Dietary poly-β-hydroxybutyrate improved the growth, non-specific immunity, digestive enzyme activity, intestinal morphology, phagocytic activity and disease resistance against Vibrio parahaemolyticus of Pacific white shrimp, Penaeus vannamei

doi:10.21203/rs.3.rs-3582264/v1

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Dietary poly-β-hydroxybutyrate improved the growth, non-specific immunity, digestive enzyme activity, intestinal morphology, phagocytic activity and disease resistance against Vibrio parahaemolyticus of Pacific white shrimp, Penaeus vannamei

https://doi.org/10.21203/rs.3.rs-3582264/v1

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published 22 Apr, 2024

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This study assessed the effects of dietary supplementation of poly-β-hydroxybutyrate (PHB) on growth performance, feed efficiency, non-specific immunity, digestive enzyme capacity, phagocytic activity, hemocyte count, intestinal morphology and disease resistance against Vibrio parahaemolyticus of Pacific white shrimp (Penaeus vannamei). Six diets were prepared by supplementing graded levels of PHB at 0.00, 0.25, 0.50, 1.00, 2.00 and 4.00% (Con, P0.25, P0.5, P1.0, P2.0 and P4.0, respectively). Triplicate groups of 25 shrimps (initial body weight: 0.25 ± 0.01 g) per treatment were randomly assigned and fed an experimental diet for 56 days. Growth performance of shrimp was significantly improved by 1% dietary PHB supplementation. PHB-included diets fed shrimp showed significantly improved hepatopancreatic trypsin, chymotrypsin and pepsin activities. Villus height was significantly increased with dietary PHB supplementation and villus width was increased at a 1% inclusion level. P0.25, P0.5 and P4.0 groups significantly increased phenoloxidase activity and the P2.0 group significantly increased anti-protease activity compared to that in Con group. The survival of shrimp challenged against V. parahaemolyticus were higher in P0.5, P1.0 and P2.0 groups than in the Con diet. According to the above observations, the optimal dietary PHB supplementation level for maximum weight gain would be 1% for Pacific white shrimp.

Poly-β-hydroxybutyrate

Penaeus vannamei

Vibrio parahaemolyticus

Growth performance

non-specific immunity

Pacific white shrimp (Penaeus vannamei) is widely cultured in global shrimp aquaculture owing to its favorable attributes, such as rapid growth, popularity among consumers and tolerance to high salinity and diseases (FAO 2020). However, the health and survival of Pacific white shrimp have been significantly compromised by infectious diseases, resulting in substantial economic losses for the aquaculture industry (Han et al. 2020). Vibrio spp. bacteria and viruses are the most virulent causative agents for a wide variety of diseases in shrimp culture. Among them, Vibrio parahaemolyticus, which carries the binary toxin plasmids VpPirA/VpPirB, is responsible for causing acute hepatopancreatic necrosis disease in shrimp and the high virulency of this infectious disease can result in drastic economic losses (Li et al. 2017). V. parahaemolyticus colonize in the shrimp intestine and release toxins that trigger the shedding of hepatopancreatic tubule epithelial cells (Soto-Rodriguez et al. 2022). Although antibiotics are used as prophylactic agents against bacterial diseases in shrimp culture, excessive usage and improper practices can result in numerous complications, such as the emergence of antimicrobial-resistant microbes, bioaccumulation, immune suppression, disruption of intestinal microbiota of aquatic animals and potential impact on consumer health (Shamsuzzaman and Biswas 2012). Therefore, it is imperative to identify environmentally friendly alternative approaches that can stimulate immunity and increase the resistance of Pacific white shrimp against virulent pathogens in high stress farming conditions, ultimately ensuring sustainable farm productivity.

Previous studies suggest short-chain fatty acids (SCFAs) and their derivatives as potential alternative non-antibiotic antimicrobial agents to enhance immunity in aquatic animals against pathogenic Vibrio, Salmonella and Shigella species (Hoseinifar et al. 2017). SCFAs are organic acids with low molecular weight and are produced during bacterial fermentation (Koh et al. 2016). They inhibit the growth of pathogenic bacteria by disrupting cytosolic pH balance, deterring the adhesion of toxin-producing bacteria and damaging bacterial cell membranes (Defoirdt et al. 2009). Additionally, previous studies indicated that formate, acetate, propionate, butyrate, lactate, valerate and their salts could enhance the growth, feed utilization and digestive enzyme activities of aquatic farm animals (Silva et al. 2016a). Although SCFAs are beneficial for growth improvement and antimicrobial activities, there are challenges in directly applying them in aquaculture feed owing to their high-water solubility, characteristic odor and taste (Becattini et al. 2016; De Schryver et al. 2010).

