Lipid-based protein sparing in juvenile whiteleg shrimp (Penaeus vannamei Boone, 1931) under reduced-protein diets and the limits of functional additives

Abstract

Dietary protein is the primary cost and environmental driver of shrimp feeds, motivating efforts to enhance protein utilization through lipid substitution and functional additives. Despite extensive interest in reducing dietary protein through lipid-based energy substitution, the physiological limits of protein sparing in whiteleg shrimp (Penaeus vannamei, Boone, 1931) remain poorly understood. This study evaluated whether lipid-based protein sparing could be achieved in juvenile P. vannamei using a blend of lipid-utilization additives. Two feeding trials were conducted under intensive recirculating conditions. Trial 1 evaluated four dietary treatments consisting of a high-protein low-lipid diet and a reduced-protein high-lipid diet, each with or without additive supplementation (choline chloride, L-carnitine, glycerol monolaurate, and lysophospholipids). Trial 2 assessed additive effects under a nutritionally adequate high-protein diet. Growth performance metrics, including feed conversion ratio, specific growth rate, weight gain, survival, and condition factor, were unaffected by dietary treatments. Reduced-protein high-lipid diets did not improve growth and resulted in significantly lower whole-body protein content and increased lipid deposition regardless of additive inclusion, indicating that protein sparing did not occur. Under adequate dietary protein supply, additive supplementation significantly improved protein efficiency ratio and reduced whole-body lipid accumulation, demonstrating enhanced nutrient utilization efficiency rather than growth promotion. Apparent protein and lipid retention exhibited positive but non-significant responses to additive supplementation, while salinity stress tolerance remained unchanged. These findings demonstrate that dietary protein adequacy, rather than additive supplementation, was the primary determinant of nutrient utilization efficiency. Protein reduction in P. vannamei feeds is therefore constrained by macronutrient balance, emphasizing the importance of nutrient partitioning and metabolic indicators in evaluating sustainable feed strategies.

1 Introduction

Aquaculture has become the dominant source of aquatic food for human consumption, driven by increasing demand for animal protein and continued intensification of production systems (FAO, 2022). Despite this growth, the sector faces persistent challenges related to feed cost and sustainability, with feed accounting for up to 70% of total production expenses and dietary protein representing the most costly and environmentally impactful component (Das et al., 2012; Taukhid et al., 2021). Improving protein utilization efficiency is therefore a central objective in aquaculture nutrition. Beyond economic considerations, enhancing protein efficiency is critical for reducing nitrogen discharge from aquaculture operations, improving feed-use efficiency, and supporting the development of sustainable aquafeeds that minimize environmental impacts while maintaining productivity (Lazzari and Baldisserotto, 2008; Taukhid et al., 2021).

The whiteleg shrimp (Penaeus vannamei Boone, 1931) is the most widely cultured crustacean species globally and a cornerstone of intensive aquaculture systems (FAO, 2022; Rimoldi et al., 2018). However, its production relies heavily on high-protein diets, typically ranging from 20% to 45%, which elevate feed costs and contribute to nitrogenous waste when inefficiently utilized (Lee and Lee, 2018; Lazzari and Baldisserotto, 2008). Optimal protein requirements remain variable across studies, reflecting differences in culture conditions and evaluation criteria (Xie et al., 2020; Barreto et al., 2023). Both insufficient and excessive protein levels have been associated with impaired growth and physiological imbalance, highlighting the complexity of protein nutrition in penaeid shrimp (Cardona et al., 2016; Chen et al., 2018). Consequently, identifying nutritional strategies that reduce dependence on dietary protein without compromising physiological performance has become a priority for sustainable shrimp production.

Protein sparing, defined as the partial replacement of dietary protein with non-protein energy sources, has emerged as a promising strategy to reduce feed costs and environmental impacts (Wang et al., 2015; Xu et al., 2018). By supplying alternative energy substrates, protein sparing aims to preserve dietary amino acids for tissue growth and maintenance rather than catabolism for energy production. Among non-protein energy sources, dietary lipids are particularly attractive because of their high energy density and important roles in membrane structure, hormone synthesis, and metabolic regulation (Tseng and Hwang, 2008; Chen et al., 2014). Successful implementation of protein-sparing strategies could reduce reliance on costly protein ingredients, improve nitrogen utilization efficiency, and contribute to more circular and resource-efficient aquaculture systems.

However, successful protein sparing requires not only adequate non-protein energy but also the metabolic capacity to utilize alternative energy substrates efficiently. The effectiveness of protein replacement is therefore governed by nutrient partitioning, defined as the allocation of dietary nutrients among growth, maintenance, energy production, and storage pathways. In crustaceans, excess dietary lipid may not necessarily be directed toward oxidation for energy generation but may instead be preferentially stored in tissues, resulting in increased lipid deposition and reduced protein retention. Such metabolic limitations can disrupt protein-energy balance and constrain the extent to which dietary protein can be replaced without compromising nutrient utilization. Consequently, protein sparing should be viewed not simply as an issue of dietary formulation but as a physiological process influenced by nutrient metabolism and partitioning.

