Effect of the Chlorella vulgaris Bioencapsulated by Daphnia magna on the Growth and Nutritional Value of the Penaeus vannamei Cultured in a Synbiotic System

Abstract

The growing need for sustainable protein sources in aquaculture has led to interest in microalgae, such as Chlorella vulgaris, known for its high nutritional value. One promising strategy for delivering these nutrients is through bioencapsulation in Daphnia magna, a common live feed used in aquaculture. This study evaluated the effect of including D. magna bioencapsulated with C. vulgaris as live feed for marine shrimp Penaeus vannamei post-larvae. Shrimp were fed at two D. magna densities (5 and 10 per shrimp, 5DF and 10DF), offered weekly, and compared with a control group receiving only commercial feed (F) for 36 days in a synbiotic nursery system. Water quality, zootechnical performance, protein, and lipid content of the shrimp were analyzed using correlation analyses, nonlinear prediction models, and principal component analysis (PCA). Shrimp fed with the 10DF treatment exhibited superior zootechnical performance, characterized by a lower feed conversion ratio (1.01) and higher feed efficiency (99.97%), protein (70.91%), and lipid (32.45%) content in comparison with the 5DF and control. Quadratic regression predictive models indicated the possibility of further testing higher concentrations of D. magna per shrimp. The results indicates that the use of C. vulgaris bioencapsulated in D. magna as live feed for P. vannamei is a promising approach to improve shrimp diets and increase production in aquaculture.

1. Introduction

There are challenges that impact sustainability and growth, such as pressure on water resources, pollution caused by the generation of effluents from aquaculture systems, and the need to ensure animal welfare. Therefore, an ecosystem-based approach is required for the intensification and expansion of aquaculture, aiming to minimize environmental impacts, ensure animal and food safety, and promote the efficient and sustainable use of inputs and resources such as water, land, and feed [1]. In this context, the selection of a nutritious diet is crucial for achieving success in production, especially in nursery culture. Consequently, the search for alternative sources of protein for animal feed is an emerging topic to ensure food security. In this context, live feed plays a significant role in aquaculture, particularly in the early stages of development. Microalgae, such as Chlorella vulgaris, are rich in essential nutrients, including proteins, lipids, and bioactive compounds that enhance nutrition, growth, stress alleviation, and immunity in aquatic organisms [2].

In terms of nutritional composition, it is well established that C. vulgaris boasts noteworthy levels of proteins (ranging from 13.6 to 65.5%), lipids (from 5.10 to 19.7%) [3], and fatty acids (around 10% dry weight) [4]. Among its fatty acid content, approximately 40.8% comprises polyunsaturated fatty acids (PUFAs) [5]. Additionally, it contains pigments, such as lutein, known for its antioxidant properties, accumulating to approximately 0.45% of the microalga’s dry weight [6]. Essential amino acids are also abundant, including a significant presence of leucine and lysine (averaging between 9 to 10% of total amino acids) [3]. Furthermore, it has been demonstrated that C. vulgaris can thrive in culture media based on agricultural fertilizers or using aquaculture wastewater, significantly reducing the production costs [7,8,9,10].

Likewise, zooplankton, such as Daphnia magna, serves as an efficient live feed, capable of bioencapsulating and delivering high-value nutrients from microalgae to shrimp larvae, improving their nutritional intake and overall health. However, the use of cladocerans as live feed in shrimp diets is still poorly studied compared with other zooplankton groups [11,12,13]. The most studied and utilized live feeds for shrimp larvae are Artemia nauplii [13,14,15,16,17], rotifers [16,18,19], and copepods [17,20,21].

So far, no studies (according to a search in the Web of Science database) have been found reporting the use of the cladoceran water flea Daphnia magna as live feed in the diet of the shrimp P. vannamei. Nonetheless, its application as meal and even as a source of several biocompounds, including chitin and chitosan, for supplementation in the feed of aquatic animals has garnered attention from researchers in recent years [22,23,24,25,26].

The cladoceran D. magna is a freshwater microcrustacean that thrives in environments rich in organic detritus, promoting the proliferation of bacteria, yeasts, and microalgae, that are the primary feed sources for this cladoceran [27,28,29]. D. magna can be cultivated easily and inexpensively using low-cost products, such as chicken manure, rice bran, bread yeast, and quail manure [30], along with aquaculture effluents [31,32]. D. magna is also notable for its nutritional profile, containing 50–60% protein, 4–8% lipids, with higher content of unsaturated fatty acid and essential amino acids, in addition to polysaccharides, like chitin and chitosan [24,25,26,28,29,30,33]. Chitin and chitosan exhibit immune-stimulating properties and induce the production of digestive enzymes such as trypsin, lysine, and pepsin in the hepatopancreas of P. vannamei [24,34].