Poly-β-hydroxybutyrate (PHB) is a biodegradable polymer of β-hydroxybutyrate and its supplementation into animal diets reportedly yields antimicrobial effects similar to those of SCFAs (Nhan et al. 2010). A wide range of bacterial strains, such as Alcaligenes eutrophus, Methylobacterium sp., Ralstonia eutropha and Bacillus megaterium, can produce and accumulate PHB during periods of physiological stress to store carbon and energy (Sui et al. 2014; Suguna et al. 2014). Within the gastrointestinal tract of aquatic animals, PHB is broken down into monomer units, namely β-hydroxybutyrate, which can lower intestinal pH and promote the growth of probiotic bacteria (Liu et al. 2010; De Schryver et al. 2010). PHB undergoes degradation under both low and high pH conditions, with its monomeric units released gradually owing to relatively low digestibility in the animal gastrointestinal tract (Defoirdt et al. 2009). Previous studies have demonstrated the positive outcomes of dietary PHB supplementation. The effects include improved growth performance, immune responses and survival in the aquatic animals, such as giant freshwater prawn (Macrobrachium rosenbergii) (Nhan et al. 2010), European sea bass (Dicentrarchus labrax) (Franke et al. 2017) and Siberian sturgeon (Acipenser baerii) (Najdegerami et al. 2015). Duan et al. (2017) observed that dietary PHB led to an increase in beneficial intestinal bacteria populations and activation of the mammalian target of the rapamycin signaling pathway in Pacific white shrimp. Furthermore, several studies have found increased disease and environmental stress resistance as a result of dietary PHB supplementations for blue mussel (Mytilus edulis) (Van Hung et al. 2015), black tiger shrimp (Penaeus monodon) (Monica et al. 2017) and Kuruma shrimp (Marsupenaeus japonicus) (Fukami et al. 2021). In light of these findings, therefore, this study aimed to assess the effects of dietary PHB supplementation on the growth, immune responses, phagocytic capacity, hemocyte count, digestive enzyme activity, intestinal morphology and disease resistance against V. parahaemolyticus in Pacific white shrimp.

Diet preparation

Six experimental diets were prepared, including a basal diet as the control. The basal diet (Con) was formulated to contain 37.5% crude protein and 7.6% crude lipid using sardine and tuna fish meals and cod liver oil (Table 1). The other five diets were prepared by adding five different levels of PHB to the basal diet at 2.5, 5, 10, 20 and 40 g kg^− 1 levels by replacing wheat flour (designated P0.25, P0.5, P1.0, P2.0 and P4.0, respectively). PHB was provided by CJ CheilJedang White BIO (Suwon, South Korea). Dry ingredients were measured according to the formulation and mixed thoroughly using a commercial feed mixer. The dough was prepared by adding fish oil and 10% distilled water and pelleted to a 2 mm size by passing the wet dough through a feed pelletizer (SP-50, KumKang Engineering. Busan, Korea). Then, the pelletized diets were dried in a dryer (25 ℃, 8 h) and stored at -20 ℃ until use.

Table 1

Dietary formulation and proximate composition of the basal diet prepared to evaluate the effect of poly-β-hydroxybutyrate on Pacific white shrimp, *Penaeus vannamei*.
Ingredients	g/kg
Fish meal, sardine¹	100.0
Fish meal, tuna²	100.0
Soybean meal	340.0
Squid liver powder	50.0
Wheat flour³	208.0
Starch	80.0
Cod liver oil⁴	30.0
Vit/Min premix⁵	30.0
Choline chloride (50%)	20.0
Lecithin⁶	10.0
Cholesterol	2.0
Monocalcium phosphate	30.0
Proximate composition (%)
Crude protein	37.5
Crude lipid	7.6
Crude ash	11.7
^{1, 2}Orizon S.A., Corp., Santiago, Chile (crude protein: 75, 60% crude lipid: 9.5, 17.1%)
³Deahan Flour Co. Ltd, Incheon, Korea
⁴E-wha oil & fat Industry Corp., Busan, Korea
⁵Vitamin/Mineral premix (g kg^− 1of mixture): retinol, 3.0; cholecalciferol, 1.0; ascorbic acid, 20.0; tocopherol, 20.0; menadione, 2.0; thiamine, 4.0; riboflavin, 6.0; pyridoxine, 5.0; cobalamin, 6.0; inositol, 54.0; pantothenic acid, 12.0; biotin, 0.2; niacin amide, 40.0; folic acid, 2.0; ferrous sulfate, 10.0; copper sulfate, 1.0; zinc sulfate, 30; manganous sulfate, 2.0; cobalt chloride, 10.; potassium iodide, 1.0; potassium, 6.0; sodium selenite, 0.01
⁶Lysoforte™ Dry, KEMIN Korea Co. Ltd., Seongnam, Korea

Experimental setting and feeding trial

Pacific white shrimp were purchased from a local hatchery (Daedong Susan. Muan-gun, Korea) and acclimatized to the experimental conditions for 3 weeks feeding a commercial diet (CJ Cheiljedang, Seoul, Korea). A total of 30 shrimp (initial body weight, 0.25 ± 0.01 g) were introduced to each acrylic tank (220 L). The experimental diets were randomly assigned. Triplicated tanks per diet treatment were fed one of the diets five times a day (08:30, 11:00, 13:30, 16:00 and 18:30 h) for 8 weeks. Shrimp were fed with 3–5% body weight and the biomass of the tank was measured bi-weekly to adjust the feeding rate. Approximately 70% of the culture water in each tank was exchanged with filtered and preheated seawater every three days while thoroughly cleaning the bottom of the tanks. Water quality was maintained during the feeding trial period as follows; dissolved oxygen (DO) 6.52 ± 0.09 mg/L, temperature 29.5 ± 0.2 ℃, ammonia 0.05 ± 0.02 ppm, pH 7.48 ± 0.07 and salinity 31.0 ± 0.5 ppt. The water samples were collected at the same location in each tank to measure pH, salinity and ammonia. During the feeding trial period, water temperature and DO of each tank were measured daily and pH and salinity were measured twice a week. Water temperature, DO, salinity and pH were measured using a regular thermometer, DO meter (Pro20, YSI, Yellow Springs, Ohio, USA), refractometer (ATAGO Co. LTD. Tokyo, Japan) and portable pH meter (Seven Compact, Mettler Toledo, Columbus, Ohio, USA), respectively. Verdouw et al. (1978) method was used to determine the water ammonia level.