Consistent with this perspective, lipid-based protein sparing in P. vannamei remains inconsistent. Reported lipid requirements vary widely (50–120 g/kg), and excessive lipid inclusion has been associated with reduced growth performance, altered lipid metabolism, excessive fat accumulation, and oxidative stress (Zhu et al., 2010; Xu et al., 2018). These findings suggest that protein sparing through lipid substitution is constrained by physiological and metabolic limits rather than representing a straightforward energy replacement strategy. Moreover, most studies continue to evaluate protein-sparing success primarily through growth performance metrics, with limited consideration of whole-body composition, nutrient retention, and other indicators of nutrient partitioning. As a result, the mechanistic boundaries governing lipid-driven protein sparing in shrimp remain poorly understood.

Functional feed additives offer a potential approach to improve lipid utilization efficiency and thereby enhance protein-sparing outcomes. Compounds such as L-carnitine, choline chloride, glycerol monolaurate, and lysophospholipids have been shown to enhance lipid digestion, transport, and oxidation, as well as modulate metabolic and antioxidant responses in aquatic species (Li et al., 2019; Liu et al., 2019; Lan et al., 2021; Wang et al., 2022). These additives may improve the efficiency with which dietary lipids are mobilized and utilized, potentially shifting nutrient partitioning away from storage and toward productive metabolic processes. Such effects could improve nutrient-use efficiency and reduce waste outputs, thereby supporting more sustainable feed formulations. However, evidence in P. vannamei remains limited, particularly for combined additive formulations evaluated under reduced-protein, high-lipid conditions where protein-sparing strategies are most relevant.

The present study addresses this knowledge gap by evaluating whether lipid-based protein sparing can be enhanced through the inclusion of lipid-utilization and emulsification additives in P. vannamei. Specifically, we examined the effects of reduced-protein, high-lipid diets with and without additive supplementation on growth performance, feed utilization, whole-body composition, nutrient retention, and salinity stress tolerance. We tested three hypotheses: (H1) reduced-protein high-lipid diets would alter nutrient partitioning and reduce body protein deposition; (H2) functional additives would improve nutrient-use efficiency under protein-adequate conditions; and (H3) additives would not fully compensate for dietary protein deficiency. By integrating conventional performance metrics with indicators of nutrient partitioning and metabolic efficiency, this study aims to clarify the physiological boundaries of lipid-driven protein sparing and provide insights for the development of more sustainable shrimp feed strategies.

2 Materials and methods

2.1 Conceptual design

The study evaluated the conditions and limits under which lipid substitution and functional feed additives influence protein utilization in Penaeus vannamei, rather than assuming effective protein sparing. Two experiments were conducted to separate dietary and additive effects.

Experiment 1 tested reduced-protein, high-lipid diets with and without additive supplementation. Experiment 2 evaluated additive effects under nutritionally adequate protein conditions. Growth performance, feed utilization, whole-body composition, nutrient retention, condition factor, and stress tolerance were assessed as integrative indicators of metabolic response.

The tank served as the experimental unit for all statistical analyses, with treatments assigned to replicate tanks and each tank considered an independent observation for performance, nutrient utilization, and physiological response variables.

However, because all tanks were connected to a common recirculating aquaculture system, complete environmental independence among replicates cannot be assumed. Accordingly, treatment effects should be interpreted with this limitation in mind.

2.2 Experimental diets, formulation, and production

Experimental diets were produced at VDS N.V. (Belgium). In Experiment 1 (Table 1), four diets were formulated: a high-protein low-lipid control (POS), a reduced-protein high-lipid diet (NEG), and their additive-supplemented counterparts (POS+ADD and NEG+ADD). Protein levels were targeted at 36% (POS) and 32% (NEG). Additives included choline chloride, L-carnitine, glycerol monolaurate, and lysophospholipids. Experiment 2 included two diets (Table 2): a high-protein control (CON) and an additive-supplemented version (CON+ADD).

The additive blend was formulated to target complementary aspects of lipid digestion, transport, and utilization. Choline chloride functions as a lipotropic agent involved in phospholipid synthesis and hepatic lipid transport, helping prevent excessive lipid accumulation. L-carnitine facilitates mitochondrial transport of long-chain fatty acids and promotes β-oxidation, thereby increasing lipid-derived energy production. Lysophospholipids enhance lipid emulsification and digestion through improved micelle formation and nutrient absorption. Glycerol monolaurate has been associated with improved lipid digestion, intestinal health, and nutrient absorption. Collectively, these additives were combined to improve lipid utilization efficiency and potentially enhance protein-sparing effects by directing dietary lipids toward energy production rather than storage.

