Effects of functional additive blend containing choline chloride, L-carnitine, glycerol monolaurate, and lysophospholipids on protein efficiency ratio, growth performance, and stress tolerance in juvenile Penaeus vannamei

1 Introduction

Aquaculture has become the dominant source of aquatic food for human consumption, driven by increasing global demand for high-quality animal protein and the continued intensification of production systems (FAO, 2022). As aquaculture production expands, improving feed efficiency has become a major priority because feed use strongly influences production economics and environmental sustainability (FAO, 2022). Consequently, nutritional strategies that enhance nutrient-use efficiency while maintaining animal performance are increasingly important for sustainable aquaculture development.

The whiteleg shrimp (Penaeus vannamei Boone, 1931) is the most widely cultured crustacean species worldwide and contributes substantially to global shrimp production (FAO, 2022; Ravuru and Mude, 2014). Commercial production depends on nutritionally balanced diets capable of supporting rapid growth, efficient feed utilization, and resilience to environmental stressors. Traditionally, dietary interventions have been evaluated using growth performance indicators such as weight gain, survival, and feed conversion ratio (FCR). However, these metrics alone may not fully capture nutritional responses because animals with similar growth rates can differ considerably in nutrient-use efficiency, body condition, growth uniformity, and physiological resilience.

Functional feed additives have emerged as promising tools for improving nutrient utilization and metabolic efficiency in aquaculture species. Among the most widely studied are choline chloride, L-carnitine, lysophospholipids, and glycerol monolaurate, which have been associated with improvements in lipid metabolism, nutrient absorption, intestinal function, and physiological performance (Li et al., 2019; Liu et al., 2020; Lan et al., 2021; Wang et al., 2022). Choline functions as a lipotropic factor involved in phospholipid synthesis and lipid transport, L-carnitine facilitates the mitochondrial transport of long-chain fatty acids for β-oxidation and has been associated with the regulation of lipid and energy metabolism in aquatic animals (Dias et al., 2001; Li et al., 2017b; Li et al., 2019). Lysophospholipids enhance lipid emulsification and nutrient absorption and may improve feed utilization and intestinal morphology in aquatic animals (Liu et al., 2020). Through these complementary mechanisms, additive blends may improve the efficiency with which dietary nutrients are converted into growth and maintenance processes.

Despite increasing interest in functional additives, reported responses in shrimp remain inconsistent. Some studies have demonstrated improvements in feed efficiency and nutrient utilization, whereas others have reported limited effects on growth performance (Li et al., 2019; Liu et al., 2020; Wang et al., 2022). Such variability suggests that dietary supplementation may influence physiological processes that are not fully reflected by conventional growth metrics. Improvements in nutrient-use efficiency may occur without detectable increases in biomass accumulation, particularly when shrimp are already receiving nutritionally adequate diets. From a production perspective, improved nutrient utilization is especially relevant because it may reduce feed costs and decrease nitrogen waste outputs, both of which are major economic and environmental concerns in shrimp farming.

Consequently, there is increasing recognition that nutritional responses should be evaluated using a broader range of biological indicators. Growth uniformity is commercially important because heterogeneous populations can reduce harvest efficiency and complicate management practices. Likewise, morphometric indicators such as body length, condition factors, and length–weight relationships provide additional information regarding body form, growth patterns, and nutrient allocation. Environmental resilience is also an important component of shrimp performance. Whiteleg shrimp are routinely exposed to salinity fluctuations during production, transport, and water-management operations, making tolerance to osmotic stress an important indicator of physiological robustness (Laramore et al., 2001; Li et al., 2007; Li et al., 2017a). Nutritional interventions that improve metabolic efficiency may therefore influence stress tolerance as well as growth-related responses.

Although individual effects of choline chloride, L-carnitine, lysophospholipids, and glycerol monolaurate have been reported in aquatic species, limited information exists regarding their combined effects on nutrient-use efficiency, morphometric development, growth uniformity, and stress resilience in juvenile P. vannamei. Furthermore, few studies have evaluated these responses simultaneously using both conventional production metrics and complementary phenotypic indicators. Addressing this knowledge gap may improve understanding of how functional additive blends influence shrimp performance beyond growth alone.

