The giant salamander (Andrias davidianus), commonly known as the baby fish, is the largest amphibian, with a history of more than 350 million years, and is a key species for the development of agricultural industrialization in China (Wang et al., 2004). It has been listed in the World Conservation Union Red List and Appendix I of the Washington Convention (CITES) (Zhang et al., 2002). Additionally, this species holds significant nutritional and medicinal benefits, as its skin, secretions, muscles, blood, and bones are rich in proteins, amino acids, and trace elements, often referred to as ginseng in water (He et al., 2018; Wang et al., 2021). At present, the industrialization of artificial breeding and cultivation of A. davidianus has taken place in various provinces across China; nevertheless, challenges remain regarding feed supply, and there has been limited research conducted on this topic (Cunningham et al., 2015). As the scale of farming continues to grow, the rising cost of breeding and decreasing market price of A. davidianus have made it imperative to advance the development of compound feed and conduct nutrition research.

Lipid has a variety of physiological functions in the metabolic process of fish, such as serving as a component of tissues and cells, supplying energy to the body, promoting the absorption and transportation of lipid-soluble vitamins, and providing essential fatty acids, etc (De Carvalho and Caramujo, 2018). Carnivorous aquatic animals have a poor ability to utilize carbohydrates, therefore protein and lipid are the main source of sustaining their life activities (Guo et al., 2023; Xie et al., 2021). Maintaining suitable dietary lipid level in the feed is beneficial for conserving protein, enhancing growth performance, and improving feed utilization efficiency (Chatzifotis et al., 2010; Mata-Sotres et al., 2022). However, long-term intake excessive lipid could result in liver disorders, cause oxidative stress (Shearer et al., 2012), induce oxidative damage (Yin et al., 2020), suppress immune responses, promote structural abnormalities in both liver and intestine (Zhu et al., 2017), and trigger inflammation (Naiel et al., 2023). Deficiency in dietary intake may result in metabolic disorders, decreased feed utilization efficiency, and inadequate nutrient provision for fish (Meng et al., 2019; Picolo et al., 2021; Qiang et al., 2017; Yan et al., 2015). Overall, the capacity to effectively use lipid differs significantly among aquatic species, as both excessive and inadequate consumption of lipid can affect ideal growth of fish.

Our research team has developed compound feed for A. davidianus, which is under development. Research on nutritional requirements and feed for A. davidianus by our research team has included evaluating protein requirement (Wang et al., 2020), functional feed additives (Lu et al., 2021), and herbal extract (Xu et al., 2021). However, the lipid requirement of A. davidianus and the impact of lipid on their growth and metabolism remain unreported. Considering this, we devised the experiment to examine the effects of varying dietary lipid levels on juvenile A. davidianus. Given the characteristics of feed for A. davidianus, it is essential to store it for extended periods. In this context, soybean oil emerges as a preferable option due to its ease of preservation and significantly lower cost compared to fish oil, aligning more closely with actual production demands. Therefore, we formulated six group of experimental feeds using soybean oil as the main lipid source to assessed their growth, body composition, digestive and absorptive capacity, and antioxidant under different lipid levels. This study aims to elucidate the lipid requirement of juvenile A. davidianus and provide valuable insights for the formulation of artificial feeds tailored to their needs.

Numerous studies have evidenced that the ideal dietary lipid level differ among various fish species (NRC, 2011). This research indicated that the dietary lipid level had a considerable impact on the growth performance of juvenile A. davidianus. Comparable results were observed in Rainbow Trout, Oncorhynchus mykiss (Chen et al., 2023), Rockfish, Acipenser schrenckii (Li et al., 2023a), white seabass, Atractoscion nobilis (López et al., 2009), silver barb, Puntius gonionotus (Mohanta et al., 2008). Notably, the WGR and SGR reached their peak at the dietary lipid level of 16.21%. Juvenile A. davidianus fed high lipid diet appear growth depression may because of the damage to the intestinal structure resulting in decreased digestion and absorption (Chang et al., 2018). In addition, high-lipid diet could cause abnormal lipid metabolism in liver, resulting in pathological changes of liver. Through quadratic regression analysis of weight gain rate (WGR), muscle RNA/DNA ratio, lipase activity, total cholesterol and skin collagen content with dietary lipid level concluded that the optimal dietary lipid level for juvenile A. davidianus is 13.3%-17.5%.

