Effects of Three Feed Additives in Low Fish Meal Diet on Growth, Antioxidant Capacity and Intestinal Microbiota of Penaeus monodon

Abstract

The effects of three feed additives in a low fish meal diet on growth, antioxidant capacity and intestinal microbiota of Penaeus monodon were studied to enhance the understanding of these effects and to provide basic data and a reference for the formulation and optimization of feed for P. monodon. A total of 630 healthy, homogenous shrimp (4.58 ± 0.05 g) were randomly divided into seven groups with three replicates per group and 30 shrimp per replicate in a breeding barrel (500 L). The additive names and amounts were 0 (CT); vitamin B₆, 100 mg/kg (V1); vitamin B₆, 200 mg/kg (V2); phytase, 1000 U/kg (P1); phytase, 2000 U/kg (P2); 0.2% yucca extract (Y1); and 0.4% yucca extract (Y2). The experiment lasted 8 weeks. The results showed that compared to CT, V1 and Y1 had a significant increasing tendency toward weight gain (WG) (p < 0.05) and had a significant decreasing tendency in the food conversion ratio (FCR) (p < 0.05). P2 had a significant increasing tendency in α-amylase activity (p < 0.05), and P1 had a significant increasing tendency in total superoxide dismutase (T-SOD) (p < 0.05). The next-generation sequencing of intestinal microbiota showed that Proteobacteria was the most abundant phylum in the seven groups, accounting for 29.33%, 56.67%, 55%, 45.33%, 73%, 39.33% and 64.33% of the total. Compared to CT, the Proteobacteria was significantly high (p < 0.05) in P2 and Y2, and the Bacteroidota in all other groups decreased significantly (p < 0.05). The functional prediction of FAPROTAX indicated that there was no significant difference (p > 0.05) in functional components among all groups. According to growth performance, antioxidant capacity and effects on intestinal microbiota, vitamin B6 (100 mg/kg), phytase (2000 U/kg) and yucca extract (0.2%) can be recommended as additives for the diet of P. monodon.

1. Introduction

Penaeus monodon, also known as the grass shrimp and black tiger shrimp, belongs to the genus Penaeus, family Penaeidae. P. monodon is the largest individual in the genus Penaeus, with a body mass of 500 g and an average body length of 300–350 mm at maturity. The average body mass is about 350–400 g [1]. P. monodon is a valuable edible shrimp with a large body, delicious taste and high levels of vitality. So, P. monodon is one of the most important marine shrimp breeding varieties in China. Moreover, P. monodon have relatively high requirements for feed, especially protein; the dietary protein requirement of P. monodon is more than 35%.

With the rapid development of the aquaculture industry, the demand for seafood is increasing. In aquaculture, feed costs can be as high as 50% [2]. Fish meal has become a scarce resource, and it has a high price [3]. Research has been conducted to find new and efficient dietary ingredients that can replace fish meal protein. To date, many protein sources have been used to replace fish meal; however, the quality level of protein sources is not balanced and the utilization rate is low, so excessive substitution could have negative effects on aquatic animals [4]. Improving the growth performance and health level of aquatic animals by supplementing their diet with feed additives has become a research hotspot in the aquatic feed industry [5]. It has been reported that diet supplementation with natural astaxanthin can enhance the growth performance, the immune response and the antioxidant status of L. vannamei [6]. Using 1% Arthrospira platensis nanoparticles as dietary supplementation can improve the growth, survival and feed utilization of Pacific white shrimp [7]. O. vulgare and orange peel oils can be used as antifungal and immunostimulant supplements in L. vannamei diets against Fusarium solani infection [8]. The biofloc treatments using wheat flour as a carbon source can compensate for the reduction in the diet of L. vannamei [9]. Vitamin B₆ plays an important role in the growth of aquatic animals and has an anti-stress effect, which can alleviate the stress response of animals under the effects of environmental changes, diseases and other stress factors [10]. In addition, vitamin B₆ is necessary for the proper function of the immune system of aquatic animals. One study pointed out that adding 3.4 mg/kg vitamin B₆ to the diet of Indian catfish can achieve a maximum increase in weight and survival rate [11]. Adding vitamin B₆ to the diet of abalone improved weight gain rate, but the difference was not significant compared with the control group [12]. In P. monodon, a lack of vitamin B6 will lead to slow growth rate and increased mortality [13]. Phytase exists widely in nature and is found in animals, plants and microorganisms. The addition of phytase can unbind phytic acid and endogenous protease as well as improve protease activity and protein utilization [14]. The protein digestibility can be improved by adding 1000–3000 FTU/kg of phytase to the diet of L. vannamei [15]. In a study on the phytase addition effect in aquatic animals, the optimal dosage was demonstrated to be about 500–1000 FTU/kg [16]. Yucca extract can absorb harmful gases, reduce ammonia emissions, improve the living environment of aquatic animals, improve their growth performance and immune ability and regulate their intestinal microenvironment [17]. Adding 0.2% yucca to the diet of Litopenaeus vannamei can significantly improve the weight gain rate [18]. Using liquid extract of yucca schidigera at a concentration of 0.75 mg/L can effectively reduce the ammonia concentration in ground-well-derived rearing water and can decrease the mortality rates of European seabass juveniles [19]. The three feed additives above can enhance the feeding and growth of aquatic animals, but studies on the low fish meal diets of P. monodon have not been published. This study researched the effects of three feed additives in low fish meal diet on the growth, antioxidant capacity and intestinal microbiota of P. monodon, and the results from the current study will enhance our understanding of the effect of these additives and provide basic data and a reference for the formulation and optimization of feed for P. monodon.

