As a non-nutritive feed additive, feeding promoting substances are currently a more studied category, which has the advantages of improving feed flavor and palatability of aquatic animals to feed, improving feeding volume and feed utilization, promoting aquatic animal foraging and growth, reducing bait coefficient and environmental pollution [4]. As an excellent food attractant and nutritional supplement raw material, squid paste has obvious feeding effect on fish and shrimp, and has a wide range of applications in aquatic feed. However, the fat and moisture content of squid cream is high, easy to deteriorate, in addition, due to different processing and treatment methods, the quality difference is large, especially the health safety hazards caused by heavy metals cadmium and mercury exceeding the standard, which seriously restricts its use, and it is also an inevitable need to find new alternative effective food attractant products. The black water fly, scientifically known as hermertia illucens, is an insect of the genus Hermertia illucens ( ) , a large diptera , of the family Water flies , and the genus Dipteridae. Because of its wide distribution in nature, rich resources, environmental safety, low difficulty in breeding [8] and easy industrialization, black water fly as a new type of functional animal feed has long been concerned by scholars, in recent years is the international community's attention and attention to resource insects, its larvae are rich in protein and fat. The application of black water fly powder in the aquatic feed to replace fishmeal has been reported [11-15], and the worm pulp has also been sporadically reported [16], while the author has observed that the dried insect powder of black water fly has obvious insect flavor, which can cause fish and shrimp to grab food when mixed into the feed. However, there were no reports on the feeding and application of black water flies in hybrid snakehead feed. In this study, the adsorptive substances suitable for hybrid snakeheads were screened out by comparing the effects of squid paste, black water fly larvae dry powder and fresh worm pulp on their growth performance, body indexes, muscle nutrients and serum indicators in the feed, and provided reference and theoretical basis for the optimization of its practical feed formulation.
Test feed
The diet formulation and nutrient levels of each group are shown in Table 1. Taking the addition of squid paste (purchased from the grease feed processing plant of Zhejiang Xingye Group Co., Ltd.) as the control group, 5% black water fly larvae dry powder (hereinafter referred to as insect powder, prepared by fresh insects being dried and crushed at 120 °C) and 10% black water fly fresh larval pulp (hereinafter referred to as worm pulp, dry matter 3%, directly crushed and pulped by fresh insects) were added as the test group, and the 3 groups of feed were equal nitrogen and other energy feeds. Dried black water fly worms and fresh worms are provided by Guangzhou Feixite Biotechnology Co., Ltd. Each raw material is crushed through 60 mesh sieve, accurately weighed raw materials according to the formula and mixed step by step, add an appropriate amount of tap water to stir and mix well, and then use The Valva-60-I. expansion machine (Guangzhou Vilavi Machinery Equipment Co., Ltd.) to make it into a 3.0 mm diameter puffed granular feed, dried at 55 °C, naturally cooled and put into a sealed bag and stored in a dry and cool place.
Experimental fish and rearing management
The aquaculture experiment was carried out in a fishing row cage of 1.5 m× 1.5 m×2 m in the aquaculture experimental base of the Agricultural Modernization Science and Technology Demonstration Zone of the Guangdong Academy of Agricultural Sciences. The fry were purchased from the Howard Fang Aquatic Fry Hatchery in Sanjiao Town, Zhongshan City, and after the purchased fry were temporarily reared in cages to suitable sizes, 180 hybrid fish with a healthy and consistent body mass of (83.24±0.13) g were selected, and randomly divided into 3 groups, 3 parallel in each group and 20 in parallel each. Three experimental feeds were fed separately. Feed twice a day (8:30-9:00 and 17:30-18:00). During the test, the water temperature was 19~34 °C, the pH was 8.0~8.5, the dissolved oxygen level was >5 mg/L, the ammonia nitrogen mass concentration was ≤ 0.1 mg/L, and the nitrite mass concentration was ≤ 0.05 mg/L. The rearing cycle is 10 weeks.