Increasing dietary lipid levels has been shown to promote growth and protein utilization (Ai et al., 2004; Han et al., 2014). The experimental findings indicate that the optimal levels of FCR and PRR were achieved at the dietary lipid level of 16.21%. While growth and feed efficiency indicate the protein-sparing benefits of higher lipid content, the PRR may be influenced when the dietary lipid surpasses 16.21%. Furthermore, the synthesis of proteins relies on RNA, which normally maintains a consistent level of DNA within cells (Clemmesen, 1994). The RNA/DNA ratio governs the ability of cell to generate RNA and synthesize protein, and the decrease of RNA/DNA ratio indicates that there are factors influencing growth of juvenile A. davidianus. In the present experiment, the RNA/DNA ratio increased to 16.21% with the dietary inclusion of lipid. This finding implies that such dietary lipid level favor optimal protein synthesis in A. davidianus, subsequently enhancing growth performance. This result is consistent with the earlier studies in largemouth bass, Micropterus salmoides (Guo et al., 2019), totoaba, Totoaba macdonaldi (Perez-Velazquez et al., 2017). Appropriate lipid intake can increase the metabolic energy of A. davidianus, improve protein utilization efficiency, and promote growth.

Collagen, abundantly in the skin, bones, muscles, and various other tissues, represents the predominant structural protein found in all animal species (Shoulders and Raines, 2009). The result of this experiment shows that the collagen of A. davidianus skin is higher than that of muscle. This study has demonstrated that optimal dietary lipid level is conducive to the synthesis of skin collagen, while excessive or insufficient lipid levels exert adverse effects. This phenomenon may be attributed to the fact that when lipid level is too low, the body primarily uses lipid to provide energy for the synthesis of skeletal muscles and other proteins (Guillard and Schwörer, 2024). Conversely, excessively high lipid level could lead to significant damage to the intestinal and liver structures, impairing intestinal digestion and absorption, as well as causing liver dysfunction. This impairment reduces the ability to metabolize lipid, consequently decreasing the availability of dietary fatty acids and inhibiting collagen synthesis (Meng et al., 2006). The result of this experiment showed a quadratic relationship between skin collagen and dietary lipid levels, with an optimal lipid level of 16.0%. Nevertheless, the exact mechanism by which appropriate lipid level promote collagen formation requires further investigation.

After being digested and absorbed, the lipid is used for tissue construction and energy storage, mainly in viscera, liver, muscles and other tissues (Guo et al., 2019). In contrast, the lipid of A. davidianus is mainly concentrated in the tail, in which unsaturated fatty acids account for 71.3%. Numerous earlier studies have shown that excessive dietary lipid level in lumpfish, Cyclopterus lumpus (Berge et al., 2023), rockfish, Sebastes schlegeli (Li et al., 2023b), and red-tailed catfish, Hemibagrus wyckioides (Hung et al., 2017), can lead to an accumulation of lipid in the visceral cavity and liver tissue, which in turn raises the whole body lipid concentration. This findings indicated that higher dietary lipid level resulted in reduced lipid content in both the liver and body of juvenile A.davidianus. This phenomenon may be due to the damage of liver function caused by metabolic disorder and the obstruction of lipid synthesis, as confirmed by the liver antioxidant indices. The lipid content of muscle and uropygial gland remained stable following elevated dietary lipid, suggesting that the liver of A. davidianus is more sensitive compared to other tissues. This sensitivity represents a significant limiting factor in its capacity to effectively utilize lipid resources.

