A Review of Research and Application of Non-nutritive Additives in Fish and Shrimp Feed

Research and Application of Non-nutritive Additives in Fish and Shrimp Feed


Introduction:

Fish feed additives are divided into two categories: nutritive and non-nutritive additives. Nutritive additives include amino acids, vitamins, minerals and trace elements; non-nutritive additives refer to additives added to feed beyond the main substance components of the feed to promote growth and development, improve feed structure, maintain feed quality, aid digestion and absorption, and prevent and treat fish diseases, including growth promoters, digestion promoters, probiotic preparations, feeding attractants, binders, antioxidants, etc. The rational use of non-nutritive metabolic regulators can improve the body's metabolism, enhance the efficiency of nutrient utilization, and increase the economic benefits of fish farming. With the rapid development of the aquaculture industry, research on non-nutritive additives for fish has become increasingly in-depth both domestically and internationally. Non-nutritive additives have been widely used in the production of formulated fish feed and have yielded a large number of research results.
This article reviews the functions, composition, domestic and international production technologies, and application effects of non-nutritive fish and shrimp bait additives such as growth promoters, enzyme preparations, probiotic preparations, feeding attractants, binders, antioxidants, and colorants.

Keywords: fish and shrimp, non-nutritive additives, growth promoters, enzyme preparations, probiotic preparations, feeding attractants, binders, colorants

I. I. Growth promoters

The main function of fish growth promoters is to stimulate the endocrine system of fish, regulate metabolism, and improve feed utilization, thereby promoting the growth of farmed fish.
1.1 Olaquindox
Olaquindox is a broad-spectrum antibacterial chemical drug with obvious growth-promoting effects, and is one of the most widely used growth promoters in animal feed. Its function is to affect metabolism, promote synthesis, improve the utilization of energy and nitrogen in fish feed, and promote nitrogen deposition. Wang Shengneng et al. added 150 mg/kg of olaquindox to the diet of carp and tilapia, and the growth rate increased by 75% and 65% compared with the control; Ye Jinyun added 50 mg/kg of olaquindox to the diet to feed 1-year-old grass carp, and the growth rate increased by 53.82% and the survival rate increased by 22.7%; Wen Yinliang et al.'s experiments on carp proved that adding 200 mg/kg of olaquindox promoted growth, with the relative growth rate increasing by 53.88% compared with the control group, and the fish's ability to tolerate low oxygen was enhanced and the meat quality was also improved; Zhang Liang et al. believed that adding 100 mg/kg of olaquindox to channel catfish had a good growth-promoting effect. Geng Yi et al. reported that long-term addition of olaquindox to carp feed should be controlled at a dose below 50 mg/kg, otherwise there is a risk of poisoning in the fish (ascites and hemorrhagic symptoms, intolerance to transport, and mass death during severe harvesting).
1.2 Antibiotics
Antibiotics are fermentation products of microorganisms. Their mechanism of action mainly involves changing the structure of pathogenic microorganisms and interfering with their metabolic processes, such as blocking cell wall synthesis, affecting the permeability of the cytoplasmic membrane, blocking protein synthesis, and altering nucleic acid metabolism. In addition, some scholars believe that after animals consume antibiotics, their intestinal walls become thinner and healthier, thereby improving intestinal absorption efficiency. However, whether antibiotics have a growth-promoting effect on fish remains somewhat controversial. Hashimoto (1956) and Aoe (1961), after reviewing the research results of Woef et al. on the effects of antibiotics such as penicillin, oxytetracycline, and chlortetracycline on fish growth, pointed out that antibiotics basically have no effect on promoting fish growth. Cao Weimin et al. (1993) added 18 mg of erythromycin to every 100 kg of basal feed and raised 40-60 g current-year Feng carp in tanks for 40 days, and also reached the same conclusion. It is believed that this is because the digestive tract of healthy fish is in a sterile state due to lack of food. However, some scholars hold the opposite view. Ye Yuantu (1994) added tetracycline drug residue at a ratio of 1.3% to formulated feed to feed carp, and after 50 days the average weight gain rate of the experimental group was 33.27% higher than that of the control group, and the feed coefficient was 22.05% lower than that of the control group. He believed that the growth-promoting effect of the drug residue mainly depends on the tetracycline in it, and the mechanism of action is to inhibit the growth of microorganisms harmful to carp growth.
Flavomycin is an antibiotic feed additive developed in the early 1970s. A large number of studies on pigs, broilers, and meat ducks have confirmed that flavomycin can promote the growth and reproduction of beneficial microorganisms in the animal intestinal tract, inhibit harmful microorganisms, maintain the microbial ecological balance in the digestive tract, and facilitate the absorption of nutrients. In recent years, research has shown that flavomycin is also a relatively ideal additive in fish feed. Flavomycin is a macromolecular substance with a relative molecular weight of 1582, and is not easily digested and absorbed by fish. Its main function is to purify the digestive tract environment, promote thinning of the intestinal tract, and improve the absorption rate of nutrients. Flavomycin can also promote various biochemical reactions in the fish body to regulate metabolism and enhance the protein conversion rate in the fish body. Wang Xuehong and Xia Changqing confirmed that flavomycin has a growth-promoting effect on slow-growing European eels; Ren Zelin reported that adding flavomycin to feed can promote the growth of carp; Zhou Xiaoqiu concluded that adding flavomycin to feed has no negative impact on the transport tolerance of carp, the optimal dosage of flavomycin in the early growth stage feed of carp is 3 mg/kg, and the combined effect of flavomycin and olaquindox on carp growth promotion is not obvious.
1.3 Hormones
Research on fish and shrimp growth promoters began in the 1960s. Because fish and shrimp are aquatic organisms, their digestion and metabolism mechanisms differ from those of mammals and poultry, so many antibiotics and antibacterial agents used to promote the growth of livestock and poultry have no effect in fish and shrimp bait. The growth promoter commonly used in fish and shrimp bait now is olaquindox. Since the 1980s, the most notable growth promoter studied internationally is growth hormone. Growth hormone is secreted by acidophilic cells in the anterior pituitary gland of vertebrates. Pickfold and Atz (1957) pointed out that growth hormone is a single-chain polypeptide hormone composed of 200 amino acid residues that can promote fish growth. Y. Eitomoite (1964), R. Smift et al. (1965) added pituitary growth hormone to bait, which significantly promoted the growth of rainbow trout and river perch. The development of genetic engineering has opened a path for the mass production of the proteinaceous trace active substance growth hormone. Takahashi Akiyoshi et al. (1985) used an improved separation method to isolate 50 micrograms of growth hormone from about 1000 chum salmon pituitaries, and together with the Kyowa Hakko Kogyo Tokyo Research Institute, which has the latest bioengineering technology, analyzed the amino acid sequence and the base sequence of the gene, and developed technology for mass production of growth hormone using genetic engineering. At present, growth hormones for cattle, pigs, sheep, chickens, fish, and many other animals can be produced worldwide, and excellent results have been achieved through feeding. The Institute of Hydrobiology, Chinese Academy of Sciences, is conducting this research work on fish.
However, there is also a view that growth hormone is a protein and is easily decomposed and destroyed by digestive enzymes, so it is not suitable for use as an additive. Experiments at the National University of Singapore confirmed that adding 0.01 mg/kg of thyroxine to feed can enhance the appetite of carp and accelerate growth rate.
Steroid hormones currently include more than 20 types of steroid hormones, which have been used in more than 20 fish species including salmon, trout, carp, crucian carp, and tilapia, all acting as non-nutritive growth promoters when administered orally. However, due to side effects such as residual carcinogenicity and effects on sex, growth promoters mainly based on steroid hormones have never formed commercial production. Among them, androgens have a significant promoting effect on fish growth, while whether estrogens have a growth-promoting effect on fish remains controversial. Another function of applying steroid hormones to fish is to change sex differentiation, promote sex reversal, and produce large numbers of monosex fish. For example, producing all-male tilapia can also achieve the purpose of improving economic benefits.
1.4 Chinese herbal medicines
As a natural resource, Chinese herbal medicines contain trace components beneficial to organisms and have no toxic side effects. Yang Shifan (1994) believed that any of the following functions can promote fish growth and enhance disease resistance: (1) those that can excite the circulatory system and respiratory center, activate and improve blood circulation, enhance cell membrane activity, and regulate digestive tract and endocrine functions, such as henbane, motherwort, and angelica; (2) those that can promote digestion and strengthen the stomach, stop fermentation and remove stagnation, strengthen gastrointestinal functional activity, and promote digestion, such as hawthorn, tangerine peel, green tangerine peel, and bitter orange; (3) those that can excite the nerve center, stimulate gastrointestinal peristalsis, resist bacteria and inflammation, and eliminate sores and toxins, such as bupleurum, wild chrysanthemum, dandelion, rhubarb, and coptis; (4) those that can strengthen the stomach, remove food stagnation, kill parasites, detoxify, and excite gastrointestinal peristalsis, such as pumpkin seeds and betel nut. Compound Chinese herbal medicine additives are a hot spot in the research and development of fish growth promoters. Wang Yongling reported that adding 1% garlic to feed can increase the weight gain rate of crucian carp by 7.99% and reduce the feed coefficient by 10.37%; Chen Weiyan et al. added 0.5% mugwort leaf to feed to feed carp, and the weight gain rates of summer fingerlings, 1-year-old carp, and 2-year-old carp increased by 11.65%, 22.46%, and 12.55% respectively compared with the control group; Wu Wen et al. added 0.3% gynostemma to feed to feed grass carp, which increased weight gain by 31.25%. Duan Ming et al. used a formula of codonopsis, astragalus, and fennel, added at 1% to feed crucian carp, which increased weight gain by 21.77%; Chen Tielang et al. used a formula of fleeceflower root and astragalus, added at 1% to feed, and the weight gain rate of Oreochromis niloticus increased by 95.40%, and the weight gain rate of catfish increased by 42.20%; Zhang Zhaohua et al. added a compound Chinese herbal medicine additive composed of 30 Chinese herbs including mugwort leaf, bitter orange, and medicated leaven to turtle feed, and the results showed that the experimental animals gained more than 30% weight compared with the same period at the end of the previous year. Wu Defeng et al. (2001) used a prescription composed of Chinese herbs such as astragalus, codonopsis, bighead atractylodes rhizome, poria, licorice, medicated leaven, hawthorn, angelica, rhubarb, figwort root, isatis root, and scutellaria as a disease-resistant and growth-promoting additive for European eels. The experimental results showed that European eels fed with Chinese herbal medicines in the daily diet gained significantly more weight than the control group, and also had varying degrees of resistance to several common infectious diseases, indicating that Chinese herbal medicine feed additives improved the breeding effect of European eels. Liu Hongbai et al. (2004) found that adding scutellaria and astragalus to feed can significantly promote carp growth and improve feed conversion efficiency, and adding poria, houttuynia, and isatis root also had certain effects. Yan Youli et al. (1994) reported that using a prescription composed of Chinese herbs such as rhubarb, sophora flavescens, tangerine peel, astragalus, and licorice for protecting the liver and promoting bile secretion, benefiting qi and nourishing blood, and clearing heat and detoxifying, added at 0.3% to carp basal feed, the results showed that the daily growth rate of the experimental group was 30.6% higher than that of the control group, and the feed coefficient was 18.91% lower. Similar conclusions were also reached by adding Chinese herbal medicine additives such as garlic, codonopsis, fennel, and licorice to the feed of crucian carp, tilapia, grass carp, silver carp, carp, carp, and prawns. This indicates that Chinese herbal medicines have obvious growth-promoting effects on aquatic animals.
Research on Chinese herbal medicines as fish growth-promoting additives is still not mature enough. Current research mainly focuses on comparing additive effects, and issues such as medication and formula, dosage, active ingredients, mechanism of action, and safety testing still need to be further explored.

