Comparative Effects of Arthrobacter bussei-Derived Powder and Probiotics, and Haematococcus pluvialis Powder, as Dietary Supplements for Pacific White Shrimp (Litopenaeus vannamei)

Abstract

This study evaluated how dietary supplementation with Haematococcus pluvialis powder (HPP), Arthrobacter bussei powder (ABP), and A. bussei probiotics affects growth, whole-body composition, non-specific immunity, antioxidant capacity, and nutrient digestibility in Pacific white shrimp (Litopenaeus vannamei). Juvenile shrimps were fed for 8 weeks with five diets: a control diet (CON), H. pluvialis powder (HPP, 1%), A. bussei powder (ABP, 1%), or A. bussei probiotics at 10⁵ (ABL) or 10⁸ (ABH) CFU g⁻¹ feed. Shrimp fed the ABP diet exhibited the highest final body weight, weight gain, and protein efficiency ratio, with a significantly improved feed conversion ratio than that of CON, ABL, and ABH groups. The HPP group exhibited significantly better growth than that of the control. Regarding immunity and antioxidant responses, lysozyme and phenoloxidase specific activities, as well as glutathione peroxidase and superoxide dismutase specific activities, were significantly enhanced in shrimp fed ABP and HPP diets, whereas malondialdehyde levels were significantly reduced compared with those in CON. Apparent crude protein digestibility was significantly higher in all supplemented diets than those in the CON group, with ABP showing the highest value. ABP and HPP supplementation improved growth, protein digestibility, and immune-antioxidant responses in L. vannamei, whereas probiotic forms showed limited effects. ABP is a superior functional feed additive than its probiotic form for enhancing productivity and health in shrimp aquaculture.

1. Introduction

The Pacific white shrimp (Litopenaeus vannamei) has become a cornerstone species in global aquaculture owing to its rapid growth rate, desirable flavor, and rich nutritional profile [1]. From 1990 to 2023, production of this species expanded from 1.0 to 6.7 million tonnes, representing nearly 85% of global shrimp output [1]. As intensive farming expands, dietary management is critical because it directly affects survival, growth, feed efficiency, and product quality [2,3]. Inadequate or poorly balanced diets can compromise nutrient assimilation and resilience, leading to weakened immunity and impaired production efficiency [3,4,5]. To address these challenges, modern feed formulation has increasingly focused not only on balancing essential nutrients (e.g., digestible protein with indispensable amino acids, essential fatty acids, vitamins and minerals) but also on incorporating functional additives that provide benefits beyond basic nutrition [4,5,6,7].

Among such additives, bioactive compounds, particularly carotenoids and specific lipids (e.g., phospholipids, long-chain PUFA), have received attention for promoting growth, strengthening antioxidant defenses, improving digestive efficiency, and enhancing immune function [6,7,8]. Within this context, microalgae are recognized as sustainable feed ingredients. The green microalga Haematococcus pluvialis is notable for its high astaxanthin content and well-documented antioxidant and immunomodulatory activities [6,7,8,9,10]. Dietary H. pluvialis has been shown to improve pigmentation, stress tolerance, and immune responses in L. vannamei and other aquatic species, including under low-fishmeal or environmental-stress conditions [6,8,9,10].

In parallel, bacterial feed additives have emerged as a major category. Probiotics (e.g., Bacillus spp. and lactic acid bacteria) have been widely studied for modulating gut microbiota, enhancing digestion, and stimulating immune responses in shrimp; however, their effectiveness can be constrained by processing stability, storage, and gastrointestinal survival [4,5,11,12,13]. To overcome these limitations, non-viable microbial preparations (“postbiotics,” “paraprobiotics,” or microbial powders) have been explored as stable alternatives that deliver bioactive components (cell-wall fragments, metabolites, pigments) without relying on viability [11,12,13]. Comparative evidence suggests that such preparations can match or surpass certain functional effects of live probiotics while offering greater consistency and ease of feed incorporation [11,13]. Within this framework, Arthrobacter bussei has recently gained attention as a candidate microbial additive. This bacterium synthesizes bacterioruberin, a C₅₀ carotenoid with potent antioxidant properties and emerging antimicrobial relevance [14,15,16]. Although A. bussei has only been recently described taxonomically [14], the biological activities of bacterioruberin across microbial sources have been increasingly substantiated [15,16]. What remains unclear for shrimp nutrition is whether benefits from A. bussei are best realized through stable powder formulations or through live-cell probiotics [11,12,13].

Therefore, the present study was designed to systematically compare the effects of H. pluvialis powder, A. bussei powder, and two concentrations of live A. bussei probiotics on growth performance, whole-body composition, non-specific immunity, antioxidant capacity, and nutrient digestibility in Pacific white shrimp. By testing both powder and probiotic A. bussei alongside H. pluvialis, we aimed to clarify the relative advantages of microalgal versus bacterial bioactives and to determine whether stable powder or live-cell probiotic delivery represents the more effective strategy for shrimp aquaculture. We expected that supplemented diets would outperform the unsupplemented control across growth and innate immune antioxidant measures, and, without an a priori preference, we compared whether the stable powder or the live cell probiotic format confers greater efficacy.

2. Materials and Methods

2.1. Preparation of Experimental Diets

Five experimental diets were prepared: one unsupplemented control (CON) and four treatments supplemented with either H. pluvialis powder (HPP, 1% w/w, as-fed), A. bussei powder (ABP, 1% w/w, as-fed), or live A. bussei probiotics at two target inclusion levels (ABL: 1 × 10⁵ CFU g⁻¹ feed; ABH: 1 × 10⁸ CFU g⁻¹ feed) (

Table 1. Dietary formulation and proximate composition of the five experimental diets for Litopenaeus vannamei (% dry matter).

