Fat source for promoting survival and metamorphosis development of zoea of macrobrachium rosenbergii and feeding domestication method

Abstract

The invention discloses a fat source for promoting survival and metamorphosis development of zoeae of macrobrachium rosenbergii and a feeding training method, and belongs to the technical field of aquaculture. The fat source is soybean lecithin, and the addition amount of the fat source is 4-8% of the total mass of the feed. According to the feeding training method, feed and artemia are alternately fed one week after incubation, and the grain size of the feed is gradually transited from 80 meshes to 20 meshes. The high emulsibility and palatability of soybean lecithin improve the fat utilization rate of the feed, a gradient feeding domestication scheme is matched to promote the ingestion adaptability of larvae, and the fat source advantage is combined with a progressive domestication strategy, so that the technical bottleneck that artemia is replaced by artificial feed is broken through, the pathogen risk and the breeding cost are reduced, and the breeding efficiency is improved. A reproducible standardized and large-scale seedling raising scheme is formed.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to a fat source and a feeding method for promoting the survival and metamorphosis of Macrobrachium rosenbergii zoeae. Background Art

[0002] Macrobrachium rosenbergii is an important economic species in my country's freshwater aquaculture. Its aquaculture scale has continued to expand in recent years, but the seedling cultivation link still faces severe challenges. The zoea hatched from fertilized eggs need to undergo 11 molting and metamorphosis to develop into shrimp larvae. This process is called zoea cultivation, which is a key stage that determines the quality of seedlings and aquaculture benefits. However, in actual production, the survival rate and metamorphosis rate of zoea are generally low, which directly restricts the large-scale supply of high-quality seedlings. Fundamentally, traditional seedling cultivation technology relies too much on biological baits such as rotifers and Artemia, but such live baits have significant defects: on the one hand, their nutritional composition is unstable and lacks comprehensiveness, making it difficult to meet the metabolic needs of the rapid development of larvae; on the other hand, live baits are easy to carry pathogenic microorganisms, resulting in an increased risk of disease transmission, further exacerbating the mortality rate of larvae. With the growing demand for intensive aquaculture, the development of efficient and stable artificial compound feeds to replace live baits has become an urgent need for industrial development.

[0003] Although studies have long confirmed the feasibility of compound feeds to replace Artemia (Lazo et al., 2000; Kovalenko et al., 2002), it has not yet been completely replaced in the larvae of Macrobrachium rosenbergii. The reason is that the digestive system of larvae is not yet fully developed, and the secretion pattern of digestive enzymes is significantly different from that of adults, resulting in low nutrient absorption efficiency. In addition, larvae are highly sensitive to the types and proportions of key nutrients such as fat and protein.

[0004] Among them, fat, as the core energy source for larvae, not only undertakes the energy supply function, but also has the functions of regulating membrane structure, promoting the absorption of fat-soluble vitamins and saving protein. However, the biological effect of fat is highly dependent on its source and fatty acid composition. For example, fish oil is rich in n-3 highly unsaturated fatty acids (HUFA) such as EPA and DHA, which can significantly improve the growth performance of crustacean larvae; soybean oil is mainly composed of n-6 polyunsaturated fatty acids, which show the advantage of promoting survival rate in certain species; the monounsaturated fatty acids in rapeseed oil may indirectly supplement n-3 long-chain polyunsaturated fatty acids (LC-PUFA) through biotransformation; and phospholipids, as key components of cell membranes, play an irreplaceable role in the metamorphosis of larvae.

[0005] Existing studies have shown that the optimal selection of fat sources can significantly affect the growth, immunity and stress resistance of crustaceans. For example, the survival rate of red claw crayfish (Cherax quadricarinatus) larvae fed with soybean oil and peanut oil is better than that of fish oil and lard groups; the metamorphosis rate of Chinese mitten crab (Eriocheir sinensis) larvae is positively correlated with the HUFA content in the feed; Japanese shrimp (Penaeus japonicus) larvae cannot complete metamorphosis if they lack phospholipids. These findings highlight the importance of fat source adaptability research in larval nutrition.

[0006] However, the current research on lipid nutrition of Macrobrachium rosenbergii zoea is still blank. Existing research on aquatic lipids mostly focuses on the late larval or adult stages. Due to their special physiological state (such as intestinal dysplasia and enzyme activity fluctuations) and high-density molting requirements, zoea may have unique requirements for the type of fat source, fatty acid ratio and phospholipid content. For example, the demand for HUFA in the early stage of larvae may be higher than that of adults, but excessive saturated fatty acids (such as lauric acid in coconut oil) may inhibit their metabolic efficiency; the proportion of phospholipid addition needs to be optimized in coordination with the main lipid source to avoid developmental stagnation caused by abnormal membrane structure. In addition, the difference in palatability of different fat sources may further affect the feeding rate, thereby indirectly restricting growth performance. Therefore, systematic analysis of the regulatory mechanism of fat sources such as fish oil, soybean oil, rapeseed oil, coconut oil and phospholipids on the growth, antioxidant, immune and stress resistance of zoea is a core scientific issue to break through the bottleneck of artificial feed replacement technology. Summary of the invention

[0007] In view of the low survival rate, delayed metamorphosis, poor stress resistance and other problems caused by the insufficient adaptability of traditional fat sources (such as fish oil, soybean oil, etc.) in the cultivation of Macrobrachium rosenbergii zoeae, the purpose of the present invention is to provide a nutritional regulation program with soybean lecithin as the core fat source, which can significantly improve the survival rate, metamorphosis rate, immunity and stress resistance of Macrobrachium rosenbergii zoeae by optimizing the fat composition of the feed of Macrobrachium rosenbergii, thereby realizing Artemia replacement and large-scale seedling cultivation.