Diets were prepared by mixing ingredients, adding water (350 mL kg^-¹), extruding through a 2.0-mm die (70–90 °C), drying at 50–60 °C, and sieving to 1.4–2.2 mm. Analyzed composition was used for interpretation.

2.3 Proximate analysis of shrimp diets

All diets were analyzed following AOAC standard methods (Horwitz and Latimer, 2006) for moisture (AOAC 950.46), crude protein (AOAC 928.08), crude fat with prior acid hydrolysis (AOAC 991.36), ash (AOAC 920.153), and crude starch (polarimetric method).

The vitamin–mineral premix composition is presented in Table 3. The analyzed proximate composition of diets used in Experiments 1 and 2 are shown in Tables 4, 5, respectively. Analyzed crude protein levels were slightly lower than formulated targets (approximately 34–35% vs. 36% for POS diets and 31–32% vs. 32% for NEG diets), which is typical of commercial feed production and analytical variation. Similarly, small differences in starch content between additive-supplemented and non-supplemented diets with otherwise identical macro-ingredient composition reflect normal analytical variability rather than intentional formulation differences.

2.4 Shrimp source and acclimation

P. vannamei postlarvae (PL) were sourced from White Panther Hatchery, Rottenmann, Austria. In the first experiment, PLs were cultured in the Recirculated Aquaculture System (RAS) of VDS, N.V., Belgium until 10.56 ± 1.76 g. In the second experiment, PLs were acclimated and grown for 38 days to an average body weight of 0.90 ± 0.87 g. Water quality parameters, including salinity, temperature, pH, dissolved oxygen (DO), oxidation-reduction potential (ORP), total ammonia, nitrite, nitrate, and KH, were monitored daily or weekly as appropriate (Tables 6, 7).

2.5 Shrimp culture conditions and feeding

Shrimp were haphazardly distributed into 500-L HDPE tanks (30 per tank in the first trial; 80 per tank in the second trial) and cultured for 52 days (experiment 1) and 42 days (experiment 2) under a single RAS system with drum filter, protein skimmer, biological filtration and automatic water temperature control. Each treatment was assigned to four replicate tanks. Feeds were administered using feeder trays continuously for 16 hours per day. Daily feed amounts were calculated to achieve 70% of the desired Daily Protein Intake (Magallón Barajas et al., 2021), adjusted weekly based on body weight, and validated using a 1-hour tray test to estimate feed consumption (Table 8). Tank cleaning, including removal of molts and sludge, was performed daily. This protocol ensured consistent culture conditions while allowing evaluation of protein-sparing effects under controlled, industry-relevant intensive aquaculture conditions.

2.6 Sampling

In the first experiment, weekly growth was monitored by sampling five shrimps per tank (~17% of the population). Samples were gently patted dry, individually weighed, and returned to the culture water within two minutes. At the end of the experiment, all shrimps were euthanized using an ice-salt slurry bath (<0 °C). Post-euthanasia, shrimps were patted dry, weighed using a Kern EMB 500-1S School Balance (0.1 g precision), and photographed for total length measurement (rostrum to telson) using ImageJ software (version 1.53k, NIH, USA). For metabolic analyses, nine shrimps per tank were haphazardly selected and immediately frozen (< -18 °C) prior to analyses for whole-body protein and lipid content after acid hydrolysis.

In the second experiment, weekly growth was monitored by sampling eight shrimps per tank (~10%). Samples were patted dry, weighed, photographed for length measurement, and returned within two minutes. At the end of the trial, all shrimp were weighed, and ten shrimp per tank were haphazardly selected for a salinity stress test. For the salinity stress test, the initial water conditions were salinity: 0.35 ± 0.00 ppt, temperature: 13.86 ± 0.19 °C, pH: 8.01 ± 0.20, DO 9.27 ± 0.77 ppm, and ORP: 142.90 ± 8.77 mV. Mortalities were recorded every 15 minutes over 7 hours.

The challenge protocol involved simultaneous exposure to low salinity and low temperature and therefore represents a combined environmental stress test rather than a salinity-only challenge.

2.7 Growth performance calculations

Growth and feed utilization were calculated as follows:

• Weight gain (WG, g) = W_final − W_initial.

• Specific growth rate (SGR, % day^-¹) = [(ln W_final − ln W_initial)/days] × 100.

• Feed conversion ratio (FCR) = total feed intake (g)/biomass gain (g).

• Survival (%) = (final number/initial number) × 100.

• Protein efficiency ratio (PER) = weight gain (g)/protein intake (g).