II. Enzyme preparations

1. Application and classification of enzyme preparations in aquatic feed
With the development of the aquatic feed industry, feed additives currently dominated by antibiotics and chemically synthesized antibacterial agents will inevitably withdraw from the market because they easily cause drug residues in aquatic animals and thus endanger human health. Enzyme preparations, as environmentally friendly green feed additives with no drug residues, no toxic side effects, and no environmental pollution, will certainly receive increasing attention. Feed enzyme preparations are currently considered the safest type of feed additive to use. With advances in technology, improvements in enzyme activity, production processes, and stabilization technology of enzyme preparations, and reductions in production costs, feed enzyme preparations will be widely used in solving the shortage of aquatic animal feed resources, improving feed utilization, reducing environmental pollution, and increasing economic benefits.
Enzymes are protein catalysts produced by living cells and play a vital role in digestion and metabolism in animals. They can promote the decomposition of proteins, fats, starch, and cellulose through biochemical reactions, eliminate anti-nutritional factors in feed, enhance the conversion rate of feed by fish, and promote fish growth. At present, proteases, amylases, saccharifying enzymes, cellulases, and compound enzyme preparations have been widely used in livestock production.
Compound feed for animals contains a variety of nutrients, most of which are composed of biological macromolecules. Before animals can digest and utilize these nutrients, they must be enzymatically hydrolyzed. Since enzymes have strict specificity and selectivity, the effect of using a single enzyme system is lower than that of using a multi-enzyme system. By utilizing the synergistic effects of various digestive enzymes and non-digestive enzymes, the utilization rate of nutrients such as energy, protein, and cellulose in feed can be maximized, thereby achieving the goals of weight gain and reduced feed consumption. At present, feed compound enzyme preparations produced worldwide mainly fall into the following categories due to their different functional characteristics:
(1) Feed compound enzymes mainly based on B-glucanase
This type of enzyme preparation is mainly used in North America, Europe, and countries where barley is the main feed ingredient.
(2) Feed compound enzymes mainly based on protease and amylase
This type of enzyme preparation is mainly used to supplement the deficiency of endogenous enzymes in animals.
(3) Feed compound enzymes mainly based on cellulase and pectinase
The main function is to destroy plant cell walls, release nutrients from cells for further digestion and absorption, eliminate anti-nutritional factors in feed, reduce the viscosity of gastrointestinal contents, and promote animal digestion and absorption.
(4) Feed compound enzymes mainly based on cellulase, protease, amylase, saccharifying enzyme, and pectinase
This type of compound enzyme combines the joint effects of the enzyme systems in 2 and 3 above and has a stronger digestion-aiding effect.
The research and application results of compound enzyme preparations in fish bait have shown obvious effects in assisting digestion, reducing bait coefficient, improving water quality, and reducing liver and digestive tract diseases. Although carnivorous fish have a strong ability to digest protein and lipids, under high-density artificial farming conditions, their ability to digest artificial bait is limited. Adding protease and lipid-degrading enzymes to bait can improve the digestibility of protein and lipids in bait and reduce the impact of undigested matter on water quality. Starch is the main component of plant feed and is also the most commonly used safe and economical binder in artificial bait. However, fish (especially carnivorous fish) have low amylase activity in their bodies and low ability to digest starch, so starch in bait cannot be fully utilized. This not only causes waste of nutrients and affects water quality, but also produces indigestion and liver disease, resulting in stunted growth and even death in severe cases. Adding amylase to bait can significantly improve starch digestibility, reduce pollution to water quality, and ensure the health of aquatic animals.
2. Mechanism of action of enzyme preparations
(1) Directly decomposing nutrient substrates, improving metabolic levels, and reducing bait coefficient
Biologically active enzyme preparations can effectively decompose and digest some macromolecular polymers in feed into nutrients easily absorbed by aquatic animals or into small fragments of nutrients for further digestion by other digestive enzymes. Some macromolecular substances that aquatic animals themselves find difficult to decompose and absorb can be promoted by adding enzyme preparations to decompose and digest nutrients in feed, improving feed utilization. Adding 0.1% enzyme preparation to grass carp feed increased the growth rate of grass carp in the experimental group by 6.6% compared with the control group, and reduced the bait coefficient by 0.29; the crude protein content of the fish in the experimental group was slightly higher than that in the control group, while the crude fat was slightly lower, and the feed cost per kilogram of fish in the experimental group was 8.93% lower than that in the control group. This enzyme preparation improved the conversion and utilization of inorganic salts by grass carp and made the absorption of various nutrients balanced and effective, indicating that the enzyme preparation can promote various biochemical reactions in the fish body and improve the protein conversion rate in the fish body. Yan Youli et al. (1992) added 0.1% cellulase to carp basal feed and raised them for 27 d. The weight gain rate of the experimental group increased by 11.96% compared with the control group, the feed coefficient decreased by 16.36%, and the feed cost decreased by 14.63%. Ye Yuantu et al. (1995) used protease, amylase, and lipase isolated from pig gastric mucosa and pancreas to form a compound enzyme preparation in a certain proportion, and added it at 0.5% and 1.0% to carp basal feed respectively, increasing the average weight gain rate of carp by 12.3%-27.5% and reducing the feed coefficient by 18.70%-10.89%, with no significant adverse effects on carp quality. The experimenters believed that the mechanism of action was that exogenous enzymes participated in the digestion process of carp feed, enhanced the conversion rate of feed by fish, and thereby promoted fish growth rate.
(2) Supplementing exogenous digestive enzymes, activating endogenous digestive enzyme secretion, and improving feed utilization
There are many studies on the effects of adding exogenous enzymes to livestock and poultry feed on animal endogenous enzyme activity. Xi Gang et al. (1999) showed in experiments adding exogenous enzymes to pig and chicken feed that exogenous enzymes can promote the secretion of endogenous digestive enzymes and improve feed utilization. The digestive system of aquatic animals is not as complete as that of livestock and poultry, and endogenous enzyme secretion is not as complete as that of livestock and poultry. The digestive tract of fish does not contain xylanase or cellulase, and amylase activity is also very low. Although fish and shrimp have a relatively high digestion and absorption rate for protein, endogenous proteases cannot fully exert their effects. After adding exogenous proteases, the deficiency of endogenous proteases can be compensated (especially in larvae), increasing the protein absorption rate by about 10%, which can achieve the purpose of saving protein. Carbohydrates are cheap feed ingredients. Although aquatic animals have a very low digestion and absorption rate for starch, in order to ensure safety and reduce costs, the amount added to feed still reaches 20%-50%. After adding amylase and saccharifying enzyme, the amylase seriously lacking in aquatic animals can be supplemented, increasing the digestion and absorption rate of starch from 20%-40% to more than 80%, thereby increasing feed energy, improving feed utilization, and reducing the use of protein as energy consumption, thus achieving the purpose of reducing feed costs. Therefore, exogenous digestive enzymes are very important for aquatic animals. Exogenous digestive enzymes can also stimulate more secretion of endogenous digestive enzymes in aquatic animals and increase the activity of endogenous digestive enzymes in chyme, accelerating the digestion and absorption of nutrients and thereby improving feed utilization. Qiao Xiuting et al. (2002) studied the effects of enzyme preparations on the digestive enzyme activity of carp. After adding compound enzyme preparations, the protease and amylase activities in the intestine and hepatopancreas of each experimental group were higher than those of the control group, reducing the consumption of protein as energy and indirectly saving protein. Zhong Jun et al. (1995) showed in experiments on prawns that exogenous enzyme systems can enhance the effect of endogenous enzymes by activating the animal's own endogenous zymogens, promote the digestive function of the hepatopancreas, and improve the total digestibility and protein digestibility of feed.