Ingredients (%)	Experimental Diets
	CON	HPP	ABP	ABL	ABH
Anchovy fish meal a	25.0	25.0	25.0	25.0	25.0
Squid liver powder b	10.0	10.0	10.0	10.0	10.0
Soybean meal b	25.0	25.0	25.0	25.0	25.0
Wheat flour b	19.5	19.5	19.5	19.5	19.5
Starch b	11.0	10.0	10.0	10.0	10.0
Haematococcus pluvialis powder	0	1.0	0	0	0
Arthrobacter bussei powder	0	0	1.0	0	0
A. bussei probiotics (1 × 105 CFU mL−1)	0	0	0	1.0	0
A. bussei probiotics (1 × 108 CFU mL−1)	0	0	0	0	1.0
Fish oil c	1.5	1.5	1.5	1.5	1.5
Monocalcium phosphate b	3.0	3.0	3.0	3.0	3.0
Lecithin	1.0	1.0	1.0	1.0	1.0
Choline chloride	1.0	1.0	1.0	1.0	1.0
Vitamin and Mineral premix d	3.0	3.0	3.0	3.0	3.0
Chemical composition (%, dry mater)
Moisture	9.27	9.35	9.41	10.80	10.95
Crude protein	37.27	37.10	37.21	37.25	37.33
Crude lipid	5.98	6.02	5.89	6.06	6.04
Crude ash	7.21	7.04	7.64	7.98	7.01

^a Supplied by Suhyup feed, Uiryeong, South Korea. ^b Supplied by The feed, Goyang, South Korea. ^c Supplied by Ewha Oil & Fat Industrial Co., Ltd., Busan, South Korea. ^d Contains (as mg kg⁻¹ in diets): Ascorbic acid, 300; dl-Calcium pantothenate, 150; Choline bitate, 3000; Inositol, 150; Menadion, 6; Niacin, 150; Pyridoxine⋅HCl, 15; Rivoflavin, 30; Thiamine mononitrate, 15; dl-α-Tocopherol acetate, 201; Retinyl acetate, 6; Biotin, 1.5; Folic acid, 5.4; Cobalamin, 0.06; NaCl, 437.4; MgSO₄•7H₂O, 1379.8; ZnSO₄•7H₂O, 226.4; Fe-Citrate, 299; MnSO₄, 0.016; FeSO₄, 0.0378; CuSO₄, 0.00033; Calciumiodate, 0.0006; MgO, 0.00135; NaSeO₃, 0.00025.

). HPP was sourced commercially (Hunan Can-Better Biological Co., Ltd., Changsha, China) and used as supplied. Preparation of A. bussei powders and probiotics followed Kim et al. (2022) with minor modifications [17]. Briefly, the strain was grown in Luria–Bertani (LB) broth at 28 °C with orbital shaking (180 rpm) to mid-log phase, harvested via centrifugation, washed, and either (i) freeze-dried to obtain a biomass powder designated non-viable (absence of colony formation on LB agar confirmed after 48 h at 28 °C), or (ii) resuspended in sterile saline to prepare viable probiotic suspensions. Viability of probiotic suspensions was verified using standard plate counts immediately before use. All base-diet ingredients were finely ground, dry-blended, and mixed with distilled water and fish oil to form a homogeneous mash, then pelleted using a laboratory pelletizer (MN-22S; Hankook Fuji Industries, Hwaseong-si, Republic of Korea). Pellets were cut to a 2.0 mm diameter suitable for the experimental shrimp size, air-dried at 25 °C for 12 h, sealed in opaque polyethylene bags, and stored at 4 °C until use. HPP and ABP were incorporated into the mash to achieve the stated 1% (w/w, as-fed) inclusion. For the probiotic diets (ABL and ABH), freshly prepared A. bussei suspensions were uniformly spray-coated onto pre-dried pellets using a minimal oil-based carrier, followed by gentle tumbling to ensure even distribution. To isolate the effect of bacterial cells, the same carrier type and volume were applied to CON, HPP, and ABP pellets (vehicle control). Coated probiotic pellets were used immediately after preparation to preserve viability. To confirm target dosing and homogeneity, triplicate subsamples from each probiotic batch (top, middle, and bottom of the mixing vessel) were assayed for CFU g⁻¹ through serial dilution and plating. Detailed formulations and proximate composition of all diets are provided in Table 1.

2.2. Experimental Design

Juvenile Pacific white shrimp (0.01 g) were obtained from a local hatchery (Daesang Aquaculture Industry, Taean, Republic of Korea). The feeding trial was conducted at the specialized shrimp-rearing facility of Kunsan National University. Before the experiment, shrimp were acclimated to laboratory conditions for approximately 3 weeks under the same temperature and photoperiod as the trial and were fed a commercial shrimp diet (Woosung Feed Co., Ltd., Daejeon, Republic of Korea). The acclimation diet followed the manufacturer’s specification and contained ≥40% crude protein. Following acclimation, shrimp with an initial mean body weight of 0.54 ± 0.02 g (juvenile stage) were randomly distributed into 15 fiberglass tanks (50 L each), corresponding to five dietary treatments with three replicate tanks per treatment, at a stocking density of 20 shrimp per tank. In total, 300 shrimp were stocked across 15 tanks (five diets × three replicates; 20 shrimp per tank). Tanks were supplied with aerated seawater and maintained under continuous aeration. Throughout the 8-week trial, water temperature was controlled at 29.7–30.5 °C using submersible heaters; dissolved oxygen was maintained at 5.9–8.2 mg L⁻¹ and pH at 6.4–7.2, monitored using a multiparameter analyzer (MultiLab 4010-3; YSI, Yellow Springs, OH, USA). A 12 h light:12 h dark photoperiod was maintained using overhead fluorescent lighting. Shrimp were fed three meals a day (08:00, 13:00, 18:00) at a restricted ration totaling 6–14% body weight per day, adjusted to apparent appetite as biomass increased. Uneaten feed and feces were siphoned daily, and the displaced volume was replaced with filtered seawater. In addition, 60% of the tank volume was exchanged once weekly to maintain water quality. Shrimp were fasted overnight (~12 h) before initial and final bulk weighing. The tank was considered the experimental unit for all statistical analyses. Animal handling and sampling procedures followed institutional guidelines for the humane treatment of aquatic animals.

