Preparation method and application of marine biological bait for fry and juvenile fish

Abstract

The present application relates to the field of aquaculture technology, and particularly relates to a preparation method and application of seawater fry biological bait. The preparation method comprises the following steps: mixing Schizochytrium sp. powder, bile acid and sunflower phospholipid according to a mass ratio of 50:10-20:10-20 to obtain a nutrition enhancer; diluting the nutrition enhancer to a concentration of 40g / t-50g / t to obtain a diluent; adding 130-150 ordinary rotifers per milliliter of the diluent, and culturing for 12-18 hours to collect enhanced rotifers; adding 30-50 ordinary artemia per milliliter of the diluent, and culturing for 12-18 hours to collect enhanced artemia; and combining the enhanced rotifers and the enhanced artemia to obtain the fry biological bait. The fry biological bait prepared by the present application has uniform fry size and high survival rate during the bait conversion period, and has significant economic and ecological benefits.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a method for preparing and applying a biological feed for marine fry and juvenile fish, and more specifically, the application refers to its application in seedling cultivation. Background Technology

[0002] With the development of aquaculture, the supply and demand imbalance of high-quality fry and juvenile fish has become prominent. How to efficiently produce fry and juvenile fish is a key technical challenge in aquaculture research. The technical difficulties faced in the cultivation of marine fish fry include: uneven fry size and high mortality during the transition to a new diet. Specifically, these manifest as follows:

[0003] In existing technologies, larvae aged 7-19 days exhibit significant size differentiation due to individual developmental differences, with body length variations reaching up to 30%. Traditional single-feeding strategies, such as using common rotifers throughout the entire feeding period, exacerbate this uneven growth. Larger larvae overgrow due to strong feeding competition, while smaller larvae suffer from insufficient food intake and reduced survival rates. Furthermore, large size differences in fry exacerbate cannibalism, further reducing fry survival rates. In actual production, grading and screening are complex and prone to causing fish damage. Therefore, there is an urgent need for a technology that can achieve uniform size in fry populations without manual sorting. In addition, practice has shown that the digestive system of larvae aged 20-30 days is not fully developed, resulting in low protein utilization of artificial feed. Traditional methods use common artichoke nauplii as live feed, but these feeds generally have insufficient DHA content, leading to nutritional imbalances and a fry survival rate of only 5%-20% during the transition period.

[0004] In summary, there is an urgent need to develop a strategy that can improve the problems of uneven seedling size and high mortality during the transition period. Summary of the Invention

[0005] This invention addresses the problems of uneven seedling size and low survival rate during the transition feeding period in the existing technology for raising juvenile fish by providing a biological feed for juvenile fish, its preparation method, and its application.

[0006] The purpose of this invention is to provide a method for preparing live feed for juvenile fish, comprising:

[0007] Mix the powder of Schizochytrium, bile acids, and sunflower phospholipids in a mass ratio of 50:10-20:10-20, filter through a 100-120 mesh to remove large particles, and obtain a nutrient fortifier.

[0008] The nutrient fortifier is diluted to a concentration of 40g / t to 50g / t to obtain a diluted solution;

[0009] Add 130 to 150 common rotifers per milliliter of diluent, incubate for 12 to 18 hours, and collect the enhanced rotifers.

[0010] Add 30 to 50 common artichokes to each milliliter of diluent, incubate for 12 to 18 hours, and collect the enhanced artichokes.

[0011] A combination of enhanced rotifers and enhanced artichokes is used as a live food for juvenile fish.

[0012] Technical effects: On a dry weight basis, the enhanced rotifers had a DHA / FA (docosahexaenoic acid as a percentage of total fatty acids) ≥14.16% and an EPA / FA (eicosapentaenoic acid as a percentage of total fatty acids) ≥9.41%. After 12-18 hours of culture, the density of rotifers was at least 51.74% higher than that of the control group, and the average body length was 0.2 mm. On a dry weight basis, the enhanced artichokes had a DHA / FA ≥8.69% and an EPA / FA ≥3.63%, with a survival rate ≥88.58%, crude protein content ≥55%, total amino acid content ≥41%, and an average body length of 1 mm.

[0013] Preferably, the DHA / FA content of the Schizochytrium powder is ≥35% on a dry weight basis, and it is frozen and stored at -20°C before use.