(3) Eliminating anti-nutritional factors in feed and improving digestive function
Plant-based raw materials contained in compound feed often contain some non-starch polysaccharides, pectin, and cellulose polymers, which have certain anti-nutritional effects on the digestion and absorption of aquatic animals. They partially dissolve in the intestine, thereby increasing the viscosity of chyme, slowing the dissolution rate of nutrients, slowing the diffusion rate of nutrients and endogenous digestive enzymes, weakening the intestine's ability to mechanically mix chyme, and reducing feed digestibility. At the same time, soluble non-starch polysaccharides can also thicken the immobile water layer on the surface of the intestinal mucosa, hindering the diffusion of nutrients and affecting the digestion and absorption of effective nutrients by aquatic animals. In addition, fecal output increases due to the increase in undigested matter, which can also lead to increased microbial reproduction in the animal gastrointestinal tract and adversely affect the quality of aquatic products. Various enzymes in enzyme preparations, especially β-glucanase, fructosidase, and cellulase, can decompose these substances into small molecules, thereby reducing the viscosity of substances in the digestive tract, promoting animal intestinal peristalsis, effectively eliminating the adverse effects of these anti-nutritional factors, and improving the digestive function of aquatic animals. For example, cellulase is composed of three highly synergistic enzymes: glucan exo-enzyme, glucan endo-enzyme, and β-glucanase. It can decompose plant fibers in rapeseed cake, cottonseed meal, and soybean meal into glucose, destroy plant cell walls, and release contents, thereby comprehensively improving feed utilization. Yu Fengnian et al. (2001) added 2400 IU/kg cellulase to feed mainly based on soybean cake and rapeseed cake to feed blunt snout bream, improving the utilization rate of feed nutrients by fish. Zhou Xiaoqiu et al. (2001) added enzyme preparations to feed mainly containing soybean meal, rapeseed meal, and cottonseed meal, improving feed utilization and reducing the viscosity of carp digestive tract chyme, which is conducive to the absorption and utilization of nutrients. In addition, cake and meal feeds such as soybean meal contain a variety of anti-nutritional factors, such as trypsin inhibitors, plant lectins, and α-galactosides. Trypsin inhibitors combine with trypsin in small intestinal fluid to form inactive complexes, reducing trypsin activity and leading to decreased feed protein digestibility. At the same time, due to binding with trypsin, endogenous protein consumption occurs in the animal body, causing the loss of sulfur-containing amino acids in the body and exacerbating metabolic disorders in the animal body caused by the shortage of sulfur-containing amino acids in soybean meal, resulting in stunted or arrested growth. Yang Lijie et al. (2000) reported that exogenous proteases can inactivate anti-nutritional factors in soybeans at room temperature and improve their utilization. Adding α-galactosidase to legume feed can increase the digestion of oligosaccharides in legume feed and reduce or eliminate digestive disorders.
(4) Improving immune resistance and enhancing aquatic animal health
Adding enzyme preparations to feed can promote the elevation of various hormone levels in the body, thereby affecting the functional activity of lymphocytes and macrophages. Enzyme preparations can also degrade polysaccharides in feed to produce oligosaccharides and their derivatives. Some oligosaccharides, such as mannan oligosaccharides, not only prevent pathogenic bacteria from aggregating in the intestine and reduce the toxic effects of pathogenic bacteria on the body, but also participate in immune regulation and improve immune capacity. In addition, nutrients are one of the decisive factors for the animal body to produce immunity. Nutrition greatly affects cellular immunity, humoral immunity, complement function, and leukocyte phagocytosis. Adding enzyme preparations can release more nutrients, thereby strengthening the body's immunity. Liu Wenbin et al. (1997) conducted experiments on Chinese soft-shelled turtles with added enzyme preparations. The enzyme preparation contents of the three experimental groups were 0.1%, 0.2%, and 0.3% respectively. The results showed that all three experimental groups had higher survival rates than the control group, and the survival rate increased with the increase in enzyme preparation addition, indicating that enzyme preparations can strengthen the turtle's physique and improve disease resistance. The reason may be that enzyme preparations contain enzymes and other active substances that can decompose harmful substances in the animal body and catalyze the conversion of harmful substances into harmless substances, thereby strengthening the physique and improving the survival rate. Zhou Siquan et al. (2000) added enzyme preparations to turtle feed, and the results improved the survival rate of turtles, especially juvenile turtles, indicating that enzyme preparations have the effect of enhancing the disease resistance of turtles.
(5) Promoting the digestion and absorption of phytate phosphorus and reducing environmental pollution
In aquatic animal feed containing plant-based raw materials such as soybean cake and rapeseed meal, the content of phytate is about 3%, of which phytate phosphorus accounts for about 60%-80% of the total phosphorus in feed. In particular, the presence of phytic acid not only makes phytate phosphorus unavailable, but also forms stable complexes with calcium, magnesium, amino acids, and starch that are difficult to digest. In addition, fish have short digestive tracts and food stays in them for a short time, resulting in incomplete digestion. According to research, the average digestibility of phytate phosphorus by stomachless fish is 8%, and that of fish with stomachs is slightly higher, such as 19% for rainbow trout. Therefore, a large amount of phytate phosphorus is wasted, resulting not only in insufficient phosphorus absorption but also requiring supplementation with inorganic phosphorus, thereby causing water environmental pollution. In recent years, many studies have shown that adding phytase to aquatic animal feed can greatly reduce the concentration of phosphorus and other nutrients in excreta, reduce pollution to water bodies, and improve the rearing environment. It is generally believed that adding phytase can increase the utilization rate of phosphorus by 20%-50%. Riche et al. (1996) added 1000 IU/kg of phytase to rainbow trout feed, and the results increased the utilization rate of phosphorus in soybean meal from 25% to 57%. Sugiura et al. (2001) reported that adding phytase to rainbow trout feed based on soybean meal, when fish consumed 1 kg of feed, the phosphorus excretion in feces was 0.32 g, which was 95%-98% less than the phosphorus excretion when fed commercial fish feed. Zeng Hong et al. (2001) added 1000 IU/kg of neutral phytase to carp feed with a cake and meal mass fraction of 5.0%, which could increase the phosphorus utilization rate of carp for feed by 41%-59.4% and reduce phosphorus excretion per unit weight gain by 32.0%. In practice, feed can be pretreated with phytase in advance. Yu Fengnian et al. (2000) first treated soybean meal with phytase in vitro, then added it to the remaining feed ingredients, mixed thoroughly and analyzed. The results showed that in vitro in gibel carp, phytate phosphorus had already undergone effective degradation under the action of phytase, and the addition of phytase promoted the absorption of nutrients in feed by gibel carp and increased the weight gain rate.
3. Research on the application of enzyme preparations in aquatic animal feed
(1) Development of feed enzyme preparations for aquatic animals