2. Materials and Methods

2.1. Experiment Materials

The experiment was conducted at the Shenzhen Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. The shrimp used in the experiment were from a new strain selected by the research team. They have the characteristics of fast growth and high survival rate. Shrimp were taken out in the breeding pond and temporarily acclimated in an 8 m³ tank for three days, and commercial feed (main raw dietary ingredients: fish meal, soybean meal, shrimp bran, squid powder, flour, minerals, trace elements, multivitamins, immune enhancers, growth promoters. Guangdong Dongteng Feed Co., Ltd., Guangzhou, China) was provided during this period until the day before the experiment. The body mass of the shrimp was 4.58 ± 0.05 g.

2.2. Experiment Feeds

According to the nutritional requirements of P. monodon, seven kinds of iso-nitrogen and iso-lipid diets were provided, and the additives are control (CT); vitamin B6, 100 mg/kg (V1) and 200 mg/kg (V2); phytase, 1000 U/kg (P1) and 2000 U/kg (P2); and yucca extract, 0.2% (Y1) and 0.4% (Y2), and additional crystal amino acids were added to meet the amino acid requirements of the shrimp. Methionine and cystine were hydrolyzed using oxidative acid hydrolysis, and the rest of the amino acids were hydrolyzed via acid hydrolysis. The raw materials were purchased from the company Guangdong Kingkey Smart Agri Technology Co., Ltd., Guangzhou, China. All the ingredients were ground into powder, sieved through an 80-unit mesh and thoroughly mixed with oil and water; the 1.5 mm diameter long doughs were extruded using a twin screw extruder (F-26, South China University of Technology, Guangzhou, China), cut into pelletized feeds using a pelletizer (G-500, South China University of Technology, Guangzhou, China), steamed in a 90 °C electric oven for 2 h, dried in an air-conditioned room and then stored at −20 °C in a refrigerator until use. The formulation and the nutritional compositions of the experimental diets are shown in

Table 1. Formulation and nutrient levels of the experimental diets (% dry matter).

	CT	V1	V2	P1	P2	Y1	Y2
Fish meal	5.00	5.00	5.00	5.00	5.00	5.00	5.00
Soybean meal	24.50	24.50	24.50	24.50	24.50	24.50	24.50
Chicken meal	8.50	8.50	8.50	8.50	8.50	8.50	8.50
Peanut hull	14.00	14.00	14.00	14.00	14.00	14.00	14.00
Wheatmeal	27.46	27.45	27.44	27.44	27.42	27.26	27.06
Beer yeast	3.00	3.00	3.00	3.00	3.00	3.00	3.00
Shrimp med	4.00	4.00	4.00	4.00	4.00	4.00	4.00
Soy protein concentrate	2.00	2.00	2.00	2.00	2.00	2.00	2.00
Soybean Lecithin	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Fish oil	1.75	1.75	1.75	1.75	1.75	1.75	1.75
Soybean oil	1.75	1.75	1.75	1.75	1.75	1.75	1.75
Vitamin C polyphosphate	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Cholesterol	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Vitamin premix (prawn)	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Mineral premix (prawn)	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Ca(H2PO4)2	2.45	2.45	2.45	2.45	2.45	2.45	2.45
Lysine hydrochloride (78%)	0.41	0.41	0.41	0.41	0.41	0.41	0.41
Methionine (99%)	0.19	0.19	0.19	0.19	0.19	0.19	0.19
Threonine (98%)	0.18	0.18	0.18	0.18	0.18	0.18	0.18
Carboxymethyl cellulose	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Vitamin B6	0	0.01	0.02	0	0	0	0
Phytase	0	0	0	0.02	0.04	0	0
Yucca extract	0	0	0	0	0	0.2	0.4
Taurine (99%)	0.21	0.21	0.21	0.21	0.21	0.21	0.21
Total	100.00	100.00	100.00	100.00	100.00	100.00	100.00
Nutrient levels
Crude protein	35.52	35.52	35.52	35.52	35.52	35.52	35.52
Crude lipid	8.60	8.60	8.60	8.60	8.60	8.60	8.60
Nitrogen free extract	29.90	29.90	29.90	29.90	29.90	29.90	29.90
Energy	16.92	16.92	16.92	16.92	16.92	16.92	16.92