[0014] Preferably, the marine larvae and juvenile fish live feed also includes an enhancer, which is alginate, sodium butyrate, cysteine ​​or γ-aminobutyric acid;

[0015] The amount of brown algae oligosaccharides added per kilogram of nutrient fortifier is 1 mg to 2 mg;

[0016] The dosage of sodium butyrate in each kilogram of nutrient fortifier is 100mg to 200mg.

[0017] The amount of cysteine ​​added per kilogram of nutrient fortifier is 10mg to 20mg;

[0018] The amount of γ-aminobutyric acid added to each kilogram of nutrient fortifier is 10mg to 20mg.

[0019] Preferably, the nutrient fortifier is mixed with ordinary rotifers to a concentration of 130 to 150 rotifers per milliliter, and the concentration of the nutrient fortifier is 50 g / t. The mixture is then cultured in a suitable environment for 12 hours to collect the fortified rotifers.

[0020] The above-mentioned nutrient fortifier was mixed with ordinary Artemia nauplii to a concentration of 30-50 nauplii per milliliter and the nutrient fortifier concentration was 40 g / t. The mixture was then cultured for 12 hours under suitable conditions, and the fortified Artemia nauplii were collected.

[0021] This invention provides a biological feed for juvenile fish prepared by the aforementioned preparation method.

[0022] The live food for juvenile fish is suitable for marine juvenile fish such as striped sea bream, black sea bream, yellow croaker or large yellow croaker.

[0023] This invention provides an application of the aforementioned live feed for larvae and juvenile fish, dividing the feeding process into two stages based on the physiological characteristics and nutritional needs of larvae and juvenile fish at different developmental stages:

[0024] (1) During the biological feed feeding stage (7 days to 30 days old), the larvae and juveniles are fed differentiatedly using the aforementioned marine larval biological feed, including:

[0025] Early metabolic window: Fry and juveniles aged 7 to 14 days are fed with ordinary rotifers (DHA / FA = 0%) to meet their basic nutritional needs; fry and juveniles aged 15 to 19 days are fed with a mixture of enhanced rotifers (DHA / FA ≥ 14.16%) and ordinary artemia (DHA / FA = 0%), with a wet weight ratio of enhanced rotifers to ordinary artemia of 1:1. Smaller fry and juveniles prefer to feed on enhanced rotifers (0.2 mm in body length) to accelerate growth, while larger fry and juveniles feed on ordinary artemia (1 mm in body length) to slow down their growth rate, thereby achieving uniformity in size and improving seed uniformity.

[0026] Late metabolic window: 20-30 day old larvae are fed the fortified Artemia esculents to ensure uniform larvae and juveniles transition to a new diet simultaneously. Fortified Artemia esculents are characterized by a DHA / FA ratio ≥ 8.69% and a crude protein content ≥ 55%. Additionally, during this stage (late metabolic window), the following supplements can be added: fucoidan 1 mg / kg-2 mg / kg, sodium butyrate 100 mg / kg-200 mg / kg, cysteine ​​10 mg / kg-20 mg / kg, and γ-aminobutyric acid 10 mg / kg-20 mg / kg, to improve intestinal function and increase larval and juvenile survival rates.

[0027] (2) During the micro-particle feed feeding stage (31 days to 60 days old), fishmeal substitute feed is provided. The dry weight concentration of non-fishmeal protein in the fishmeal substitute feed is 40% to 60%, including the use of a three-stage fishmeal substitution program:

[0028] For juvenile fish aged 31 to 36 days (early stage of micro-particle feed feeding), the fishmeal substitute feed formula is as follows: by dry weight percentage, fishmeal, enzymatically hydrolyzed soybean peptides, black soldier fly protein, and yeast protein are 60%, 20%, 10%, and 10%, respectively, with an additional 1% to 2% of neutral protease equivalent to the total dry weight of fishmeal, enzymatically hydrolyzed soybean peptides, black soldier fly protein, and yeast protein. A key feature of this stage is that protein digestibility can reach 65%.

[0029] For larvae and juveniles aged 37 to 45 days (late stage of micro-particle feed feeding), the fishmeal substitute feed formula is as follows: by dry weight percentage, fishmeal, black soldier fly protein, and yeast protein are 50%, 25%, and 25%, respectively, with the addition of chitinase equivalent to 1% to 2% of the total dry weight of fishmeal, black soldier fly protein, and yeast protein; the characteristic of this stage is that the utilization rate of black soldier fly and yeast protein reaches 85%.

