Marine fish oil is rich in high-energy and essential omega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These fatty acids play vital roles in cell membrane structure and function, which are essential for the growth, health, and reproduction of aquatic species (NRC, 2011). Fish oil also contains other essential nutrients, such as cholesterol and phospholipids, which improve lipid digestibility and contribute to superior growth performance and efficient nutrient utilization in aquatic animals, especially crustaceans. Cholesterol, a precursor of steroid hormones and bile acids, constitutes a key component of the cell membrane (Liang et al., 2023). Bile acids and phospholipids, key components of cell membranes, act as natural emulsifiers that enhance lipid digestion and absorption in aquatic animals (Lordan and Blesso, 2023; Lee et al., 2024). However, the availability of marine fish oil in the aquaculture industry is declining owing to increasing consumer demand. Moreover, the impacts of climate change have led to increases in the cost of fish oil, making it less economically viable (IFFO, 2024). Therefore, it is crucial to explore sustainable alternative oils for aquatic feed formulations.

Currently, alternative oils, such as soybean oil, are increasingly being utilized to partially replace fish oil in commercial aquatic feed, offering a sustainable and cost-effective solution (Hodar et al., 2020; Radhakrishnan et al., 2024). Previous studies have shown that combining dietary soybean oil with fish oil in freshwater fish feed can support optimal growth performance and feed utilization without adverse effects. For example, soybean oil can replace a significant portion of fish oil in the diets of various freshwater fish species, such as tilapia and carp, without compromising growth rate or feed efficiency (Xu et al., 2016; Oliva-Teles et al., 2022). However, several factors must be considered when using soybean oil in marine shrimp diet. Unlike freshwater fish, marine shrimp has specific nutritional requirements and physiological characteristics that make the effects of soybean oil less predictable and dependent on various conditions. For example, Pacific white shrimp (Litopenaeus vannamei) require specific long-chain polyunsaturated fatty acids, such as EPA and DHA, which are abundant in fish oil but not in soybean oil (Lima and Figueiredo-Lima, 2016). Additionally, marine shrimp has a unique digestive system with a short gut passage period and limited bile acid synthesis. Therefore, it is necessary to use fish oil because it is more easily digested and absorbed than soybean oil (Li et al., 2023). Moreover, fish oil provides a more comprehensive nutrient profile that supports optimal growth and health in marine shrimp than soybean oil (NRC, 2011). Although complete replacement of fish oil with vegetable oils reduced shrimp growth due to essential fatty acid (EFA) imbalances (Xie et al., 2020), partial substitution with soybean oil has shown no adverse effects (Chen et al., 2022; Xu et al., 2016). Diets containing soybean oil or soybean and lecithin oil combined with fish oil (2% total dietary lipid) showed higher survival rates than those fed 3% fish oil or 3% soybean oil without soy lecithin or phospholipids (Hu et al., 2011). Although combined administration of fish oil, soybean oil, and soy lecithin did not significantly affect growth performance, it improved survival rate, total cholesterol, and phospholipid concentrations in the hemolymph. Lecithin and phospholipids are natural emulsifiers generally used in animal feed, especially for shrimp. Phospholipids consist of two lipophilic fatty acid tails, which play key roles as efficient emulsifiers under water-in-oil conditions. However, phospholipids function as weak emulsifiers in oil-in-water emulsions, such as those in the digestive tract of shrimp (Lin et al., 2021).