Sampling and Analyses

At the end of 8-week feeding trial, the weight of individual shrimp in each tank was measured and total number of shrimp was counted. Three shrimp were collected from each tank and their hemolymph were collected from the ventral sinus of the shrimp using a 1 mL 25-gauge syringe. Hemolymph was diluted with an anticoagulant solution (Alsever's solution (A3551), Sigma-Aldrich. St. Louis, USA) at a 1:1 ratio. The hemolymph was stored at -80°C until analysis was performed.

Proximate compositions of the experimental diets and shrimp whole-body were analyzed following AOAC (2005) methods. The moisture contents of samples were analyzed by drying at 105 ℃ in a drying oven until obtaining a constant weight and ash content was measured by incinerating in a muffle furnace for 6 h at 550 ℃. Crude lipid level was determined by the Soxhlet extraction method (SOX406 fat analyzer. Jinan Hanon Instruments. Shandong, China) and crude protein content was determined by the Kjeldahl method (Kjeltec™ 2300, FOSS Analytical, Hilleroed, Denmark).

Phenoloxidase (PO) activity was assessed by Hernández-López et al. (1996) method. Lysozyme and antiprotease activities were measured following the method described by Hultmark et al. (1980) and Ellis (1990), respectively. The phagocytic activity during respiratory burst was measured by nitroblue tetrazolium (NBT) assay following the method described by Munoz et al. (2000). Commercial assay kits purchased from Sigma-Aldrich (St. Louis, Missouri, USA) were used to determine the superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activities.

Flow cytometry was performed immediately after hemolymph collection. Flow cytometry analysis was used to determine the hemocyte count and phagocytosis using a flow cytometer (CytoFlex, Beckman Coulter Life Science. Indianapolis, USA) equipped with two lasers (488 nm and 639 nm) and four fluorescence channels. Phagocytosis was evaluated based on the ingestion of 1.0 µm diameter fluorescent beads (Sigma-Aldrich. St. Louis, Missouri, USA). The hemolymph (30 µL) was diluted with the same amount of phosphate-buffered saline and incubated with beads (final concentration at 2%) in dark condition at 25 ℃ for 30 min to induce phagocytosis. The percentage of beads engulfed hemocytes among total hemocytes was used to express phagocytosis capacity. A two-parameter cytogram was constructed using the size scatter height and forward scatter height of unlabeled cells. A total of 10,000 events were counted for each hemocyte sample and results were presented as dot plots indicating total hemocyte count (THC) and hyaline cell count (HCC).

Three hepatopancreas samples were separated from three shrimp, homogenized separately by a hand-held homogenizer in distilled water, centrifuged at 10,000 ×g for 30 min at 4 ℃ and the supernatant was separated and stored at -20 ℃ for digestibility enzyme analysis. The protein content of the supernatant was estimated according to Bradford (1976) using a commercial total protein assay kit (Bio-Rad, Hercules, CA, USA). Amylase activity was assayed as described by Medagoda et al. (2023). Trypsin and chymotrypsin activities were determined according to Erlanger et al. (1961) and pepsin activity was assessed using Anson (1938) method. Lipase activity was analyzed following the procedure described by Kuepethkaew et al. (2017a).

The mid-gut samples were dissected from three shrimp in each tank and fixed in Davidson's AFA (alcohol, formalin and acetic acid) solution for 24 hours. Samples were dehydrated in a graded ethanol solution series and embedded in paraffin blocks. They were sectioned at 6-µm thickness using a microtome (Leica, Germany) and stained with hematoxylin and eosin. Then, the stained sections were examined under a light microscope (ICC50, Leica, Germany) at ×40 magnification. Intestinal villus height (VH) and villus width (VW) were measured by an image analyzing software (Leica Application Suite, version 4.13.0, Switzerland).

Challenge test

After the feeding trial, 17 shrimp were randomly selected from each tank and randomly arranged into 18 tanks (120 L) in triplicated tanks per treatment in a quarantine room for the challenge experiment. Another three tanks were set up as a negative control treatment (Broth) to inoculate with 50 mL of broth solution which was used for the preparation of the bacterial solution. Randomly selected 51 shrimp from all tanks of the feeding trial were distributed into the negative control tanks. V. parahaemolyticus was used as the pathogen and confirmed by PCR targeting pirA- and pirB-like genes. The bacteria were cultured in tryptic soy broth (TSB+) media containing 2% NaCl shaking at 150 rpm at 30 ℃ for 24 hours to achieve a concentration of 1.8 × 10⁶ CFU mL^− 1. The bacterial solution of 50 mL was introduced into each tank (except for negative control). Then, the shrimp mortality was monitored hourly and recorded. Shrimp were reinoculated with 3.6 × 10⁶ CFU mL^− 1 bacterial solution at 43rd h because there was no mortality. During the challenge period, each respective experimental feed was supplied three times a day (08:30, 13:30 and 18:30). The challenge test was continued for 256 h until no further mortality was observed in each treatment.