Because all additives were administered as a single dietary blend, this study was designed to evaluate the overall response to the combined formulation rather than the individual effects of its constituent components. Accordingly, the present study investigated the effects of a dietary additive blend containing choline chloride, L-carnitine, glycerol monolaurate, and lysophospholipids in juvenile P. vannamei. Growth performance, feed utilization, growth uniformity, morphometric condition, allometric growth, and low-salinity stress tolerance were assessed following a 42-day feeding trial. Nutritional interventions that improve nutrient utilization efficiency may influence physiological robustness, animal welfare, and resilience to environmental stressors that can compromise health and increase susceptibility to disease under commercial farming conditions. Consequently, functional feed additives are increasingly being evaluated not only for their effects on growth performance but also for their potential contribution to broader health-management strategies in aquaculture. We hypothesized that dietary supplementation with the additive blend would primarily enhance nutrient-use efficiency, as reflected by improvements in protein efficiency ratio (PER) and feed conversion metrics. Furthermore, we hypothesized that improved nutrient utilization would be associated with favorable changes in growth uniformity, morphometric condition, and tolerance to acute low-salinity stress, even in the absence of substantial differences in overall growth performance.

2 Materials and methods

2.1 Experimental design

This study evaluated the effects of a functional additive blend on growth performance, feed utilization, morphometric condition, growth uniformity, allometric growth, and low-salinity stress tolerance in juvenile whiteleg shrimp (Penaeus vannamei). The experiment consisted of two dietary treatments: a control diet (CON) and an additive-supplemented diet (CON+ADD). Each treatment was assigned to four replicate tanks, and the tank was considered the experimental unit for all tank-level analyses.

A total of 80 shrimp were stocked per tank. At stocking, shrimp had an initial mean body weight of 0.90 ± 0.87 g, with no significant differences between treatments (p > 0.05).

2.2 Experimental diets

Experimental diets were manufactured by VDS N.V. (Belgium). Both diets were formulated to contain approximately 42% crude protein and 7% crude fat. The CON+ADD diet contained a functional additive blend composed of choline chloride (0.30%), L-carnitine (0.05%), glycerol monolaurate (0.07%), and lysophospholipids (0.05%), whereas the control diet contained no additives.

The additive blend was designed to support lipid digestion, transport, and utilization. Choline chloride functions as a lipotropic factor involved in phospholipid synthesis and lipid transport. L-carnitine facilitates mitochondrial transport of long-chain fatty acids for β-oxidation. Lysophospholipids enhance lipid emulsification and nutrient absorption, while glycerol monolaurate has been associated with improved intestinal health and nutrient utilization.

Diets were produced by mixing dry ingredients, adding water (350 mL kg^-¹), extruding through a 2.0-mm die at 70–90 °C, drying at 50–60 °C, and sieving to obtain pellets between 1.4 and 2.2 mm. Diets were analyzed for crude protein, crude fat, moisture, ash, and starch according to AOAC methods (Horwitz and Latimer, 2006). The diet composition and analyzed nutrient composition of the experimental diets is presented in Tables 1, 2.

Table 1. Percent ingredient composition of the experimental diets of Penaeus vannamei.

Values are presented as recorded manufacturing inclusion rates. The CON diet consisted solely of the basal formulation, whereas the CON+ADD diet consisted of the recorded basal formulation supplemented with a functional additive blend containing choline chloride, L-carnitine, glycerol monolaurate, and lysophospholipids. Basal ingredients and additive inclusions are presented separately to distinguish the underlying diet formulation from the supplemental additive blend. Minor differences among subtotals reflect the recorded formulation values and rounding.

Table 2. Proximate composition of the experimental diets.

2.3 Shrimp source and culture conditions

Juvenile P. vannamei were obtained from White Panther Hatchery (Rottenmann, Austria) and acclimated in a recirculating aquaculture system (RAS) at VDS N.V., Belgium. Shrimp were cultured in 500-L HDPE tanks supplied by a common RAS equipped with mechanical filtration, biological filtration, protein skimming, and temperature control.

