations (D’Ambrosio et al., 2019). Prior studies have demonstrated notable negative impacts of inbreeding on the weight of female rainbow trout and the weight of their spawn (Kincaid, 1983), as well as on the number of eggs produced and the age at which spawning occurs (Su et al., 1996). In addition, Chilean studies have shown that the breeding effort has shown significant genetic improvement in body weight at harvest, with an increase of approximately 10 to 13% per generation (Neira et al., 2006; Yañez et al., 2014).

Shape differences can be found in both farmed and wild fish, but they are studied more in aquaculture because they can lower the number of fish that are produced and cause financial losses (Kause et al., 2005). Genetic diversity in populations usually goes down when culture systems are in place. This can cause inbreeding depression and other problems (Aulstad and Kittelsen, 1971). Populations with inbreeding experience slower rates of growth and reproduction as well as increased vulnerability to externally caused mortality. Inbreeding can be bad for natural populations because it lowers the genetic diversity within a population (Kause et al., 2005) and increases the number of harmful alleles that people with two copies of the recessive gene often have (Keller, 2002). Although the exact cost of inbreeding is still uncertain and subject to debate (Pusey & Wolf, 1996; Crnokrak and Roff, 1999; Frommen et al., 2008), its repercussions potentially heighten the risk of extinction (Aulstad and Kittelsen, 1971; Keller, 2002; Kerniske et al., 2021).

In Chile, the calculated genetic diversity among rainbow trout (Oncorhynchus mykiss) was estimated to be between 22% (Cárcamo et al., 2015) and 15.6-28.1% (Gajardo et al., 1998), which falls within the range previously reported for this species (Currens et al., 1990). Additionally, the average heterozygosity of 0.070 is higher than the value of 0.059 reported previously (Hershberger, 1992) for the same species. According to previous research on rainbow trout, the estimates show that the populations being studied have a lot of room to adapt and/or split apart. n these studies, genetic diversity, measured as heterozygosity, has been linked to key fitness characteristics (Danzmann et al., 1988).

Blood analysis was helpful in evaluating the physiological state of all species, especially fish. Testing constitutes a minimally invasive procedure and is also useful for evaluating new diets, changes in culture conditions, immune status, or stress related to fish farming management (Kaneko, 1997; Bellier, 2010; Li et al., 2010; Servicio Nacional de Pesca y Acuicultura (SERNAPESCA), 2023). The metabolic parameters obtained in this study showed differences between total protein content (53.26 g/L vs. 41.83 g/L, p<0.01), mainly in the globulin fraction (25.23 g/L vs. 12.69 g/L, p<0.01). This contrast was linked to a nutritional deficit in fish with mandibular deformity because they are unable to feed properly, resulting in general deterioration with decreased size and weight and a drastic loss of visceral fat.

The increased alkaline phosphatase levels reported here imply a signal of phosphorus recovery at the systemic level (10.44 mmol/L and 7.57 mmol/L, p< 0.001). In addition, it relates to renal reabsorption and removal by the action of enzymes (Fernández-Tresguerres et al., 2006). Calcium levels presented differences between the control (9.96 mmol/L) and the deformed fish group (9.14 mmol/L, p<0.001). While calcium is absorbed from the diet and through the gills, skin, and fins from the water, the soluble phosphorus forms are absorbed from the water, therefore, the most crucial pathway of phosphorus uptake comes from food, and its deficiency has been associated with demineralization and deformity (Organización de la Naciones Unidas para la Alimentación y la Agricultura (FAO), 2023; Witten et al., 2015; Rojas et al., 2016; Baejverjord et al., 2019).

The quantification of magnesium did not show significant differences between groups because, as with calcium, uptake in the gastrointestinal tract, gills, skin, and fins from the water is completed by a similar transport system (Organización de la Naciones Unidas para la Alimentación y la Agricultura, 2014). We also found decreased iron levels in deformed fish, which has been related to the development and growth problems of the fish, and microcytic anemia due to poor nutrition (4, Desjardins et al., 1987; Lall and Kaushik, 2021).

Salmonids, such as trout, can spend seasons without feeding, and their physiological state tends to be normal (Godoy et al., 2022). However, microelement deficiency, such as calcium and phosphorus, causes skeletal alterations (Witten et al., 2015; Rojas et al., 2016; Baejverjord et al., 2019; Godoy et al., 2022). Likewise, we found higher glucose values in the mandibular deformity group (5.17 mmol/L), suggesting stress in the fish (Polakof et al., 2011; Godoy et al., 2022). The microstructural and mineralogical characterization of mandibular bone reveals a loss of mineralization that has been primarily associated with lower phosphorus levels than with any other mineral in the mandibular bone. This loss of mineralization was discrete ( Figures 4 , 5 ). The SEM-EDX analysis exhibited higher mineral values because homogenization by reduction to powder impacts the quantification in areas of interest (porosity or fractures). The porosity and surface area analysis showed a larger pore size, while the available surface area was much smaller, indicating that controls were less likely to develop microfractures (Godoy et al., 2022).