Vitamin premix: VA, 18 mg/kg; VD₃, 5 mg/kg; VE, 150 mg/kg; VC, 500 mg/kg; VB₁, 16 mg/kg; VB₆, 20 mg/kg; VB₁₂, 6 mg/kg; VK₃, 18 mg/kg; riboflavin, 40 mg/kg; inositol, 320 mg/kg; calcium-D-pantothenate, 60 mg/kg; niacinamide, 80 mg/kg; folic acid, 5 mg/ kg; biotin, 2 mg/kg; ethoxyquin, 100 mg/kg. Mineral premix; Na, 30 mg/kg; K, 50 mg/kg; Mg, 100 mg/kg; Cu, 4 mg/kg; Fe, 25 mg/kg; Zn, 35 mg/kg; Mn, 12 mg/kg; I, 1.6 mg/kg; Se, 0.2 mg/kg; Co 0.8, mg/kg.

.

2.3. Feeding Management

A total of 630 shrimps of uniform size, normal body color and healthy body mass were randomly selected and divided into breeding barrel (500 L). Each group was set up with 3 replicates and 30 shrimps in each replicate. The shrimp were fed three times daily at 8:00, 15:00 and 22:00. The uneaten pellets and feces were removed by siphon method, the exuviae and dead shrimp were removed by a dredge. After feeding, the feed surplus in the pellet tray was observed at 1 h and 2 h. The feeding amount was increased if there were surplus remaining pellets, and otherwise, it was decrease. The daily feeding amount was 2% to 5% of the body mass of the shrimp. The water was treated using filtered sand. During the feeding experiment, the water temperature was maintained at 27–32 °C, the salinity at 28–32 ppt, the pH at 7.5–8.0, the ammonia nitrogen level at 0–0.2 mg/L, and the dissolved oxygen level at 6–7 mg/L. The feeding experiment lasted for 8 weeks.

2.4. Sample Collection and Index Measurement

After the test, the shrimp were starved for 24 h, and the surface moisture of the shrimp was dried with a towel. The number of shrimp in each glass fiber bucket was calculated and the total weight (accurate to 0.01 g) was measured to calculate the survival rate, weight gain rate and feed conversion rate of the shrimp in each feed group. The calculation formula was as follows:

$$\mathrm{S}\mathrm{u}\mathrm{r}\mathrm{v}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{l} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} \left(\mathrm{\%}\right)=100\times \frac{\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}}{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r}}$$

$$\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{g}\mathrm{a}\mathrm{i}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} \left(\mathrm{\%}\right)=100\times \frac{\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l} \mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \left(\mathrm{g}\right)-\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \left(\mathrm{g}\right)}{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \left(\mathrm{g}\right)}$$

$$\mathrm{F}\mathrm{C}\mathrm{R}=\frac{\mathrm{f}\mathrm{e}\mathrm{e}\mathrm{d} \mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \left(\mathrm{g}\right)}{\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l} \mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \left(\mathrm{g}\right)-\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{a}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \left(\mathrm{g}\right)}$$

2.5. Composition Analysis of Whole Shrimp

After the experiment, five shrimp were taken from each breeding barrel and stored at −20 °C for a future assessment of whole-body composition. Moisture content was measured after oven-drying at 105 °C to a constant weight. The ash content was measured by burning in a muffle furnace for at least 5 h at 550 °C. Crude protein content (N × 6.25) was measured using the Kjeldahl method (Kjeltec™8400; FOSS, Copenhagen, Denmark). Crude lipid was measured by soxhlet extraction using the soxhlet system HT (Soxtec System HT6, Tacator, Stockholm, Sweden) [20].