2.3. Growth Performance

At the conclusion of the 8-week feeding trial, shrimp were fasted for 18 h before measurements to standardize physiological status and were sampled at fixed daytime hours. All shrimp were included in the measurements, and no perimolt signs (e.g., soft exoskeleton) or fresh exuviae were observed at the time of sampling. Individual body weights for all shrimp in each tank were recorded biweekly using an electronic balance (0.01 g precision); data were summarized as tank means. Daily feed intake was recorded per tank and summed over the trial; when mortalities occurred, biomass and feed intake were corrected per tank to avoid bias in feed-use indices. Growth and feed-use metrics were computed as follows (all on a per-tank basis): Weight gain (WG, %) = 100 × (Final mean body weight − Initial mean body weight)/Initial mean body weight; Feed conversion ratio (FCR) = Dry feed intake (g)/Wet WG (g); Specific growth rate (SGR, %/day) = {[ln(Final body weight) − ln(Initial body weight)]/Days} × 100; Protein efficiency ratio (PER) = Wet WG (g)/Total protein intake (g); Survival rate (SR, %) = (Initial number − Mortalities) × 100/Initial number.

2.4. Sample Collection and Biochemical Analyses

At the end of the feeding trial, eight shrimp per replicate tank were randomly selected and anesthetized in ice-chilled seawater to minimize stress and metabolic activity. Sampling was performed at fixed daytime hours across all tanks, and no perimolt signs were observed at collection. Hemolymph was withdrawn from the ventral sinus at the cephalothorax–abdomen junction using pre-chilled, sterile syringes preloaded with Alsever’s solution (anticoagulant; Sigma-Aldrich, St. Louis, MO, USA) and transferred to microtubes kept on ice. Samples were centrifuged at 5000× g for 10 min at 4 °C to obtain the plasma fraction (supernatant). Plasma was analyzed immediately or stored at −80 °C until assay. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were quantified using an automated Fuji DRI-CHEM 3500i dry-chemistry analyzer (Fuji Photo Film Ltd., Tokyo, Japan) following the manufacturer’s instructions. All measurements were performed in duplicate, with instrument quality controls and routine calibrations applied.

2.5. Proximate Composition Analysis

Proximate composition analysis of the experimental diets and whole-body shrimp samples was performed in triplicate to quantify moisture, crude protein, crude lipid, and crude ash content using standardized AOAC methods [18]. All results were expressed as percentages on a dry-weight basis. Moisture content was determined gravimetrically. Briefly, approximately 2 g of homogenized sample was weighed into pre-dried porcelain crucibles and dried in a forced-air drying oven (DO-150; Nexus Co., Ansan, Republic of Korea) at 105 ± 2 °C for 24 h or until constant weight was achieved. Moisture percentage was calculated as follows: Moisture (%) = (Weight loss during drying/Initial sample weight) × 100. To determine crude protein content, samples (0.5 g) were digested in concentrated sulfuric acid (H₂SO₄, 98%) with a catalyst mixture (potassium sulfate:copper sulfate, 10:1) at 420 °C for 2 h using a digestion system (FOSS Kjeltec 8400; FOSS Analytical, Hillerød, Denmark). Liberated ammonia was distilled into boric acid (4%) and titrated with 0.1 N HCl. Protein content was calculated using a nitrogen-to-protein conversion factor of 6.25: Crude Protein (%) = (Titrant volume × Normality of HCl × 14.01/sample weight) × 6.25. Total lipid content was determined using Soxhlet extraction [19]. Briefly, samples (3 g) were wrapped in fat-free filter paper and continuously extracted with anhydrous diethyl ether (C₄H₁₀O) for 6 h using a Soxtec™ 8000 system (FOSS Analytical). The solvent was evaporated at 60 °C, and the residual lipids were quantified gravimetrically: Crude Lipid (%) = (Weight of extracted fat/Initial sample weight) × 100. Ash content was determined by incineration following the AOAC Method [18]. Pre-weighed samples (1 g) were placed in pre-ashed porcelain crucibles and combusted in a muffle furnace (FHP-03 WiseTherm; Daihan Scientific Co., Ltd., Namyangju, Republic of Korea) at 550 ± 5 °C for 6 h. After cooling in a desiccator, the percentage of ash was calculated as follows: Crude Ash (%) = (Residual ash weight/Initial sample weight) × 100.