A total of 80 shrimp were stocked per tank and cultured for 42 days. Water-quality parameters were monitored throughout the feeding trial to ensure suitable culture conditions and comparable environmental conditions between treatments. Water-quality summary statistics were verified against the archived monitoring records prior to analysis and are presented in Table 3.

Table 3. Water-quality parameters monitored during the 42-day feeding trial.

Shrimp were fed continuously for 16 h day^-¹ using feeding trays. Feeding rates were adjusted weekly based on biomass estimates and tray observations. Uneaten feed was assessed after 1 h to refine feed allocation throughout the experiment.

2.4 Feed utilization calculations

Feed-utilization indices were calculated at the tank level, with each tank considered the experimental unit. Feed conversion ratio (FCR) and protein efficiency ratio (PER) were calculated using the following equations:

FCR = Feed intake (g)/Wet weight gain (g)

Protein efficiency ratio (PER)= Wet weight gain (g)/Protein intake (g)

Protein intake (g) = Feed intake (g) × dietary crude protein fraction

Protein intake was calculated using the analyzed crude protein content of each experimental diet. Feed intake was estimated from cumulative feed-allocation records maintained for each tank throughout the 42-day feeding trial.

Shrimp were fed using feeding trays, and feeding rates were adjusted weekly based on biomass estimates and tray observations to minimize feed wastage and maintain feeding to apparent satiation. Feeding trays were used as a management tool to assess feeding activity and guide subsequent feed allocations; however, uneaten feed was not quantitatively recovered or weighed. Consequently, feed intake values represent apparent feed intake derived from cumulative feed allocation rather than direct measurements of feed consumption.

Feedwater stability and nutrient leaching characteristics were evaluated separately during the feed-quality assessment; however, no correction for nutrient leaching losses was applied when calculating FCR or PER. Because all diets were managed under identical feeding protocols and environmental conditions, these procedures were applied consistently across all experimental units and treatments, allowing valid comparative evaluation of feed-utilization responses between dietary groups.

2.5 Low-salinity stress challenge

At the end of the feeding trial, ten shrimp were randomly selected from each tank and subjected to an acute low-salinity challenge. Shrimp were transferred directly to test tanks containing water at 0.35 ± 0.00 ppt salinity. This salinity level was selected to induce acute osmotic stress and maximize the ability to detect potential treatment-related differences in physiological resilience. Initial water conditions were 13.86 ± 0.19 °C, pH 8.01 ± 0.20, dissolved oxygen 9.27 ± 0.77 mg L^-¹, and oxidation–reduction potential 142.90 ± 8.77 mV. Mortality was recorded at 15-min intervals over a 7-h exposure period. Time-to-event data were subsequently used for survival analyses. Because challenge water temperature (13.86 ± 0.19 °C) was substantially lower than the culture temperature maintained during the feeding trial (27.7 °C), shrimp were exposed simultaneously to acute osmotic and thermal stress. Consequently, the challenge should be interpreted as a combined low-salinity and temperature-shock test rather than a purely osmotic stress challenge.

2.6 Exploratory modeling analyses

Exploratory analyses were conducted to identify variables associated with dietary treatment responses. Standardized path analysis was used to evaluate potential relationships among treatment, protein efficiency ratio (PER), feed conversion ratio (FCR), growth uniformity, and morphometric condition.

Random Forest classification was additionally performed to rank variable importance for treatment discrimination. Because only eight experimental units were available (four tanks per treatment), these analyses were considered exploratory and were not used for formal hypothesis testing. Due to the limited sample size, results from these analyses should be interpreted as hypothesis-generating rather than inferential.

2.7 Statistical analyses

Statistical analyses were performed using GraphPad Prism 8 (v8.02) and JASP 0.18.3 software. The tank was considered the experimental unit for all tank-level analyses. Data were assessed for normality using the Shapiro–Wilk test and for homogeneity of variance using the Brown–Forsythe test. Treatment effects on growth performance, feed utilization, growth uniformity, morphometric traits, and condition indices were evaluated using Welch’s independent-samples t-tests. Results are presented as mean ± standard deviation (SD), and statistical significance was accepted at p< 0.05.