Enzyme preparations added to livestock and poultry feed should use xylanase, β-glucanase, cellulase, and amylase as the main enzyme systems, with acid protease and lipase as auxiliary enzyme systems. The digestive system of aquatic animals is not as complete as that of livestock and poultry. Most farmed fish have no stomach and only have a digestive tract, and the digestive tract is very short compared with livestock and poultry, with a pH generally neutral or slightly alkaline. Therefore, feed enzyme preparations for aquatic animals should be compound enzyme preparations mainly based on neutral or alkaline proteases, amylases, and saccharifying enzymes, supplemented by acid proteases, lipases, xylanases, β-glucanases, and cellulases. In addition, special compound enzyme preparations suitable for specific conditions should be developed according to the specific conditions of aquatic animal species, age, feeding habits, physiological digestion characteristics, and feed specificity. Fish with different feeding habits also have different compositions of digestive enzymes in their digestive tracts. Among endogenous digestive enzymes, the order of amylase activity from high to low is herbivorous > omnivorous > carnivorous fish. The protease in eel stomach tissue is significantly higher than that in intestinal tissue, and the foregut > midgut > hindgut. The amylase activity in intestinal tissue is foregut<中肠<后肠，这种趋势与草鱼、鲤中的结果一致。肠蛋白酶最适pH7.8；肠淀粉酶最适pH7.6；甲鱼的胃、胰组织蛋白酶活性显著高于肠组织蛋白酶活性，且胃>pancreas > foregut > midgut > hindgut; the optimal pH of proteases in stomach, pancreas, and intestine tissues is 2.2, 6.2, and 7.6 respectively; this is similar to the digestive characteristics of fish with stomachs. The protein digestion and absorption of soft-shelled turtles occurs in the stomach and foregut, which is extremely similar to monogastric mammals, but generally monogastric mammals do not carry out protein digestion in the hindgut, which seems to be different. This can be explained as the protein digestion characteristics of amphibians. The enzyme activity of the prawn digestive tract is inversely proportional to body weight. That is to say, smaller shrimp have stronger digestive tract protease activity, while larger shrimp have weaker digestive tract protease activity. The reason is that small shrimp in the vigorous growth stage need more protein to maintain growth, so they need stronger enzyme activity to digest and utilize protein; larger shrimp gradually tend to grow relatively stable, require relatively less protein, so enzyme activity gradually decreases. Under the same body length or body weight, female shrimp have greater protease activity than male shrimp. This indicates that female shrimp have stronger digestive power and utilize more protein, so female shrimp grow faster than male shrimp. Kawai et al. studied the digestive enzymes in the pharynx and esophagus of carp and found that there were relatively weak trypsin-like and maltase activities in the pharynx, and relatively strong amylase, maltase, and trypsin-like activities in the esophagus. This study indicates that in stomachless fish like carp, the enzymes in the esophageal region play a digestive role. It also indicates that the highest activity of maltase and amylase is at the end of the intestine, while the highest activity of sucrase is in the middle of the intestine. The action of digestive enzymes varies significantly depending on fish species, tissue, season, and growth stage. Higo et al. compared the activities of various digestive enzymes in carp, sweetfish, eel, and rainbow trout. The results showed that trypsin activity was strongest in rainbow trout, about 1/2 of that in carp and sweetfish, and weakest in eel, only 1/9 of that in rainbow trout. However, pepsin was strongest in eel, about 4 times that of rainbow trout, and almost absent in carp and sweetfish. Amylase was strongest in carp, followed by sweetfish, and the amylase of rainbow trout was weaker than that of both carp and sweetfish. This can be fully seen from the difference in starch digestibility between carp and rainbow trout. The starch digestibility of carp is 46%, while that of rainbow trout is 26%, but much stronger than that of eel. Moreover, there are certain differences in the demand for feed enzymes at different growth stages. The digestive system of larvae is not fully developed, and endogenous digestive enzyme secretion is insufficient. Adding exogenous digestive enzymes to feed can promote the digestion of protein, fat, and starch in food by larval aquatic animals and improve growth rate.
In general, the use of compound enzyme preparations for aquatic animals should comprehensively consider the animal's species, feeding habits, growth stage, and digestive enzyme secretion characteristics for compounding, and only after verification through rigorous animal growth experiments can relatively good results be achieved.
(2) Quantitative research on feed enzyme preparations for aquatic animals
Compared with livestock and poultry feed enzyme preparations, there is not much research on the application of enzyme preparations in aquatic animals, and most of it remains qualitative, that is, whether adding enzyme preparations has a growth-promoting effect on aquatic animals, whether it can improve feed utilization, reduce bait coefficient, and reduce phosphorus excretion, while there is less quantitative research on enzyme preparations. Quantitative research is very important. All enzyme preparations have a certain suitable addition range. Only within their suitable addition range can growth reach the optimal state. Too low or too high is not good. Liu Wenbin et al. (1997) reported that 0.1%, 0.2%, and 0.3% of domestic compound enzyme preparations were added to juvenile turtle feed respectively, and the growth effects were observed. The experimental results showed that the addition amount of 0.1% had the best breeding effect, with the weight gain rate increased by 75% compared with the control group and the bait coefficient reduced by 14.5%; followed by the addition amount of 0.2%, with the weight gain rate increased by 20% compared with the control group and the bait coefficient reduced by 5.9%; the addition amount of 0.3% had a breeding effect even worse than the control group. This is consistent with the experimental results of Liu Wenbin et al. (1999) on gibel carp, where the 0.75% enzyme preparation addition amount was better than both 0.5% and 1%. The reasons may be: (1) too high exogenous enzyme addition may inhibit endogenous enzyme secretion, reducing endogenous enzyme secretion and affecting the digestion and absorption rate of feed nutrients; (2) too high exogenous enzyme addition reduces the viscosity of chyme, shortens the retention time of chyme in the intestine, accelerates the loss of nutrients, reduces the digestion and absorption of nutrients by animals, and causes their production performance to decline. Zhou Xiaoqiu et al. (2001) added enzyme preparations to carp bait, and the results showed that adding 500 g/t of enzyme preparation to the diet could significantly improve the growth rate and bait utilization of carp, but when the addition amount exceeded 700 g/t, the growth rate and bait utilization of carp decreased significantly. Han Qing et al. (2002) showed in experiments on yellow catfish that adding 0.15% enzyme preparation to the diet was better than adding 0.1% and 0.25%.
4. Current problems in the application of enzyme preparations
(1) Stability of feed enzyme preparations for aquatic animals
At present, there are problems in the application of enzyme preparations regarding thermal stability during feed processing, storage stability, and stability in water.
Enzymes are proteins with biological activity and, like other proteins, are very sensitive to temperature. The suitable temperature for general feed enzymes is 35-40 °C. During the pelleting process of aquatic animal feed, the temperature is around 90 °C, especially in extruded feed, where the temperature can reach 140 °C. At the same time, under conditions of high pressure and high humidity during pelleting, enzyme activity suffers great loss, and some enzyme activities are even almost completely lost.
At present, domestic enzyme preparations also have inactivation problems during storage at room temperature. The storage time is generally only a few months, otherwise the activity loss is serious.
The difference between the use of aquatic enzyme preparations and livestock and poultry enzyme preparations is that aquatic enzyme preparations must be able to remain in water for a long time without dissolving and being lost.
(2) Deepening of research on the mechanism of action and formulation of standards and specifications
At present, the mechanism of action of enzyme preparations is not particularly clear, and in-depth research should be conducted on the effects of enzyme preparations on the physiology and endocrine system of aquatic animals. Adding enzyme preparations to feed is different from general enzyme application methods. Because enzyme reactions are related to the enzyme systems present in the aquatic animal body, the reaction is extremely complex. Which enzyme to add and how much to add vary according to different aquatic animals, different raw material types, and their proportions. Therefore, application experiments should be emphasized, and the type of enzyme to add and the amount of enzyme activity should be determined through experiments. Relative to the activity of foreign enzyme preparations, the activity of domestic enzyme preparations is still very low. The improvement of enzyme preparation activity depends on technological progress. Fermentation processes should be improved, and suitable strains should be selected through genetic engineering. A substantial increase in enzyme product activity will certainly reduce costs, thereby promoting the large-scale promotion and application of enzyme preparations in China.
There is no unified standard for the determination of enzyme preparation activity, which is not conducive to academic exchange and research and limits the development and application of enzyme preparations. In addition, the determination of enzyme activity in vitro cannot fully reflect the true activity of enzymes in the aquatic animal body, because there are many factors affecting enzyme preparation activity, such as the reaction substrate, temperature, pH value, etc. Therefore, standardizing the definition of enzyme activity and unifying the determination standards for enzyme preparations are quite important for the development of feed enzyme preparations.