2.6. Determination of Antioxidant Enzymes in Hepatopancreas of Shrimp

After the experiment, five shrimp were taken from each breeding barrel. The Hepatopancreas and intestine were taken and frozen with liquid nitrogen. After that, they were stored at −80 °C; for the determination of antioxidant enzyme measurement. Hepatopancreas and intestine samples were homogenized entirely with nine volumes of physiological saline (1: 9 dilution, w:v). The homogenate was then centrifuged for 15 min (4 °C, 3500 rpm) and the aliquots of the supernatant were used for antioxidant- and immune-related enzymes analysis. Acid phosphatase (ACP), alkaline phosphatase (AKP), total superoxide dismutase (T-SOD) and total antioxidant activity (T-AOC) were tested using a commercial test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

2.7. Determination of Intestinal Microbiota in Shrimp

After the experiment, three shrimp were taken from each breeding barrel. The intestines were removed and frozen with liquid nitrogen, and then stored at −80 °C. Microbial DNA from intestinal samples was extracted using the Stool Genomic DNA Extraction Kit (DP712) (TIANGEN, Beijing, China) and the magnetic bead soil method, according to the manufacturer’s protocols. Using 16 S rRNA or ITS (Internal Transcribed Spacer) universal primers for PCR amplification, and then sequencing the highly variable region and strain identification, the microbial diversity in the sample was analyzed through sequencing. We obtained classification tables for species annotation by comparing the current ASV sequences with those in the green gene (16SrRNA) database. Based on the results of a recent study, the mi-biome diversity of seven groups of experimental animals was analyzed by evaluating α-diversity indices as well as β-diversity metrics (using the principal coordinate analysis method). FAPROTAX [21] is a database manually constructed by Louca et al. It is more suitable for functional annotation in the biochemical processes of the sea and lakes (sulfur, nitrogen, hydrogen and carbon cycles).

2.8. Data Statistics and Analysis

Statistical analysis was performed using SPSS 21.0 (SPSS Inc., Michigan Avenue, Chicago, IL, USA) for Windows. The effect was tested by one-way ANOVA. When there were significant differences (p < 0.05), the group means were further compared by Duncan’s multiple range test. The results were presented as the means ± SD (n = 3).

3. Results

3.1. Growth Performance and Feed Utilization of P. monodon

The effects of different additives on the nutritional composition of P. monodon are shown in

Table 2. Nutritional composition of P. monodon.

Items	Diets
	CT	V1	V2	P1	P2	Y1	Y2
Moisture	74.88 ± 0.42	75.56 ± 0.55	75.62 ± 0.94	75.18 ± 0.3	75.41 ± 0.47	75.23 ± 0.82	75.41 ± 0.37
Crude protein	72.07 ± 2.43	74.2 ± 1.26	72.68 ± 4.82	71.68 ± 2.21	73.69 ± 0.96	72.44 ± 2.62	75.46 ± 0.91
Crude fat	6.20 ± 0.64	6.09 ± 0.76	5.67 ± 0.66	5.06 ± 0.74	6.19 ± 0.58	5.56 ± 1.06	5.94 ± 0.36
Crude ash	16.53 ± 0.51	16.82 ± 0.59	16.03 ± 0.19	16.18 ± 0.46	15.90 ± 0.86	16.14 ± 0.81	15.38 ± 0.70

Note: Unit: %. Means with different superscripts were significantly different (p < 0.05).

. There was no significant difference in moisture, crude protein, crude fat and crude ash among all experiment groups (p > 0.05). The effects of different additives on growth performance of P. monodon are shown in

Table 3. Effects of different additives on growth performance of P. monodon.