Weekly body-weight measurements were collected to monitor growth progression throughout the feeding trial. These data were used to fit Gompertz growth models and describe treatment-specific growth trajectories. Repeated-measures inferential analyses were not performed because the study was designed and powered using the tank as the experimental unit, and trajectory analyses were intended to provide descriptive characterization of temporal growth patterns rather than formal hypothesis testing.

Survival during the low-salinity challenge was evaluated using Kaplan–Meier survival analysis and the Mantel–Cox (log-rank) test. Mortality risk was assessed using a Cox proportional hazards model with hazard ratios and 95% confidence intervals. Because analyses were conducted at the individual-shrimp level despite shrimp originating from replicated feeding-trial tanks, results were interpreted as exploratory rather than confirmatory tank-level inferences.

For allometric analyses, ANCOVA was used to compare length–weight relationships between treatments. PCA was used for multivariate visualization of treatment responses. Path analysis and Random Forest modeling were conducted as exploratory analyses intended to identify variables potentially associated with treatment responses rather than to provide confirmatory inference.

3 Results

3.1 Growth performance, feed utilization, and growth uniformity

Growth performance, feed utilization, and growth uniformity responses are summarized in Table 4 and Figure 1. Dietary supplementation with functional additives did not significantly affect survival, final biomass, or final mean body weight of juvenile Penaeus vannamei. Survival remained high in both treatments, averaging 93.75 ± 2.70% in CON+ADD and 92.19 ± 4.83% in CON. Likewise, final biomass (938.27 ± 50.45 vs. 929.53 ± 42.34 g) and final mean body weight (12.50 ± 0.35 vs. 12.61 ± 0.28 g) were comparable between treatments (Table 4). Effect sizes for these variables were small to moderate, indicating limited biological differences between treatments despite natural tank-to-tank variation.

Table 4. Tank-level performance and morphometric endpoints.

Values are mean ± SD (n = 4 tanks per treatment). Means within a row sharing the same superscript letter are not significantly different (Welch’s t-test, p > 0.05). Different superscript letters indicate significant differences between treatments (p< 0.05).

Feed-utilization responses differed from growth outcomes. Protein efficiency ratio (PER) was significantly higher in shrimp fed the additive-supplemented diet than in those receiving the control diet (0.0237 ± 0.0008 vs. 0.0221 ± 0.0006; Figure 1A), representing approximately a 7.2% increase in protein utilization efficiency. This was the only significant treatment effect detected among the measured growth and feed-utilization variables (Table 4). In contrast, feed conversion ratio (FCR) was numerically lower in CON+ADD (1.35 ± 0.08) than in CON (1.46 ± 0.07), representing an improvement of approximately 7.5%; however, the difference was not statistically significant (Figure 1B).

Growth uniformity was not affected by dietary treatment. Weight coefficient of variation (CV) averaged 22.48 ± 2.96% in CON+ADD and 23.12 ± 1.90% in CON, while length CV averaged 8.91 ± 3.34% and 7.45 ± 2.67%, respectively (Figures 1C, D). No significant differences were detected between treatments for either weight or length uniformity (Table 4). Overall, dietary supplementation improved protein utilization efficiency but did not significantly influence growth performance, feed conversion efficiency, or growth uniformity.

3.2 Morphometric condition and allometric growth

Morphometric condition and allometric growth responses are presented in Figure 2 and Table 5. Dietary supplementation with functional additives did not significantly affect final body length, modified condition factor (K), or the allometric growth coefficient (b). Mean final body length was 11.37 ± 0.24 cm in CON+ADD and 11.92 ± 0.49 cm in CON (Figure 2C), while modified K values averaged 0.848 ± 0.059 and 0.801 ± 0.041, respectively (Figure 2B). Similarly, the allometric coefficient did not differ between treatments, averaging 3.01 ± 0.18 in CON+ADD and 3.03 ± 0.23 in CON (Figure 2A).

Table 5. Length–weight relationship.

Length–weight relationships were described by the equation W = aL^b. Growth was considered isometric when the estimated allometric coefficient (b) did not differ significantly from 3.0 (Student’s t-test, p > 0.05). ANCOVA detected no significant treatment effect and no significant treatment × length interaction, indicating similar length–weight relationships between dietary treatments.