III. Probiotic preparations

1. There are many methods for classifying microecological preparations.
(1) According to purpose of use, there are growth promoters, immune promoters, therapeutic agents, water quality improvers, etc.;
(2) According to different strains, there are Bacillus preparations, lactic acid bacteria preparations, yeast preparations, Bdellovibrio, photosynthetic bacteria, etc.;
(3) According to the number of strains, there are single-strain preparations and compound-strain preparations.
Most microecological preparations have the following three basic characteristics: they can antagonize pathogenic bacteria or rapidly degrade organic matter in in vitro experiments; they can survive in the intestinal tract of farmed animals and in aquaculture water; and in infection experiments, they can improve the resistance of farmed animals to pathogens and promote animal growth.
According to different strains, they can be classified as:
(1) Lactic acid bacteria, which grow anaerobically or facultatively anaerobically and can decompose sugars to produce lactic acid; in animals, they lower pH through biological antagonism, prevent and inhibit the invasion and colonization of pathogenic bacteria, and maintain intestinal microecological balance.
(2) Bacillus, mainly including Bacillus subtilis, Bacillus licheniformis, Bacillus cereus, etc. Bacillus has the advantages of acid resistance, salt resistance, and high temperature resistance (100 °C), can lower the pH in the jejunum, and promote the decomposition of starch, cellulose, etc.
(3) Yeast, an aerobic bacterium that prefers acidic environments and can multiply in large numbers in the intestinal tract. Yeast is rich in a variety of nutrients essential to animals, is a source of vitamins and protein, can increase digestive enzyme activity, and can increase the activity of the nonspecific immune system.
(4) Photosynthetic bacteria, heterotrophic microorganisms with photosynthetic capacity. They mainly use small molecule organic matter to synthesize nutrients needed for their own growth and reproduction, and under anaerobic conditions can convert hydrogen sulfide into harmless substances. They are not only a safe energy source but can also produce bioactive substances such as anti-active virus factors.
(5) Bdellovibrio, Gram-negative, short rod-shaped, with extremely long single flagella, and very small in size. Its main biological characteristic is that it parasitizes certain bacteria and can cause their lysis. It has strong lytic activity against most Gram-negative bacteria such as Shigella, Salmonella, Escherichia, Vibrio, Pseudomonas, Leptospira, and other microorganisms; it is preliminarily believed that the suitable pH for Bdellovibrio to form plaques on bacteria is 7-8, the temperature range for growth and reproduction in liquid is 20-30 °C, and it is inactivated at 50 °C for 30 minutes.
2. Mechanism of action of microecological preparations
(1) Microecological balance mechanism:
The microecological balance in healthy animals can be destroyed due to pathogen invasion, changes in environmental factors, and other reasons. If the degree of destruction exceeds the adaptive capacity of the host animal, the animal's immunity will decline, leading to nutritional and growth disorders and the occurrence of disease. At this time, supplementing appropriate microorganisms can repair the microecological environment in a timely manner and allow the animal to return to a healthy state. Biological oxygen scavenging is also part of this mechanism. Some aerobic bacterial microecological preparations, especially Bacillus, can consume oxygen in the intestinal tract, creating an anaerobic environment that helps the growth of anaerobic microorganisms, thereby restoring the imbalanced flora to a normal state.
(2) Disease prevention and control mechanism:
Some probiotics regulate the host's internal flora structure through competition, including competition for adhesion sites, competition for chemicals or available energy, and competition for iron. Some probiotics produce inhibitory substances during growth, such as lactic acid, lactobacillin, and hydrogen peroxide produced by lactic acid bacteria, which have inhibitory effects on pathogenic microorganisms. Some probiotics have immunostimulatory effects and are good immune activators, such as yeast cell walls. Bdellovibrio first collides violently with the host cell, and the end without flagella attaches to the surface of the host cell. The bacterial body rotates at high speed and releases various enzymes, causing small pores to form in the host cell wall. Bdellovibrio then enters the host body, rapidly replicates DNA, and then the bacterial body elongates and undergoes equal binary fission to form progeny and is released from the host cell body, ultimately causing the host cell to disintegrate, thereby achieving the effect of sterilization and disease prevention.
(3) Growth-promoting mechanism:
Many probiotics used as bait additives contain large amounts of nutrients in their bacterial bodies. At the same time, as they multiply and metabolize in the animal digestive tract, they can produce nutrients needed for animal growth and can also produce digestive enzymes to assist animals in digesting bait and improve bait conversion rate.
(4) Water purification mechanism:
After probiotics enter the aquaculture pond, they can participate in the most basic material cycle of the water body, degrading organic matter into nitrates, phosphates, and carbon dioxide, etc., providing nutrients for the growth and reproduction of single-celled algae; and the photosynthesis of unicellular algae in turn provides dissolved oxygen for the oxidative decomposition of organic matter, the respiration of microorganisms and cultured animals, forming a benign ecological cycle. Some probiotics such as photosynthetic bacteria have unique photosynthetic capabilities and can directly consume and utilize organic matter, ammoniacal nitrogen, hydrogen sulfide, etc. in water; while denitrifying bacteria can remove nitrite nitrogen in water through denitrification, thereby improving water quality.
3. Application effects of microecological preparations in aquaculture
Kazasa (1986) was the first to apply probiotics in aquaculture, treating Japanese eel (Anguilla japonica) with a Bacillus strain isolated from soil, reducing mortality caused by Edwardsiella. Austin and Akifumi et al. reported that in shrimp and shellfish production, Vibrio alginolyticus and pigment-producing bacteria were used to control various pathogenic Vibrio species during the larval stage. Moriarty reported that experiments conducted in Indonesia showed that adding certain specific Bacillus species to aquaculture water could greatly reduce the number of harmful Vibrio species such as luminous Vibrio in the water, and the survival rate of shrimp in experimental ponds was much higher than in control ponds. Experiments have shown that using yeast together with lactic acid bacteria can improve the growth rate and survival rate of Indian prawn (Penaeus indicus) larvae. Zhang Xinming et al. reported that a strain of Micrococcus luteus had good antagonistic effects against pathogenic Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio nereis, and Vibrio natriegens. Chen Jiazhang et al. used Bdellovibrio preparations to splash river crab (Eriocheir sinensis) culture ponds for a period of 3 months; the total bacterial count in crab ponds decreased exponentially, and ammonia nitrogen, COD, and sulfide all decreased to varying degrees. Crab ponds using Bdellovibrio had no disease outbreaks throughout the year, river crab growth rate accelerated, and survival rate improved. He Yijin et al. in allogynogenetic crucian carp (Carassius auratus gibelio) culture experiments added pre-cultured Aeromonas punctata, the pathogen of crucian carp hemorrhagic ascites disease, then added Bdellovibrio, and regularly detected the number of pathogenic bacteria in the water. The results showed that with the extension of time, the number of pathogenic bacteria in the water body decreased significantly, and after one week they could not be detected. Zhang Shuhua et al. added probiotics to shrimp compound feed to feed shrimp. The results showed that adding 0.1% probiotics to the feed could increase the survival rate of shrimp by 5.03%-6.18% and increase unit yield by 8.76%-12.45%. Wang Xianghong, based on a systematic study of the intestinal microflora of healthy adult cultured and wild-caught Chinese shrimp (Fenneropenaeus chinensis), screened out multiple beneficial strains, among which two beneficial bacteria strains added to shrimp larval culture water could increase the metamorphosis rate and survival rate of shrimp larvae at different developmental stages, while the disease resistance and low-salinity tolerance of shrimp fry were also improved, and average body weight and body length both increased. Li Zhuojia et al. reported that the use of microecological preparations in shrimp culture has the effects of purifying water quality, improving shrimp immune function, and increasing culture survival rate.
Lactic acid bacteria are considered the main probiotics, and in addition, there are more than 40 species including yeast, Bacillus subtilis, Bifidobacterium, Bacillus, and Streptococcus. Probiotics used in fish production are mostly composite strains. Wu Yin reported that probiotic microecological preparations can improve the overwintering ability of hybrid carp, with mortality decreasing from 65% to 52%.
Zhou Dongming et al. measured the effects of temperature, pH, and antibiotics on the activity of live bacterial preparations. Bacillus has strong heat resistance, with essentially no loss after treatment at 100°C for 2 min, while yeast and lactic acid bacteria lose much activity when the temperature exceeds 80°C. The temperature during fish pellet feed production is generally 80-100°C, which has no effect on Bacillus but has a relatively large effect on lactic acid bacteria and yeast. Within the pH range of 2.2-7.0, various bacteria are basically not damaged. The pH in the fish stomach is generally 2-3, and the intestinal pH is 5-7; such a pH range is suitable for the growth of the above bacteria. When using probiotic preparations, attention should be paid to the combination with disease-resistant and growth-promoting substances. Furazolidone and chloramphenicol have very strong inhibitory effects on the above various bacteria, followed by chlortetracycline, erythromycin, and oxytetracycline; maduramicin, bacitracin zinc, and olaquindox basically have no inhibitory effect on Bacillus. They have weak inhibitory effects on lactic acid bacteria and yeast.