Consequently, the combined nutritional benefits found in both C. vulgaris and D. magna suggest potential as a super live feed for aquatic animal larvae, particularly for shrimp species like P. vannamei. In this synergy, the water flea serves as a bioencapsulating agent, effectively transferring the nutritional advantages of C. vulgaris to the cultured animals. Moreover, the nutritional content of C. vulgaris is readily bioavailable due to the breakdown of its cell wall facilitated by specific enzymes contained in D. magna. Microalgae often pose a challenge for proper digestion and nutrient assimilation due to their rigid cell walls, hindering the access of digestive enzymes to the intracellular components [2,35].

Hence, it is essential to assess the effect of supplementing the diet of marine shrimp P. vannamei post-larvae with D. magna bioencapsulated with the microalga C. vulgaris as live feed. Such an investigation aims to identify potential live feed options that can yield significant production outcomes for the aquaculture sector, particularly in shrimp farming industry.

2. Materials and Methods

2.1. Chemicals

Analytical-grade chemicals (purity ≥ 98%) were purchased from Dinâmica Química Contemporânea Ltda (Indaiatuba, São Paulo, Brazil), Êxodo Científica (Sumaré, São Paulo, Brazil), and Sigma-Aldrich Co. (St. Louis, MO, USA), and used without further purification.

2.2. Experimental Design

To assess the influence of offering Chlorella vulgaris bioencapsulated in the water flea Daphnia magna as live feed to Penaeus vannamei post-larvae, the following experimental design was outlined: (1) nursery cultivation of P. vannamei without the addition of D. magna bioencapsulated with C. vulgaris (F), meaning only commercial feed acting as the control; (2) nursery cultivation of P. vannamei with the addition of D. magna bioencapsulated with C. vulgaris at a concentration of 5 Daphnia per shrimp plus commercial feed (5DF); (3) nursery cultivation of P. vannamei with the addition of D. magna bioencapsulated with C. vulgaris at a concentration of 10 Daphnia per shrimp plus commercial feed (10DF). All 3 treatments were randomized with 4 replicates each (n = 4), totaling 12 experimental units, in order to minimize data variability and ensure that the results were more representative and reproducible.

2.3. Experimental Conditions

This study was conducted at the Sustainable Mariculture Laboratory (LAMARSU) of the Department of Fisheries and Aquaculture (DEPAq) at the Federal Rural University of Pernambuco, Brazil.

Initially, the synbiotic system was prepared 40 days before cultivation. In a production tank (1.2 m³), seawater (30 g L⁻¹ salinity) was chlorinated with 13 mg L⁻¹ of active chlorine. Then, a single inorganic fertilization was performed with urea (4.5 g m⁻³ N), triple superphosphate (0.30 g m⁻³ P), and sodium silicate (0.23 g m⁻³ Si). Organic fertilization was carried out through 15 applications of a synbiotic compound consisting of rice bran (30 g m⁻³), molasses (3 g m⁻³), sodium bicarbonate (6 g m⁻³), and a bacterial product (0.5 g m⁻³) containing Bacillus subtilis, B. licheniformis, Saccharomyces sp., and Pseudomonas sp. at a total of 5.5 to 6.5 × 10⁷ CFU g⁻¹ (Kayros Environmental and Agricultural), and seawater (30 g L⁻¹ salinity) at a ratio of 1:10 (w:v) of the amount of rice bran, following a protocol adapted from Lima et al. [36]. The synbiotic was prepared with a 24 h anaerobic phase followed by a 24 h aerobic phase and added every two days.

Post-larvae of shrimp (PL₁₃), with an average weight of 0.011 ± 0.006 g, were stocked at a density of 3000 PL m⁻³, cultivated for 36 days in 15L circular tanks, with a useful volume of 10 L (30 PL per tank). Feeding followed the methodology of Van Wyk [37], based on four daily offerings (at 08:00, 11:00, 14:00, and 16:00), with commercial feed containing 45% crude protein and 9.5% ether extract, adjusted daily according to shrimp consumption estimates and mortality rates. The first biometric measurement was taken on the 12th day of cultivation, with subsequent measurements every 8 days (0th, 12th, 20th, 28th, and 36th) to determine shrimp growth and adjust the amount of feed offered.