[0030] For larvae aged 46-60 days (early juvenile stage), the fishmeal substitute feed formula is as follows: by dry weight percentage, fishmeal, black soldier fly protein, and yeast protein are 40%, 30%, and 30%, respectively. An additional 1%-2% of thermotolerant lactic acid bacteria, equivalent to the total dry weight of fishmeal, black soldier fly protein, and yeast protein, is added. Thermotolerant refers to tolerance to temperatures above 40℃. Characteristics of this stage: intestinal colonization rate >60%, and short-chain fatty acid production increased by 40%.

[0031] Existing aquatic feed technologies have long relied on fishmeal, but declining fishery resources have led to a shortage of fishmeal supply. Plant proteins (such as soybean meal) have a digestibility of only 38%–45% due to anti-nutritional factors (trypsin inhibitors, phytic acid); insect proteins (such as black soldier flies and yeast), while rich in chitin, are difficult for larvae and juveniles to digest. Furthermore, existing alternatives often use a single protein source and do not consider the metabolic differences at different developmental stages of larvae and juveniles, such as changes in digestive enzyme activity around 21 days of age, making it difficult to achieve a fishmeal replacement rate exceeding 50%. The micro-particle feed provided by this invention is a fishmeal substitute for the feeding stage, focusing on metabolic reprogramming to adjust the metabolism of larvae and juveniles and reduce their dependence on fishmeal.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] In terms of nutritional fortification: the DHA / FA ratio of rotifers increased from 0% to 14.16%, and the EPA / FA ratio increased from 5.28% to 9.41%; the DHA / FA ratio of artichokes increased from 0% to 8.69%, and the EPA / FA ratio increased from 0.99% to 3.63%; the survival rate of larvae and juveniles at 30 days of age increased from 53% to 66%.

[0034] Regarding the uniformity of seedling size: the coefficient of variation (CV) of body length of juveniles aged 15 to 19 days decreased from 23.85% to about 11% (P<0.01), and the standardization rate of 20-day-old fish (body length ±10%) increased to over 82.47%; the synchronization of feeding was improved, which increased the acceptance rate of micro-particle feed by 18% at 31 days old.

[0035] Regarding fishmeal substitution: In the early juvenile stage, the fishmeal substitution rate can reach 60%; feed costs are reduced by 18.7%; nitrogen emissions from feces are reduced by 22.41%; and the success rate of switching to new feed increases from 23% to 44%.

[0036] In terms of growth performance: specific growth rate (SGR) reached 3.2% / d; intestinal villus height increased by 32.65%, reaching 325±18μm; goblet cell density increased by 20%; in terms of metabolic regulation: PPARα and TOR pathways were activated; liver alanine aminotransferase (ALT) activity decreased by 40%; lipase activity increased by 35%. Detailed Implementation

[0037] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0038] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0039] Example 1

[0040] A method for preparing live feed for juvenile fish includes:

[0041] Step 1, Material Preparation

[0042] Common rotifer: Brachionus plicatilis, initial culture density was 170 rotifers / mL;

[0043] Schizochytrium limacinum powder (DHA / FA≥35%), bile acids, and sunflower phospholipids were purchased from Qingdao Yueyang Import & Export Co., Ltd., Hebei Pengyu Biotechnology Co., Ltd., and Zhengzhou Yuhe Food Additives Co., Ltd., respectively, and were stored at -20℃ before use.

[0044] Step 2, Preparation of nutritional fortifiers

[0045] Accurately weigh 100g of *Schizochytrium* powder, add 250mL of purified water at 50℃, and grind thoroughly using a stirrer. Add 20g of bile acid and 20g of sunflower phospholipid until a uniform, particle-free suspension is formed. Add room temperature purified water to bring the suspension to a final volume of 500mL to obtain the nutrient fortifier stock solution. Filter the suspension through a 120-mesh nylon mesh to remove large particles. Aliquot the filtrate into centrifuge tubes and store at -20℃ for later use to obtain the nutrient fortifier.

[0046] Step 3, Preparation of enhanced rotifers

[0047] (1) Preparation

[0048] The nutrient fortifier was diluted to concentrations of 0 g / t, 20 g / t, 50 g / t, and 80 g / t to obtain diluted solutions;

[0049] Add 150 common rotifers to each milliliter of diluent, and incubate for 0h, 3h, 6h, 9h, 12h, and 18h ​​to collect enhanced rotifers;

[0050] The controlled culture conditions were: temperature 25±1℃, salinity 28±1, light intensity 2000Lux, and continuous aeration.

[0051] (2) Enhancement effect detection

[0052] The enhancement effect was detected using the following methods:

[0053] a) DHA content was determined by gas chromatography-mass spectrometry (GC-MS);

[0054] b) Biological indicators include rotifer density, oviposition rate, and survival rate.