Statistical analysis

A complete randomized block design was employed to assign treatments in the experiment tank system and percentage data were arcsine transformed before analysis. The resulting data were analyzed using a one-way ANOVA in SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). Duncan's multiple range test (P < 0.05) was used to compare the mean differences between groups.

Dietary PHB supplementation significantly improved weight gain (WG) and specific growth rate (SGR) of shrimp (Table 2). WG and SGR were significantly higher in P1.0 group compared to those of Con group. Dietary PHB did not significantly affect final body weight (FBW), feed conversion ratio (FCR) and protein efficiency ratio (PER), but they were numerically improved with increasing PHB levels up to 1% level. Significantly higher survival was observed in P0.25 group than compared to the highest two inclusion levels (P2.0 and P4.0).

Table 2

Growth performance, feed utilization of Pacific white shrimp, *Penaeus vannamei*, fed different levels of poly-β-hydroxybutyrate included experimental diets for 56 days.
Diets	FBW¹	WG²	SGR³	FCR⁴	PER⁵	Survival (%)
Con	7.22 ± 0.25	2786 ± 88^b	6.00 ± 0.05^b	1.36 ± 0.04	1.86 ± 0.06	92.2 ± 1.9^ab
P0.25	7.39 ± 0.15	2845 ± 59^ab	6.04 ± 0.04^ab	1.32 ± 0.03	1.90 ± 0.04	96.7 ± 0.0^a
P0.5	7.57 ± 0.36	2929 ± 144^ab	6.09 ± 0.09^ab	1.32 ± 0.06	1.89 ± 0.09	92.2 ± 1.9^ab
P1.0	7.71 ± 0.22	2986 ± 87^a	6.12 ± 0.05^a	1.30 ± 0.06	1.93 ± 0.09	92.2 ± 5.1^ab
P2.0	7.46 ± 0.32	2885 ± 132^ab	6.06 ± 0.02^ab	1.32 ± 0.03	1.91 ± 0.04	91.1 ± 1.9^b
P4.0	7.37 ± 0.19	2833 ± 70^ab	6.03 ± 0.04^ab	1.34 ± 0.03	1.89 ± 0.05	88.9 ± 3.9^b
Con, P0.25, P0.5, P1.0, P2.0 and P4.0 are 0, 0.25, 0.5, 1.0, 2.0 and 4.0% poly-β-hydroxybutyrate incorporated experimental diets. Initial body weight of shrimp was 0.25 ± 0.01 g. Values are means of triplicates (n = 3) and presented as mean ± S.D. Values with different superscript letters in the same column are significantly different (P < 0.05).
¹Final body weight (g)
²Weight gain (%) = [(final body weight - initial body weight)/ initial body weight] × 100
³Specific growth rate (%) = [(log_e final body weight - log_e initial body weight)/days] × 100
⁴Feed conversion ratio = Dry feed fed/Wet weight gain
⁵Protein efficiency ratio = Wet weight gain/ Total protein given

Trypsin activity was significantly increased with dietary PHB supplementation (Table 3). Chymotrypsin activity was significantly increased at the above inclusion levels over 0.5% and P4.0 group showed the highest activity. P0.25, P1.0 and P2.0 groups showed significantly higher pepsin activities than Con group. Dietary PHB did not affect amylase activity. P0.5 group showed significantly higher lipase activity than P4.0 group. Intestinal VH of the shrimp was significantly taller in all the PHB supplemented groups except for P2.0 group compared to that in Con group (Table 4). The PHB supplementation increased VW of the shrimp, however, P1.0 group only had significantly increased VW compared to that of Con group. Figure 1 shows representative histological cross-sectional images of the intestines, selected based on the mean villus heights for each treatment. In all experimental groups, the structural integrity of the intestinal villus of shrimp remained intact and there was an observed increase in VH with the addition of PHB.