Consistent with what we obtained in TGA-DSC and mapping the elements in SEM (phosphorous was more affected than calcium), we found evidence of the inorganic forms of co-precipitation or different chemical structures, which was reflected in the XRD diffractograms and FT-IR spectra ( Figures 8 , 9 ). In the XRD analysis ( Figure 8 ), two peaks were identified in all samples, one of greater intensity at 2θ = 32-33° and a secondary peak at 2θ = 25°. According to the analysis, they correspond to HPA and TCP, respectively. This coincides with what was reported in the literature for HPA crystal, but deformed fish showed a slightly stronger signal to 2θ = 29° which was not identified (Raina et al., 2019). We report another peak 2θ= 26-27°, which was positively identified as apatite (A), indicating tissue formation. The functional groups’ analysis of FT-IR ( Figure 9 ) compared to commercial HPA with control and deformed fish, revealed characteristic signals of structural compounds, such as hydroxyl and phosphate groups, in all samples and showed the presence of carbonate groups probably bound to HPA. We detected peaks at 2800-3000, 1553.5, and 1241.9 cm^-1, which correspond to the presence of N-H (amide), and another peak at 1645.5 that corresponded with carbonyl groups (C=O). Both N-H and C=O groups indicated the presence of collagen in the samples (Nesseri et al., 2020).

The gene expression analysis showed decreased Runx2 levels in deformed fish vs. normal fish ( Figure 10A ), reducing Ocn and Alp expression ( Figure 10C ), two proteins directly related to bone mineralization. Therefore, if Ocn decreases, calcium fixation also decreases. Consequently, phosphorus migrates for HPA formation and deposition in the bone matrix to form new mineralized tissue. ALP data indicate a lack of signal for phosphorus resorption in bones, which explains why systemic levels increase. On the other hand, we observed increased levels of type I collagen, which could indicate bone tissue’s resistance to rupture or large fractures. Instead, they progressively deform. This increase in collagen may be due to a compensatory response to preserve the structure. The increased expression of Osx, although it has not a relevant pathway in juvenile states, can be related to Bmp2 induction ( Figure 10B ) and is independent of the expression of Runx2 ( Figure 10A ) (Lee et al., 2003; Javed et al., 2008). Like increased collagen, this drastic increase in Bmp2 levels compared to Bmp4 can be considered an adaptive or compensatory response to interrupting the primary signal mediated by Runx2. Bone remodeling or repair processes are absent due to low Bmp4 expression ( Figure 10B ) (Dumic-Cule et al., 2018). Likewise, studies in mice indicate that over-expression of Bmp4 translates into less bone formation (Fernández-Sánchez and Mayani, 2008).

According to previous results in rainbow trout (Nesseri et al., 2020), the authors report a consistent Ca/P ratio due to the crystallographic formation of HPA and TCP ( Figure 8 ). Given its size (nm), the porosity (indicated in Table 5 ) does not interfere with cell adhesion. However, it affects its biomechanical properties, as increased porosity decreases the surface area, making the bone more susceptible to microfracture. Also, deregulated Runx2 expression, and therefore that of Ocn and Col-I ( Figures 10A, C ), may be directly associated with stress suffered by the fish under culture conditions. We also discovered elevated glucose levels in deformed fish, implying that this skeletal alteration can occur at any stage of development in freshwater.

Regarding the mechanisms involved in decreased Runx2 expression, the role of Bmp2 and its influence on the induction of bone mineralization is not entirely clear. Studies indicate that even when Bmp2 expression is high, the start of mineralization has been associated with Smad/Runx2 interaction in the presence of Zn (Javed et al., 2008). This coupling loss may be caused by stress, resulting in decreased Runx2 levels. Even though Osx expression was stimulated (Fernández-Sánchez and Mayani, 2008), it was insufficient to generate mineralization in deformed fish. In addition, endogenous BMP2 induction replaces the effect of Runx2, causing loss of mineralization (Kacem et al., 2000; Kacem and Meunier, 2003), increased porosity and microfractures, and advancing disease progression with deformities due to the increased type I collagen. Finally, the fish’s inability to feed due to mandibular deformation will ultimately lead to death (Fernández-Tresguerres et al., 2006; Witten et al., 2015; Godoy et al., 2022).

The etiology of skeletal abnormalities remains an enigmatic and contentious matter in fish populations. Skeletal deformities in juvenile rainbow trout are driven by bone demineralization associated with phosphorus deficiency rather than calcium. Decreased iron and phosphorus levels are caused by poor nutrition due to an impediment to eating properly (reflected in the low content of total proteins and their fractions). This nutritional deficit does not affect calcium, as it can be absorbed through the skin and gills from the water, leaving other minerals such as magnesium unaffected. Microstructurally, demineralized bone shows microfractures and becomes more porous. Current evidence suggests it generates crystallographic formations different from HPA and TCP. Demineralization has been rather discreet or slight, which implies that there was an incomplete pathway interruption.

There was deregulation between the signals of exogenous bone tissue formation, Bmp2, and the expression of Runx2, and therefore in the expression of bone matrix proteins, such as OCN and ALP, inhibiting the formation of inorganic matrix by interfering with calcium and phosphorus fixation while also promoting an increase in the expression of organic matrix by type I collagen as a compensatory measure. Stress under culture conditions may be the most probable cause for this deregulation. It only affects a subset of the specimens in the tank and has nothing to do with developmental affections (malformations). However, genetic factors and inbreeding could also be considered as potential causes of abnormalities.

The original contributions presented in the study are publicly available. This data can be found here: https://doi.org/10.6084/m9.figshare.24561739.v1.

The Scientific Ethics Committee of the Universidad de La Frontera has approved the experimental protocol (N°061_20). The study was conducted in accordance with the local legislation and institutional requirements.

KG: Conceptualization, Formal Analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing. CS: Writing - original draft, Writing - review & editing. CV: Methodology, Writing - review & editing. CM: Methodology, Writing - review & editing. BT: Methodology, Writing - review & editing. JC: Methodology, Writing - review & editing. CB: Formal analysis, Writing - review & editing. MB: Formal analysis, Writing - review & editing. SV: Formal analysis, Writing - review & editing. MR: Supervision, Writing - review & editing. LS: Conceptualization, Investigation, Supervision, Writing - review & editing.