Items	Diets
	CT	V1	V2	P1	P2	Y1	Y2
IW (g)	4.59 ± 0.03	4.57 ± 0.07	4.54 ± 0.03	4.55 ± 0.03	4.59 ± 0.03	4.61 ± 0.07	4.61 ± 0.05
FW (g)	9.44 ± 0.36 a	12.29 ± 0.27 ab	9.34 ± 0.89 a	10.66 ± 2.15 ab	11.29 ± 0.95 ab	14.29 ± 1.05 b	10.21 ± 0.63 a
WG (%)	105.80 ± 8.62 a	168.83 ± 10.68 bc	105.47 ± 24.58 a	133.66 ± 55.38 ab	145.95 ± 25.34 ab	210.04 ± 28.79 c	121.13 ± 16.21 ab
SR (%)	84.44 ± 0.05	82.22 ± 0.01	84.44 ± 0.05	90.00 ± 0.02	92.22 ± 0.11	91.11 ± 0.05	91.11 ± 0.04
FCR	2.01 ± 0.20 b	1.19 ± 0.03 a	2.09 ± 0.51 b	1.98 ± 0.52 b	1.54 ± 0.18 ab	1.39 ± 0.16 ab	1.89 ± 0.08 b

IW: initial body weight; FW: final body weight; SR: survival rate; WG: weight gain rate; FCR: feed coefficient ratio; data are expressed as mean ± SD (n = 3). Means with different superscripts were significantly different (p < 0.05).

. For WG, V1 and Y1 were significantly higher than CT (p < 0.05), while there was no significant difference among other groups (p > 0.05). There was no significant difference in SR among all groups (p > 0.05). As for FCR, V1 was significantly lower than CT (p < 0.05), and P2 and Y1 were lower than CT, but there were no significant differences (p > 0.05).

3.2. Antioxidant Capacity and Digestive Enzyme Activity of P. monodon

The activities of α-amylase in P. monodon are shown in Figure 1. In the stomach, there were no significant differences (p > 0.05) among all experiment groups. In the intestines, there were no significant differences (p > 0.05) in vitamin B₆ group and yucca extract group. In the phytase group, compared to CT, there was no significant difference between CT and P1 (p > 0.05), but the α-amylase activity in P2 was significantly high (p < 0.05). In the hepatopancreas, the α-amylase activity in vitamin B₆ group showed an increasing trend, but there was no significant difference (p > 0.05). In phytase and yucca extract group, the α-amylase activity was higher than CT, but there was no significant difference (p > 0.05).

The antioxidant capacity of P. monodon is shown in Figure 2. For T-SOD, there was an increasing trend in the vitamin B₆ group and the yucca extract group, but there were no significant differences (p > 0.05). There was a significant difference in T-SOD between P1 and CT (p < 0.05). For T-AOC, there were no significant differences (p > 0.05) among all experimental groups.

3.3. Composition and Rationality Analysis of Intestinal Microbial ASV in P. monodon

We use Venn [22] plots to show the number of common and unique amplicon sequence variants (ASV) among samples (Figure 3a), a total of 449 ASV numbers were obtained by 16SrDNA high-throughput sequencing. According to the analysis of the Venn diagram, the seven test groups had 50 ASVs in common; there was one unique ASV in the P1 group, and no unique ASV was found in all the other groups. Shannon–Wiener curve (Figure 3b) [23] considers both species evenness and richness. Flat curve indicates that the amount of sequencing data is large enough. Species accumulation curve can tell whether the species increased along with the increase in sample size, which is an effective tool to determine whether a sample size sufficient. The curve gradually inclines to a flat line, proving that the species in the samples did not increase with the increase in sample size (Figure 3c); the foundation of ASV in the data analysis. The results indicated that the microbial diversity was sufficient to be fully detected and the sequencing data were reasonable.

3.4. The Richness and Diversity of Intestinal Microbiota in P. monodon

Alpha richness and the plurality of intestinal microorganisms were evaluated using the Chao1, Simpson and Shannon indices. Alpha diversity analysis (

Table 4. Changes in intestinal microbial alpha-diversity of P. monodon.

Items	Diets
	CT	V1	V2	P1	P2	Y1	Y2
Chao index	124.28 ± 0.39 a	124.48 ± 47.98 a	168.12 ± 11.10 a	165.91 ± 5.64 a	95.20 ± 44.83 a	116.95 ± 68.82 a	106.14 ± 65.10 a
Shannon index	4.04 ± 0.95 a	3.69 ± 1.17 a	4.92 ± 0.30 a	4.42 ± 0.81 a	3.73 ± 1.12 a	4.23 ± 1.39 a	4.13 ± 1.67 a
Simpson	0.83 ± 0.13 a	0.83 ± 0.09 a	0.92 ± 0.26 a	0.84 ± 0.09 a	0.82 ± 0.10 a	0.87 ± 0.11 a	0.87 ± 0.14 a

Note: data are expressed as mean ± SD (n = 3). Means with different superscripts were significantly different (p < 0.05).