[0055] (3) Experimental Results

[0056] The main results are shown in Table 1.

[0057] Table 1. Results of Nutritional Index Tests on Enhanced Rotifers

[0058]

[0059] The main results in Table 1 are analyzed as follows:

[0060] 20g / t group: DHA / FA reached 10.59% (dry weight) and EPA / FA reached 8.05% (dry weight) at 12h.

[0061] 50g / t group: DHA / FA reached 14.16% (dry weight) and EPA / FA reached 9.41% (dry weight) at 12h.

[0062] Group 80g / t: DHA / FA reached 15.88% (dry weight) and EPA / FA reached 7.47% (dry weight) at 12h.

[0063] Biological indicators:

[0064] Fertility rate: At 12 hours, the fertility rates of the 20g / t group, 50g / t group, and 80g / t group reached 28.34%, 36.93%, and 44.61%, respectively, which were significantly higher than the 19.1% of the control group.

[0065] Survival rate: At 12 h, the density of the 50 g / t group reached 349 rotifers / mL, which was 51.74% higher than that of the control group.

[0066] (4) Optimal process parameters

[0067] Taking into account both the culture time and the nutritional value of the enhanced rotifers, the optimal process parameters were determined to be: 150 ordinary rotifers per milliliter of diluent, a nutrient fortifier concentration of 50 g / t, and a fortification time of 12 h. The enhanced rotifers prepared under these conditions exhibited the following characteristics: DHA / FA ratio ≥ 14.16%, EPA / FA ratio ≥ 9.41%, rotifer density increased by 51.74%, and average body length 0.2 mm.

[0068] Based on these optimal process parameters, experiments were conducted to investigate the effects of different nutrient fortifier formulations on the nutrient fortification results. In Table 2, apart from the differences in nutrient formulations, the preparation method of marine larvae and juvenile fish live feed is the same as in Example 1.

[0069] Table 2 Formulas of Different Nutrient Fortifiers

[0070]

[0071] In Table 2, " / " indicates that it has not been added.

[0072] Table 3. Nutritional composition test results of fortified rotifers

[0073]

[0074]

[0075] The results in Table 3 show that the nutritional components of the nutrient-fortified rotifers prepared by the nutrient fortifier formulations of each embodiment are all good, and the embodiments with additional fortifiers added in Examples 4-7 are better than those in Examples 1-3.

[0076] (5) Application instructions

[0077] The average body length of enhanced rotifers is 0.2 mm, which is suitable for feeding larvae aged 7 to 14 days. It can complement the average body length of enhanced artichokes of 1 mm, and achieve a differentiated feeding strategy for larvae aged 15 to 19 days.

[0078] Step 4, Preparation of Nutritionally Fortified Artemia

[0079] (1) Material preparation

[0080] Dormant eggs of Artemia tibetiana were purchased from Shuanghu County, Nagqu City, China, and stored at -20℃ for later use. Schizochytrium powder, bile acids, and sunflower phospholipids were purchased from Qingdao Yueyang Import & Export Co., Ltd., Hebei Pengyu Biotechnology Co., Ltd., and Zhengzhou Yuhe Food Additives Co., Ltd., respectively, and were also stored at -20℃ before use. Enhancement experiments were conducted at the Xixuan Fishery Science and Technology Island Aquaculture Base in Zhoushan City, Zhejiang Province.

[0081] (2) Preparation of nutritional fortifiers

[0082] Accurately weigh 100g of *Schizochytrium* powder, add 250mL of purified water at 50℃, and grind thoroughly using a stirrer. Add 20g of bile acid and 20g of sunflower phospholipid until a uniform, particle-free suspension is formed. Add room temperature purified water to bring the suspension to a final volume of 500mL to obtain the nutrient fortifier stock solution. Filter the suspension through a 120-mesh nylon mesh to remove large particles. Aliquot the filtrate into centrifuge tubes and store at -20℃ for later use.

[0083] (3) Artemia hatching

[0084] Hatching conditions: Place 1000g of dormant Artemia larvae in a 350L conical hatching tank and add seawater with a salinity of 30%. Hatching conditions are as follows: pH 7.5, surface light intensity 2000Lx, continuous aeration for 24 hours, and water temperature maintained at 27℃ by a heater.