Effect of black water fly on growth performance of hybrid snakehead
The survival rate of hybrid snakeheads in each experimental group reached 100%. The average quality and quality increase rate of hybrid squid in the dried insect powder group and the fresh worm pulp group increased compared with the squid paste group (control group), among which the dried insect powder group was significantly higher than that in the control group (P<0.05), the fresh worm pulp group was higher than that in the squid paste group (P=0.055), the specific growth rates of the dried insect powder group and the fresh worm pulp group were significantly higher than that in the control group (P<0.05), and the bait coefficients in the dry insect powder group and the fresh worm pulp group were significantly lower than those in the control group (P<0.05) (Table 2). The differences in feed intake, body fullness, visceral ratio, liver-to-body ratio and intestinal body ratio of each group were not significant (P>0.05).
Effects of black water fly on the nutritional composition of the dorsal muscles of hybrid snakeheads
Compared with the control group, the crude fat content of the hybrid spinach back muscle in the dried insect powder group and the fresh worm pulp group increased, of which the dry insect powder group was significantly higher than that in the control group (P<0.05), and the differences in the dry matter, crude protein, ash, calcium and total phosphorus content of the hybrid spinach back muscles of each group were not significant (P>0.05) (Table 3).
Effect of black water fly on serum biochemical indexes of hybrid snakehead
The serum cholesterol content of the dried worm powder and fresh worm pulp group was significantly lower than that of the control group (P<0.05), and the serum triglycerides of the dried worm powder and fresh worm pulp group were higher than those in the control group, of which the dried worm powder group was significantly higher than that of the control group (P<0.05); the difference in blood glucose, alanine aminotransferase, aspartate aminotransferase, albumin, globulin and total protein content in the serum between the test groups was not significant (P>0.05) (Table 4).
Table 2 Feeding and growth performance of 3 hybrid snakeheads of different feeding combinations
Note: The difference in lowercase letters of the shoulder label of the peer indicates that the difference is significant (P<0.05), the same below.
Table 3 Nutritional composition of hybridized dorsal muscles of 3 different feeding combinations %
Table 4 Serum content of hybrid snakeheads of 3 different feeding combinations
Effect of black water fly on serum antioxidant and intestinal barrier function indicators of hybrid water fly
The content of serum immune antioxidant index glutathione peroxidase, superoxide dismutase and alkaline phosphatase was not significant (P>0.05) between the groups, and the content of diamine oxidase, endotoxin and D-lactate in the serum barrier function index was not significant (P>0.05) (Table 5).
Table 5 Serum antioxidant and intestinal barrier indicators of 3 different feeding combinations
The results of this experiment showed that the addition of dried larval powder and fresh worm pulp of black water fly to the feed was no difference between the control group of squid paste, and the growth of hybrid snakeheads and the reduction of bait coefficients were added to the feed, indicating that the dried insect powder and fresh worm pulp of black water fly had the same effect as squid paste in feeding. Compared with the control group, the quality increase rate was significantly increased by 38.19%, the specific growth rate was significantly increased by 25.47%, and the feed coefficient was significantly reduced by 28.22%. This is inconsistent with the fact that the substitution of fishmeal in Pelteobagrus fulvidraco, Cyprinus carpio var. jian and koi (C. carpio koi) does not have a significant effect on growth performance, and the reasons for the analysis are related to the amount of insect meal used and its quality (general fish meal is superior to black water fly meal, insect meal is better than soybean meal) and fish species. The study found that the larvae of black water fly are not only rich in protein, essential amino acids, unsaturated fatty acids and odd carbon fatty acids, but also contain bioactive ingredients such as antimicrobial peptides, chitin and chitosan, which can be used as a new type of feed raw material in aquatic feed. In the test of replacing fishmeal with dried larval powder of black water fly, the growth performance of jian carp and koi carp appeared at the maximum value of 75% and 70% of the replacement group, respectively, but there was no difference with the fishmeal control group, indicating that the effect of black water fly larval powder was better than that of the same amount of fishmeal when using the appropriate dose, and the analysis was related to its containment of antibacterial peptides, chitin and other active substances. Studies have reported that 20% fresh fly maggot pulp can significantly improve the mass increase rate of Monopterus albus and reduce the feed coefficient, and the effect is better than the stored 180 days and frozen fly maggot pulp. Compared with the control group, the addition of fresh worm pulp of black water fly in this experiment significantly improved the growth parameters of hybrid snakeheads, significantly reduced the bait coefficient, and indicated that the appropriate amount of fresh worm pulp could improve the growth of fish. However, it is inconsistent with the report that there were no differences in growth performance and body composition in the feed of juvenile carp (the highest group of 10%) for 8 weeks of cultivation of black water fly worm pulp (the highest group), which may be related to the quality of the test animal breed and the black water fly worm pulp.