) showed that the P2 had the lowest Chao index, but there was no significant difference in Chao index among all experiment groups (p > 0.05). There was no significant difference in Shannon index and Simpson among all experiment groups (p > 0.05). The microbial samples from the V1 and V2, Y1 and Y2 groups were clearly distinguishable based on the PCoA plot. The CT group could only be distinguished from the V2 and Y2 groups and not from the other experimental groups (Figure 4).

3.5. Intestinal Microbiota Composition of P. monodon

The analysis of the intestinal contents of P. monodon encompassed multiple taxonomic levels, specifically, phylum, order, family and genus. Phylum was chosen as the representative taxonomic level for this study. As shown in Figure 5, at the phylum level, Proteobacteria, Bacteroidota and Actinobacteriota formed the core microbiota. Proteobacteria were the most abundant phyla among the seven groups, accounting for 29.33%, 56.67%, 55%, 45.33%, 73%, 39.33% and 64.33%, respectively (

Table 5. Distribution of the top nine microbial phyla in the intestinal contents of P. monodon in different treatment groups.

Phylum	Group
	CT	V1	V2	P1	P2	Y1	Y2
Proteobacteria	29.33 ± 5.92 a	56.67 ± 1.52 a	55.00 ± 12.07 a	45.33 ± 27.50 a	73.00 ± 11.00 b	39.33 ± 16.25 a	64.33 ± 13.50 b
Bacteroidota	66.00 ± 4.00 a	23.00 ± 9.00 b	24.33 ± 7.02 b	23.33 ± 14.64 b	13.00 ± 12.00 b	37.33 ± 0.58 b	22.50 ± 4.50 b
Actinobacteriota	7.20 ± 6.80	0.70 ± 0.30	10.00 ± 13.00	9.33 ± 5.03	7.50 ± 3.50	6.43 ± 10.89	1.60 ± 1.40
Firmicutes	0.33 ± 0.27	12.25 ± 12.75	7.00 ± 5.56	17.00 ± 21.00	3.15 ± 2.85	12.67 ± 21.07	4.50 ± 1.50
Verrucomicrobiota	2.00 ± 0.50	4.40 ± 3.60	1.33 ± 0.58	3.00 ± 1.00	1.50 ± 0.50	2.00 ± 2.00	2.60 ± 2.40
Desulfobacterota	0.10 ± 0.10	0.15 ± 0.06	0.69 ± 1.13	0.63 ± 0.38	0 ± 0	0.22 ± 0.33	1.37 ± 2.28
Planctomycetota	0.67 ± 0.35	0.41 ± 0.39	0.18 ± 0.28	0.14 ± 0.14	0.15 ± 0.15	0.27 ± 0.25	0.40 ± 0.31
Dadabacteria	0.40 ± 0.30	0.25 ± 0.16	0.73 ± 0.25	0.40 ± 0.35	1.15 ± 0.85	0.60 ± 0.53	1.15 ± 0.85
Dependentiae	0.22 ± 0.19	0.11 ± 0.10	0.29 ± 0.22	0.15 ± 0.13	0.35 ± 0.34	0.05 ± 0.04	0.35 ± 0.35

Note: data are expressed as mean ± SD (n = 3). Means with different superscripts were significantly different (p < 0.05).

). Compared with CT, Proteobacteria increased significantly in P2 and Y2 (p < 0.05). There were significant differences in Bacteroidota between CT and other groups (p < 0.05). There were no significant differences in other bacteria among the seven groups (p > 0.05).

3.6. Functional Prediction of FAPROTAX

The functional predictions of FAPROTAX are shown in Figure 6 and

Table 6. FAPROTAX functional abundance in seven groups.