[0085] Separation and Collection: After 24 hours of incubation, the eggs were kept in a closed-loop, light-proof environment for 30 minutes to allow the nauplii of common artichokes to separate from the eggshells. The nauplii were then collected through a 120-mesh filter screen fitted over the lower outlet.

[0086] (4) Nutritional fortification experiment

[0087] Density enhancement: Common artichoke nauplii were placed in 5L PP tanks at densities of 10, 30, 50, and 70 nauplii / mL, with three replicates per group. Survival rate and body length were measured at 0h, 4h, 8h, 12h, 18h, and 24h. Results showed that at a density of 50 nauplii / mL, the survival rate reached 85% within 12h, and the body length growth was moderate, making it suitable for subsequent enhancement.

[0088] Fortifier concentration and time: At a density of 50 individuals / mL, nutrient fortifiers of 0 g / t, 20 g / t, 40 g / t, 60 g / t, and 80 g / t were added respectively (corresponding to 0, 5, 10, 15, and 20 mL of stock solution), with 3 replicates per group. Samples were taken at 0 h, 4 h, 8 h, 12 h, 18 h, and 24 h to determine the survival rate and nutrient composition.

[0089] (5) Nutritional composition analysis

[0090] Standard components: Moisture, crude protein, crude fat and ash content were determined using the AOAC method.

[0091] Fatty acid analysis: The fatty acid composition was determined by gas chromatography.

[0092] (6) Results

[0093] Table 4 shows the test results for indicators that differed significantly from the control group.

[0094] Table 4. Results of nutritional index tests for fortified Artemia esculenta.

[0095]

[0096]

[0097] The results showed that prolonged fortification time significantly increased ash and moisture content (P<0.05), but slightly decreased crude protein and crude fat content. The fortifier significantly increased DHA (P<0.001) and EPA (P<0.05) content. After 12 hours of fortification, the DHA / FA ratio in the 40g / t group reached 8.69%, while DHA was undetectable in the unfortified group (common artichokes); the EPA / FA ratio reached 3.63%, an increase of 266.67% compared to the unfortified group. Total amino acid content decreased slightly with prolonged fortification time, but the fortification dose had no significant effect.

[0098] The results showed that when the nutrient fortifier was applied at a dose of 40g / t for 12h, the survival rate of Artemia esculents was 88.58%, the DHA content increased to 8.69% of the total fatty acids, and the body length increased from 0.65mm at hatching to 1.00mm, which met the feeding requirements of larvae.

[0099] (7) Conclusion

[0100] The optimal enrichment conditions determined in this embodiment are: a density of 50 Artemia filaments / mL, a nutrient fortifier concentration of 40 g / t, and an enrichment time of 12 h. Under these conditions, the enriched Artemia filaments exhibit significant DHA enrichment, high survival rate, and moderate body length, making them suitable as live feed for larvae and juvenile fish.

[0101] Experiment 1: Verification of the effect of size control on juvenile rock seabream

[0102] (1) Materials and Methods

[0103] Experimental fish: 7-day-old striped rock seabream (Oplegnathus fasciatus) larvae and juveniles with an initial body length of 3.4±0.3 mm. They were taken from a self-bred population and randomly divided into 2 groups, with 4 replicates in each group and 200 fish in each replicate.

[0104] Feeding program:

[0105] Control group: Feed the animals with common rotifers from 7 to 19 days old. Common rotifers contain 0% DHA.

[0106] Example group: 7-14 day old rotifers were fed with a mixture of enhanced rotifers and ordinary artichokes from 15-19 days old, with a wet weight ratio of enhanced rotifers to ordinary artichokes of 1:1. Enhanced rotifers were prepared using different nutritional formulas as described in Table 2, and were used to investigate the effects of different nutritional fortifier formulations on the size regulation of striped rock seabream larvae. The enhanced rotifer conditions used were: 150 ordinary rotifers per milliliter of diluted solution, a nutritional fortifier concentration of 50 g / t, and a fortification time of 12 hours.

[0107] Management: Feed 3 times a day at 8:00, 12:00 and 16:00, with a water temperature of 28±1℃ and dissolved oxygen ≥6mg / L.

[0108] (2) Results

[0109] See Table 5.

[0110] Table 5 Results of differentiated feeding of striped rock seabream larvae with rotifers and artemia.

[0111]

[0112] Growth uniformity: At 19 days of age, the coefficient of variation (CV) of body length in the example group was less than 11.53%, which was significantly lower than the CV of 23.85% in the control group (P<0.01); more than 82.47% of individuals in the example group had a body length in the range of 6.03mm to 7.37mm, while only 52.08% of individuals in the control group had a body length in the range of 6.03mm to 7.37mm.