Initial whole-body composition was determined from representative shrimp sampled immediately before the start of each feeding trial. Baseline whole-body protein and lipid values obtained from these samples were used in the calculation of apparent nutrient retention.

Apparent nutrient retention:

• ANPR (%) = [(final body protein − initial body protein)/protein intake] × 100.

• ANLR (%) = [(final body lipid − initial body lipid)/lipid intake] × 100.

Condition factor:

• K = 100 × W/L³.

where W = body weight (g) and L = total length (cm).

2.8 Metabolic parameters

Nine shrimp per diet were haphazardly sampled for whole-body protein and lipid content. Crude fat was measured using Soxhlet extraction (AOAC 991.36) after acid hydrolysis with HCl, and petroleum ether was used as the extraction solvent. Crude protein was analyzed using the micro-Kjeldahl procedure (AOAC 928.08).

2.9 Statistical analyses

All statistical analyses were performed using GraphPad Prism 8 (v8.02) and JASP 0.18.3. The tank was considered the experimental unit for all analyses. Data were first assessed for normality using the Shapiro–Wilk test and for homogeneity of variance using the Brown–Forsythe test.

In Experiment 1, the four dietary treatments (POS, NEG, POS+ADD, and NEG+ADD) were analyzed as independent dietary groups because the primary objective was to compare the biological responses to complete feed formulations representing distinct nutritional strategies. Therefore, treatment effects were evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) test for pairwise comparisons when significant differences were detected.

Prior to statistical analysis, data were evaluated for normality using the Shapiro–Wilk test and for homogeneity of variance using the Brown–Forsythe test. When both assumptions were satisfied, data were analyzed using one-way ANOVA followed by Tukey’s multiple-comparison test. When normality was met but homogeneity of variance was violated, Welch’s ANOVA followed by Games–Howell multiple-comparison tests was applied. When both assumptions were violated, data were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple-comparison procedure.

For Experiment 2, comparisons between the control and additive-supplemented diets were performed using independent-samples t-tests when assumptions of normality and homogeneity of variance were satisfied. When assumptions were violated, the Mann–Whitney U test was used.

Salinity stress tolerance was evaluated using Kaplan–Meier survival analysis, and treatment differences were assessed using the Mantel–Cox (log-rank) test. Statistical significance was established at α = 0.05.

In addition to significance testing, effect sizes were calculated to quantify the magnitude of treatment effects. Eta-squared (η²) values were reported for ANOVA-based analyses and interpreted according to conventional thresholds as small (< 0.06), moderate (0.06–0.14), and large (> 0.14) effects. Effect size estimates were used to complement p-values and provide additional insight into the biological relevance of observed treatment responses.

3 Results

3.1 Growth parameters

Growth performance of juvenile P. vannamei was largely unaffected by dietary treatment in both experiments (Tables 9, 10). In Experiment 1, shrimp fed reduced-protein, high-lipid diets (NEG and NEG+ADD) exhibited growth performance comparable to those receiving high-protein diets (POS and POS+ADD). No significant differences were observed in feed conversion ratio (FCR; p = 0.1951), average weight gain (AWG; p = 0.6187), specific growth rate (SGR; p = 0.5318), survival rate (SR; p = 0.7440), protein efficiency ratio (PER; p = 0.7885), body weight variability (%CV Weight; p = 0.2822), or body length variability (%CV Length; p = 0.8987). Although average body weight (ABW; p = 0.0148) and average body length (ABL; p = 0.0182) differed significantly among treatments, the magnitude of these differences was small, with treatment means differing by less than 1.5 g and 0.3 cm, respectively. Corresponding effect sizes were low (η² < 0.20), indicating limited biological relevance. Overall, neither dietary protein reduction nor additive supplementation produced meaningful improvements in growth performance under the conditions tested.

In Experiment 2, additive supplementation under adequate dietary protein conditions significantly increased PER (2.37 ± 0.08 vs. 2.21 ± 0.06; p = 0.0178), with a large effect size (η² = 0.6356), indicating improved protein utilization efficiency. ABL was also greater in CON+ADD than in CON (11.92 ± 0.18 vs. 11.37 ± 0.52 cm; p = 0.0211); however, the associated effect size was small (η² = 0.0672), and this difference was not accompanied by corresponding increases in ABW (p = 0.7698), AWG (p = 0.6553), or SGR (p = 0.6139). FCR showed a numerical improvement in CON+ADD (1.35 ± 0.08) relative to CON (1.46 ± 0.07), although the difference was not statistically significant (p = 0.0871). Survival rate (p = 0.6637), body weight variability (p = 0.7257), and body length variability (p = 0.5203) were likewise unaffected by additive supplementation.