In the animal feed industry, several emulsifiers are used to enhance lipid digestion and absorption of essential nutrients. Key emulsifiers, such as phospholipids, lysophospholipids (LPLs), bile salts, glycerol polyethylene glycol ricinoleate (GPEGR), and sodium steroyl-2-lactylate (SSL), help improve growth performance and feed efficiency, especially in young terrestrial animals and crustaceans with limited bile acid- and lipase-producing ability (Siyal et al., 2017; Saleh et al., 2020). LPLs are natural emulsifiers derived from the enzymatic hydrolysis of phospholipids, which removes one fatty acid tail. LPLs are more hydrophilic than phospholipids and exhibit enhanced emulsification under oil-in-water conditions for lipid digestion in the hepatopancreas and midgut of shrimp (Lin et al., 2021). Additionally, the key characteristics of effective emulsifiers are assessed based on the hydrophilic–lipophilic balance (HLB), critical micelle concentration (CMC), emulsion stability index (ESI), safety, non-cytotoxicity, and cost-effectiveness (Nayak et al., 2021; Ravera et al., 2021). In terms of emulsifying properties, higher HLB values indicate enhanced hydrophilicity, which facilitates the formation of micelles that are essential for emulsification and solubilization processes. Generally, LPLs, phospholipids, bile acids, GPEGR, and SSL have HLB values of approximately 10–20, 4–6, 18, 12–14, and 10–12, respectively, indicating the superior hydrophilicity of LPLs (Smejkal et al., 2024; Van Hoogevest, 2017). A comparison of CMC values, the concentration of each emulsifier at which micelles start to form, showed that LPLs have a lower CMC value (0.01–0.1 mM) (Alkademi, 2020) than phospholipids (0.3–2 mM) (Khan et al., 2018), bile acids (2–20 mM) (Pavlović et al., 2018), GPEGR (0.531 mM) (Held, 2014), and SSL (0.214 mM) (Wang and Marangoni, 2015). Additionally, ESI indicates the ability of the compound to maintain stable emulsions. A high ESI can promote digestive enzymes, especially lipase, to break down fats and other nutrients in the digesta by reducing the size of fat droplets and increasing their surface area. LPLs are key emulsifiers with high ESIs of approximately 200–300 min and strong emulsion stability (Calvano et al., 2020). In contrast, other emulsifiers, such as bile acids, GPEGR, and SSL, generally have lower ESIs of approximate 150–250 min (Shen et al., 2022), 150–250 min (Li et al., 2021), and 100–200 min (Li et al., 2019), respectively. Additionally, safety, cost effectiveness, and sustainability should be considered when selecting emulsifiers for shrimp feed.

LPLs have advantages, including cost effectiveness, safety, non-cytotoxicity, compatibility with various biological systems, and sustainability. LPLs have been studied for their numerous benefits in shrimp, such as increased nutrient digestion and absorption, improved feed utilization, and improved growth performance and feed efficiency. For example, the use of LPLs promoted emulsifying capacity, improves fatty acid digestibility, and increases productivity in tiger shrimp (Penaeus monodon) (Khan et al., 2018). Therefore, LPLs could replace dietary emulsifiers in the diet of Pacific white shrimp (Lin et al., 2021). For example, replacement of 1%–2% dietary soybean lecithin oil with 0.1% dietary LPLs improved survival rate, growth performance, and immunity in oriental river prawn (Macrobrachium nipponense) (Guoliang et al., 2023). Additionally, LPL supplementation enhanced the antioxidant system and color in shrimp, suggesting that LPLs could enhance emulsification and promote cholesterol transport and dietary astaxanthin efficiency, resulting in improved growth, survival rate, stress resistance, and pigmentation in juvenile Pacific white shrimp (Song et al., 2024a).

Despite the benefits of LPLs, the optimal supplementation level necessary to maximize growth performance and nutrient utilization in shrimp fed fish oil-reduced diets remains unknown. Additionally, the long-term effects of LPL supplementation on shrimp health and the overall sustainability of using LPLs as substitutes for fish oils are not fully understood. Therefore, the aim of this study was to investigate the effects of varying LPLs levels in partial fish oil reduction diet on shrimp growth and nutrient absorption, especially digestive enzyme activity and the expression of genes related to lipid metabolism. Overall, it is anticipated that this study will provide valuable insights on strategies to reduce fish oil dependence in aquafeeds.