Length–weight relationships were strongly described by the power function (W = aL^b) in both dietary treatments (Table 5). Estimated allometric coefficients were 2.948 for CON+ADD and 3.126 for CON, with coefficients of determination (R²) of 0.865 and 0.897, respectively. In both cases, the estimated coefficients did not differ significantly from the theoretical isometric value of 3.0, indicating approximately isometric growth patterns.

Analysis of covariance detected neither a significant treatment effect (p = 0.111) nor a significant treatment × length interaction (p = 0.134), demonstrating that dietary supplementation did not alter the scaling relationship between body length and body weight. Collectively, these results indicate that functional additives did not affect body condition, body morphology, or allometric growth characteristics in juvenile Penaeus vannamei.

3.3 Low-salinity stress tolerance

Responses to acute low-salinity exposure are summarized in Table 6 and Figure 3. A total of 40 mortality events were recorded in CON+ADD and 39 in CON. Mean time to death was 273.0 ± 78.9 min in shrimp fed the additive-supplemented diet and 293.5 ± 76.9 min in the control group, while median survival times were 285 and 300 min, respectively (Table 6).

Table 6. Survival responses of juvenile whiteleg shrimp (Penaeus vannamei) during an acute low-salinity challenge.

Kaplan–Meier survival curves showed substantial overlap throughout the challenge period (Figure 3). Consistent with this observation, survival distributions did not differ significantly between treatments (log-rank p = 0.149). Cox proportional hazards analysis likewise detected no significant treatment effect on mortality risk. Although the point estimate of the hazard ratio (1.34; 95% CI: 0.86–2.09) suggested a numerically higher mortality risk in CON+ADD, the confidence interval was wide and encompassed unity, indicating substantial uncertainty and no statistically significant treatment effect. Overall, dietary supplementation did not improve tolerance to acute osmotic stress under the conditions of the low-salinity challenge. .

3.4 Growth dynamics and allometric growth relationships

Weekly average body-weight measurements were accurately described by Gompertz growth models in both dietary treatments, with coefficients of determination exceeding 0.99 (Figure 4A), indicating comparable growth dynamics throughout the 42-day feeding period. Consistent with these parameter estimates, no evidence of treatment-specific divergence in growth trajectories was observed.

Length–weight relationships likewise showed substantial overlap between treatments (Figure 4B). Estimated allometric coefficients were 2.948 for CON+ADD and 3.126 for CON, with corresponding coefficients of determination (R²) of 0.865 and 0.897, respectively. Mixed-effects allometric modeling indicated that body weight increased predictably with body length across all shrimp, while the treatment × length interaction was not significant (ANCOVA p = 0.134). Together, these analyses indicate that dietary supplementation did not alter temporal growth trajectories or body-size scaling relationships in juvenile Penaeus vannamei.

3.5 Multivariate phenotypic responses to dietary treatments

Multivariate phenotypic responses are summarized by principal component analysis in Figure 5. The first two principal components explained 75.1% of the total variance, with PC1 accounting for 45.2% and PC2 accounting for 29.8%. Variables contributing most strongly to the multivariate structure included protein efficiency ratio (PER), feed conversion ratio (FCR), weight coefficient of variation (Weight CV), modified condition factor (K), and final body weight.

The PCA biplot revealed partial separation between dietary treatments along the primary axes of variation, although substantial overlap among tanks remained (Figure 5). Tanks receiving the additive-supplemented diet tended to be associated with higher PER values and lower FCR, whereas variation in body condition and growth uniformity contributed to differences among individual tanks regardless of treatment.

Overall, the PCA was consistent with univariate results, indicating that dietary supplementation primarily influenced nutrient-use efficiency while exerting comparatively limited effects on growth performance, morphometric condition, and population uniformity. Given the limited number of experimental units available for multivariate analysis (n = 8 tanks), these results should be interpreted as exploratory visualizations of data structure and variable relationships rather than confirmatory evidence of treatment effects.