Items	Group
	CT	V1	V2	P1	P2	Y1	Y2
chemoheterotrophy	12,748 ± 787 a	10,716 ± 3041 a	11,618 ± 760 a	9394 ± 2306 a	11,781 ± 2056 a	9167 ± 4482 a	11,838 ± 1933 a
aerobic_chemoheterotrophy	6717 ± 1966 a	9641 ± 3981 a	10,102 ± 16 a	7808 ± 1300 a	10,823 ± 2424 a	7007 ± 2590 a	11,132 ± 2808 a
fermentation	983 ± 732	5847 ± 3713	3618 ± 2087	1400 ± 546	5075 ± 4164	2798 ± 1088	5233 ± 1752
nitrate_reduction	667 ± 886	4176 ± 4812	3040 ± 1624	594 ± 323	4641 ± 4544	2394 ± 1388	5006 ± 2109
intracellular_parasites	105 ± 60	100 ± 79	135 ± 58	268 ± 154	120 ± 121	193 ± 302	189 ± 300
animal_parasites_or_symbionts	137 ± 98	50 ± 54	20 ± 9	80 ± 99	50 ± 84	88 ± 77	89 ± 107
human_pathogens_all	92 ± 66	19 ± 12	3 ± 4	68 ± 80	51 ± 84	4 ± 6	38 ± 28
human_associated	92 ± 66	19 ± 12	3 ± 4	68 ± 80	51 ± 84	4 ± 6	38 ± 28

Note: data are expressed as mean ± SD (n = 3). Means with different superscripts were significantly different (p < 0.05).

. In all groups, the abundance of chemoheterophya and aerobic chemoheterotrophy were high, while fermentation and nitrate-reduction were relatively low, and there were no significant differences in these two functional traits (p > 0.05). Intracellular parasite-, aromatic compound degradation- and human-associated factors were found to be very low, and there were no significant differences (p > 0.05).

4. Discussion

The growth performance of aquatic animals is generally expressed by WG, SR, FCR, etc. [24]. Both a deficiency and excess of vitamins can affect the growth of shrimp [25]. Studies had shown that adding 80–160 mg/kg of vitamin B₆ into the diet of L. vannamei could significantly improve the weight gain rate and did not affect the survival rate [26]. Different aquatic animals have different requirements for vitamin B₆. The specific growth rate and survival rate of Carassius auratus gibelio can be significantly improved by adding vitamin B₆ into their diet, and their specific growth rate reached the highest value when the addition of vitamin B₆ was 13.3 mg/kg [27]. In our experiment, the WG of P. monodon in V1 was significantly higher than that in CT (p < 0.05), while the WG in CT and V2 did not show a significant difference (p > 0.05). The FCR of V1 was significantly lower than that of CT (p < 0.05), but there was no significant difference (p > 0.05) between CT and V2. The performance of V1 was similar to that seen in Wang et al. [26]. The reason for the decrease in V2 may be that the added amount of vitamin B₆ was too high, and the research of Xu found a similar result [25]. Phytase is increasingly used in aquatic animals. Biswas found that the WG and FCR displayed no significant differences when adding phytase into the diet of P. monodon [28]. In the research of Chen [29], a dietary supplementation of 500–2000 FTU/kg of phytase had no significant effects on the weight gain rate and survival rate of L. vannamei. In this paper, the WG and FCR in P1 and P2 both showed no significant differences (p > 0.05) compared with CT, which is the same as in the previous study [28]. The study of yucca extract in aquatic animals is in its infancy. Yucca extract can significantly increase the growth rate of Ictalurus punctatus [30] but had no significant difference on FCR according to the study of Kelly and Khole. Adding 750 mg/kg of yucca extract into the diet of Oreochromis niloticus can significantly improve growth performance [31]. In this paper, the WG in Y1 was significantly higher than that in CT (p < 0.05), but there were no significant differences (p > 0.05) in FCR between CT, Y1 and Y2. This was the same as the results of Kelly and Kohle.