2.6. Non-Specific Immunity and Antioxidant Assays

Non-specific immunity and antioxidant status were assessed in hemolymph prepared as described above. Lysozyme activity was determined turbidimetrically using a suspension of Micrococcus lysodeikticus (0.2 mg mL⁻¹ in 0.05 M phosphate buffer, pH 6.2). Briefly, 10–20 µL hemolymph was added to 200 µL of substrate in 96-well plates, and the decrease in absorbance at 450 nm was recorded for 3–5 min at 25 °C. Antiprotease activity was quantified by inhibition of trypsin in 50 mM Tris-HCl (pH 8.2) using BAPNA (1 mM) as a chromogenic substrate. After a 10 min pre-incubation of hemolymph (10–20 µL) with trypsin at 25 °C, the rate of p-nitroaniline release was read at 410 nm. Phenoloxidase activity was measured as the rate of dopachrome formation from L-DOPA by monitoring the increase in absorbance at 490 nm for 5–10 min. Glutathione peroxidase (GPx) activity was determined with a coupled NADPH-oxidation assay kit (Biomax, Seoul, Republic of Korea) at 340 nm. Superoxide dismutase (SOD) activity was analyzed using a WST-based SOD assay kit (Biomax) in which SOD inhibits formazan formation. Absorbance was measured at 450 nm, and activities were calculated from the kit standard curve. As an index of lipid peroxidation, malondialdehyde (MDA) was quantified using a TBARS kit (Biomax). Reactions were developed at 95 °C, cooled, and measured at 532 nm (with 600 nm background correction when applicable). All reactions were run at 25 °C using a microplate spectrophotometer, performed in triplicate with blanks and standards.

2.7. Digestibility Test

Apparent digestibility was determined by adding 1.0% chromium(III) oxide (Cr₂O₃) (Sigma-Aldrich) to each experimental diet as an inert marker (as-fed basis). Juvenile shrimp (initial mean body weight 0.60 ± 0.02 g) were randomly stocked into three 50 L acrylic tanks at 15 shrimp per tank and fed the marker diets twice daily (08:00 and 15:00) for 8 weeks. Uneaten feed and debris were siphoned 30 min after each meal. Feces were collected twice daily (09:00 and 16:00) by gentle siphoning, pooled by tank, immediately frozen at −80 °C, then freeze-dried, finely ground, and passed through a 0.5 mm sieve before analysis. Chromium contents in diets and feces were determined after acid digestion using atomic absorption spectrophotometry according to standard procedures. Dry matter and crude protein (Kjeldahl N × 6.25) were analyzed following AOAC proximate methods. Apparent digestibility coefficients (ADCs) were calculated per tank as follows: for dry matter, ADC = 100 − 100 × (Cr₂O₃ in diet/Cr₂O₃ in feces); for protein, ADC = 100 − 100 × (Cr₂O₃ in diet/Cr₂O₃ in feces) × (protein in feces/protein in diet). All determinations were performed in triplicate.

2.8. Statistical Analyses

Data are presented as mean ± SD, with the tank as the experimental unit. Treatment effects were tested by one-way ANOVA, followed by Tukey’s HSD for multiple comparisons (p < 0.05). Percentage data (e.g., survival, ADCs when expressed as %) were arcsine square-root transformed before analysis. Statistical analyses were conducted using SPSS Statistics v26.0 (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Growth Performance and Whole-Body Composition

The growth performance of L. vannamei fed the experimental diets is summarized in

Table 2. Growth performance of Pacific white shrimp (Litopenaeus vannamei) fed the CON, HPP, ABP, ABL, and ABH diets during the experimental period. ¹.

		CON	HPP	ABP	ABL	ABH
Initial mean weight (g)		0.54 ± 0.00 a	0.54 ± 0.00 a	0.54 ± 0.00 a	0.54 ± 0.00 a	0.54 ± 0.00 a
Final mean weight (g)		5.65 ± 0.12 c	6.50 ± 0.19 b	6.99 ± 0.15 a	5.95 ± 0.19 c	6.18 ± 0.21 bc
WG (%) 2		952.6 ± 22.0 c	1109.6 ± 35.8 b	1201.2 ± 27.0 a	1006.8 ± 38.0 c	1053.1 ± 40.2 bc
FCR 3		1.47 ± 0.03 c	1.24 ± 0.04 ab	1.14 ± 0.04 a	1.39 ± 0.05 bc	1.33 ± 0.05 b
SGR (%/day) 4		3.84 ± 0.08 a	4.12 ± 0.11 a	4.28 ± 0.32 a	3.95 ± 0.15 a	4.11 ± 0.19 a
PER 5		1.83 ± 0.04 c	2.14 ± 0.07 b	2.31 ± 0.05 a	1.94 ± 0.07 c	2.02 ± 0.07 bc
SR (%) 6		81.7 ± 2.9 a	86.7 ± 2.9 a	90.0 ± 5.0 a	88.3 ± 2.9 a	90.0 ± 0.0 a

¹ Values are means from triplicate groups of shrimps, where the values in each row with different superscript letters are significantly different (p < 0.05). ² Weight gain (WG, %) = (final weight − initial weight) × 100/initial weight. ³ Feed conversion ratio (FCR) = dry feed fed (g)/wet weight gain (g). ⁴ Specific growth ratio (SGR, %/day) = (log_e final weight − log_e initial weight) × 100/days. ⁵ Protein efficiency ratio (PER) = Wet weight gain/total protein given. ⁶ Survival rate (SR, %) = (initial number of shrimp − dead shrimp) × 100/initial number of shrimps.