3.6 Exploratory modeling of treatment-response pathways and variable importance

Exploratory modeling analyses were performed to identify variables most strongly associated with dietary treatment effects (Figure 6). Standardized path coefficients indicated that treatment was most strongly associated with protein efficiency ratio (PER), whereas pathways involving feed conversion ratio (FCR), growth uniformity, and morphometric condition showed weaker relationships (Figure 6A).

Random Forest analysis produced similar results, identifying PER and FCR as the most influential variables for treatment discrimination, followed by final body weight and growth-uniformity metrics (Figure 6B). Morphometric traits, including the allometric coefficient (b) and modified condition factor (K), contributed comparatively little to model performance.

Although these analyses should be interpreted cautiously because of the limited number of experimental units (n = 8 tanks), both approaches consistently identified nutrient-use efficiency as the primary response associated with dietary supplementation. These findings support the univariate and multivariate analyses, which likewise indicated that functional additives primarily influenced protein utilization rather than growth performance, morphometric condition, or low-salinity stress tolerance. Given the small sample size, however, the Random Forest and pathway analyses should be regarded as hypothesis-generating tools and not as predictive or confirmatory evidence of treatment effects.

4 Discussion

The present study evaluated the effects of a dietary additive blend containing choline chloride, L-carnitine, glycerol monolaurate, and lysophospholipids on growth performance, feed utilization, morphometric condition, growth uniformity, allometric growth, and low-salinity stress tolerance in juvenile Penaeus vannamei. The principal finding was that dietary supplementation significantly improved protein efficiency ratio (PER), whereas most other measured responses, including growth performance, morphometric traits, allometric growth characteristics, and stress tolerance, remained unchanged. Collectively, these results indicate that the additive blend primarily influenced nutrient-use efficiency rather than inducing broad changes in growth or physiological performance.

The improvement in PER suggests that shrimp receiving the additive-supplemented diet utilized dietary protein more efficiently than those fed the control diet. The observed PER response may reflect the collective action of the additive blend or interactions among its components. Because choline chloride, L-carnitine, glycerol monolaurate, and lysophospholipids were administered together in a single formulation, the present experimental design does not allow attribution of the response to any individual additive. Consequently, the potential mechanisms discussed below should be regarded as plausible hypotheses based on previous literature rather than direct evidence generated by the present study. Choline chloride, L-carnitine, lysophospholipids, and glycerol monolaurate have all been associated with enhanced lipid digestion, nutrient absorption, and metabolic efficiency in aquatic animals (Li et al., 2019; Liu et al., 2020; Lan et al., 2021; Wang et al., 2022). Choline functions as a lipotropic factor involved in phospholipid synthesis and lipid transport, whereas L-carnitine facilitates the mitochondrial transport of long-chain fatty acids for β-oxidation. Lysophospholipids can improve lipid emulsification and nutrient absorption, while glycerol monolaurate has been linked to improved intestinal health and nutrient utilization. Through these complementary mechanisms, the additive blend may have improved the efficiency with which dietary nutrients were digested, absorbed, and utilized, thereby enhancing protein-use efficiency. Improved lipid utilization may also have reduced reliance on amino acids as energy substrates, resulting in a protein-sparing effect whereby a greater proportion of dietary protein was retained for growth and maintenance rather than catabolized for energy production.

An additional consideration when interpreting the PER response is the slight difference in analyzed crude protein content between the experimental diets. Although both diets were formulated to be isonitrogenous (42% crude protein), proximate analyses indicated crude protein contents of 42.06% in the CON diet and 42.54% in the CON+ADD diet. This difference was small (0.48 percentage units) and is within the range commonly encountered during feed manufacture and analytical determination. Nevertheless, because PER calculations are based on protein intake, a minor contribution of this difference to the observed PER response cannot be completely excluded. Therefore, the improved PER observed in the supplemented group should be interpreted in the context of both the additive inclusion and the small variation in analyzed dietary protein content.