Collectively, these results demonstrate that functional feed additives improved protein utilization efficiency when dietary protein was adequate but did not consistently enhance growth performance. Furthermore, additive supplementation did not compensate for reduced dietary protein levels, as growth responses remained similar between supplemented and unsupplemented reduced-protein diets.

3.2 Weekly growth trends

Weekly growth trends for the two experiments are illustrated in Figures 1–3. In Experiment 1 (Figure 1), all treatments showed a consistent increase in average body weight (ABW) over time, with broadly similar trajectories among groups. Exponential curve fitting was used descriptively to visualize growth patterns, and the resulting curves showed substantial overlap, indicating no apparent differences among treatments. POS and POS+ADD exhibited slightly higher values at later stages, although these differences were not statistically significant.

Figure 1

Figure 2

Figure 3

In Experiment 2, both average body weight (Figure 2) and average body length (Figure 3) increased steadily in CON and CON+ADD groups, with comparable growth trajectories across treatments. Exponential curve fitting was likewise applied for descriptive visualization, and no clear separation between treatment groups was observed. Variability increased over time but remained comparable between treatments.

3.3 Condition factor

Condition factor (K) and length–weight relationship parameters (a and b) are presented in Tables 11, 12. In Experiment 1, K ranged from 0.785 ± 0.050 (NEG) to 0.860 ± 0.042 (POS+ADD), with no significant differences among treatments (p = 0.307). The length–weight exponent b varied between 2.232 and 2.560, indicating negative allometric growth (b < 3), indicating that length increased proportionally faster than body weight. Parameters a and b did not differ significantly across diets (p > 0.10).

In Experiment 2, K values were similar between CON (0.812 ± 0.012) and CON+ADD (0.899 ± 0.190; p = 0.468), while b-values ranged from 2.912 to 3.200, reflecting approximately isometric growth (b ≈ 3). The difference in b between trials likely reflects ontogenetic stage or experimental conditions, with smaller juvenile shrimp in Experiment 2 exhibiting more proportionate growth.

3.4 Whole-body protein and lipid content

Whole-body protein and lipid composition of juvenile P. vannamei differed significantly among dietary treatments (Figures 4, 5, respectively). Shrimp fed the high-protein diet (POS) had the highest protein content (73.54% ± 1.13), whereas the low-protein diet (NEG) produced the lowest (70.45% ± 0.11), with intermediate values observed in POS+ADD and NEG+ADD. These patterns indicate differences in whole-body nutrient composition across dietary treatments.

Figure 4

Figure 5

Lipid content was highest in NEG and NEG+ADD (7.91–8.43%) and lowest in POS+ADD (6.44%), while POS remained similar to initial values (6.34%). These results indicate that reduced-protein, high-lipid diets were associated with increased lipid deposition, whereas additive supplementation under adequate protein supply was associated with lower lipid accumulation.

Despite intermediate protein levels in NEG+ADD, apparent net protein retention (ANPR) did not differ significantly among treatments (Table 13). This indicates that observed differences in whole-body composition are not directly reflected in retention indices and may instead reflect differences in nutrient partitioning. Overall, these results suggest that functional additives may influence nutrient partitioning, although retention indices were not significantly affected.

3.5 Apparent net protein and lipid retentions

Apparent Net Protein Retention (ANPR) and Apparent Net Lipid Retention (ANLR) are summarized in Table 13. ANPR was numerically highest in POS+ADD (128.9 ± 14.26%) and lowest in NEG (114.5 ± 7.06%), while ANLR followed a similar numerical pattern (POS+ADD > POS > NEG+ADD > NEG). However, neither ANPR (p = 0.6225) nor ANLR (p = 0.4122) differed significantly among treatments.

These results indicate that, although numerical trends were observed, dietary treatments did not significantly affect protein or lipid retention under the conditions tested.

3.6 Salinity stress

Survival of juvenile P. vannamei under acute low-salinity stress for the second experiment (CON and CON+ADD) is presented in Figure 6. Mortality began earlier in the CON+ADD group (120 min) compared to CON (165 min); however, cumulative survival at the end of the 420-min test was similar. Also, there is no significant difference between the survival curves of the two treatment (p = 0.2531), indicating no statistically significant survival advantage from additive supplementation.

Figure 6

Water quality parameters were consistent across treatments (salinity 0.35 ppt; temperature 13.8–13.9 °C; pH 7.93–8.09; dissolved oxygen 8.82–9.72 ppm; ORP 140–145 mV; Table 7), confirming that observed survival patterns were not influenced by environmental variation.