Post-larvae were fed once a week with the respective concentrations of water flea bioencapsulated with C. vulgaris per shrimp from each treatment (5DF and 10DF), adapted from Aravind et al. [38]. Water fleas bioencapsulated with C. vulgaris were offered in vivo at 15:00, between two feedings with commercial feed (14:00 and 16:00). The D. magna bioencapsulated with C. vulgaris and the quantities of protein and lipids in the diet (commercial feed, D. magna, and C. vulgaris) are detailed in Figure 1.

2.4. Maintenance of C. vulgaris and D. magna Stock Cultures

The stock production of C. vulgaris was carried out in a semi-continuous system using 5 L polyethylene containers. The culture medium consisted of NPK fertilizer (at a 20:10:20 ratio) at a concentration of 2 mL L⁻¹, supplemented with a solution of B-complex vitamins (cyanocobalamin and biotin) (0.2 mL L⁻¹), and a trace metals solution (1 mL L⁻¹). The nutrient composition of the medium was based on the BBM medium [39], with the metal solution adjusted according to Renstrom et al. [40] with some modifications. The microalgae were subjected to continuous illumination (24 h of light) with an irradiance of 30 µmols photons m⁻² s⁻¹ (from 10 W LED lamps), continuous aeration, and maintained at pH 7.2–7.8 and a temperature of 25–27 °C.

The stock culture of D. magna was maintained in 15 L circular tanks with a working volume of 12 L, in a mixotrophic system involving the fermentation of chicken manure (0.3 g L⁻¹) and dry bread yeast (Saccharomyces cerevisiae) (0.3 g L⁻¹), as adapted from Herawati et al. [30]. Environmental conditions were maintained with alkalinity between 100 and 120 mg L⁻¹ CaCO₃, pH 7–8, temperature 26–28 °C, constant aeration, and a photoperiod of 12 h of light provided by 10W LED lamps at 30 µmol photons m⁻² s⁻¹. The bioencapsulation involved feeding D. magna with C. vulgaris ad libitum, allowing the ingestion and temporary retention of microalgal cells within their digestive tract. To ensure efficient microalgal transfer and maximize the bioavailability of key compounds, Daphnia were harvested and offered as live feed to shrimp within one hour after feeding.

2.5. Water Quality Monitoring

Dissolved oxygen (DO), temperature, pH, and salinity were measured using a YSI multiparameter probe (Model 556 MPS, Yellow Spring Instruments, Yellow Springs, OH, USA), while settleable solids (SS) were assessed twice a week using an Inhoff cone [41]. Total ammonia nitrogen (TAN) (mg L⁻¹), N-nitrite (NO₂⁻-N) (mg L⁻¹), N-nitrogen (NO₃⁻-N) (mg L⁻¹), and orthophosphate (PO₄⁻³) (mg L⁻¹) levels were analyzed at the beginning and end of the culture following standard methodologies [42,43,44,45]. Total alkalinity was measured once a week using the method outlined by APHA [46].

2.6. Zootechnical Performance Variables of Shrimp

Biometry of reared shrimp was conducted weekly to determine the zootechnical performance. At the end, the following parameters were calculated: weight gain (g), biomass gain (g), final weight (g), final biomass (g), survival rate (%), specific growth rate (in weight) SGR (%/day), feed conversion ratio (FCR), feed efficiency (%) (FE), productivity (kg·m⁻³), and protein efficiency ratio (PER) according to the following equations:

Weight gain (g) = final weight − initial weight

(1)

Biomass gain (g) = (final weight − initial weight) × survival rate% × N shrimp

(2)

Final weight (g) = biomass (g)/N shrimp

(3)

Final biomass (g) = Final weight (g) × N shrimp

(4)

Survival rate (%) = (N_f shrimp/N_i shrimp) × 100

(5)

Specific growth rate − SGR (%/day) = [(Ln final weight − Ln initial weight) × 100]/days of culture

(6)

Feed conversion ratio − FCR = feed offered per tank/live shrimp weight gain × live shrimp number per tank

(7)

Feed efficiency (%) − FE = [(shrimp final average weight × final live shrimp number per tank − shrimp initial average weight × initial live shrimp number per tank)/feed offered per tank] × 100

(8)

Productivity (kg m⁻³) = biomass (kg)/tank volume (m³)

(9)

PER = Biomass gain (g)/feed offered per tank × percentage of crude protein in the feed (g)

(10)

2.7. Analysis of Proteins and Lipids

The protein (Kjeldahl method 945.01, conversion factor 4.78) and lipid (Soxhlet method 920.39C) content of C. vulgaris, D. magna, and shrimp fed on the respective diets were quantified using dry matter following the methodologies described by AOAC [47].