. The initial mean body weight of shrimp was uniform across all treatments (0.54 g, p > 0.05). At the end of the feeding trial, shrimp fed the ABP diet exhibited the highest final mean weight (6.99 ± 0.15 g) and WG (1201.2 ± 27.0%), which were significantly greater than those of the CON (5.65 ± 0.12 g; 952.6 ± 22.0%), HPP (6.50 ± 0.19 g; 1109.6 ± 35.8%), and ABL (5.95 ± 0.19 g; 1006.8 ± 38.0%) groups (p < 0.05). No significant differences were detected between the CON and ABL groups. Feed utilization efficiency also differed among treatments. Shrimp fed the ABP diet showed significantly improved FCR (1.14 ± 0.04) compared with that in the CON (1.47 ± 0.03), ABL (1.39 ± 0.05), and ABH (1.33 ± 0.05) groups, whereas no significant difference was observed relative to the HPP group (1.24 ± 0.04). Similarly, PER was highest in the ABP group (2.31 ± 0.05), followed by the HPP (2.14 ± 0.07) and ABH (2.02 ± 0.07) groups, all of which had significantly greater values than that of CON (1.83 ± 0.04) (p < 0.05). No significant differences were observed in SGR among dietary groups, whose values ranged from 3.84 ± 0.08 to 4.28 ± 0.32% day⁻¹ (p > 0.05). Survival rates ranged from 81.7 to 90.0% and were not significantly affected by dietary treatments (p > 0.05).

The whole-body proximate composition of L. vannamei at the end of the feeding trial is presented in

Table 3. Whole-body proximate composition (%) of Pacific white shrimp (Litopenaeus vannamei) fed the CON, HPP, ABP, ABL, and ABH diets at the end of the feeding trial. ¹.

		CON	HPP	ABP	ABL	ABH
Moisture (%)		75.80 ± 1.05	74.90 ± 1.23	75.03 ± 1.77	74.50 ± 1.77	75.60 ± 2.59
Crude protein (%)		18.13 ± 1.05	18.27 ± 0.60	18.97 ± 1.76	18.90 ± 0.61	18.60 ± 1.08
Crude lipid (%)		1.21 ± 0.29	1.20 ± 0.12	1.17 ± 0.11	1.29 ± 0.12	1.25 ± 0.09
Crude ash (%)		1.57 ± 0.15	1.50 ± 0.26	1.47 ± 0.25	1.31 ± 0.23	1.40 ± 0.10

¹ Values are means ± SD (n = 3). No significant differences were detected among treatments within each row.

. No significant differences were observed among dietary treatments in moisture, crude protein, crude lipid, or crude ash contents (p > 0.05). Moisture content ranged from 74.50 ± 1.77% to 75.80 ± 1.05%, crude protein from 18.13 ± 1.05% to 18.97 ± 1.76%, crude lipid from 1.17 ± 0.11% to 1.29 ± 0.12%, and crude ash from 1.31 ± 0.23% to 1.57 ± 0.15%. Although crude protein content tended to be higher in shrimp fed the ABP (18.97 ± 1.76%) and ABL (18.90 ± 0.61%) diets compared with that in the CON group (18.13 ± 1.05%), the differences were not significant (p > 0.05). Overall, dietary supplementation with HPP, ABP, ABL, or ABH did not induce significant changes in whole-body proximate composition relative to that of the control, indicating that improvements in growth performance with ABP supplementation were not accompanied by marked alterations in body nutrient composition under the present conditions.

3.2. Non-Specific Immunity, Antioxidant Capacity, and Biochemical Parameters

The effects of the experimental diets on non-specific immunity, antioxidant capacity, and biochemical indicators in L. vannamei are summarized in

Table 4. Non-specific immunity and antioxidant parameters of Pacific white shrimp (Litopenaeus vannamei) fed the CON, HPP, ABP, ABL, and ABH diets. ¹.

		CON	HPP	ABP	ABL	ABH
Innate immunity parameters
Lysozyme (U mL−1) 2		7.55 ± 0.32 b	9.09 ± 0.23 a	9.16 ± 0.19 a	7.73 ± 0.42 b	8.70 ± 0.64 b
Antiprotease		27.4 ± 0.9 a	28.2 ± 1.2 a	29.3 ± 0.6 a	28.0 ± 1.3 a	28.1 ± 1.4 a
PO 3		0.306 ± 0.023 b	0.448 ± 0.042 a	0.482 ± 0.017 a	0.328 ± 0.049 b	0.426 ± 0.045 ab
Antioxidant capacity parameters
GPx (mU mL−1) 4		44.6 ± 0.7 b	57.6 ± 3.8 a	58.0 ± 5.2 a	46.5 ± 5.5 ab	53.2 ± 6.4 ab
SOD (U mL−1) 5		22.3 ± 2.9 b	31.1 ± 0.4 a	31.3 ± 2.3 a	27.6 ± 2.8 ab	28.2 ± 2.5 ab
MDA (U mL−1) 6		5.37 ± 0.50 b	3.63 ± 0.23 a	3.57 ± 0.50 a	4.77 ± 0.42 ab	4.10 ± 0.79 ab
Biochemical parameters
AST (U L−1) 7		58.5 ± 1.1 a	51.3 ± 4.0 a	52.2 ± 2.3 a	53.7 ± 4.3 a	54.3 ± 2.0 a
ALT (U L−1) 8		1.54 ± 0.31 a	0.98 ± 0.11 a	0.92 ± 0.06 a	1.41 ± 0.40 a	0.97 ± 0.16 a

¹ Values are means from triplicate groups of shrimps, where the values in each row with different superscript letters are significantly different (p < 0.05). ² Lysozyme (U mL⁻¹), One unit is the activity that produces a change of 0.001 absorbance units per minute at 450 nm. Report as U per mL; ³ PO: Phenoloxidase activity, absorbance units per minute per mg protein at 490 nm; ⁴ GPx: Glutathione peroxidase activity, 1 mU equals oxidation of 1 nmol of NADPH per minute at 340 nm under the assay conditions; ⁵ SOD: Superoxide dismutase, one unit is defined as 50% inhibition of the reduction reaction; ⁶ MDA: Malondialdehyde, Thiobarbituric-acid–reactive substances (TBARS) index reported as nmol of MDA per mL of fluid; ⁷ AST: Aspartate Aminotransferase, 1 μmol oxaloacetate formed per minute at the stated assay temperature; ⁸ ALT: Alanine Aminotransferase, 1 μmol pyruvate formed per minute at the stated assay temperature.