IV. Feeding attractants

Feeding attractants, also called attractants or feeding stimulants, function to improve the palatability of fish compound feed, induce and promote fish feeding on feed, stimulate the secretion of digestive enzymes in fish and shrimp, thereby improving feed utilization rate and obtaining better aquaculture benefits. To enhance fish and shrimp appetite, shorten the residence time of food in water, and reduce nutrient loss, the application of feeding attractants has attracted increasing attention. Since the 1970s, scholars from Japan, the United States, and the former Soviet Union, starting from exploring the responses of fish and shrimp olfaction and gustation to chemical substances, have conducted in-depth research on the effects of adding feeding attractants to bait. Research shows that most fish and shrimp feeding attractants are amino acids, betaine, fatty acids, and certain volatile substances. Different fish and shrimp species have different types of feeding attractants, mainly including the following categories:
1. Amino acids
Amino acids have quite obvious olfactory stimulation effects on fish and are good feeding attractants, with great differences among different fish species. According to fish feeding habits, carnivorous fish are sensitive to alkaline and neutral amino acids, while herbivorous fish are sensitive to acidic amino acids. At the same time, studies by some domestic and foreign scholars have shown that the feeding attraction effect produced by combining amino acids with other feeding attractants has a synergistic effect.
(1) Amino acids;
Current research shows that the amino acids with feeding attraction activity for fish are mainly L-type amino acids, and the feeding attraction effect of amino acids on fish is specific, among which the activity of glycine, alanine, and proline is of great significance. For example, alanine has feeding attraction activity for Japanese eel but no feeding attraction activity for rainbow trout; another example is that proline and alanine have particularly obvious feeding attraction effects on Atlantic salmon fry, but the effect is not obvious on one-year-old fish (Mearns, 1996). Adding methionine to rainbow trout feed has the best effect, adding cystine to channel catfish (Ictalurus punctatus) feed has the best effect, adding arginine and glutamic acid to carp feed has the best effect, and adding cystine to grass carp feed has a significant effect. Generally speaking, the feeding attraction effect of mixing two or more amino acids is better than that of a single amino acid. For example, the complex of L-tyrosine, phenylalanine, and histidine has a very good feeding attraction effect, but a single amino acid has poor effect. There are also some amino acids that have an inhibitory feeding attraction effect on certain fish when present alone, but when mixed with several other amino acids, they have feeding attraction activity; the relevant mechanism is still unclear.
(2) Amino acids and nucleotides;
Nucleotides are usually used together with amino acids and betaine and can improve bait palatability. Foreign experiments have shown that many nucleotides have feeding attraction activity for fish. Nucleotides, such as inosine and its derivatives, are feeding attractants for turbot (Scophthalmus maximus), and still have activity even at very low concentrations. Machie et al. (1978) tested 47 compounds as feeding attractants for turbot, and the results showed that only 6 hypoxanthine and quanine compounds were effective at the test concentrations, especially inosine, which had a strong stimulating effect.
(3) Amino acids with trimethylamine lactone and certain salts or organic acids;
Larezack (1963) proved that trimethylamine lactone has a feeding attraction effect on various crustaceans (including crayfish, crabs, lobsters, and Cambarus). Cazz (1978), in chemoreception research on shrimp, pointed out that an artificial mixture of trimethylamine lactone and amino acids accounted for 60%-100% of the stimulatory activity of crab, oyster, and sea urchin extracts. Machie (1973) found that a mixture of amino acids, nucleotides, and quaternary amines had a feeding attraction effect on American lobster (Homarus americanus), while single components in this mixture were ineffective.
Taurine, also known as taurocholic acid or taurocyamine, is a small-molecule sulfur-containing amino acid and becomes a conditionally limiting amino acid during the fry stage of some marine fish when the diet is deficient. Taurine usually exists in the form of free amino acids or small peptides in aquatic animals; it is a non-proteinogenic amino acid and cannot be metabolized as an energy substance. The most important physiological role is that it can stimulate fish feeding and has stimulating effects on olfaction and gustation. Sola & Tosi (1993) reported that taurine has an attracting effect on the feeding of Arctic charr, salmon, trout, eel, American lobster, and tiger shrimp, and can increase feeding rate. Takeuch (2001) observed that adding taurine could quickly calm Japanese flounder (Paralichthys olivaceus) and red sea bream (Pagrus major) larvae in aquarium tanks and cause them to settle to the bottom and quickly forage. Experiments by Jose Brotons Martinez et al. on Japanese sea bass (Lateolabrax japonicus) further proved the feeding attraction effect of taurine. Qiu Gaofeng et al. also reported that taurine is a very good feeding promoter for shrimp and crabs, with a stronger feeding promotion effect than asparagine, proline, and glycine. Numerous studies have shown that adding taurine at the juvenile stage of marine fish can promote their growth, but for freshwater fish and crustaceans, the addition effect is not obvious.
2. Betaine
Betaine is glycine trimethylamine lactone extracted from sugar beet processing by-products, and is a crystalline quaternary ammonium alkaloid. Betaine has a sweet and umami taste that fish are sensitive to and is an ideal feeding attractant. At the same time, its feeding attraction effect is also manifested in a synergistic effect with some amino acids. Experiments by the Finnish Sugar Company showed that betaine increased rainbow trout body weight and feed conversion rate by nearly 20% (Clayfon jill, 1989). Mixing 0.5%-1.5% by mass fraction of betaine into feed has an extremely strong stimulating effect on the olfaction of all fish and crustaceans such as shrimp and crabs. Yan Xizhu reported that betaine can enhance fish digestive fluid secretion, increase food intake, and reduce feeding time. Li Yongmei et al. experiments showed that adding 0.3% by mass fraction of pure betaine and compound betaine to feed could significantly promote tilapia growth and reduce feed coefficient; at the same time, it was observed that betaine had obvious feeding attraction effects on carp and wild carp (Labeo), but not obvious on grass carp. Liang Mengqing et al. research showed that adding betaine had no effect on promoting the feeding of red sea bream, but could promote the growth of red sea bream. Sun Yuxiu added 0.3%-0.5% by mass fraction of betaine to compound feed for 2-year-old carp, increasing daily weight gain rate by 41%-49% and reducing feed coefficient by 14%-24%. A carp experiment conducted by the Beijing Chaoyang District Fisheries Bureau showed that adding 0.1%, 0.2%, and 0.3% by mass fraction of betaine and 0.1% betaine compound agent to the same basal feed increased the daily weight gain of carp by 16.5%, 17.4%, 21.5%, and 34.6%, respectively. Yan Youli et al. reported that betaine added to carp feed at mass fractions of 0.3%-0.5% increased weight gain by 41.78%-49.32% and reduced feed coefficient by 14.13%-24.16%.
3. Sulfur-containing compounds
Allicin is a mixture of diallyl trisulfide, diallyl disulfide, and methyl allyl disulfide. It has good feeding attraction effects on many aquatic animals and can also sterilize and resist disease and improve feed utilization rate. Allicin can be extracted from garlic or synthesized artificially.
Japanese scholars have also developed a sulfur-containing compound (DMPT) with excellent feeding attraction effects. This compound has feeding attraction effects on carp, crucian carp, Japanese flounder, etc., and can also enhance fish tolerance to changes in the water environment. Recent studies have found that the feeding attraction effect of DMPT differs from other substances such as L-amino acids or nucleotides; it has feeding attraction and feeding promotion effects on many marine fish, freshwater fish, crustaceans, and shellfish. Nakajima et al. (1989) reported that using 1.0 mmol/L DMPT and other sulfur-containing compounds mixed into various test baits, with the control group mixed with distilled water instead, a feeding response test was conducted on crucian carp. The results found that in the first 4 groups of experiments, the DMPT group had an average of 126 more bait-biting frequency than the control group; in the second 5 groups of experiments, the DMPT group had an average of 262.6 more bait-biting frequency than the control group. In a comparison test with glutamine, it was found that at a concentration of 1.0 mmol/L, the bait-biting frequency of crucian carp reached a maximum. The bait-biting frequency of crucian carp for DMPT-containing bait was about 2 times higher than for glutamine-containing bait, and this was true at different concentrations. In experiments feeding crucian carp and carp with semi-natural bait and natural bait, it was found that adding 1.0 mmol/L DMPT could increase the feeding frequency of crucian carp by about 0.4 times and carp by 0.3-0.5 times. From olfactory tract electrical response stimulation tests in carp, DMPT was found to be a strong olfactory nerve excitant. Nakajima et al. (1992) reported that in experiments with goldfish, the feeding stimulation effect of DMPT was 2.55 times higher than that of glutamine. In experiments with natural bait on crucian carp, the number of bites by crucian carp on bait added with DMPT increased by 1.3-1.4 times compared with control bait. Nakajima et al. (1992) also reported that DMPT has feeding stimulation effects on common shellfish as well as special shellfish such as abalone and oysters. Liang Mengqing et al. (2001) studied the feeding attraction and growth promotion effects of 5'-inosine monophosphate, betaine, fish meat hydrolysate, trimethylamine oxide, and DMPT on shrimp. The results showed that DMPT had the strongest feeding attraction effect, followed by fish meat hydrolysate and betaine, and 5'-inosine monophosphate and trimethylamine oxide had the worst effects.
3. Fatty acids
Fish growth experiments have proved that fish oil has strong feeding attraction effects and unknown growth factors, mainly because it is rich in essential fatty acids that fish need to maintain normal physiological activities and growth. Chen Changfu et al. (1989) conducted gustatory stimulation tests on Japanese eel using water-soluble low-grade fatty acids and found that Japanese eel showed certain olfactory responses and strong gustatory responses. The experiment confirmed that as molecular weight increases, the feeding attraction effect of fatty acids increases.
4. Extracts of certain animals (Cladocera, chironomid larvae, mollusks, crustaceans, various animal tissues, etc.).
In the process of studying the utilization of plant protein in compound feed for red sea bream, Ina Kazuo, after identifying the active components of feeding-promoting substances in natural bait for red sea bream, found that the digestive fluids of the viscera of fish such as albacore tuna and purpleback flying squid contain feeding-promoting substances for red sea bream. Therefore, in the experiment, compound feed was studied in which these fish viscera were processed and mixed with plant protein that red sea bream does not like to eat, and the effect of feeding red sea bream with these feeds was observed. The results showed that replacing animal protein with more than half plant protein that fish do not like to eat produced feeding effects such as growth rate roughly the same as feeding natural bait or standard feed. This shows that as long as fish feeding-promoting substances are appropriately used in compound feed, it can not only effectively improve feeding effects but also expand feed sources and achieve the purpose of developing new feeds.
Research in this field started relatively late in China. However, research work in this area has now begun, such as reports on the feeding attraction effect of Artemia on shrimp and the feeding attraction effect of fish meat extracts or animal processing factory scraps. Liu Defang reported that eels have preferences for chocolate flavor, cheese flavor, milk flavor, and fish meat flavor, and eel fry feed often has shellfish or shrimp extracts added. However, problems generally exist such as active ingredients not being quantified and enrichment of toxic and harmful substances, which limit the use of such feeding attractants.
5. Chinese herbal medicines
Existing research shows that adding various Chinese herbal medicines such as parsley, cardamom, fragrant mint, hawthorn, Phellodendron amurense, Polygonum hydropiper, garlic, pine needle powder, rhubarb, Coptis chinensis, dried tangerine peel, etc. to feed has feeding attraction effects on fish and can also prevent and treat fish diseases. According to Zeng Hong et al., garlic has good feeding attraction effects on loach, rainbow trout, tilapia, etc., which is related to the volatile low-molecular organic compounds with special odor contained in garlic; Tong Shengying et al. reported that hawthorn contains a large amount of organic acids and has an obvious feeding attraction effect on Haliotis discus hannai. Chen Zhenkun et al. reported that feed containing 0.25% dried tangerine peel had the highest feeding attraction efficiency for grass carp, and the number of bites and pecking force of grass carp were 7.25 times and 2.1 times those of the control group, respectively. Ding Guang et al. reported that adding 0.05%, 0.1%, 0.25%, 0.5%, 1.0%, and 3.0% citrus or sweet orange outer peel to basal feed, and using a recorder to record the frequency and strength of carp pecking at feed balls, showed that citrus and sweet orange outer peel are feeding attractants for carp, and the feeding attraction effects of the two on carp are basically the same, with suitable addition amounts of 0.25% and 0.1%, respectively. Guo Yongjun et al. (2005) selected 8 Chinese herbal medicines including dried tangerine peel, clove, cinnamon, Angelica dahurica, star anise, gardenia, Kaempferia galanga, and Ferula as feeding attractants for carp feed, and found through experiments that clove and dried tangerine peel had significant feeding attraction effects on carp. Han Yanyan et al. (2003), based on Chinese herbal medicine pharmacological design, made 6 compound additives from Astragalus membranaceus, hawthorn, Chinese yam, goji berry, dried tangerine peel, etc., and conducted feeding attraction activity tests on crucian carp and Amur sturgeon (Acipenser schrenckii). The results showed that the feeding attraction effects of the 6 Chinese herbal medicine compound additives on the two fish species were not completely the same. The compound additives all had extremely significant feeding attraction effects on crucian carp, and only two compound additives had significant feeding attraction effects on Amur sturgeon. This further proves that the feeding attraction effect of Chinese herbal medicine feeding attractants is related to the animal's preference for their odor and taste, and also related to the composition of the Chinese herbal medicine additives. Overall, the mechanism of the feeding attraction effect of Chinese herbal medicine feed additives on aquatic animals is still unclear and needs further research.