2.8. Statistical Analysis

Homoscedasticity (Bartlett’s test) and normality (Shapiro–Wilk) tests were employed to evaluate the data. One-way analysis of variance (ANOVA) was conducted using Tukey’s test to identify any statistically significant differences in water quality parameters, zootechnical performance metrics, and nutritional composition. Prior to analysis, the data were subjected to a logarithmic transformation (log (x + 1)) for Pearson correlation analysis and principal component analysis (PCA). Quadratic regression analysis was conducted on the variables of final weight, productivity, protein content, and lipid content concerning the concentration of water flea D. magna bioencapsulated with C. vulgaris per cultivated shrimp. All statistical analyses were performed using the free R Core Team software, version 4.2.3 [48]. For all analyses, a level of 5% of significance was adopted.

3. Results

3.1. Water Quality Monitoring

Overall, water quality data showed no statistical differences between treatments, except for the concentrations of orthophosphate (PO₄⁻³), where 10DF had a higher mean value compared with the others (

Table 1. Water quality parameters (mean ± standard deviation) measured during the cultivation of Penaeus vannamei post-larvae in a synbiotic system supplemented with Chlorella vulgaris bioencapsulated in live Daphnia magna.

Parameter	F	5DF	10DF
Temperature (°C)	28.42 ± 1.06	28.49 ± 1.10	28.57 ± 1.13
pH	8.50 ± 0.10	8.52 ± 0.09	8.52 ± 0.08
Salinity (g L−1)	30.43 ± 2.09	29.37 ± 2.55	29.56 ± 2.52
DO (mg L−1)	7.26 ± 0.88	7.66 ± 0.98	7.58 ± 0.91
SS (ml L−1)	1.96 ± 1.58	3.64 ± 2.55	4.97 ± 2.45
Alkalinity (mg CaCO3 L−1)	152 ± 23.71	161 ± 18.33	165 ± 14.26
TAN (mg L−1)	0.96 ± 0.47	0.73 ± 0.28	0.80 ± 0.26
NO2−-N (mg L−1)	0.06 ± 0.04	0.09 ± 0.07	0.08 ± 0.05
NO3−-N (mg L−1)	0.65 ± 0.24	0.45 ± 0.26	0.51± 0.15
PO4−3 (mg L−1)	5.89 ± 0.60 b	6.56 ± 0.54 ab	6.72 ± 0.68 a

Treatments: F = P. vannamei shrimp fed exclusively with commercial feed; 5DF = P. vannamei shrimp fed with commercial feed plus C. vulgaris bioencapsulated in live D. magna (5 individuals per shrimp); 10DF = P. vannamei shrimp fed with commercial feed plus C. vulgaris bioencapsulated in live D. magna (10 individuals per shrimp). DO—dissolved Oxygen; SS—settleable solids; TAN—total ammonium nitrogen. Different letters within columns indicate significant differences between treatments (p < 0.05), according to Tukey’s test.

). The average amounts of nitrogenous compounds (TAN, NO₂⁻-N, and NO₃⁻-N) remained below 1 mg L⁻¹. The temperature was reported around 28 °C, dissolved oxygen (DO) at 7 mg L⁻¹, pH at 8.5, and salinity at 30 g L⁻¹. The variables TSS and alkalinity exhibited greater variation among treatments, ranging from 1.96 to 4.97 mL L⁻¹ and 152 to 165 mg CaCO₃ L⁻¹, respectively (Table 1).

3.2. Zootechnical Performance Variables of Shrimp

When analyzing the zootechnical results presented at the end of the cultivation, it was possible to identify significant differences (p < 0.05) for the variables of final biomass, final weight, SGR, biomass gain, weight gain, FCR, FE, and productivity (

Table 2. Zootechnical performance parameters (mean ± standard deviation) during the cultivation of Penaeus vannamei post-larvae in a synbiotic system supplemented with Chlorella vulgaris bioencapsulated in live Daphnia magna.