Despite the significant improvement in PER, no corresponding increases in final biomass, final body weight, survival, or feed conversion ratio were observed. Similar responses have been reported in nutritional studies where improvements in nutrient utilization did not necessarily translate into measurable gains in growth performance (Cuzon et al., 2004; Xie et al., 2020). One possible explanation is that the experimental diets already met the nutritional requirements of juvenile P. vannamei, limiting the extent to which improved nutrient utilization could stimulate additional growth. Under such conditions, additive supplementation may optimize nutrient use without substantially altering growth trajectories or biomass accumulation over the relatively short duration of the feeding trial. In addition, modest improvements in growth may have remained undetected because of the limited number of tank replicates available for statistical comparison. These findings reinforce the concept that nutrient-use efficiency and growth performance are related but distinct aspects of nutritional response. The present study utilized four replicate tanks per treatment, which is consistent with many controlled shrimp nutrition trials but limits statistical power to detect small-to-moderate treatment effects. Consequently, non-significant responses, including the numerical improvement observed for FCR (approximately 7.5%), should be interpreted cautiously because biologically relevant differences may have remained undetected. Although statistical significance was not achieved, the magnitude of the observed FCR response suggests that potential production-relevant effects cannot be excluded. Future studies employing larger numbers of experimental units would improve statistical sensitivity and strengthen inference regarding the magnitude and consistency of additive effects.

Morphometric condition and body-size scaling were similarly unaffected by dietary supplementation. Final body length, modified condition factor (K), and allometric growth coefficient (b) did not differ significantly between treatments, while length–weight analyses indicated approximately isometric growth in both groups. The absence of treatment effects on these parameters suggests that the additive blend did not alter patterns of morphological development or nutrient allocation sufficiently to influence body form. This conclusion is further supported by the ANCOVA analyses, which demonstrated similar length–weight relationships between treatments. Although improvements in nutrient-use efficiency were evident, these changes were not accompanied by measurable alterations in growth morphology or body-size scaling.

Growth uniformity also remained unchanged. Weight and length coefficients of variation were similar between treatments, indicating that dietary supplementation did not affect population heterogeneity. Uniformity is an important production characteristic because highly variable populations can reduce harvest efficiency and complicate management practices. The lack of treatment effects suggests that the additive blend neither enhanced nor disrupted competitive interactions among shrimp within culture tanks. Consequently, while nutrient-use efficiency improved, additive supplementation did not produce detectable changes in population-level growth structure.

The low-salinity challenge revealed no significant effects of dietary supplementation on stress tolerance. Kaplan–Meier survival curves showed substantial overlap among treatments, while both the log-rank test and Cox proportional hazards model indicated comparable mortality risks. Although shrimp fed the control diet exhibited slightly longer mean and median survival times, these differences were not statistically significant. Acute osmotic stress imposes considerable energetic demands related to ion regulation, osmoregulation, and cellular homeostasis (Li et al., 2007; Li et al., 2017a). Because the challenge salinity (0.35 ppt) approached freshwater conditions, the severity of the osmotic stress may have exceeded any modest physiological benefits associated with improved nutrient-use efficiency, limiting the detection of treatment-related differences in survival. Similar findings have been reported in previous studies where nutritional interventions improved feeding performance without consistently enhancing tolerance to environmental stressors (Laramore et al., 2001). Collectively, these results suggest that the additive blend did not confer measurable protection against severe low-salinity stress.

Interpretation of these findings should be made cautiously because shrimp were exposed simultaneously to abrupt reductions in both salinity and temperature. Although the challenge was intended to assess tolerance to acute low-salinity exposure, the approximately 14 °C decrease in water temperature relative to culture conditions likely imposed an additional physiological stressor. Consequently, mortality responses cannot be attributed solely to osmotic stress, and any effects of dietary supplementation on salinity tolerance may have been masked by concurrent thermal shock. Future studies should evaluate salinity-challenge responses under temperature-controlled conditions to better isolate osmotic stress effects.

Although the challenge conditions were intentionally severe, abrupt reductions in salinity may occur during extreme rainfall events, emergency water exchanges, harvesting, transport, and acclimation procedures in commercial shrimp farming. Consequently, evaluating shrimp responses under severe osmotic stress provides insight into physiological resilience under worst-case conditions. Nevertheless, future studies should incorporate moderate and stepwise salinity challenges to determine whether dietary supplementation influences stress tolerance under conditions more representative of routine farm management.