In this study, we hypothesized that supplementing an energy-low diet with LPLs would compensate for the optimal nutrients required for growth performance in shrimp. Shrimp fed diets supplemented with 0.03–0.06% LPLs showed comparable performance with those in the PC group (higher energy and higher fish oil diet). Shrimp have insufficient bile acids, which are essential for lipid digestion and absorption (Shi et al., 2023; Kumar et al., 2021). Therefore, the efficiency of the lipid digestion processes, including emulsification, lipase activation, and micelle formation, may be suboptimal in shrimp, indicating the need for diets with essential additives that enhance lipid digestion. LPLs, which are derived from the enzymatic hydrolysis of phospholipids, promote emulsification and enhance fat digestion in animal feed and nutrient absorption (Song et al., 2024a; Thornhill, 2020). Research findings indicate that dietary LPLs improve growth performance, health, and feed efficiency in various species, including largemouth bass (Lu et al., 2022), rainbow trout (Taghavizadeh et al., 2020), turbot (Li et al., 2019), tiger shrimp (Khan et al., 2018), and Pacific white shrimp (Wang et al., 2024). LPLs can promote growth and lipid metabolism, improve astaxanthin deposition and body coloration, and enhance lipid and cholesterol transportation, especially in aquatic animals such as Pacific white shrimp (Wang et al., 2024). Research on the substitution of dietary fish oil with LPLs in crustacean nutrition remains relatively limited. However, existing studies have reported various beneficial effects associated with LPL supplementation. For instance, Wang et al. (2024) demonstrated that dietary inclusion of LPLs significantly improved growth performance, specifically weight gain and SGR, as well as enhanced lipid metabolism, antioxidant enzyme activity, and immune responses in Pacific white shrimp (Wang et al., 2024). Furthermore, the inclusion of 0.1% LPLs was found to mitigate the negative effects of a 1% reduction in dietary phospholipids, and 0.2% LPLs was recommended to compensate for a 1% reduction in fish oil. In this experiment, dietary inclusion levels of 0.03% and 0.06% LPLs were selected based on prior research in aquatic species. For instance, Xu et al. (2022) reported that similar concentrations of lysophosphatidylcholine (LPC) in LPLs significantly enhanced growth performance and modulated the expression of lipid metabolism-related genes in juvenile turbot (Xu et al., 2022). Additionally, Song et al. (2024b) demonstrated that LPL supplementation may contribute to a 15% decrease in dietary lipid levels without adverse effects on growth performance in Pacific white shrimp, indicating the potential of LPLs to improve feed efficiency under lipid-restricted conditions (Song et al., 2024b). In the present study, supplementation with 0.03%–0.06% LPLs improved growth performance parameters, including Tprod, WGR, SGR, FCR, and PER, compared with those in the NC group. Notably, similar results were observed in shrimp in the PC group. Additionally, the growth-regulating effects of LPLs may be dose-dependent. Collectively, these findings suggest that supplementing a low-energy diet (50 kcal/kg; 1% fish oil) with 0.03%–0.06% LPLs can reduce dietary lipid requirements and enhance lipid digestibility and metabolic efficiency. Furthermore, the observed improvements in growth performance with LPL supplementation can be attributed to improved lipid digestion and enhanced energy utilization efficiency. However, further studies are necessary to investigate the optimal inclusion level of 0.06% LPLs to minimize the use of dietary fish oil, promote sustainability, and reduce production costs without affecting growth and production quality.

Dietary LPLs significantly increased the crude protein and lipid contents in shrimp muscle. Similarly, previous studies have shown that LPLs can enhance protein and lipid deposition in various species, including large yellow croaker (Yi et al., 2014), rainbow trout (Adhami et al., 2021), and yellow drum (Wang et al., 2016). LPLs play important roles in cellular functions, including increasing protein synthesis and cellular size and upregulating fatty acid synthetase (FAS) mRNA expression, thereby contributing to improved lipid metabolism and utilization (Li et al., 2019). Our findings indicate that LPLs can increase the permeability and fluidity of hepatopancreatic epithelial cells, improving nutrient digestion, transportation, and absorption in the midgut, particularly the utilization of essential nutrients such as lipids and proteins, and promoting nutrient deposition in shrimp muscle.

Although several studies have examined the effects of LPLs on lipid metabolism in aquatic animals, research on their effects on cholesterol levels in hemolymph, hepatopancreas, and shrimp muscle remains scarce. Song et al. (2024a) reported that dietary LPLs increased astaxanthin deposition, regulated cholesterol transportation, increased high-density lipoprotein cholesterol (HDL-C) levels, and decreased low-density lipoprotein cholesterol (LDL-C) levels in the hemolymph. LDL transports cholesterol from the hepatopancreas to peripheral tissues, whereas HDL removes cholesterol by transporting it back to the hepatopancreas. HDL-C plays a crucial role in lipid metabolism by mediating reverse cholesterol transport and conversion into bile acids in the hepatopancreas (Shi et al., 2023). A study showed that a 1% decrease in fish oil in the diet of Pacific white significantly decreased total cholesterol (T-CHO) level in the hemolymph and that LPL supplementation mitigated this reduction and enhanced lipid metabolism. Importantly, similar findings have been reported in turbot and rainbow trout (Taghavizadeh et al., 2020; Li et al., 2019; Wang et al., 2024). Cholesterol is a biosynthetic precursor of ecdysteroid hormones in crustaceans that supports molting and cellular functions (Su et al., 2022). Khan et al. (2018) reported that dietary LPL supplementation in a fishmeal- and fish oil-reduced diet significantly decreased cholesterol content in the tail muscle and non-muscle portions, promoting growth performance and carcass fatty acid composition in tiger shrimp. In the present study, LPL supplementation in energy-reduced diets increased cholesterol levels in the hemolymph and hepatopancreas, supporting growth, development, and molting by promoting lipid storage and utilization. Particularly, shrimp in the NC+0.03% LPLs group showed increased cholesterol levels in the hepatopancreas and hemolymph, which could be due to the ability of LPLs to promote lipid digestion, absorption, and transportation. Future studies should investigate the relationship between LPL supplementation and its benefits in lipid digestion and cholesterol utilization for growth and molting, with the aim of reducing cholesterol accumulation in shrimp meat. Although research on this function remains limited, the use of LPLs as valuable natural emulsifiers in aquaculture could help produce healthier and more nutritious shrimp for consumers.