4 Discussion

4.1 Failure of lipid-driven protein sparing: evidence from nutrient partitioning

This study evaluated whether lipid substitution combined with functional feed additives could enhance protein utilization in juvenile Penaeus vannamei. Across both experimental phases, conventional growth indicators, including feed conversion ratio (FCR), average weight gain (AWG), specific growth rate (SGR), survival, and growth variability, were largely unaffected by dietary treatments. These findings indicate that short-term growth performance was relatively insensitive to additive supplementation and moderate alterations in dietary protein and lipid content when baseline nutritional requirements were largely met. Growth responses in P. vannamei are strongly dependent on adequate dietary protein supply (Lee and Lee, 2018; Xie et al., 2020), and modifications in dietary energy sources do not necessarily translate into measurable improvements in biomass accumulation over short production periods (Cuzon et al., 2004).

More importantly, the reduced-protein high-lipid diets failed to achieve effective protein sparing. Shrimp fed these diets exhibited lower whole-body protein content and increased lipid deposition without corresponding improvements in growth performance or feed efficiency. These responses suggest that the additional dietary lipid was preferentially directed toward storage rather than supporting protein accretion. Such findings highlight the importance of nutrient partitioning in determining the success of protein-sparing strategies.

Protein sparing depends not only on the provision of alternative energy sources but also on maintaining an appropriate protein-to-energy ratio that supports amino acid utilization for tissue growth. When dietary protein becomes limiting, excess dietary energy cannot fully compensate for deficiencies in essential amino acids required for protein synthesis. Under these conditions, surplus lipid may accumulate in tissues while protein deposition declines. Similar responses have been reported in penaeid shrimp, where increased dietary lipid did not consistently improve growth and frequently resulted in altered nutrient utilization and greater lipid storage (Rosas et al., 2001; González-Félix et al., 2002).

Recent studies further support the view that successful protein-sparing strategies in P. vannamei are constrained by dietary protein adequacy, amino acid balance, and nutrient partitioning rather than by energy supply alone. Barreto et al. (2023) reported that post-larval P. vannamei cultured in clear-water recirculating systems required relatively high dietary protein levels to achieve optimal growth and feed utilization, while reductions in dietary protein resulted in poorer biological performance. Likewise, Yuan et al. (2023) demonstrated that dietary protein level significantly influenced growth performance, antioxidant capacity, intestinal morphology, and physiological condition, highlighting that protein reduction must remain within species-specific nutritional limits. These findings reinforce the concept that dietary protein serves functions beyond energy provision and remains essential for sustaining tissue accretion and physiological homeostasis.

Evidence from recent amino acid nutrition studies further suggests that the success of protein-sparing strategies depends not only on total dietary protein concentration but also on amino acid adequacy. Ko et al. (2024) demonstrated that dietary valine level significantly affected growth performance, feed conversion ratio, protein efficiency ratio, whole-body composition, and immune-related gene expression in juvenile P. vannamei. Similarly, Zheng et al. (2023) reported that methionine supplementation improved growth performance, protein metabolism, antioxidant status, and hepatopancreatic condition in shrimp fed low-fishmeal diets. Collectively, these studies indicate that maintenance of essential amino acid balance is critical when dietary protein levels are reduced and that growth performance may be compromised when amino acid supply becomes limiting despite adequate dietary energy availability.

Taken together, both classical and recent evidence support the interpretation that effective protein sparing requires more than the provision of alternative energy sources. When dietary protein or essential amino acid availability becomes limiting, additional dietary lipid may be preferentially directed toward storage rather than productive protein deposition. The lower whole-body protein content and increased lipid accumulation observed in the present study are consistent with this nutrient-partitioning framework and suggest that lipid-based protein sparing was not achieved under the dietary protein and lipid levels tested. These findings indicate that moderate dietary protein reduction cannot be fully compensated by increased lipid inclusion under the present experimental conditions.

An additional limitation is that dietary amino acid profiles were not analyzed. Consequently, it is not possible to determine whether reduced body protein deposition in the low-protein diets resulted solely from altered protein–energy balance or was partly influenced by reduced availability or imbalance of essential amino acids required for protein synthesis. Given the growing evidence that amino acid adequacy strongly influences nutrient utilization efficiency in P. vannamei (Ko et al., 2024; Zheng et al., 2023), future studies should incorporate detailed amino acid profiling when evaluating protein-sparing strategies.

4.2 Conditional effects of functional additives on nutrient utilization

Although additive supplementation did not improve growth performance, clear effects on nutrient utilization were observed under protein-adequate conditions. The significant increase in protein efficiency ratio (PER) in the additive-supplemented control diet indicates that shrimp utilized dietary protein more efficiently when adequate protein was available. Notably, this improvement occurred without corresponding increases in body weight gain or specific growth rate, suggesting that additive supplementation influenced nutrient allocation rather than overall growth potential.