Another limitation is that shrimp used in the survival analysis originated from replicated culture tanks, yet tank identity was not incorporated into the survival models. Although mortality was recorded at the individual level, ignoring potential tank-level clustering may have affected variance estimates and statistical inference. Therefore, the survival results should be regarded as exploratory. Future studies should retain tank identity throughout stress testing and apply tank-clustered survival analyses, frailty models, or tank-level survival summaries to account for experimental unit effects.

The multivariate and exploratory analyses provided additional insight into treatment responses. Principal component analysis indicated that PER and FCR contributed most strongly to the primary axes of variation, whereas morphometric traits, growth uniformity metrics, and condition indices contributed comparatively less. Similarly, exploratory path analysis and Random Forest modeling consistently identified PER as the variable most strongly associated with dietary supplementation. Although these analyses should be interpreted cautiously because of the limited number of experimental units, their agreement with the univariate results supports the conclusion that nutrient-use efficiency was the primary response to additive supplementation. Because these multivariate analyses were conducted using only eight tank-level experimental units, the PCA, path analysis, and Random Forest outputs should be interpreted as exploratory assessments of variable relationships and data structure rather than confirmatory evidence of treatment effects. These approaches were included primarily to facilitate data integration and hypothesis generation for future research.

Taken together, the results demonstrate that functional additives can influence physiological efficiency even when conventional growth metrics remain unchanged. This finding highlights the importance of evaluating nutritional interventions using multiple complementary endpoints rather than relying exclusively on growth performance. Improvements in nutrient-use efficiency may represent meaningful biological responses that are not immediately reflected in body weight gain or feed conversion, particularly over relatively short production periods.

From a practical perspective, the observed increase in PER suggests that the additive blend may improve the efficiency with which dietary protein is utilized by juvenile P. vannamei. Enhanced protein utilization could contribute to improved protein retention, reduced nitrogen excretion, and more efficient use of dietary protein, which is typically the most expensive component of shrimp feeds. Consequently, even modest improvements in nutrient-use efficiency may have economic and environmental value in intensive production systems. However, the absence of significant effects on growth performance, morphometric condition, growth dynamics, and low-salinity stress tolerance indicates that the benefits of supplementation are likely to be subtle and primarily related to nutrient utilization rather than broad enhancements of shrimp performance. Future studies incorporating longer feeding periods, larger sample sizes, and direct measurements of digestive, metabolic, and osmoregulatory processes would help clarify the mechanisms underlying these responses and determine whether improvements in protein-use efficiency can translate into economically meaningful production benefits under commercial culture conditions.

5 Conclusion

Dietary supplementation with a blend of choline chloride, L-carnitine, glycerol monolaurate, and lysophospholipids significantly improved protein utilization efficiency in juvenile Penaeus vannamei, as reflected by a higher protein efficiency ratio. However, supplementation did not significantly affect growth performance, feed conversion ratio, growth uniformity, morphometric condition, allometric growth, growth dynamics, or tolerance to acute low-salinity stress. Multivariate and exploratory analyses consistently identified protein efficiency ratio as the primary response associated with dietary supplementation, suggesting that nutrient-use efficiency may be more sensitive to dietary intervention than conventional growth metrics. Although improved protein utilization indicates a biological response to supplementation, the absence of corresponding improvements in growth and stress tolerance suggests that practical production benefits remain uncertain under the conditions tested. Because the stress challenge involved simultaneous exposure to low salinity and reduced temperature, conclusions regarding salinity tolerance should be interpreted cautiously. Future studies using larger sample sizes, longer feeding periods, temperature-controlled stress challenges, and factorial or dose–response designs are needed to clarify the mechanisms underlying the observed responses and determine whether improved nutrient-use efficiency translates into economically meaningful benefits under commercial farming conditions.

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Footnotes

Edited by: Mohamed Faisal, Michigan State University, United States

Reviewed by: Carlo C. Lazado, Norwegian Institute of Food, Fisheries and Aquaculture Research (Nofima), Norway

Eman Zahran, Mansoura University, Egypt