[0113] Survival rate: At 19 days old, the survival rate of the control group exceeded 88.46%, which was 26.74% higher than that of the control group (61.72%) (P<0.05).

[0114] Histological analysis:

[0115] Intestinal development: In Example 1, the height of intestinal villi in group 1 (above 280±15μm) increased by 27% compared with the control group (220±12μm) (P<0.01);

[0116] Liver metabolism: In the first example group, the lipid droplet area of ​​hepatocytes decreased by 30%, and ALT activity decreased by 25% (P<0.05).

[0117] (3) Conclusion

[0118] This invention achieves uniformity in size (CV < 15%) among juvenile fish populations through differentiated feeding strategies and significantly improves adaptability to new feeds. The high mortality rate during the transition period lays the foundation for subsequent metabolic reprogramming and fishmeal substitution.

[0119] Experiment 2: Evaluation of the effects of nutritionally fortified Artemia on the growth performance of striped rock seabream larvae and juveniles.

[0120] (1) Materials and Methods

[0121] a) Source of experimental fish: 19-day-old striped rock bream fry that had been regulated to the size of Experiment 1 were selected, with an initial body length of 6.7±0.67mm, and randomly distributed into 16 50L water buckets, with 135 fish in each bucket.

[0122] b) Experimental design: Divided into 4 groups, with 4 replicates in each group:

[0123] Control group: fed with unenhanced Artemia (common Artemia) nauplii;

[0124] Example group: The enhanced artichokes prepared by adding 50 common artichokes per milliliter of diluted solution, 40 g / t of nutrient fortifier, and fortification for 12 h; different nutrient fortifier formulations are shown in Table 2, used to explore the effects of different nutrient fortifier formulations on the growth performance of striped rock seabream fry.

[0125] c) Feeding and management: Feeding was conducted at 7:00 and 18:00 daily, with an artemia density of 5 individuals / mL. 70% of the water was changed daily, and the number of deaths was recorded. The experiment lasted for 12 days.

[0126] (2) Results and Analysis

[0127] See Table 6.

[0128] Table 6 Results of nutritionally fortified Artemia fowl-fed juvenile and larval striped sea bream

[0129]

[0130] a) Growth performance:

[0131] Survival rate: After 12 days, the survival rate of each group in the example was at least 20.2% higher than that of the control group (P<0.05).

[0132] Body length and weight: On day 12 of the example, the body length of each group increased by more than 12.17% compared with the control group (P<0.05); the weight at the end of the period increased by more than 49.27% ​​compared with the control group (P<0.05).

[0133] b) Intestinal histology: In the examples, the height of intestinal villi, the thickness of the muscle layer and the number of goblet cells in each group were significantly increased (P<0.05), and the microvilli density increased by more than 20%, indicating enhanced digestive and absorptive capacity.

[0134] c) Liver structure and metabolism: Vacuolization was observed in the liver of the control group, while in the example groups, hepatocytes were more tightly packed and lipid droplet area was reduced by more than 35% (P<0.05). Metabolomics showed that the glycerophospholipid metabolic pathway was significantly enriched in the example groups, and DHA-containing lipids such as TG (20:5 / 18:3 / 22:6) were upregulated.

[0135] (3) Conclusion

[0136] The enhanced Artemia prepared by the formula in Table 2 can significantly improve the growth performance, intestinal development and liver health of striped rock sea bream fry and juveniles, providing an optimized solution for seedling cultivation.

[0137] Experiment 3: Evaluation of the effect of fishmeal substitution

[0138] (1) Materials and Methods

[0139] a) Experimental design:

[0140] Experimental fish species: 30-day-old striped rock bream larvae and juveniles who had completed nutritional fortification in Experiment 1 and Experiment 2 were selected, with an initial average weight of 6±0.5g, and randomly divided into several groups (4 replicates per group, 200 fish per replicate); Note that the formulas of different fortifiers are as shown in Table 2, and in each group in Table 7, the formulas of nutritional fortifiers used in Experiment 1 and Experiment 2 for the 30-day-old striped rock bream larvae and juveniles are the same.