The reduction in whole-body lipid content observed in additive-supplemented shrimp further supports this interpretation. Functional additives such as L-carnitine and lysophospholipids facilitate fatty acid transport, lipid emulsification, and nutrient absorption (Li et al., 2019). The reduced whole-body lipid content and improved protein efficiency ratio observed in this study are consistent with enhanced nutrient utilization; however, no direct measurements of lipid metabolism, digestive enzyme activity, gene expression, or hepatopancreatic histology were performed, and the underlying metabolic mechanisms therefore remain unconfirmed. Similar improvements in lipid utilization and metabolic efficiency following supplementation with lipid-metabolism enhancers have been reported in other aquatic species (Khan et al., 2018; Liu et al., 2024).

Recent studies in P. vannamei provide additional support for this interpretation. Chen et al. (2024) reported that dietary lysophospholipid supplementation improved growth performance, digestive capacity, hepatopancreatic condition, lipid metabolism, and intestinal microbial balance in shrimp fed low-fishmeal diets. Likewise, Limwachirakhom et al. (2025) demonstrated that lysophospholipid supplementation enhanced protein efficiency ratio, digestive enzyme activity, and lipid-metabolism-related gene expression, suggesting improved dietary nutrient utilization. Studies evaluating glycerol monolaurate and other functional lipid-utilization additives have also reported improvements in intestinal health, nutrient absorption, and metabolic efficiency (Lan et al., 2021; Wang et al., 2022). Collectively, these findings support the hypothesis that functional additives may improve the efficiency with which dietary nutrients are processed and utilized, thereby reducing lipid accumulation and enhancing protein-use efficiency even when growth responses remain unchanged.

Because digestive enzyme activity, mitochondrial β-oxidation, lipid catabolism, gene expression, and digestibility coefficients were not measured, the mechanisms underlying the observed responses remain speculative. Consequently, it cannot be determined whether the responses resulted from differences in nutrient absorption, post-absorptive nutrient partitioning, or both. The findings should therefore be interpreted as evidence of improved nutrient utilization and nutrient partitioning rather than confirmation of specific metabolic pathways.

Furthermore, because choline chloride, L-carnitine, glycerol monolaurate, and lysophospholipids were administered as a combined additive blend, the contribution of individual components could not be distinguished. Accordingly, the observed effects should be attributed to the additive blend as a whole. While previous studies have demonstrated positive effects of each of these additives individually, potential synergistic or antagonistic interactions among components remain poorly understood and warrant further investigation.

4.3 Physiological limits of nutritional compensation

A key finding of this study is that functional additives improved nutrient utilization only when dietary protein was sufficient. Under reduced-protein high-lipid conditions, additive supplementation failed to improve growth performance, protein retention, or body protein content. Although additives may enhance digestion and nutrient utilization, they could not compensate for the fundamental nutritional constraints imposed by reduced dietary protein availability.

This finding provides important insight into the physiological limits of protein-sparing strategies. Additives can improve the efficiency with which nutrients are utilized, but they cannot replace essential nutrients that are absent or insufficient in the diet. Once dietary protein falls below the threshold required to support optimal amino acid supply and tissue protein synthesis, improvements in lipid digestion or energy utilization alone are unlikely to restore normal protein deposition. Recent studies support this interpretation. Yuan et al. (2023) showed that dietary protein level influenced growth performance, body composition, intestinal digestion, and microbiota in Litopenaeus vannamei, while Ko et al. (2024) demonstrated that dietary valine balance affected growth performance, feed utilization efficiency, protein efficiency ratio, whole-body protein synthesis, and immune-related responses in juvenile P. vannamei. These findings reinforce the view that protein and essential amino acid adequacy remain primary constraints on nutrient utilization, even when energy supply or functional additives are provided.

The lack of significant differences in apparent net protein retention (ANPR) and apparent net lipid retention (ANLR) further supports this interpretation. Although additive-supplemented treatments showed numerical improvements, retention indices did not differ significantly among diets. Therefore, evidence for additive effects is more strongly supported by the significant improvement in protein efficiency ratio (PER) and changes in whole-body composition than by retention metrics alone. Collectively, these findings suggest that additives act as supportive enhancers of nutrient-use efficiency rather than mechanisms capable of overcoming dietary protein deficiency. This interpretation is consistent with recent lysophospholipid studies in L. vannamei, where supplementation improved digestive capacity, lipid metabolism, antioxidant responses, and PER under suitable dietary conditions, but did not indicate that additives can replace adequate protein or amino acid supply (Chen et al., 2024; Limwachirakhom et al., 2025).

Apparent net protein retention values exceeding 100% should be interpreted cautiously, as they were estimated from whole-body composition and nutrient intake and do not account for factors such as digestibility, endogenous nitrogen losses, microbial contributions, feed leaching, or analytical variability. Consequently, values above 100% likely reflect limitations of the apparent retention calculation rather than true biological protein retention.