V. Binders

Binders are additives in fish pellet feed that play a binding and shaping role. Their function is to bind various components together, prevent feed nutrients from dissolving and disintegrating in water, facilitate fish feeding, improve feed efficiency, and prevent water quality deterioration. Fish feed binders can be roughly divided into two major categories: natural substances and chemically synthesized substances. Natural substances include wheat flour, corn flour, gluten, tapioca starch, α-starch, dextrin, sodium alginate, kelp gum, and brown algae gum; dragon gum, carrageenan, bone glue, hide glue, fish paste, etc. Chemically synthesized substances include carboxymethyl cellulose, sodium polyacrylate, etc.
An ideal binder should have the following characteristics: ideal adhesion to various components in feed, ensuring complete nutrition and preventing loss and pollution; easy to prepare without hindering nutrient absorption; relatively high chemical stability and thermal stability without adverse chemical reactions with feed components; low cost, small dosage, wide source, easy to mix, non-toxic, no undesirable odor, good palatability, strong water stability, etc. Binders each have their advantages and disadvantages, and in application they are generally used in combination; the effect in application is also affected by minerals, temperature, and other components. Although many new binders have been introduced one after another, due to cost and other reasons they are limited to use in scientific research. The binders for carp, tilapia, and rainbow trout feed in China still mostly use starch gelatinization to achieve the binding purpose, so the development of new binders with high efficiency, low price, and nutritional value is becoming increasingly urgent.

VI. Antioxidants

The oils and vitamins contained in feed are very easily oxidized and decomposed, causing nutrient deficiency and producing toxic substances, so antioxidants need to be added. Antioxidants themselves are easily oxidized and combine with the active free radicals of easily oxidized substances to generate inactive antioxidant free radicals, interrupting the oxidation reaction, thereby stopping or slowing the oxidation process. Because antioxidants can play a stabilizing role, they can extend the preservation period of fish feed.
Currently commonly used antioxidants include ethoxyquin (EQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT), and there are also gallic acid esters, tocopherols, and ascorbic acid, etc. The addition amount of BHA, BHT, and EQ in general fish feed is 100-200 mg/kg. When the fat content in fish feed is relatively high, the addition amount should be appropriately increased. If BHT and BHA are used simultaneously with ascorbic acid, citric acid, glucose, or other reducing agents, the dosage is 1/4-1/2 of that when BHT is used alone, and the antioxidant effect is particularly significant. To prevent accumulation of antioxidants in animal bodies, the U.S. FDA stipulates that when adding ethoxyquin to feed, its dosage based on the final product must not exceed 150 mg/kg; BHT and BHA, based on the fat content in feed, must not exceed 200 mg/kg.