Table 3

Digestive enzyme activity in hepatopancreas of Pacific white shrimp, *Penaeus vannamei*, fed different levels of poly-β-hydroxybutyrate included experimental diets for 56 days.
Diets	Trypsin (U/ protein)	Chymotrypsin (U/ protein)	Pepsin (U/ protein)	Amylase (U/ protein)	Lipase (U/ protein)
Con	0.90 ± 0.13^b	4.04 ± 0.14^d	4.57 ± 0.67^c	43.1 ± 0.1	193 ± 7^ab
P0.25	1.26 ± 0.06^a	4.12 ± 0.10^d	10.3 ± 1.77^a	45.1 ± 7.9	213 ± 22^ab
P0.5	1.25 ± 0.10^a	4.36 ± 0.10^c	8.53 ± 1.96^abc	52.7 ± 6.8	232 ± 39^a
P1.0	1.38 ± 0.08^a	4.62 ± 0.10^ab	10.4 ± 3.49^a	48.7 ± 2.2	197 ± 28^ab
P2.0	1.25 ± 0.02^a	4.44 ± 0.09^bc	9.88 ± 3.99^ab	49.0 ± 6.5	228 ± 11^ab
P4.0	1.33 ± 0.08^a	4.74 ± 0.01^a	5.41 ± 1.07^bc	50.4 ± 1.9	179 ± 35^b
Con, P0.25, P0.5, P1.0, P2.0 and P4.0 are 0, 0.25, 0.5, 1.0, 2.0 and 4.0% poly-β-hydroxybutyrate incorporated experimental diets. Initial body weight of shrimp was 0.25 ± 0.01 g. Values are means of triplicates (n = 3) and presented as mean ± S.D. Values with different superscript letters in the same column are significantly different (P < 0.05).

Table 4

The intestinal morphology of Pacific white shrimp, *Penaeus vannamei*, fed different levels of poly-β-hydroxybutyrate included experimental diets for 56 days.
Diets	Villus height (µm)	Villus width (µm)
Con	10.2 ± 1.0^b	15.7 ± 1.8^b
P0.25	21.6 ± 8.0^a	18.3 ± 1.2^ab
P0.5	21.0 ± 2.9^a	18.9 ± 1.6^ab
P1.0	21.5 ± 1.0^a	22.5 ± 0.7^a
P2.0	19.6 ± 4.6^ab	17.7 ± 2.7^ab
P4.0	22.9 ± 4.2^a	19.2 ± 3.1^ab
Con, P0.25, P0.5, P1.0, P2.0 and P4.0 are 0, 0.25, 0.5, 1.0, 2.0 and 4.0% poly-β-hydroxybutyrate incorporated experimental diets. Initial body weight of shrimp was 0.25 ± 0.01 g. Values are means of triplicates (n = 3) and presented as mean ± S.D. Values with different superscript letters in the same column are significantly different (P < 0.05).

Dietary inclusion of 0.25, 0.50 and 4.0% PHB showed significantly improved PO activity than Con diet (Table 5). All the PHB supplementation levels increased antiprotease activity of shrimp, but P2.0 group only significantly higher than Con group. Lysozyme and NBT activities showed no significant differences among the treatments. For the antioxidant capacity (Table 6), P2.0 and P4.0 groups showed significantly higher catalase activities than Con group. SOD and GPx activities were not significantly affected by PHB. Phagocytic activity was significantly increased in P0.5 and P1.0 groups compared to that in Con group (Table 7). P0.25 group showed significantly increased HCC than Con group and dietary PHB did not affect THC. Whole-body crude protein, crude lipid and ash levels showed no significant differences among the treatments (Table 8). During the V. parahaemolyticus challenge test, infected shrimp showed symptoms, such as a red body, pale appearance of the abdominal region and soft carapace. At the end of 256-h challenge period, P2.0 group showed the highest survival (59.3%) followed by P0.5 diet (57.4%) (Fig. 2). Survival percentages of P0.25, P1.0 and P4.0 groups were 44.4, 55.6 and 50.0%, respectively. Con group had the lowest survival (42.6%), whereas the negative control (Broth) showed no mortality.

Table 5

Non-specific immune responses of Pacific white shrimp, *Penaeus vannamei*, fed different levels of poly-β-hydroxybutyrate included six experimental diets for 56 days.
Diets	PO¹	Lysozyme²	NBT³	Anti-protease⁴
Con	0.17 ± 0.00^b	2.08 ± 0.19	3.18 ± 0.43	10.2 ± 1.7^b
P0.25	0.25 ± 0.07^a	2.38 ± 0.59	3.13 ± 0.66	13.5 ± 1.0^ab
P0.5	0.25 ± 0.02^a	2.32 ± 0.32	3.20 ± 0.69	13.6 ± 2.4^ab
P1.0	0.23 ± 0.04^ab	3.00 ± 0.42	3.30 ± 0.64	12.7 ± 2.7^ab
P2.0	0.22 ± 0.04^ab	2.23 ± 0.91	3.24 ± 0.29	15.1 ± 2.6^a
P4.0	0.25 ± 0.01^a	2.40 ± 0.48	2.97 ± 0.20	13.0 ± 2.1^ab
Con, P0.25, P0.5, P1.0, P2.0 and P4.0 are 0, 0.25, 0.5, 1.0, 2.0 and 4.0% poly-β-hydroxybutyrate incorporated experimental diets. Initial body weight of shrimp was 0.25 ± 0.01 g. Values are means of triplicates (n = 3) and presented as mean ± S.D. Values with different superscript letters in the same column are significantly different (P < 0.05).
¹Phenoloxidase activity (absorbance)
²Lysozyme activity (µg ml^− 1)
³Nitro blue tetrazolium activity (absorbance)
⁴Antiprotease activity (% inhibition)

