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 B
6 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 B
6 is necessary for the proper function of the immune system of aquatic animals. One study pointed out that adding 3.4 mg/kg vitamin B
6 to the diet of Indian catfish can achieve a maximum increase in weight and survival rate [
11]. Adding vitamin B
6 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.
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:
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).
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 B
6 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 B
6. The specific growth rate and survival rate of
Carassius auratus gibelio can be significantly improved by adding vitamin B
6 into their diet, and their specific growth rate reached the highest value when the addition of vitamin B
6 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 B
6 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 B
6 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 B
6 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 B
6 group compared with CT. We speculate that the reason may be the high amount of vitamin B
6 supplementation, since Xu demonstrated that excessive vitamin B
6 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.