The salinity stress challenge provided additional evidence of these physiological constraints. Additive supplementation did not improve survival under acute low-salinity exposure, despite previous indications of improved nutrient utilization during feeding. Severe osmotic stress imposes immediate energetic demands associated with ion regulation, osmoregulation, antioxidant defense, immune regulation, and metabolic adjustment that may override nutritional advantages acquired during growth (Li et al., 2007, 2017). Similar reductions in survival following abrupt salinity change have been reported in juvenile shrimp (Laramore et al., 2001). Recent work further indicates that salinity stress in L. vannamei is closely linked to osmoregulatory and antioxidant demands, and that nutritional interventions may improve stress tolerance only when they directly support these physiological pathways (Su et al., 2023). The lack of treatment effects observed here suggests that the stress conditions exceeded the capacity of dietary interventions to confer measurable resilience. Because low salinity and low temperature were imposed simultaneously, the relative contribution of each stressor to mortality cannot be determined.

4.4 Implications for sustainable aquafeeds

The present findings have important implications for sustainable shrimp feed development. Reducing dietary protein remains a major objective in aquaculture because protein ingredients represent the largest feed cost and a major source of nitrogen discharge. Effective protein-sparing strategies could therefore improve feed efficiency, reduce environmental impacts, and enhance production sustainability.

However, the results indicate that dietary protein reduction is constrained by physiological limits associated with nutrient partitioning and protein–energy balance. Although dietary lipid can serve as an alternative energy source, excessive reliance on lipid substitution may promote lipid storage rather than productive protein deposition. Consequently, feed formulation strategies should prioritize adequate dietary protein levels and balanced amino acid profiles before implementing substantial protein reductions through increased lipid inclusion.

The positive responses to additive supplementation under protein-adequate conditions suggest that functional additives can improve nutrient-use efficiency and reduce unnecessary nutrient accumulation. Nevertheless, these additives should be regarded as complementary tools rather than substitutes for adequate protein nutrition. From a practical perspective, optimizing nutrient partitioning appears to be a more effective strategy for sustainable feed formulation than maximizing dietary lipid inclusion.

Although growth performance remained largely unaffected, the observed reduction in whole-body protein content and increased lipid deposition in shrimp fed reduced-protein diets may have practical implications for commercial production. Changes in carcass composition can influence product quality, nutrient utilization efficiency, and feed cost-effectiveness. From a feed formulation perspective, these findings suggest that maintaining adequate dietary protein and amino acid supply remains essential, even when alternative energy sources are provided. Consequently, evaluation of protein-sparing strategies should include body composition and nutrient partitioning indicators in addition to conventional growth metrics.

Overall, this study demonstrates that dietary protein adequacy is the primary determinant of nutrient utilization efficiency in P. vannamei. Functional additives can enhance nutrient-use efficiency when protein requirements are met but cannot fully compensate for protein deficiency. These findings highlight the value of nutrient partitioning as a framework for evaluating protein-sparing strategies and support the development of more environmentally and economically sustainable shrimp feeds.

Several limitations should be considered when interpreting the results. The use of a shared recirculating aquaculture system may have reduced the environmental independence of experimental units, while the use of only four replicate tanks per treatment may have limited statistical power to detect smaller treatment effects. Consequently, non-significant numerical trends should be interpreted cautiously. In addition, amino acid and fatty acid profiles of the experimental diets were not determined, preventing evaluation of the potential contribution of nutrient composition to the observed responses. Future studies should incorporate detailed nutrient profiling to better elucidate the mechanisms underlying protein-sparing responses and nutrient partitioning in shrimp.

5 Conclusion

This study demonstrates that lipid-based protein sparing was not achieved under the dietary conditions evaluated. Further studies employing broader protein and lipid gradients are required to define the physiological limits of protein sparing in P. vannamei. Reduced-protein, high-lipid diets resulted in lower whole-body protein content and greater lipid deposition, indicating that lipid substitution cannot fully replace dietary protein without altering nutrient partitioning. Functional lipid-utilization additives improved protein-use efficiency and reduced lipid accumulation under protein-adequate conditions, but these benefits did not translate into improved growth performance and were absent under reduced-protein diets. This suggests that additives can enhance nutrient utilization only when dietary protein requirements are met and cannot compensate for protein deficiency. Importantly, whole-body composition and protein efficiency ratio (PER) proved more sensitive indicators of nutritional responses than growth performance alone, revealing treatment effects that would not have been detected through conventional growth metrics. Overall, dietary protein adequacy was the primary determinant of nutrient utilization efficiency, highlighting the importance of balanced protein–energy formulation in sustainable shrimp feeds. Future studies should integrate metabolic, transcriptomic, and enzymatic indicators to identify the physiological mechanisms limiting protein sparing in penaeid shrimp.

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