In crustaceans, the hepatopancreas is an important organ not only for absorbing and storing nutrients but also for synthesizing digestive enzymes to digesting food [32]. Different diets can affect the digestive enzyme activity of aquatic animals [33]. Oxidative stress is one of the response mechanisms of animal organisms to environmental stress. Changes in the diet of aquatic animals can affect their antioxidant capacity and immuno-enzyme activity. Under environmental stress, many reactive oxygen species will be produced in the body, resulting in damage to the organism [34]. P. monodon has a relatively complete antioxidant system that can maintain the homeostasis of an organism [35]. SOD plays a crucial role in the balance between oxidation and antioxidants in the body and is an important antioxidant oxidase in maintaining a normal metabolism [36]. Studies have reported that vitamin B₆ can enhance the secretion of digestive enzymes by promoting the growth and development of digestive organs, thereby improving the digestion and absorption capacity [37]. It has been reported that adding vitamin B6 into the diet of Apostichopus japonicus can improve the expression of intestinal amylase [38]. A study by Wei showed that vitamin B₆ can improve the ability of Rhodeus sinensis Gunther to resist hypoxia [39]. In the present study, we did not find any significant difference in vitamin B₆ group compared with CT. We speculate that the reason may be the high amount of vitamin B₆ supplementation, since Xu demonstrated that excessive vitamin B₆ could reduce the hepatopancreatic amylase activity of Fenneropenaeus chinensis [40]. Our results were similar to those of Xu. Phytic acid can be complexed with proteins and inhibit the activity of digestive enzymes, such as pepsin and amylase, and the addition of phytase can release the digestive enzymes that bind to it and improve the activity of digestive enzymes. Phytase can also improve the efficiency of protein utilization and make full use of protein in feed. Ma showed that the addition of 1000 U/kg of phytase can improve the amylase activity of Ctenopharyngodon Idella [41]. Yao also found that adding 1000 U/kg of phytase to the diet of Oreochromis niloticus × O. aureus can significantly improve intestinal amylase activity [42]. Moreover, phytase can improve the AKP activity of Ictalurus punctatus as well [43]. In the present study, the intestinal α-amylase activity was significantly improved in P2 compared to CT and P1, which was similar to the above studies. The reason why P1 did not have significant difference may be that the fish and shrimp have different requirements, and the amount of phytase added in shrimp is not enough. There is not much research on the use of yucca extract in aquatic animals. Yucca extract can improve the α-amylase activity in aquatic animals, the study of Ma showed that adding 0.25% of yucca extract is able to significantly improve hepatopancreas α-amylase activity [44]. Zhang proved that the addition of 0.2% yucca powder can improve the T-SOD and T-AOC of Megalobrama terminalis [45]. In our study, the α-amylase activity in hepatopancreas in Y1 was improved in Y1 compared to CT, but there was no significant difference; thus, we hypothesized that the same concentration of yucca extract would have different effects on fish and shrimp. The T-SOD in P1 was significantly higher than CT, proving that appropriate phytase can improve the antioxidant capacity of P. monodon. There were no significant differences (p > 0.05) in T-AOC among all experimental groups. This proved that these three additives did not negatively affect the antioxidant capacity of P. monodon.

Intestinal microbiota refers to the large number of microorganisms present in the intestinal tract of animals that they depend on to live and help them perform a variety of physiological and biochemical functions. Changes in aquatic animal diets may have some effect on the intestinal microbiota [46]. 16SrRNA is located on the small subunit of the ribosome of prokaryotic cells, including ten conserved regions and nine hypervariable regions, among which the conserved regions have little difference between bacteria and the hypervariable regions are specific to the genus and vary between types. Therefore, 16SrDNA can be used as a characteristic nucleic acid sequence to reveal biological species and is the most suitable index for bacterial phylogeny and classification identification. Although the OTU clustering method can effectively overcome sequencing errors, it reduces the accuracy of classification, and some sequences below a set threshold cannot be accurately distinguished. The de-noising method is recommended. OTUs have been given a new name, ASVs (amplicon sequence variants) [47,48]. Intestinal probiotics can promote the body’s digestion and absorption of food and competitively inhibit the growth of intestinal harmful microorganisms, thus maintaining the ecological balance in the body and promoting the healthy growth of the body [49]. The functional prediction of FAPROTAX in this paper does not detect the functional components that are harmful to the body. Many studies have shown that Proteobacteria are the dominant bacteria in the gut of shrimp [50,51], and some members of this phyla are involved in the nitrogen cycling and the mineralization of organic compounds [52,53]. In this study, Proteobacteria were the most abundant bacteria among the seven groups. Compared with the CT group, the abundance of Proteobacteria was significantly improved in P2 and Y2 and the increasing abundance of Proteobacteria caused some diseases; thus, we hypothesized that the reason may be the excessive amounts of phytase and yucca extract. And compared with CT, the abundance of Bacteroidota was significantly improved in all other groups. Bacteroidota can actively improve the intestinal environment, the high abundance of Bacteroidota in CT may be due to excessive intestinal workload and the additive can alleviate the workload.

5. Conclusions

The addition of these three additives can, respectively, improve some of the properties of P. monodon. However, attention should be paid to the amounts of these additives used, since high amounts have negative impacts on P. monodon. Using them in combination may give a better result, but more research needs to be carried out to obtain more precise dosages. Combined with growth performance, antioxidant capacity and intestinal microbiota, it is feasible to add around 100 mg/kg of vitamin B6, 2000 U/kg of phytase and 0.2% yucca extract to the diet of P. monodon.