Parameter	F	5DF	10DF
Final biomass (g)	14.53 ± 0.65 a	16.67 ± 1.81 ab	18.05 ± 1.44 b
Final weight (g)	0.575 ± 0.033 a	0.623 ± 0.071 a	0.750 ± 0.065 b
Weight gain (g)	0.564 ± 0.033 b	0.612 ± 0.071 ab	0.709 ± 0.088 a
Biomass gain (g)	14.2 ± 0.65 b	16.34 ± 1.81 ab	17.72 ± 1.44 a
FCR	1.57 ± 0.19 a	1.44 ± 0.32 ab	1.01 ± 0.14 b
SGR (% day−1)	11.43 ± 0.12 a	12.24 ± 0.37 ab	12.47 ± 0.64 b
Survival rate (%)	84.44 ± 6.28 a	89.33 ± 0.94 a	80.25 ± 3.09 a
Productivity (kg m−3)	1.45 ± 0.06 a	1.66 ± 0.18 ab	1.80 ± 0.14 b
FE (%)	64.03 ± 7.50 a	71.95 ± 10.97 a	99.97 ± 14.54 b
PER	0.78 ± 0.03 a	0.81 ± 0.09 a	0.88 ± 0.07a

Treatments: F = P. vannamei shrimp fed exclusively with commercial feed; 5DF = P. vannamei shrimp fed with commercial feed plus C. vulgaris bioencapsulated in live D. magna (5 individuals per shrimp); 10DF = P. vannamei shrimp fed with commercial feed plus C. vulgaris bioencapsulated in live D. magna (10 individuals per shrimp). SGR—specific growth rate; FCR—feed conversion ratio; FE—feed efficiency; PER—protein efficiency ratio. Different letters within columns indicate significant differences between treatments (p < 0.05), according to Tukey’s test.

). The variables of survival and PER showed no significant differences between treatments (p > 0.05). Overall, treatments with the addition of C. vulgaris bioencapsulated in the D. magna in the shrimp diet exhibited higher results in comparison with the control (Table 2).

Analyzing the growth curve of shrimp post-larvae throughout the cultivation period revealed statistically significant differences (p < 0.05) among the treatments, particularly on days 20, 28, and 36, with 10DF exhibiting the highest values (Figure 2).

Another noteworthy aspect pertains to the feeding behavior of shrimp during the predation of D. magna bioencapsulated with C. vulgaris, which exhibited distinctions between the early PL13 and late stages of cultivation. Initially, the post-larvae could only ingest the internal contents of the water fleas, leaving their exoskeletons at the tank’s bottom. However, as the cultivation period progressed from the middle to the end, the shrimp were capable of fully consuming the Daphnia, including their exoskeletons. The feeding process commences from the organisms’ heads until complete ingestion. A video illustrating this feeding behavior has been recorded and is accessible via digital version (Supplementary Material Video S1). Readers of the print version are kindly encouraged to access the digital version of the manuscript.

3.3. Protein and Lipid Content

The protein and lipid quantities (dry weight) showed significant differences among the treatments (p < 0.05), with the highest amounts observed in the shrimp from the 10DF treatment, reaching protein and lipid quantities of 70.91% and 32.45%, respectively. Conversely, the lowest quantities were reported in the F treatment, reaching 65.80% and 23.05% for protein and lipids, respectively (Figure 3).

3.4. Correlation, Regression, and PCA Analysis

After applying Pearson’s correlation analysis to the zootechnical performance variables and protein and lipid contents, significant direct correlations (p < 0.05) were identified. Inverse correlations between the survival variable and the others were not considered significant (p > 0.05). High correlations were identified among the zootechnical performance variables, reaching an r of up to 1 (final biomass and productivity) (Figure 4).

Quadratic regression analysis was applied to the final weight, productivity, protein, and lipid contents in relation to the concentration of water fleas bioencapsulated with C. vulgaris per cultivated shrimp. Significant statistical differences (p < 0.05) were identified, with R² = 0.68 for final weight (Figure 5A), R² = 0.59 for productivity (Figure 5B), R² = 0.86 for protein quantities (Figure 5C), and R² = 0.91 for lipids (Figure 5D). The generated equations for each nonlinear model are displayed in the graphs of Figure 5.

PCA ordination explained 89.36% of the data variability in the PC1 (76.04%) and PC2 (13.32%) axes (Figure 6). The variables that primarily contributed to explaining PC1 included final biomass (0.327), productivity (0.328), feed efficiency (FE) (0.333), biomass gain (0.327), weight gain (0.331), final weight (0.338), protein efficiency ratio (PER) (0.305), and specific growth rate (SGR) (0.2811). Meanwhile, survival (0.710), protein (−0.282), and lipids (−0.363) variables made the most significant contributions to PC2.

Upon grouping by treatments, most variables exhibited a stronger association with treatment 10DF, except the survival variable, which displayed a closer relationship with treatment 5DF. In contrast, treatment F did not demonstrate prominence for any of the analyzed variables (Figure 6).