Liver-to-body ratio is an important indicator to evaluate the health of fish, liver index is low, indicating that the liver load of the fish body can be controlled within the scope of its own liver function regulation, and when the visceral ratio, liver-to-body ratio, intestinal ratio increases, indicating that the internal organs due to fat deposition and then cause hypertrophy, not only affect its function, but also reduce the feed utilization efficiency. The results of this test showed that the differences in the visceral ratio, hepatic body ratio and intestinal body ratio of the hybrid snakeheads were not significant, indicating that the addition of black water fly powder and black water fly worm pulp to the feed had no effect on the liver function regulation ability of hybrid snakeheads as when using squid paste. Studies have shown that certain protein substitutes do not have a significant effect on the liver-body ratio of aquatic animals, which is consistent with the results of the trial study of black water fly powder in this trial.
In this test, the differences in the content of water, crude protein, ash, calcium and total phosphorus of hybrid snakeheads between groups were not significant, indicating that the nutritional level of hybrid snakeheads (except crude fat) was not greatly affected by these kinds of precipitating substances (at this added level) and was in a relatively balanced state. The study found that the addition of 1% to 2% squid paste to the feed had no significant effect on the nutritional composition of the hybrid squid back muscle, which was consistent with this test. In the animal production process, the increase of intramuscular fat has an important role in improving the quality and flavor of meat, the results of this test in the hybrid water fly powder group hybrid shrimp muscle crude fat content is significantly higher than that of squid paste and fresh worm pulp group, which may be related to the black water fly powder itself has a higher fat content, and the black water fly fat is conducive to the deposition of muscle fat, and the black water fly fat content to reach a certain amount to be able to have an effect, which may be the black water fly fresh worm pulp group (converted into black water fly dry matter is 3%, less than the amount of 5% of the dry worm powder group The reason why the fat content of the back muscle of hybrid snakeheads is lower than that of the dry worm powder group. The study found that adding a certain amount of fly maggot pulp to the feed can reduce the fat content in the muscle of the yellow eel, and it is not related to the freshness of the fly maggot pulp, which is inconsistent with the results of the test, and may be related to the nutritional composition of insect species and worm pulp, especially the composition and content of fatty acids.