Table 6

Antioxidant enzyme activities of Pacific white shrimp, *Penaeus vannamei*, fed different levels of poly-β-hydroxybutyrate included experimental diets for 56 days.
Diets	SOD¹	GPx²	Catalase³
Con	68.3 ± 3.8	124 ± 21	0.08 ± 0.04^c
P0.25	71.3 ± 6.4	126 ± 21	0.08 ± 0.00^c
P0.5	70.4 ± 3.2	168 ± 37	0.14 ± 0.03^bc
P1.0	69.6 ± 3.4	141 ± 27	0.16 ± 0.10^abc
P2.0	69.7 ± 1.5	129 ± 07	0.28 ± 0.12^a
P4.0	74.0 ± 5.8	126 ± 25	0.27 ± 0.03^ab
Con, P0.25, P0.5, P1.0, P2.0 and P4.0 are 0, 0.25, 0.5, 1.0, 2.0 and 4.0% poly-β-hydroxybutyrate incorporated experimental diets. Initial body weight of shrimp was 0.25 ± 0.01 g. Values are means of triplicates (n = 3) and presented as mean ± S.D. Values with different superscript letters in the same column are significantly different (P < 0.05).
¹Superoxide dismutase (% inhibition)
²Glutathione peroxidase (mU ml^− 1)
³Catalase (mU ml^− 1).

Table 7

Phagocytic activity and hemocyte counts of Pacific white shrimp, *Penaeus vannamei*, fed different levels of poly-β-hydroxybutyrate included experimental diets for 56 days.
Diets	Phagocytosis¹	THC²	HCC³
Con	5.77 ± 1.49^b	4.13 ± 2.35	4.40 ± 1.70^b
P0.25	9.96 ± 2.84^ab	3.89 ± 1.23	7.38 ± 0.15^a
P0.5	12.5 ± 2.51^a	5.14 ± 4.03	5.03 ± 1.40^ab
P1.0	13.9 ± 2.50^a	4.76 ± 2.01	6.14 ± 0.78^ab
P2.0	11.3 ± 3.98^ab	4.50 ± 2.02	5.27 ± 1.35^ab
P4.0	10.6 ± 4.21^ab	2.19 ± 0.42	5.25 ± 1.35^ab
Con, P0.25, P0.5, P1.0, P2.0 and P4.0 are 0, 0.25, 0.5, 1.0, 2.0 and 4.0% poly-β-hydroxybutyrate incorporated experimental diets. Initial body weight of shrimp was 0.25 ± 0.01 g. Values are means of triplicates (n = 3) and presented as mean ± S.D. Values with different superscript letters in the same column are significantly different (P < 0.05).
¹Phagocytosis capacity (% inhibition)
²Total hemocyte count (×10⁷)
³Hyaline cell count (×10⁶)

Table 8

Whole-body composition (%, wet basis) of Pacific white shrimp, *Penaeus vannamei*, fed different levels of poly-β-hydroxybutyrate included experimental diets for 56 days.
Diets	Crude protein	Crude lipid	Ash
Con	74.8 ± 1.1	4.67 ± 0.26	13.5 ± 1.8
P0.25	73.6 ± 1.5	4.65 ± 0.45	13.4 ± 0.3
P0.5	73.2 ± 0.2	4.82 ± 0.39	14.7 ± 0.4
P1.0	74.8 ± 0.3	4.90 ± 0.17	13.5 ± 0.4
P2.0	74.7 ± 2.0	4.76 ± 0.51	14.4 ± 0.4
P4.0	75.4 ± 1.0	4.72 ± 0.48	13.6 ± 0.5
Con, P0.25, P0.5, P1.0, P2.0 and P4.0 are 0, 0.25, 0.5, 1.0, 2.0 and 4.0% poly-β-hydroxybutyrate incorporated experimental diets. Initial body weight of shrimp was 0.25 ± 0.01 g. Values are means of triplicates (n = 3) and presented as mean ± S.D.

In this study, dietary PHB supplementation enhanced the growth performance of Pacific white shrimp, which aligns with the findings of previous studies investigating the effects of PHB supplementation on different shrimp and fish species (Duan et al. 2017; Situmorang et al. 2020). Duan et al. (2017) and Situmorang et al. (2020) reported that FBW, WG and SGR of Pacific white shrimp (5.66 and 0.07 g) were significantly enhanced at 10–50 g kg^− 1 and 0.5 g kg^− 1 PHB-supplementations, respectively. SCFAs, including butyrate, are vital substrates that contribute to ATP production, fulfilling the energy requirement of shrimp (Romano et al. 2015; Calzada et al. 2020). Notably, Duan et al. (2017) demonstrated how dietary PHB upregulated gene expression related to the mTOR signaling pathway, which governs cell growth, angiogenesis, bioenergetics and cell survival. This suggests that monomeric units of PHB might provide energy to animals, similar to SCFAs. The degradation of PHB produces monomeric units (β-hydroxybutyric acid) that lower the pH in the intestine, supply energy to intestinal epithelial cells, increase the availability of amino acids and nucleotide derivatives and suppress the growth of pathogenic intestinal microbiota (Defoirdt et al. 2007; De Schryver et al. 2010). Kiran et al. (2014, 2016) reported the anti-adhesive properties of PHB and the anti-infective effects of its degradation intermediates against Vibrio spp. These effects play a pivotal role in improving the intestinal health of aquatic organisms and by extension, positively impact their growth. Although these observations suggest potential mechanisms for the improved growth performance of Pacific white shrimp, the exact underlying mechanism has yet to be fully elucidated.