VII. Colorants

1. Classification of commonly used colorants
Colorants commonly used in feed are generally divided into two types by source, namely natural colorants and synthetic colorants. Natural colorants refer to certain yellow, red, and purple animals rich in carotenoids and xanthophylls and their extracts, such as krill and Euphausia; plants and their extracts such as corn, carrot, alfalfa meal, and orange peel; microorganisms and their extracts such as yeast, Rhodospirillum in photosynthetic bacteria, and Dunaliella in microalgae; Spirulina and its extracts. Among synthetic colorants, there are 8 feed colorants permitted for use in China, namely β-carotene; β-apo-8'-carotenal; capsanthin; β-apo-8'-carotenoic acid ethyl ester; β-carotene-4,4-dione (canthaxanthin); xanthophyll; marigold flower extract (natural xanthophyll); astaxanthin.
Olsen et al. (1994), when adding 0-192 mg/kg astaxanthin to Arctic charr feed, found that the degree of redness of Arctic charr muscle was positively correlated with the amount of astaxanthin added, and when the addition amount reached 70 mg/kg, it reached a stable period of pigment formation. Choubert et al. (1996) added 100 mg/kg astaxanthin, 100 mg/kg canthaxanthin, and 100 mg/kg astaxanthin extracted from yeast respectively to rainbow trout feed, and found that carotenoids in rainbow trout muscle all increased to varying degrees. Wei Wanquan et al. (2003) used three commercial pigments (Carophyll Yellow, Carophyll Red, and Carophyll Pink) and corn gluten meal containing yellow xanthophyll as pigment sources for coloring, and explored the effects of pigments on coloring of walking catfish (Clarias fuscus). The results showed that adding Carophyll Yellow, Carophyll Red, and Carophyll Pink to feed could effectively color walking catfish. To achieve better coloring, the effective content of carotenoid pigments in feed (i.e., the content of apoester, canthaxanthin, or astaxanthin) should not be less than 50 mg/kg, and the coloring time should not be less than 8 weeks. At the same time, in the culture process of tilapia, carp, red sea bream, crucian carp, carp, and shrimp, adding appropriate amounts of carotenoids such as astaxanthin and zeaxanthin can improve fish body color and flesh quality and increase the deposition amount of pigments in the fish body surface, skin, and muscle.
2. Mechanism of action
Fish body color is mainly determined by a class of pigments called carotenoids (melanin, purines, and iridocytes also participate in body color formation), and is also related to the fish's physiological state and environmental factors. Carotenoids appear yellow, orange, and red, etc. According to whether their molecules contain oxygen, they can be divided into two major categories: one is carotenes without oxygen, which have relatively poor coloring effect; the other is xanthophylls containing oxygen, such as astaxanthin, lutein, and zeaxanthin, which are the main pigments that play a coloring role in fish and shrimp bodies. Different species of fish and shrimp have different types of xanthophylls that mainly play a coloring role in their bodies. For example, crustaceans such as shrimp and crabs and salmonid fish use astaxanthin, goldfish mainly use lutein and astaxanthin, and sunfish mainly use lutein. Different species of fish and shrimp also have different metabolic capacities for carotenoids, and can be divided into three categories: the first category is the red carp type, such as goldfish, red carp, and koi, which can convert lutein and zeaxanthin into astaxanthin and then deposit it in tissues; the second category is the prawn type, including almost all crustaceans, which can convert β-carotene, lutein, and zeaxanthin into astaxanthin; the third category is the sea bream type, including salmonid fish and sea bream, which cannot convert the above substances into astaxanthin but can directly store them in the body (skin, adipose tissue, and eggs). To achieve ideal coloring effects in cultured fish and shrimp, it is necessary to select suitable carotenoid types according to the pigment composition of wild individuals and their metabolic capacity for pigments.
3. Factors affecting fish body coloring
Coloring is a very complex process. It is affected by many exogenous factors (such as the source, type, level, and feeding duration of pigments in feed, water quality, stress, etc.) and endogenous factors (genetic performance, physiological status, growth rate, etc.), involving the digestion of pigments in feed, absorption of pigments in the digestive tract, transport in the body, metabolism, and deposition by binding with related tissues.
(1) Influence of other components in feed. The content of fat, vitamin A, and vitamin E in feed as well as certain drugs and mycotoxins can also affect coloring effects. Studies have found that increasing the fat level in feed helps improve the digestion, absorption, and body retention efficiency of carotenoid pigments in Atlantic salmon and rainbow trout. This is because carotenoids in the digestive tract need to be dissolved in fat before they can be effectively absorbed. Transport in the blood also requires the assistance of lipoproteins. The structures of vitamin A and carotenoids are interrelated (vitamin A and the carotenoid ring). When vitamin A supply in feed is insufficient, animals can convert certain carotenoids into vitamin A, thereby reducing coloring. However, when vitamin A is excessive, compounds with similar molecular structures will produce competitive absorption, likewise reducing coloring effects. The suitable addition level of vitamin A in walking catfish feed is 8000 IU/kg; too high or too low vitamin A in feed will inevitably have adverse effects on coloring. A relatively high level of vitamin E in feed helps enhance coloring effects. This is mainly because the strong antioxidant properties of vitamin E help protect carotenoids in feed. Certain drugs in feed (such as sulfonamides, etc.) and aflatoxin can also affect the coloring effect of carotenoids.
(2) Culture environment. Many organisms, including fish, have learned the ability to change color over the long course of evolution, and can to a certain extent change their body color according to changes in the external environment to more effectively evade natural enemies or capture prey, such as Japanese flounder, turbot, and large yellow croaker. Although this body surface color change mechanism (mainly caused by the aggregation and dispersion of pigment cells) may be completely different from the pigment coloring mechanism, practice has shown that the color of the culture environment can affect animal body color.
(3) Stress response. Body color changes are related to stress response, but pigment deposition levels can also resist body color changes caused by stress. An effective method to reduce body color changes during harvesting and transportation is to add high levels of vitamin C and vitamin E to feed.
4. Common pigment-containing feed ingredients
Common pigment-containing feed ingredients include yellow corn, corn gluten meal, pine needle powder, algae (such as Spirulina), Phaffia yeast, shrimp and crab shell powder, alfalfa, and marigold, etc. The effective coloring components contained in these ingredients are not the same. The content also varies greatly from batch to batch (for example, the xanthophyll content in yellow corn is 12-25 mg/kg), and changes will occur during raw material processing and storage as well as feed processing and storage (for example, stored at 27°C for 9 months, the xanthophyll content in corn gluten meal can drop from 350 mg/kg to about 130 mg/kg). According to data provided by Kemin Industries of the United States, the xanthophyll contents of new corn and corn gluten meal in China are (18±6) mg/kg and (157±81) mg/kg, respectively. Stored at room temperature for one year, their xanthophyll content will lose 50%. At the same time, it was found that the xanthophyll content of Asian corn is 25%-30% higher than that of American corn, while the xanthophyll content of American corn gluten meal is 60% higher than that of Asia, reaching 250 mg/kg. In addition, the utilization rate of pigments in these raw materials is also unstable.
5. Pigment level and coloring time.
Coloring is a pigment deposition process, and its effect is closely related to the pigment dose and coloring time. Studies have shown that within the range of 0-50 mg/kg (active ingredient), whether Carophyll Yellow, Carophyll Red, or Carophyll Pink, the coloring effect increases with increasing pigment addition. On the other hand, pigment level and coloring effect do not show a linear proportional relationship; as the pigment level in feed increases, its utilization rate tends to decrease. Coloring time is another issue worth attention, because there may be a certain "saturation" in the absorption and transport of pigments in the body, and excess pigment is wasted because it cannot be absorbed and utilized. This also explains the phenomenon that pigment level and coloring effect are not linearly proportional. Pigment deposition is a gradual process, and its duration is related to animal species, pigment level in feed and its utilization rate, and the desired depth of color. The coloring effect of a slightly smaller pigment dose over a longer time is usually better and more stable than that of a very large pigment dose over a very short time.
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Bump it!!
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Thanks to the original poster for sharing
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Bump................
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Good material, worth learning from, paying tribute to the original poster
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This material is pretty good, maybe just a bit long, tiring to read. The content inside is all what I organized and prepared for a publication; I'll post it first to see if the response is good. If everyone is interested, you can post your email address; we still have more articles that cannot be shared with everyone due to space limitations and upload format.
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Such a good article with no one bumping it, let me bump it!
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I am interested, my email is [email protected]
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Share with me, [email protected]