Effect of black water fly on serum indices of hybrid snakeheads
The physiological and biochemical indicators of blood are important indicators of animal physiological function and metabolic status, and are also indicators of animal health standards [25]. Studies have found that the pre-pupa of black water flies replaces fishmeal to reduce the plasma cholesterol content of Dicentrachus labrax [26], which may be lower than the cholesterol content of black water fly pre-pupa and degreaser powder, resulting in a decrease in the cholesterol content in the alternative feed, resulting in a low plasma cholesterol content of sea bass, and the cholesterol content in the feed directly affects the blood cholesterol content of farmed animals. Wen Yuanhong et al. [29] also found that fly maggot powder replaced fishmeal to reduce the serum cholesterol content of yellow jaw fish. The results of replacing squid paste with black water fly powder and worm pulp in this test are basically consistent with the above research results. There have also been different reports that the replacement of fishmeal by black water fly meal (3.5% and 7.0% of black water fly powder dosages) has no effect on the serum cholesterol and triglyceride content of the construction carp [13]. In this test, the triglyceride content in the plasma of the black water fly powder and worm plasma group was increased compared with that of the control group, and the serum triglyceride content reflected the development of adipose tissue and fat deposition capacity, and the increase in the content of fat deposition suggested an increase in fat deposition [30], which was also verified by the increase in the crude fat content of the back muscle in the test group. In this test, there was a downward trend in the serum alanine aminotransferase and aspartate aminotransferase after the substitution of degreasing insect meal and worm pulp with squid paste, which was consistent with the significant decrease in glutamate and the decrease in aspartate aminotransferase in koi plasma after replacing fishmeal with black water flies
Organisms in the growth and development process will produce a large number of free radicals, if these free radicals can not be removed by the body's antioxidant defense system in time, excess free radicals can induce oxidative stress, and lead to oxidative damage. The susceptibility of organs and tissues to oxidative stress depends on the body's dynamic balance of oxidation and antioxidants. Superoxide dismutase, glutathione peroxidase, etc. as important regulatory enzymes for biological antioxidant systems, their activity directly affects the body's antioxidant capacity, while malondialdehyde, as a marker of the degree of oxidative stress of the body, is positively correlated with the body's oxidative stress. Studies have shown that black water fly powder has a promoting effect on the content of koi plasma superoxide dismutase, catalase and glutathione, and the pupal peptide protein replaces 45% and 60% of the fish meal in the juvenile compound of Scophthalmus maximus, respectively, which can significantly increase the superoxide dismutase activity of the liver of turbot juveniles[33], and the addition of 20% fly maggot protein to the feed can effectively improve the litopenaeus vannamei. Serum superoxide dismutase activity[34] is similar to the addition of dry powder of black water fly larvae to this study to increase the content of superoxide dismutase and glutathione peroxidase to a certain extent, possibly because insect powder contains antioxidant proteins and antimicrobial peptides[35], and its effect is also related to the amount of insect powder. The addition of 2.5% to 10% of black water fly worm pulp to the feed can increase the serum catalase of juvenile mirror carp and significantly decrease malondialdehyde [16], while in this test, 10% of black water fly worm pulp has no significant effect on the antioxidant index of hybrid snakehead, and the analysis may be related to the quality of the worm pulp and the type of fish tested, and more test verification is needed.
The intestine is not only an important endocrine organ, regulating the digestion and absorption of nutrients, but also an important immune organ, and its barrier function can shield harmful substances (such as diamine oxidase, endotoxin, D-lactic acid) in the intestinal lumen from entering the body and causing damage to the body. The intestine communicates with the outside world at all times, and the number of exposures to external microorganisms is also the largest, so it is also more likely to produce oxidative stress, causing oxidative damage [36-37]. Diamine oxidase is an intracellular enzyme containing deamines of putrescine and histamine, most of which are found in the mucous membrane of the small intestine, and when the intestinal mucosa is damaged, the enzyme is released into the bloodstream. Endotoxins are lipopolysaccharides in the cell walls of gram-negative bacteria, and are toxic substances released after the bacteria proliferate in large quantities or are lysed by death. D-lactic acid is a metabolite of bacteria inherent in the intestine, and D-lactic acid in the blood is basically derived from the intestine. Therefore, the determination of diamine oxidase, endotoxin and D-lactate content in serum can comprehensively assess the permeability of the intestinal mucosa. The results of this experiment showed that there was no significant difference in the serum diamine oxidase, endotoxin and D-lactate content of hybrid snakeheads in each group, indicating that there was no difference in the intestinal barrier function of the fish in each group.
Comprehensive comparison of the application effects of squid paste, black water fly dried insect powder and black water fly fresh worm pulp in hybrid shrimp feed showed that 5% of black water fly dried worm powder or 10% fresh worm pulp can be used in hybrid water fly feed to replace squid paste as a feeding promoting substance, and it has a better growth promotion effect than squid paste.