4. Discussion

4.1. Water Quality

The differences found in orthophosphate concentrations among the treatments may be related to the solids concentrations present in the water, as, although not indicating significant differences, treatment 10DF obtained higher average concentrations of settleable solids (SS). Nevertheless, the levels presented in this study remained within the indicated values of shrimp farming in intensive systems. According to Emerenciano et al. [49], orthophosphate concentrations ranging from 0.5 to 20 mg L⁻¹ are within the ideal range for shrimp cultivation, along with SS concentrations of 5 to 15 mL L⁻¹. Although orthophosphate levels were significantly higher in the 10DF treatment, no negative effects on shrimp performance were observed, likely due to the assimilation of excess nutrients by phytoplankton, which helped maintain system balance and supported shrimp health [50]. The water quality in shrimp farming systems results from complex interactions among various physicochemical variables. Among these, the presence of suspended solids is particularly relevant, as they are associated with settleable organic matter, whose decomposition can consume dissolved oxygen and compromise its availability within the system.

Total ammonia and nitrite concentrations found in this study are also within the recommended range as indicated by Furtado et al. [49], who suggest that ammonia/nitrogen concentrations should be kept below 1.5 mg L⁻¹, while nitrite/nitrogen concentrations must remain below 2 mg L⁻¹. The average alkalinity and pH concentrations found in this study also align with the findings of Furtado et al. [51], who reported that elevated alkalinity, particularly above 150 to 300 mg CaCO₃ L⁻¹, is more favorable for shrimp growth due to the maintenance of pH levels and nitrifying bacteria. Therefore, strict monitoring and control of these parameters are essential to ensure a healthy and productive aquatic environment.

4.2. Zootechnical Performance

In terms of zootechnical performance, the standout observed for treatments involving the addition of C. vulgaris bioencapsulated in the water fleas once again underscores the significance of providing live feed during the early stages of cultivation. The most extensively studied and utilized zooplanktons as live feed for shrimp during their initial phase typically include Artemia nauplii (e.g., Artemia franciscana and Artemia salina) [14,15,16,17], rotifers (e.g., Brachionus plicatilis and Brachionus rotundiformis) [16,18,19], and copepods, such as Oithona rigida [21], Tisbe biminiensis [20], and Calanopia elliptica [16]. The use of cladocerans, like Moina spp. [52,53], Eurycercus beringi sp. [38], and Diaphanosoma celebensis [54], as live feed in shrimp diets remains relatively understudied as this group is more commonly utilized in fish feeding [11,12,13].

However, to date, no studies have reported the use of C. vulgaris bioencapsulated in the water flea D. magna as live feed for P. vannamei, making this study pioneering in this field. Nevertheless, studies have reported the use of D. magna meal as a substitute for fish meal in fish feeding [22,26], along with the utilization of chitin and chitosan extracted from D. similis as additives to the diet of shrimp P. vannamei [24,25]. Moreover, this organism presents several advantages for use as live feed in aquaculture, such as a high fecundity rate, the ability to reach high population densities, a broad tolerance to environmental variables, a low production cost, and the potential to thrive in wastewater conditions [55]. It also represents a more sustainable alternative to Artemia, which relies on the extraction of natural stocks to meet market demand.

According to Hoff and Snell [56], rotifers, Daphnia spp., and Artemia spp. are considered by far the most efficient, due to their ease of production and the superior zootechnical performance results of the animals that consume them. This was corroborated in this study in the shrimp that were fed C. vulgaris bioencapsulated in the D. magna at 10DF. Furthermore, other studies have reported an increase in the zootechnical performance of shrimp fed with cladocerans. For instance, Aravind et al. [38] evaluated the provision of the cladoceran Eurycercus beringi in the diet of larvae from the Missis-3 to PL₁₃ stage of Peaneus indicus and reported improved growth and survival outcomes when they combined E. beringi with Artemia sp. nauplii, achieving a final weight of 0.9 mg, an SGR of 22.5% day⁻¹, and a survival rate of 65 ± 3%.