. Lysozyme activity, an important indicator of innate immune response, was significantly higher in shrimp fed the HPP (9.09 ± 0.23 U mL⁻¹) and ABP (9.16 ± 0.19 U mL⁻¹) diets compared with that of the CON (7.55 ± 0.32 U mL⁻¹) and ABL (7.73 ± 0.42 U mL⁻¹) groups (p < 0.05). The ABH group (8.70 ± 0.64 U mL⁻¹) exhibited intermediate values without significant differences from the other groups. Antiprotease activity ranged from 27.4 ± 0.9 to 29.3 ± 0.6 and did not differ significantly among treatments (p > 0.05). Phenoloxidase (PO) activity was significantly higher in the HPP (0.448 ± 0.042) and ABP (0.482 ± 0.017) groups compared with that in the CON (0.306 ± 0.023) and ABL (0.328 ± 0.049) groups (p < 0.05). The ABH group (0.426 ± 0.045) showed intermediate values that were not significantly different from the other groups. GPx activity was significantly increased in the HPP (57.6 ± 3.8 mU mL⁻¹) and ABP (58.0 ± 5.2 mU mL⁻¹) groups compared with that in the CON group (44.6 ± 0.7 mU mL⁻¹) (p < 0.05), while ABL (46.5 ± 5.5 mU mL⁻¹) and ABH (53.2 ± 6.4 mU mL⁻¹) displayed intermediate levels. Similarly, SOD activity was higher in shrimp fed the HPP (31.1 ± 0.4 U mL⁻¹) and ABP (31.3 ± 2.3 U mL⁻¹) diets compared with that in the CON group (22.3 ± 2.9 U mL⁻¹) (p < 0.05). The ABL (27.6 ± 2.8 U mL⁻¹) and ABH (28.2 ± 2.5 U mL⁻¹) groups showed intermediate values. MDA levels were significantly reduced in shrimp fed the HPP (3.63 ± 0.23 U mL⁻¹) and ABP (3.57 ± 0.50 U mL⁻¹) diets compared with those in the CON group (5.37 ± 0.50 U mL⁻¹) (p < 0.05). Shrimp in the ABL (4.77 ± 0.42 U mL⁻¹) and ABH (4.10 ± 0.79 U mL⁻¹) groups showed intermediate values. Biochemical parameters were not significantly affected by dietary treatments. AST values ranged from 51.3 ± 4.0 to 58.5 ± 1.1, and ALT values ranged from 0.92 ± 0.06 to 1.54 ± 0.31 (p > 0.05).

3.3. Apparent Digestibility Coefficients

The ADCs of dry matter (ADCd) and crude protein (ADCp) in L. vannamei fed the experimental diets are presented in

Table 5. Apparent digestibility coefficients (ADCs) of dry matter (ADCd) and crude protein (ADCp) in Pacific white shrimp (Litopenaeus vannamei) fed CON, HPP, ABP, ABL, and ABH diets. ¹.

		CON	HPP	ABP	ABL	ABH
ADCd (%) 2		85.4 ± 1.33	84.6 ± 0.70	84.1 ± 1.77	85.5 ± 1.09	85.3 ± 2.45
ADCp (%) 3		85.5 ± 0.73 b	90.4 ± 0.68 a	91.3 ± 0.87 a	89.0 ± 1.15 a	90.2 ± 0.96 a

¹ Values are means from triplicate groups of shrimps, where the values in each row with different superscript letters are significantly different (p < 0.05). ² Apparent digestibility coefficient of dry matter (%). ³ Apparent digestibility coefficient of crude protein (%).

. No significant differences were observed in dry matter digestibility, with values ranging from 84.1 ± 1.77% to 85.5 ± 1.09% (p > 0.05). In contrast, crude protein digestibility differed significantly among treatments. Shrimp fed the HPP (90.4 ± 0.68%), ABP (91.3 ± 0.87%), ABL (89.0 ± 1.15%), and ABH (90.2 ± 0.96%) diets showed significantly higher ADCp compared with those fed the CON diet (85.5 ± 0.73%) (p < 0.05). Among the supplemented groups, the ABP diet yielded the highest crude protein digestibility, although no significant differences were detected compared with that in HPP, ABL, or ABH.