Owing to their hydrophilic and hydrophobic properties, LPLs enhance lipid metabolism and provide significant benefits, especially under oil-in-water conditions, by increasing digestive enzyme activity (Rodríguez-Viera et al., 2022). Overall, these properties improve emulsification in the shrimp gut, stimulate bile secretion, facilitate micelle formation, and increase the contact area for efficient lipase activity (Li et al., 2018). LPL supplementation enhances lipid digestion as well as improves protein digestibility by increasing protein solubility and substrate concentration, thereby accelerating digestive enzyme activity (Lu et al., 2022). Consistent with findings in turbot (Li et al., 2019) and largemouth bass (Lu et al., 2022), 0.03 and 0.06% dietary LPLs significantly increased lipase activity in the present study. Che et al. (2023) reported that 0.1% dietary LPLs increased lipase, protease, and amylase activities, particularly glucose-6-phosphate dehydrogenase (G-6-PDH), enhancing energy supply in largemouth bass. Based on these findings, the reduction of fish oil in the NC group resulted in decreased levels of long-chain polyunsaturated fatty acids (LC-PUFAs), as well as led to a reduction in other essential components naturally present in fish oil, such as phospholipids and cholesterol. These components play a crucial role in micelle formation, which is necessary for lipid solubilization and absorption in the hepatopancreas, enhancing the activity of digestive enzymes. For example, the activities of lipase and amylase were lower in the NC group, potentially affecting the digestion of nutrients such as lipids and carbohydrates. In contrast, dietary supplementation with 0.06% LPLs in the NC group significantly enhanced the activities of lipase, protease, trypsin, chymotrypsin, and amylase, suggesting improved digestive capacity under conditions of limited dietary fish oil.