Previous studies have demonstrated the potential of zooplankton enriched with microalgae as an effective live feed for shrimp post-larvae, improving both survival and growth rates. The nutritional enhancement of cladocerans through microalgae enrichment has been particularly promising, as it enhances their lipid and protein profiles, making them a valuable dietary source. Rasdi et al. [54] observed a high survival rate (87.33%) and SGR (17.14%) for post-larvae of Penaeus monodon supplemented with the cladoceran Moina sp. enriched with Chlorella sp. Similarly, Khatoon et al. [57] indicated that supplementation with Diaphanosoma celebensis enriched with Chaetoceros calcitrans had a similar effect on the growth of P. vannamei post-larvae to supplementation with Artemia nauplii, achieving a survival rate of 67% and an SGR of 15%, whereas, with the use of Artemia, the survival rate and SGR were 65% and 14%, respectively. Furthermore, the study conducted by De Andrade et al. [18] showed that the use of rotifer Brachionus plicatilis enriched with Nannochloropsis oculata at different feeding frequencies during the nursery cultivation of P. vannamei achieved results similar to those found in the present study, i.e., final weights of 0.76 ± 0.01g, productivities of 1.88 ± 0.10 kg m⁻³, and SGRs of 14.31 ± 0.18% day⁻¹. These findings reinforce the potential of C. vulgaris bioencapsulated in the D. magna as a viable and possibly superior alternative to traditional live feeds such as Artemia and rotifers, given its comparable or improved results in shrimp growth performance and survival.

4.3. Protein and Lipids Content

The results obtained in this study regarding protein and lipid contents were higher than those found in other studies using other cladocerans enriched with microalgae in shrimp feeding. For example, Rasdi et al. [54] enriched the cladoceran Moina sp. with the microalgae Chlorella sp. and offered it to post-larvae of P. monodon, obtaining protein and lipid concentrations of approximately 63.49% and 4.28%, respectively. Likewise, Khatoon et al. [56] assessed the effect of offering Diaphanosoma celebensis enriched with the microalga C. calcitrans on the protein and lipid content of P. vannamei post-larvae and found protein and lipid concentrations of 46.2% and 15.4%, respectively. Similarly, Das et al. [53] reported protein and lipid concentrations ranging from 49.35% to 49.5% and 5.4% to 5.7%, respectively, in post-larvae of Macrobrachium rosenbergii supplemented with the cladoceran Moina micrura enriched with different oil emulsions. These results highlight that the protein and lipid levels obtained in the 10DF group are among the highest reported in the literature for shrimp fed with cladocerans enriched with microalgae, reinforcing the superior nutritional value of the C. vulgaris bioencapsulated in the D. magna used in this study.

The high concentrations of protein and lipids present in the shrimp fed with C. vulgaris bioencapsulated in the D. magna at a concentration of 10 Daphnia shrimp⁻¹ may be related to both the microalga C. vulgaris and the water flea. This green alga has relevant nutritional content, presenting approximately, in dry weight, a variation of protein quantities from 13.6% to 65.5%, 5.10% to 19.7% lipids [3], and 10% fatty acids [4], where, of the total quantity of fatty acids, it can reach up to 40.8% of polyunsaturated fatty acids (PUFAs) [5]. It produces pigments such as chlorophyll a, more abundantly ranging from 2.5 to 17.5 mg g⁻¹ per dry weight [56], and carotenoids, highlighting lutein, which possess antioxidant properties and can accumulate up to 0.45% of the alga’s dry weight [6]. Additionally, C. vulgaris is rich in essential amino acids, including a significant amount of leucine and lysine (average of 9% to 10% of total amino acids) [3].

In this study, the microalga C. vulgaris presented, in dry weight, 26.87% and 14.31% of proteins and lipids, respectively. It is important to note that all algae production used in the water flea feed was carried out with agricultural fertilizer, NPK; this enables the producers to achieve lower production costs compared with using specific chemical reagents for producing the culture medium.

Therefore, the excellent results found are due not only to microalga C. vulgaris but also to the D. magna. Daphnia species generally have a crude protein percentage of 52% to 68% and 4% to 7.8% lipids [30,33], which can vary depending on cultivation and the feed provided. In this study, the water flea fed with C. vulgaris reached 61.2% protein and 6.9% lipids. Additionally, water fleas possess high-quality essential amino acids: arginine (10.26%), cystine (1.17%), histidine (2.69%), methionine (3.45%), tryptophan (3.62%), and tyrosine (4.27%) [27]; lysine (2.06%), leucine (2.69%), isoleucine (1.51%), phenylalanine (2.13%), valine (2.18%), and threonine (2.01%) [22].