4. Discussion

In the present study, L. vannamei fed diets containing 1% HPP or 1% ABP showed significantly higher WG and PER, together with a lower FCR, than those in the control. These outcomes are consistent with reports that dietary H. pluvialis (e.g., 5% GXU-A23) improves performance in L. vannamei and can support growth under low-fishmeal (10%) formulations during prolonged low-temperature exposure, whereas excessive fishmeal reduction depresses growth and immune indices [9,20]. Similarly, previous work indicates that inclusion of A. bussei powder at ≥0.4% enhances growth rate and feed efficiency in L. vannamei, supporting its practical use in formulated diets [17]. In contrast, the Arthrobacter probiotic tested in the current study did not yield growth or feed efficiency gains comparable to those of the powder forms. Nonetheless, prior studies have reported the benefits of Arthrobacter probiotics in shrimp and fish. For example, administering Arthrobacter sp. CW9 to rearing water at 10⁵–10⁷ CFU mL⁻¹ improved survival and mean body weight over 24 days in L. vannamei [21], and co-administration of Arthrobacter nicotianae with Bacillus cereus (40 mL kg⁻¹ feed) enhanced growth performance in red tilapia (Oreochromis sp.) [22]. These precedents suggest that the advantages of Arthrobacter probiotics can emerge under specific application modes or compositions, namely, waterborne dosing and/or co-formulation with Bacillus spp. In our trial, however, we evaluated a single-genus Arthrobacter preparation delivered through feed surface coating and did not include water application or Bacillus co-formulation; thus, differences in delivery route and formulation likely account for the discrepancy with those studies. Moreover, because probiotics must survive passage to, and remain active within, the gastrointestinal tract, inactivation before or during ingestion (e.g., by digestive conditions) can reduce viable recovery and effective exposure at the gut interface, leading to context-dependent efficacy [13,23]. The 8-week duration and the use of a juvenile-only age/size class limit extrapolation to long-term health outcomes and later grow-out phases; future trials should extend the duration and include subadult and grow-out stages.

Taken together, the available evidence indicates that, for A. bussei, dietary powders may provide more uniform dosing and compositional consistency (cell wall and intracellular constituents delivered together), potentially maintaining higher functional activity than live-cell preparations that can be compromised by heat or processing, thereby supporting more reliable improvements in growth and protein utilization [24]. Within this context, the literature on pigment-bearing powders provides complementary support for our powder outcomes. In shrimp, standardized H. pluvialis powders have repeatedly been associated with improved performance, with benefits commonly attributed to the accompanying astaxanthin payload [10,25]. The physicochemical properties of astaxanthin, extended conjugation and strong reactivity in lipid phases, are frequently cited as consistent with enhanced physiological efficiency under culture conditions, and these reports support the view that H. pluvialis powders can reliably deliver astaxanthin in diet-incorporated form [26]. A parallel rationale applies to A. bussei powders, which are described as containing bacterioruberin, a rare C₅₀ carotenoid with extended conjugation and favorable stability in the cellular matrix relative to many C₄₀ carotenoids [27]. Although bacterioruberin and astaxanthin were not quantified in the present study, the concordance between our 1% inclusion results and prior powder studies indicates that H. pluvialis and A. bussei powders remain promising dietary inputs for reproducible performance gains in L. vannamei. From a practical perspective, improvements in feed-use efficiency (e.g., FCR) are relevant to on-farm costs; however, the magnitude of any economic benefit depends on local prices and logistics, which were outside the scope of this trial. Accordingly, we refrain from cost modeling here and identify farm-scale validation coupled with a formal techno-economic assessment as a priority for future work.

In this trial, inclusion of 1% HPP, 1% ABP, and the probiotic treatments in the diet did not produce significant differences in whole-body moisture, crude protein, crude lipid, or crude ash of L. vannamei at the end of feeding. The absence of major compositional shifts despite improvements in WG, PER, and FCR is consistent with a previous report showing that A. bussei powder did not alter whole-body crude protein or lipid content [17]. In contrast, context-dependent changes have been reported elsewhere: supplementation with glutathione-rich yeast hydrolysate (GYH) increased whole-body crude protein, astaxanthin from Yarrowia lipolytica altered whole-body moisture and muscle protein while leaving whole-body lipid and ash unchanged, and a natural-astaxanthin study observed increases in whole-body fat and ash at higher astaxanthin inclusion [25,28,29]. Taken together, these findings indicate that whole-body proximate composition is sensitive to additive identity, the analytical matrix (whole body or tissue), inclusion level, and experimental context, and that growth enhancement does not necessarily entail re-partitioning of carcass nutrients. Carotenoid profiling was not performed owing to budgetary constraints; within available funds we prioritized replication and environmental control. Future, funded work will include graded inclusion levels with HPLC quantification to define dose–response and bioactivity links.