Intestinal digestive enzyme activity and intestinal tissue development directly reflect the ability of the intestine to digest and absorb nutrients (Ghasemi et al., 2020; Sesay et al., 2016). The findings indicated a significant inhibition of trypsin activity in the intestine with the consumption of the high-lipid diet, as evidenced by a decrease in activity when dietary lipid levels exceeded 12.90%. Furthermore, earlier research has demonstrated that increased lipid content was associated with increased lipase activity in larval sea bass, Dicentrarchus labrax (Morais et al., 2004), teleost pejerrey, Odontesthes bonariensis (Gómez-Requeni et al., 2013), and Atlantic salmon, Salmo salar (Krogdahl et al., 2015). It suggests that the A. davidianus could adapt to the increasing lipid levels in the diet by regulating their own lipase activity, but when the dietary lipid level exceeded 16.21%, the lipase activity decreased significantly. These results are related to the species of experimental animals, feed composition and living environment. Based on the liver antioxidant index and transmission electron microscopy analysis, it was observed that the liver of A. davidianus experienced impairment because of the high-lipid diet, potentially leading to compromised bile secretion and decreased availability of substrates for lipase binding. Comparable outcomes have been identified in the whiteleg shrimp, Litopenaeus vannamei investigation (Hamidoghli et al., 2019).

The intestine, as the largest immune system and digestive organ in the body, could selectively absorb essential nutrients that aid in combating external pathogens and other detrimental substances (Lee et al., 2017). The interior wall of intestine, whose morphological integrity is the basis for the function of intestine, is composed of folds, villi and microvilli. The increase in mucosal fold height and number implies an increase in nutrient-absorbing surface area (Ramos et al., 2017). In the present investigation, appropriate dietary lipid can improve the intestinal morphology of fish by increasing the width and height of intestinal mucosal fold. Additionally, the findings from the electron microscope analysis an overabundance of dietary lipid hindered the development of intestinal villi in A. davidianus, leading to a reduction in absorptive surface area. Moreover, Na+-K+-ATPases are proteins embedded in cell membranes that provide energy for ion transport by hydrolyzing ATPases (Gorini et al., 2002), thus enhancing the active transport and absorption capacity of amino acids, glucose and other nutrients in the intestine (Kopecka-Pilarczyk, 2010). It has been demonstrated that Na+-K+-ATPase activity is directly related to intestinal digestion and absorption capacity (Almansa et al., 2003). The experimental results show that appropriate lipid inclusion in the diet can improve the activity of Na+-K+-ATPase in the intestine of A. davidianus, thus promoting the absorption and deposition of nutrients.

Triglyceride (TG) is mainly involved in the production and storage of energy in the body, serving as a crucial substrate for energy metabolism. TCHO is the raw material for the synthesis of bile acids, vitamin D, and steroid hormones, and is also involved in the formation of cell membranes. TCHO and TG in plasma are regarded as key indicators connected to lipid metabolism, providing insight into the nutritional condition of fish. Earlier research efforts concerning various fish species have observed that TG and TCHO consistently rose as dietary lipid levels increased (Du et al., 2006; Hansen et al., 2008; Yan et al., 2015). In the current investigation, a markedly reduced concentration of TG and TCHO in plasma was observed in the lowest dietary lipid group. Additionally, as the dietary lipid level rose, the content of TG and TCHO exhibited a rising tendency. This observation suggests that the transport of endogenous lipid becomes more active in reaction to elevated dietary lipid level (Du et al., 2005). However, when the dietary lipid level exceeds 16.21%, the level of TCHO and TG decreases significantly. Similar conclusion to the present experiment were reached in the studies of the Hiphaestus fuliginosus) (Wang et al., 2010) and Sebastiscus marmoratus (Shi et al., 2013), in which the reduction of TG and TCHO levels in the blood could be the result of bile secretion abnormality or liver dysfunction.