LPLs act as natural emulsifiers, promoting lipid digestion and the absorption of fatty acids and other lipid-soluble substances. Additionally, they facilitate micelle formation, lipid transportation, and the transfer of absorbed lipids from the hepatopancreas into the hemolymph. To elucidate the potential mechanisms of LPL supplementation in lipid metabolism and oxidative stress response, we examined the mRNA expression of genes involved in lipid metabolism and antioxidant response. Consistent with findings in juvenile turbot (Li et al., 2019) and Pacific white shrimp (Wang et al., 2024), supplementing energy-reduced diets with 0.03% and 0.06% LPLs upregulated the expression of the lipid metabolism genes Lip and dFAD-6. Lip is crucial for triglyceride hydrolysis in the hepatopancreas, whereas dFAD-6 is essential for synthesizing polyunsaturated fatty acids, including arachidonic acid and eicosatetraenoic acid. Arachidonic acid benefits shrimp by enhancing cellular health and immune response, and both arachidonic acid and eicosatetraenoic acid improve nutrient absorption and utilization. In contrast, the absence of significant changes in HK and GPx genes expression following LPL supplementation may be attributed to the lipid-specific functional role of LPLs and the lack of substantial metabolic or oxidative stress during the experimental period. HK gene encodes a key glycolytic enzyme that phosphorylates glucose to glucose-6-phosphate, initiating energy production, and its expression often reflects cellular energy demand (Cruz-Moreno et al., 2024). GPx gene encodes the glutathione peroxidase enzyme, a selenium-dependent antioxidative stress enzyme responsible for detoxifying peroxides and protecting cells from oxidative damage. The stable expression of both genes suggests that LPLs did not induce a level of physiological stress sufficient to trigger transcriptional responses in glycolytic or antioxidant pathways (Parida and Sahoo, 2023). The unchanged expression of HK and GPx genes following LPL supplementation, as well as in the positive and negative control groups, may reflect a metabolically balanced state in which energy demands were adequately met through lipid-derived pathways, thereby minimizing the reliance on glycolysis and limiting the activation of antioxidant defense mechanisms. This observation implies that the dietary energy levels provided in the present study were presumably sufficient to prevent the onset of energy deficiency or metabolic stress, which would otherwise trigger compensatory upregulation of glycolytic or redox-regulatory genes. Consequently, the absence of transcriptional changes in these pathways may not indicate a lack of LPL efficacy, but rather a reflection of adequate energy homeostasis under the experimental conditions. This outcome suggests LPLs effectively promoted lipid metabolism and enhanced digestive enzyme activity, their influence on glycolytic and redox-regulatory pathways appeared limited under stable, non-stressful conditions. Consistent with findings in Pacific white shrimp (Wang et al., 2024), the mRNA expression of the antioxidant gene SOD was significantly upregulated in the NC+0.03% LPLs and NC+0.06% LPLs groups, enhancing growth, antioxidative stress enzyme, and immune capacity. From a statistical perspective, some response parameters did not reach statistical significance, certain values showed trends that were close to the threshold, which may indicate potential biological relevance. Duncan’s multiple range test was selected in this study due to its higher sensitivity in detecting differences, despite being more permissive and carrying a greater risk of type I error. These near-significant results were interpreted with caution. To reduce the chance of type I error in future research, the use of more conservative post-hoc tests such as Tukey’s HSD, along with increasing the sample size and considering adjusted p-values, may improve the reliability of statistical outcomes. Collectively, our findings suggest that LPL supplementation enhances the utilization of energy (fish oil)-reduced diets, promoted lipid, protein, and glucose metabolism, improved vital nutrient transportation, and upregulated lipid metabolism and antioxidative stress gene in shrimp. LPL supplementation in energy-reduced diets might provide positive benefits without adverse effects on growth performance or feed utilization. However, it is important to acknowledge that some studies have reported neutral or context-dependent effects of LPLs. For example, Al-Jebory et al. (2023) observed limited improvements in growth performance during the finisher period when dietary lipid digestibility was already sufficient (Al-Jebory et al., 2023). Similarly, Lee and Stein (2022) reported that the positive effects of LPLs on nutrient digestibility and growth performance may be more pronounced in young or developing animals with relatively underdeveloped digestive systems than in fully developed adults (Lee and Stein, 2022). These findings underscore the importance of both dietary composition and physiological status in modulating the efficacy of LPLs. Therefore, although the positive effects of LPLs are evident under specific nutritional conditions, further research is required to more precisely define the conditions and interactions that influence their effectiveness across different species and dietary formulations. In summary, these findings highlight the potential of LPLs as functional feed additives to support shrimp growth and metabolic health, particularly under energy-reduced diets. Future studies should focus on dose optimization, long-term effects, and species-specific responses to maximize the practical applications of LPLs in aquaculture nutrition.

LPL supplementation in energy-reduced diets may contribute to improved growth performance and nutrient utilization in juvenile Pacific white shrimp, potentially through enhanced lipid digestion and increased absorption and transport of essential nutrients. LPL supplementation at inclusion levels of 0.03%–0.06% increased digestive enzyme activity, elevated cholesterol concentrations in the hepatopancreas and hemolymph, enhanced nutrient retention, and upregulated genes related to lipid metabolism and antioxidative stress responses. Collectively, these findings suggest that LPLs may play a role in mitigating some of the physiological impacts associated with 1% dietary fish oil or 50 kcal/kg energy reduction and may support shrimp health and development under such conditions. Although the current study demonstrated promising results under controlled experimental conditions, it is important to exercise caution when extrapolating these findings to commercial-scale shrimp farming. The long-term effects of LPL supplementation, as well as its cost-effectiveness and performance under different farming environments remain to be fully elucidated. Further research is recommended to investigate the mechanisms of LPLs on molting physiology. A more comprehensive understanding of these factors will be essential for the effective application and optimization of LPLs in practical aquafeed formulations.