Other important nutritional components existing in the Daphnia genus are chitin and chitosan, which are polysaccharides of the beta-glucan group [56] and have been studied to improve the growth of aquatic organisms and enhance the immune system [24]. Studies report that cladocerans of the Daphnia genus exhibit a variation of 2.9% to 7% chitin (percent of total body weight) [58]. Tseng et al. [24] extracted chitin and chitosan from D. similis and added them as a supplement to the feed used in P. vannamei shrimp feeding, observing higher protein quantities (90.07%) in treatments with chitin addition, far superior to what was found in this study. Beyond their nutritional role, chitin and chitosan from D. magna exert immunomodulatory effects in P. vannamei, stimulating enzymes such as phenoloxidase and lysozyme, which enhance innate immunity [59,60,61]. These compounds also support intestinal integrity and microbial balance, contributing to improved nutrient absorption and growth performance. Moreover, chitin-derived oligosaccharides can activate immune receptors and induce the expression of antimicrobial peptides, reinforcing their functional role in shrimp health [62]. Thus, the efficiency of live feed in supplementing aquatic organisms lies in the function it performs, acting as a bioencapsulating agent of the primary feed they consume, which can also be enriched with vitamins, highly unsaturated fatty acids (HUFAs) [63], and probiotics [10], thereby transferring these components to the cultivated animals.

4.4. Interaction Between Zootechnical Performance and Nutritional Content

The high values of direct correlations found for the relationships between performance variables were expected, as they are interdependent variables. Moreover, the observed direct correlations between zootechnical performance variables and protein and lipid quantities reinforce the importance of live feed towards maintaining the quality, in terms of biological value, of reared shrimp.

From the prediction models generated by regression analysis, it was possible to identify that possibly higher concentrations of D. magna bioencapsulated with C. vulgaris per shrimp could be evaluated to optimize final weight, productivity, protein, and lipid content. For instance, according to the model, concentrations of 16 Daphnia per shrimp could provide final weight, productivity, protein, and lipid content of 1.00 g, 1.885 kg m⁻³, 70.84%, and 46.63%, respectively, while concentrations above 20 Daphnia per shrimp would already lead to a decrease in productivity (1.87 kg m⁻³) and protein concentration (69.31%). However, more trials are needed to determine the ideal concentration of water fleas to be used in the diet and also the frequency of supply, as this study provided D. magna bioencapsulated with C. vulgaris only once a week.

The good zootechnical results, along with the high quantities of proteins and lipids found for the 10DF treatment, were confirmed by PCA analysis. The formation of the three well-evidenced groups, with clear distances between them (10DF, 5DF, and F), and especially the isolation of the F treatment, once again demonstrate the importance and difference that the supply of live feed provides for the cultivation of aquatic organisms in the initial stages of shrimp growth. This practice is extremely relevant to ensure production success, reducing losses and feed costs and increasing productivity. However, it requires skilled labor to manage the entire production process and its distribution to post-larvae. Nonetheless, Daphnia production is considered relatively easy and low cost, combined with the production of C. vulgaris, which can be cultivated with agricultural fertilizer, leading to a reduction in production costs. Thus, research related to the search for new protein sources from different options of live organisms to be added to the diet of shrimp in the early development stages, along with aquatic organisms in general, strengthens the aquaculture sector and promotes its growth.

An important consideration in this context is the potential biological variability between different batches of Daphnia magna and Chlorella vulgaris. Although standardized cultivation protocols were used in this study, natural differences in growth rates and biochemical composition may occur and affect the reproducibility of the results. Future studies should investigate this variability more thoroughly, including batch-to-batch comparisons, to ensure the consistency and robustness of live feed strategies in aquaculture.

5. Conclusions

Based on the findings, it can be concluded that offering Chlorella vulgaris bioencapsulated in the Daphnia magna, as live feed in the diet of Penaeus vannamei post-larvae at a concentration of 10 Daphnia per shrimp, led to improved zootechnical performance and higher biological value in shrimp. Quadratic regression predictive models suggested the possibility of further research with higher concentrations of Daphnia bioencapsulated with C. vulgaris per shrimp to be explored. As pioneers in the utilization of C. vulgaris bioencapsulated in the D. magna as live feed for P. vannamei, our findings contribute to the evaluation of potential live feed that could be incorporated into the shrimp diet, resulting in enhanced production outcomes for the aquaculture sector, particularly in shrimp farming.

These results suggest that the strategic use of bioencapsulated microalgae in live feeds may represent an effective approach to enhance shrimp aquaculture, contributing to both sustainable production and improved product quality. Nevertheless, further studies are needed to evaluate different feeding frequencies in order to identify the most effective regimen. Assessing the scalability and economic viability of these feeding strategies under commercial conditions is also crucial. In addition, future research should investigate potential physiological and immunological changes in shrimp using more sensitive analytical approaches, including fatty acid composition, enzymatic activity, and resistance to pathogens. Exploring alternative microalgae species or their combinations for bioencapsulation may also help optimize the nutritional profile of diets and further improve shrimp growth performance.