[0141] Control group: fed a whole fish meal diet (70% fish meal, 20% soybean meal, and 10% wheat flour by dry weight percentage);

[0142] Experimental group: The fishmeal formula of this invention was used as a feed substitute, with the following formulation:

[0143] Early stage (31-36 days old): Based on dry weight percentage, fish meal 60% + enzymatically hydrolyzed soybean peptides 20% + black soldier fly protein 20%, with the addition of neutral protease equivalent to 1.5% of the total dry weight of fish meal, black soldier fly protein and yeast protein;

[0144] Later stage (37-45 days old): Based on dry weight percentage, fish meal 50% + black soldier fly protein 25% + yeast protein 25%, with the addition of chitinase and β-mannanase equivalent to 1.5% of the total dry weight of fish meal, black soldier fly protein and yeast protein.

[0145] Juvenile stage (46-60 days old): By dry weight percentage, fish meal 40% + black soldier fly protein 30% + yeast protein 30%, with the addition of thermoresistant lactic acid bacteria equivalent to 1.5% of the total dry weight of fish meal, black soldier fly protein, and yeast protein. The thermoresistant lactic acid bacteria is Enterococcus faecalis 20 billion feed-grade lactic acid bacteria, purchased from Luoyang Oukebike Biotechnology Co., Ltd.

[0146] Feeding period: 30 days.

[0147] 2. Feeding and Management:

[0148] Feed the fish three times a day, at 8:00, 12:00 and 16:00, with each feeding amount being 5% of the fish's body weight.

[0149] Maintain water temperature at 28±1℃, dissolved oxygen ≥6mg / L, and pH 7.8.

[0150] Growth indicators were measured weekly, and liver, intestinal tissue, and fecal samples were collected at the end of the experiment.

[0151] (2) Results and Analysis

[0152] Table 7 Results of the fishmeal substitution experiment

[0153]

[0154]

[0155] 1. Growth performance:

[0156] Survival rate (survival rate during the transition period): The survival rate of each group in the example (≥89.5%) was significantly higher than that of the control group (82.4%) (P<0.05).

[0157] Specific growth rate (SGR): There was no significant difference between the groups in the examples (approximately 3.2% / d) and the control group (3.1% / d) (P>0.05).

[0158] Feed conversion ratio (FCR): The FCR of each group in the example (≤1.26) was 9.35% lower than that of the control group (1.39) (P<0.05).

[0159] 2. Effects of fishmeal as a substitute:

[0160] Protein utilization: The protein efficiency PER (≥2.80) of each group in the examples was 21.21% higher than that of the control group (2.31) (P<0.01), which was attributed to the synergistic effect of enzymatic hydrolysis of soybean peptides and chitinase on insect protein.

[0161] Nitrogen emissions: The fecal nitrogen content in the experimental group (≤3.22) was 22.41% lower than that in the control group (4.15) (P<0.05).

[0162] 3. Physiological indicators:

[0163] Intestinal morphology: In the example, the height of intestinal villi (≥325μm) in each group increased by 32.65% compared with the control group (245μm) (P<0.01), and the density of goblet cells increased by 20% (P<0.05).

[0164] Liver health: In the experimental group, liver fat deposition was reduced by 40% (lipid droplet area ratio 12.5% ​​vs 20.8%), and alanine aminotransferase (ALT) activity was reduced by 38% (P<0.01).

[0165] Metabolic regulation: qPCR detection showed that the expression of PPARα pathway genes (ppara, cpt1a) in the experimental group was upregulated by 2.1-fold to 2.5-fold (P<0.01), and lipase activity was increased by 35% (P<0.05).

[0166] (3) Discussion

[0167] 1. Advantages of metabolic reprogramming replacement:

[0168] Live feed stage: High DHA-enhanced artichokes promote nerve development, laying the foundation for subsequent feed transition.

[0169] In the micro-particle feed stage: the low digestibility of a single protein source was overcome through gradual fishmeal replacement (60%→40%) and the addition of compound enzymes. For example, chitinase increased the utilization rate of black soldier fly protein from 65% to 85%.

[0170] 2. Ecological benefits:

[0171] The experimental group reduced fishmeal usage by 60%, feed costs by 18.7%, and nitrogen emissions by 22.41%, meeting the requirements of green aquaculture.

[0172] (4) Conclusion

[0173] The metabolic reprogramming fishmeal replacement scheme of this invention significantly improves protein utilization, liver health, and reduces environmental pollution while maintaining the growth performance of larvae and juveniles, providing technical support for the sustainable development of aquaculture.

[0174] This invention effectively solves key technical problems in the cultivation of larvae and juvenile fish through innovative nutritional fortification methods and metabolic reprogramming fishmeal substitution strategies, resulting in significant economic and ecological benefits.