In the present study, dietary inclusion of H. pluvialis powder (HPP, 1%) and A. bussei powder (ABP, 1%) significantly enhanced non-specific immune and antioxidant indices in L. vannamei. Relative to the control, shrimp receiving HPP or ABP exhibited higher lysozyme and PO activities, together with increased SOD and GPx activities and lower MDA levels. The probiotic groups (ABL, ABH) displayed intermediate shifts that were less consistent across markers. This pattern is consistent with prior work showing that carotenoid-bearing microalgal inputs and bacterial biomass can fortify first-line defenses and redox homeostasis in penaeids. Findings for A. bussei are consistent with reports that ABP at practical inclusion levels (≥0.4%) elevates lysozyme and antioxidant enzyme activities in shrimp [17]. Likewise, studies with H. pluvialis have documented improved anti-inflammatory status alongside increases in SOD and GPx and reductions in MDA [9]. Parallel evidence from astaxanthin interventions shows significant gains in lysozyme, PO, phagocytic activity, and expression of immune effectors such as prophenoloxidase, β-glucan binding protein, and crustins, together with strengthened antioxidant capacity [10,26]. Collectively, these data support an immuno-nutritive mechanism in which diet-derived pigments and microbe-associated bioactives augment front-line antibacterial capacity and constrain lipid peroxidation under culture conditions. A complementary body of probiotic literature reinforces this interpretation and clarifies context. In L. vannamei, Bacillus subtilis supplementation increased PO and lysozyme [25,30]. Pediococcus pentosaceus raised total hemocyte count (THC) and lysozyme [31], and P. pentosaceus combined with fructo-oligosaccharide increased THC, PO, lysozyme, and phagocytosis [32]. Bacillus firmus-based probiotic feeding enhanced SOD, acid and alkaline phosphatases, nitric-oxide synthase, and lysozyme [33]. Mixed-strain probiotic regimens elevated lysozyme, alkaline phosphatase, and acid phosphatase [34]. Paenibacillus polymyxa raised lysozyme and serum protein fractions, including globulin [35]. Broadly defined probiotics also increased SOD and lysozyme and upregulated immune-related genes [36]. Of particular relevance to functional outcomes, P. pentosaceus improved innate immunity and conferred higher survival under white spot syndrome virus challenge [37]. Against this background, the stronger and more uniform improvements observed with HPP and ABP compared with our probiotic arms are consistent with the literature trend that stable, diet-incorporated powders deliver consistent basal stimulation, whereas live probiotics often show greater context-dependence on strain, formulation, and husbandry conditions. The observed decreases in MDA align with prior reports in which H. pluvialis, astaxanthin sources, or A. bussei powder lowered lipid peroxidation in shrimp, frequently in parallel with increases in SOD, GPx, and total antioxidant capacity [9,17,25,26,38,39,40]. Taken together, the directionality across markers—higher lysozyme and PO, higher SOD and GPx, and lower MDA—is internally consistent and indicative of coordinated enhancement of innate defense and redox buffering. AST and ALT did not differ among treatments and fell within physiological ranges. This agrees with studies in which functional feed additives in shrimp or finfish improved immune and antioxidant readouts without eliciting hepatopancreatic stress, as reflected by unchanged transaminases [39]. The lack of AST/ALT perturbation in our trial, therefore, supports the dietary safety of the tested inclusion levels. HPP and ABP consistently strengthened non-specific immunity and antioxidant defenses in L. vannamei while maintaining serum biochemical homeostasis. These outcomes, together with congruent findings across astaxanthin-based and probiotic literature, support the use of diet-incorporated microalgal and bacterial powders as practical immuno-nutritional strategies in shrimp aquaculture. Improved innate and antioxidant status may contribute to reduced antibiotic reliance in intensive farming; this proposition warrants controlled challenge trials and on-farm validation. In juvenile L. vannamei, phenoloxidase and lysozyme are significantly influenced by molt stage, with phenoloxidase generally higher during inter-molt and lower during post-molt [41]. Although molt stages were not formally scored in the present study, all tanks were sampled at the same daytime hours and no perimolt signs were observed at sampling. Nevertheless, we acknowledge unassessed molt-related variability as a limitation, and future work should incorporate formal molt-stage scoring to more clearly isolate treatment effects.

Across treatments, ADCd was indistinguishable, whereas ADCp was significantly higher in all supplemented groups than those in the control. Shrimp fed HPP, ABP, ABL, and ABH exceeded the control. ABP showed the highest mean value among supplemented diets, but without statistical separation from HPP, ABL, or ABH. Although digestive enzyme activities (protease, trypsin, amylase, lipase) were not measured here, the consistent rise in ADCp suggests improved protein utilization; future work should verify this link via direct enzyme assays. The coexistence of stable ADCd and selectively elevated ADCp indicates a protein-focused improvement in digestive efficiency rather than a generalized change in dry-matter assimilation. These outcomes are directionally concordant with prior work showing that functional additives improve protein utilization in penaeids. Diets containing H. pluvialis or astaxanthin sources have repeatedly been associated with superior protein use at the whole-animal level, often reflected as a higher PER (e.g., Y. lipolytica-derived astaxanthin used with H. pluvialis) [29]. Bacterial inputs, including P. polymyxa and multi-strain probiotic blends, have similarly been linked to improved PER and upregulation of digestive enzymes such as protease, trypsin, amylase, and lipase in shrimp [35,36,37,42]. Although digestive enzyme activities were not measured in the present study, the consistent rise in ADCp across all supplemented diets provides a measurement-based explanation for the superior FCR and PER observed in the powder groups and is compatible with the enzyme-level improvements documented elsewhere. Consistent with reports that astaxanthin-rich H. pluvialis improves growth, immune responses and antioxidant activity in L. vannamei, HPP favorably shifted several endpoints relative to the control. Under the present rearing conditions, the A. bussei powder outperformed the live-cell probiotic counterpart in magnitude and consistency across growth/feed-use and immune–antioxidant indices. We interpret these differences as trial-specific efficacy contrasts; mechanisms were not profiled and require dedicated, graded-dose and compound-quantification studies. Future applications should evaluate microencapsulation to protect probiotic viability, prebiotic co-formulation, alternative delivery routes (e.g., waterborne dosing), and synbiotic combinations with established probiotic strains.

5. Conclusions

In summary, dietary supplementation with 1% HPP and 1% ABP significantly enhanced growth performance parameters, including WG and PER, while improving FCR in L. vannamei. These improvements were accompanied by significant upregulation of non-specific immune and antioxidant defenses, evidenced by elevated lysozyme, PO, SOD, and GPx activities, as well as reduced MDA levels. Importantly, supplementation did not adversely affect serum biochemical markers, with AST and ALT remaining within physiological ranges, underscoring the safety of these dietary interventions. Contrastingly, Arthrobacter probiotic supplementation showed less consistent effects, likely due to differences in delivery mode, microbial viability, and gut colonization dynamics. The enhanced protein digestibility observed with powder supplements further supports improved nutrient assimilation as a key mechanism underlying growth promotion. Taken together, these findings support the use of H. pluvialis and A. bussei powders as practical, reliable feed additives to boost shrimp growth, immunity, and oxidative stress resilience in aquaculture settings.