The process of lipid metabolism in the liver is intricate, and abnormalities at any stage can result in disorder associated with lipid metabolism (Karavia et al., 2013). Excessive lipid diet leads to cell swelling, nuclear translocation, accumulation of lipid droplets (Cao et al., 2018), and hepatocellular hypertrophy with increased cell diameters (Ragab et al., 2015). The impacts have been comprehensively documented among different fish species (Lin and Yeh, 2022; Zhao et al., 2023). These changes in liver of A. davidianus in this study also support this assertion. During cell growth and development, intracellular biomolecules and damaged organelles can be degraded and recycled by cellular autophagy (Van den Bossche et al., 2017). Under normal physiological condition, autophagy actively participates in cellular differentiation, growth, and development, as well as in the prevention of neurodegeneration and the regulation of immune responses at the cellular level (Papáčková and Cahová, 2014). Additionally, autophagy can be upregulated in response to stressors such as starvation, hypoxia, and DNA damage, thereby has offering cellular protective mechanisms. However, if cellular autophagy is overactive, it can damage normal organelles and cause autophagic death (Murray and Wynn, 2011). Significant changes in the ultrastructural composition were noted after the intake of diet excessive in lipid in the current research. As the level of feed lipid increased, the number of autophagosomes in liver cells also rose, and the mitochondria in the highest lipid level exhibited swelling and rounding. The result suggests that high-lipid diet leads to oxidative stress in the liver of A. davidianus, resulting in enhanced cellular autophagy, liver damage and significant growth inhibition. A deeper insight into these histological alterations in the liver could be essential for exploring metabolic disorders (Lu et al., 2013).

Chronic consumption of high-lipid diet results in the accumulation of lipid within cells, which in turn causes an overproduction of reactive oxygen species (ROS) (Schieber and Chandel, 2014). These ROS can trigger permanent alterations in both cells and tissues (Chen et al., 2024). This study found that MDA content in A. davidianus livers increased significantly as dietary fat content increased, suggesting that extremely high dietary lipid content increases lipid peroxidation (Mas-Bargues et al., 2021; Baskol et al., 2007). T-AOC reflects the total antioxidant level of the organism, and SOD, as an important antioxidant enzyme in the antioxidant system of aquatic animals. Reactive oxygen species, including superoxide anion radicals, free oxygen, and H₂O₂, can be efficiently scavenged by them, helping to reduce the harm caused to tissues and cells by free radicals resulting from PUFA peroxidation (Valko et al., 2004). In the current investigation, the activity of those enzymes increased when dietary lipid levels increased from 5.02% to 16.21% and declined when dietary lipid levels exceeded 16.21%. The increase in enzyme activity might be the result of maintaining the proper ratio of ROS to antioxidant, shielding the liver cells from their harmful effects (Paital and Chainy, 2010) Therefore, to mitigate significant oxidative stress, it is anticipated to uphold elevated levels of antioxidant enzyme production. When the generation of reactive oxygen species (ROS) exceeds the capacity of antioxidant defenses, organisms experience oxidative stress, leading to detrimental effects (Bize et al., 2008). Consequently, the antioxidant enzymes displayed a declining trend when the A. davidianus was fed diet beyond 16.21% lipid level, indicating the possibility of oxidative stress. Comparable findings were observed in prior studies involving different species (Deng et al., 2021; Ebadi et al., 2021; Liou et al., 2023).

Through quadratic regression analysis using WGR, skin collagen content, muscle RNA/DNA ratio, total cholesterol (TCHO), and lipase activity as indicators, it was determined that the optimal lipid level of A. davidianus is 13.3% to 17.5%. This dietary lipid level can significantly improve skin collagen content, antioxidant capacity and the health of intestine and liver, thereby positively influencing grow perform. Diet that contains either insufficient or excessive lipid can adversely affect health of A. davidianus, and excessive lipid diet has the most serious impact. Specifically, high-lipid diet result in lipid metabolism disorder, oxidative stress, and detrimental effects on the intestinal and liver health of A. davidianus.