[0175] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described in this invention to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the inventive concept of this invention, can make other changes and modifications to these embodiments, and all such changes and modifications fall within the scope of this invention.

[0176] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If such modifications and variations fall within the scope of equivalents of this invention, then this invention also intends to include these modifications and variations.

Claims

1. An application of a marine larvae / juvenile fish biological feed, characterized in that, The method for preparing the marine larvae and juvenile fish live feed includes: A nutrient fortifier is obtained by mixing Schizochytrium powder, bile acids, and sunflower phospholipids in a mass ratio of 50:10~20:10~20. The nutrient fortifier was diluted to a concentration of 40 g / t ~ 50 g / t to obtain a diluted solution; Add 130-150 common rotifers per milliliter of diluent, incubate for 12-18 hours, and collect the enhanced rotifers. When enhancing the rotifers, the nutrient fortifier also includes an enhancer, which is fucoidan, sodium butyrate, cysteine, or γ-aminobutyric acid (GABA). The amount of fucoidan added per kilogram of nutrient fortifier is 1-2 mg; the amount of sodium butyrate added per kilogram of nutrient fortifier is 100-200 mg; the amount of cysteine ​​added per kilogram of nutrient fortifier is 10-20 mg; and the amount of GABA added per kilogram of nutrient fortifier is 10-20 mg. Add 30 to 50 common artichokes to each milliliter of diluent, culture for 12 to 18 hours, and collect the enhanced artichokes. A combination of enhanced rotifers and enhanced artichokes is used as a live feed for larvae and juveniles; The marine juvenile fish mentioned are rock snapper juveniles; The marine juvenile fish live feed is used to improve the uniformity of juvenile fish fry and the survival rate of juvenile fish in aquaculture, and to reduce dependence on fishmeal.

2. The application according to claim 1, characterized in that, On a dry weight basis, docosahexaenoic acid accounts for more than 35% of the total fatty acid mass in the powder of *Schizochytrium*.

3. The application according to claim 1, characterized in that, Differentiated feeding of larvae and juveniles using the aforementioned marine larval and juvenile biological feed includes: Feed 7-14 day old fry with common rotifers; Fry and juvenile fish aged 15 to 19 days were fed a mixture of enhanced rotifers and common artichokes, with a wet weight ratio of enhanced rotifers to common artichokes of 1:

1. Feed the juvenile fish, aged 20 to 30 days, with the aforementioned enhanced artichokes.

4. The application according to claim 3, characterized in that, Feed the 7-14 day old rotifers three times a day, with a concentration of 5-8 rotifers per milliliter of seawater. Feed the infants, aged 15 to 19 days, three times a day, a mixture of the enhanced rotifers and common artichokes described above; Feed the 20-30 day old animals three times a day, with a concentration of 10 Artemia worms per milliliter of seawater.

5. The application according to claim 3, characterized in that, Fry and juvenile fish aged 31 to 60 days were fed fishmeal substitute feed, with the non-fishmeal protein dry weight percentage in the fishmeal substitute feed being 40% to 60%.

6. The application according to claim 5, characterized in that, The feeding method for fishmeal substitute feed includes the following steps: For juvenile fish aged 31 to 36 days, the fish meal substitute feed formula is as follows: by dry weight percentage, fish meal, enzymatically hydrolyzed soybean peptide, black soldier fly protein, and yeast protein are 60%, 20%, 10%, and 10%, respectively, with an additional neutral protease equivalent to 1% to 2% of the total dry weight of fish meal, enzymatically hydrolyzed soybean peptide, black soldier fly protein, and yeast protein. For juvenile fish aged 37 to 45 days, the fishmeal substitute feed formula is as follows: by dry weight percentage, fishmeal, black soldier fly protein, and yeast protein are 50%, 25%, and 25%, respectively, with the addition of chitinase equivalent to 1% to 2% of the total dry weight of fishmeal, black soldier fly protein, and yeast protein. For juvenile fish aged 46 to 60 days, the fishmeal substitute feed formula is as follows: by dry weight percentage, fishmeal, black soldier fly protein, and yeast protein are 40%, 30%, and 30%, respectively. In addition, 1% to 2% of the total dry weight of fishmeal, black soldier fly protein, and yeast protein is added, where heat resistance means resistance to temperatures above 40℃.

7. The application according to claim 6, characterized in that, The fishmeal substitute feed is used to improve the uniformity of juvenile and young fish fry and the survival rate of juvenile and young fish in aquaculture, and to reduce dependence on fishmeal.

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