Digestive enzyme activities, histomorphology and inflammatory conditions have a significant impact on the growth and feed utilization efficiency of animals (Shan et al. 2008). The primary functional digestive enzymes involved in protein degradation in penaeid shrimps are trypsin and chymotrypsin (Hernández and Murueta 2009). Additionally, lipase catalyzes the hydrolysis of triacylglycerol, releasing glycerol and free fatty acids (Kuepethkaew et al. 2017b). Nutrient absorption in the intestine depends on the integrity of the villus structure, an effective absorptive surface area and the height and width of the intestinal villus (Daniels et al. 2010; Zhang et al. 2012). A study by Liu et al. (2022) demonstrated that dietary PHB supplementation improved the mucosal barrier of the intestine in Gibel carp (Carassius auratus gibelio). This improvement was attributed to an increase in muscularis height and mucosal folds of the midgut, resulting in an evenly distributed, well-integrated and tightly connected villus structure. Likewise, Silva et al. (2016b) observed improved villus structure in Pacific white shrimp, suggesting that PHB supplementation could potentially mitigate the degradation of the intestinal epithelium by safeguarding it against adverse microbial activities and toxins. Research by Duan et al. (2017) indicated that dietary PHB supplementation (30g kg^− 1 in the diet) led to an increase in SCFAs in the intestine. These SCFAs play a crucial role in meeting the energy requirements of enterocytes, facilitating the development of the absorptive surface. The absence of SCFAs can lead to starvation of enterocytes, causing ulcerative colitis and intestinal inflammation, which in turn deteriorates the absorptive surfaces (Wächtershäuser and Stein 2000). Consistent with our findings, studies involving dietary sodium butyrate and PHB supplementation revealed improved intestinal morphology and increased activity of digestive enzymes, including amylase, lipase, trypsin and pepsin activities in Pacific white shrimp (Silva et al. 2016b; Kiran et al. 2020). These findings collectively suggest that the monomer units generated through PHB digestion could improve intestinal structure and digestive enzyme activity, thus improving the digestibility, absorption and growth performance of Pacific white shrimp.

Crustaceans rely on non-specific immune responses to defend against pathogens and stress due to the absence of an adaptive immune system. Processes such as encapsulation, phagocytosis, clotting, cell agglutination, nodule formation and the presence of antimicrobial proteins and the prophenoloxidase (proPO) system play pivotal roles within the non-specific immune system (Yang et al. 2015). The proPO system is activated when pattern recognition receptors on hemocytes are triggered by pathogenic organisms, leading proPO to convert into its active form, phenoloxidase (Vargas-Albores and Yepiz-Plascencia 2000). In this study, dietary PHB increased PO activity in Pacific white shrimp, consistent with the findings of Kiran et al. (2020). Suguna et al. (2014) reported that dietary supplementation with PHB improved serum peroxidase and protease activities in Mozambique tilapia (Oreochromis mossambicus). Duan et al. (2017) found a significant enhancement of lysozyme and antioxidative activity in the intestinal tissues of Pacific white shrimp with 5% dietary PHB inclusion. Similar results were reported by Liu et al. (2022) and Suguna et al. (2014) in sea cucumbers (Apostichopus japonicus) and Mozambique tilapia, respectively. Among the six types of lysozymes, shrimps possess invertebrate-type lysozyme (i-type), with relevant genes expressed in granular and semi-granular hemocytes. The observed increase in phagocytic activity might be linked to the rise in lysozyme activity observed in this study. NBT activity showed no significant differences among treatments with varying PHB levels. Notable, 2% dietary PHB supplementation significantly increased antiprotease activity in this study, which aligns with Suguna et al.’s (2014) findings for Mozambique tilapia fed a diet supplemented with 5% PHB. Antiprotease enzymes play a vital role in inhibiting microbial proteinases (Kanost 1999) and increased activity could bolster protection against invasive pathogens. A 30 day-feeding trial involving soiny mullet (Liza haematocheila) revealed that incorporating 0.5-2% PHB into diets increased antioxidant capacity, including catalase and SOD (Qiao et al. 2019). In our study, hemolymph catalase activity increased significantly at PHB inclusion levels over 2%, although GPx and SOD activities did not differ significantly. Reactive oxygen species (ROS) are generated during aerobic mitochondrial respiration, inflammation and stress and antioxidative enzymes like SOD, GPX and catalase function to mitigate ROS-induced cellular damage by converting superoxide anions and hydrogen peroxide into harmless water molecules (Snezhkina et al. 2019). In the context of dietary PHB supplementation, the monomeric units of PHB contribute to energy production, which could potentially elevate ROS generation. Consequently, the production of antioxidative enzymes might increase in response to increased ROS content within cells.

In crustaceans, hemocytes play a pivotal role in phagocytic activity. This process involves the identification, engulfing and destruction of pathogenic microorganisms, foreign particles and cellular debris (Johansson et al. 2000; Abnave et al. 2017). Consequently, hemocytes are key players in the humoral immune mechanism of shrimp (Yang et al. 2015). Indicators of immune response of shrimp include THC, phagocytic and antibacterial activity and phagocytosis is mainly performed by hemocytes (Zhu et al. 2018; Sun et al. 2020). Three distinct types of hemocytes can be identified: hyaline (HC), granular and semi-granular cells. These are differentiated based on their size, the number of intercellular granules and the nuclear/cytoplasm ratio (Jiravanichpaisal et al. 2006). During pathogenic invasions, antimicrobial proteins initiate antimicrobial mechanisms such as the proPO system. Additionally, the Down syndrome cell adhesion molecule, released from activated hemocytes, opsonizes pathogens and initiates phagocytosis (Low and Chong 2020). The phagocytic capacity varies across different invertebrate species. In shrimp, HC are particularly significant in this process (Low and Chong 2020). In this study, dietary inclusion of 0.5-1.0% PHB resulted in a significant increase in shrimp phagocytosis. Furthermore, the supplementation of 0.25% PHB led to a significant increase in HCC. Concurrently, PO activity also increased with PHB supplementation. These findings indicate that dietary PHB levels of 0.25-1% can enhance the antimicrobial capacity of Pacific white shrimp. However, the exact underlying mechanism remains unclear. Silva et al. (2016b) reported that supplementing Pacific white shrimp (3.96 ± 0.04 g) diets with 2% PHB improved THC, granular cell counts and HCC. This study is the first to assess the relationship between dietary PHB supplementation and phagocytosis activity and hemocyte counts in Pacific white shrimp. Thus, we propose that PHB has a direct impact on these aspects.

Dietary supplementation with PHB ranging from 0.5-2.0% significantly improved resistance against V. parahaemolyticus, as evidenced in this study. SCFAs and their derivatives can infiltrate the bacterial cytoplasm by penetrating the cell wall. Once there, they break down into anions and protons, inducing a pH decrease and resulting in anion-related toxicity. The pH shift compels bacteria to use ATP to rebalance the pH, ultimately depleting their energy reserves within the bacterial cell (Ricke 2003). For giant freshwater prawn larvae, dietary PHB supplementation led to a significant reduction in total bacteria and Vibrio spp. counts, underscoring PHB’s ability to inhibit the growth of these pathogens (Nhan et al. 2010). Various studies propose that SCFAs and their derivatives inhibit the pathogenicity of virulent intestinal bacteria. Notably, Galán (1996) and Van Immerseel et al. (2003) demonstrated that dietary SCFAs downregulate the expression of pathogenicity island 1 gene in Salmonella, which dictates invasion and virulence. Another study by Defoirdt et al. (2006) highlights SCFAs’ potential to decrease the virulence of luminescent Vibrio by modulating bacterial quorum sensing. As PHB degrades within the intestine, the monomeric units generated offer a source of energy for colonocytes. This energy infusion can help counteract the negative impact of pathogenic intestinal bacteria on intestinal morphology (Pryde et al. 2002). Kiran et al. (2014) demonstrated the substantial reduction of biofilm formation in V. vulnificus, V. fischeri, V. parahaemolyticus, V. alginolyticus and V. harveyi due to PHB. Furthermore, Kiran et al. (2020) observed a 100% survival in PHB-treated shrimp challenged with V. parahaemolyticus SF 14, compared to the control group. This underscores PHB’s antiadhesive properties and its potential to safeguard shrimp against Vibrio infections.

The findings of this study suggest that incorporating dietary PHB supplementation for Pacific white shrimp improves multiple aspects across several parameters, including growth, non-specific immunity, antioxidant capacity, diet digestibility, intestinal morphology and resistance to V. parahaemolyticus infections. Considering the results, the optimal inclusion level of PHB in Pacific white shrimp diet appears to lie within the range of 1–2%.

Funding Information

This study was funded by CJ CheilJedang Co., Ltd and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2019R1A6A1A03033553).

Competing interests

The authors declare that they have no competing or financial interests or personal relationships that could have influenced this work.

Author contributions

Suhyeok Kim participated in the feeding trial, sampling, analyses and manuscript preparation. Se Ra Choi, So Yun Park and Jeung-Yil Park designed the experiment, reviewed and edited the manuscript and provided ingredients. Jaebeom Shin and Nalin Medagoda edited the manuscript and analyzed the parameters. Kyeong-Jun Lee organized and supervised the whole study and completed the manuscript.

Ethical approval

The protocols of this feeding trial were evaluated and approved by the Institutional Animal Care and Use Committee of Jeju National University.

Data availability statement

All the datasets analyzed in this study are available from the corresponding author upon reasonable request.

ORCIDs

Suhyeok Kim: https://orcid.org/0009-0003-8521-0428

Jaebeom Shin: https://orcid.org/0000-0002-4618-9414

Nalin Medagoda: https://orcid.org/0000-0002-5835-1423

Kyeong-Jun Lee: https://orcid.org/0000-0003-0268-578X

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No competing interests reported.

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Dietary poly-β-hydroxybutyrate improved the growth, non-specific immunity, digestive enzyme activity, intestinal morphology, phagocytic activity and disease resistance against Vibrio parahaemolyticus of Pacific white shrimp, Penaeus vannamei

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