[0008] The present invention is implemented by the following technical solutions:

[0009] 1. Feed formula composition

[0010] Protein source: fish meal, dissolved fish paste powder, wheat enzymatic protein casein, sodium caseinate;

[0011] Carbohydrates: α-starch;

[0012] Fat source: mainly soybean lecithin (SL), and the control group included fish oil (FO), soybean oil (SO), rapeseed oil (RO), and coconut oil (CO);

[0013] Other ingredients: monocalcium phosphate, trace element premix, mineral premix choline chloride, cellulose, cholesterol;

[0014] Content: fish meal 30-40%; dissolved fish paste powder 8-12%; wheat enzymatic protein casein 5-10%; sodium caseinate 3-7%; α-starch 20-30%; soybean lecithin 5-10%; calcium dihydrogen phosphate 1-3%; trace element premix 2-5%;

[0015] Nutritional indicators: crude protein ≥53%, crude fat content ≥10%.

[0016] 2. Preparation method

[0017] Raw material processing: the protein source is crushed and passed through a 60-mesh sieve, and the calcium, phosphorus and trace elements are passed through an 80-mesh sieve; the fat source and water are then added and fully mixed, and then extruded and granulated by a twin-screw extruder (die aperture 1.5 mm);

[0018] Drying and sieving: After drying at 40℃, pass through 20, 40, 60 and 80 mesh sieves in sequence to adapt to the feeding diameter of the larvae in stages;

[0019] Storage conditions: Store in sealed container at -20℃.

[0020] 3. Cultivation method of Macrobrachium rosenbergii zoeae

[0021] Source of larvae: Healthy Macrobrachium rosenbergii zoeae (average weight 0.25 mg) provided by Zhejiang Zhongyi Aquatic Seed Co., Ltd.

[0022] Aquaculture system: 200L plastic barrel, water body is freshwater with salinity of 12‰, water temperature of 30-32℃, pH 7.6-8.1, dissolved oxygen>6.5mg / L;

[0023] Feeding: In the early stage of breeding, Artemia and feed are fed alternately, and then gradually transitioned to full feed;

[0024] Water quality management: Change 1 / 3 of the water daily, maintain water temperature at 30-32°C, pH 7.6-8.1, dissolved oxygen>6.5mg / L;

[0025] Anti-stress regulation: The soybean lecithin content in the feed significantly increased the survival rate of larvae exposed to air (24h survival rate ≥90%).

[0026] 4. Effect verification and data analysis

[0027] Larval survival rate and metamorphosis rate: After the breeding experiment is completed, the number of surviving shrimp fry and metamorphosis shrimp fry in each breeding bucket is counted and weighed.

[0028] Immune and antioxidant indicators: All shrimps were taken from each breeding barrel and rinsed with ultrapure water to remove the interference of fresh seawater, and then homogenized by ice bath electric homogenization with 0.86% physiological saline at a ratio of 1:9 (mass to volume ratio). The supernatant was centrifuged and analyzed with kits for alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) content.

[0029] Stress resistance: Take 30 larvae from each parallel, place them on a sieve of the same specifications, remove excess water with a paper towel, and expose them to air for 30 minutes. After the timing is over, quickly put the larvae in water, and observe their survival after 24 hours. The final survival rate is used to determine the stress resistance of the post-larvae.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The high emulsification and palatability of soybean lecithin in the present invention improve the utilization rate of feed fat, and the gradient feeding scheme combined with the gradient feeding strategy promotes the feeding adaptability of the larvae, and reduces the feed waste rate. The advantages of fat sources are combined with the progressive feeding strategy to break through the technical bottleneck of artificial feed replacing Artemia, reduce pathogenic risks and seedling costs, and form a replicable standardized and large-scale seedling plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Effects of different fat sources on the growth performance of Macrobrachium rosenbergii larvae

[0033] Figure 2 Effects of different fat sources on the immune performance of Macrobrachium rosenbergii larvae;

[0034] Figure 3 To study the effects of different fat sources on the antioxidant capacity and malondialdehyde content of Macrobrachium rosenbergii larvae.

[0035] Figure 4 The effect of different fat sources on the stress resistance of Macrobrachium rosenbergii larvae. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0037] Example 1

[0038] Preparation of fat source adapted feed:

[0039] Fish meal, dissolved fish pulp powder, wheat enzymatic protein casein and sodium caseinate were used as the main protein sources, α-starch was used as the carbohydrate source, and fish oil (FO), soybean oil (SO), rapeseed oil (RO), coconut oil (CO), and soybean lecithin (SL) were used as 5 different fat sources to prepare isonitrogenous and isolipid feeds with a crude protein content of 53% and a crude fat content of 10%. The experimental feed formula and nutritional components are shown in Table 1.

[0040] Table 1 Experimental feed formula and nutritional composition

[0041]

[0042]

[0043] The vitamin premix formula (g / kg): VA 6.4g, VC 151.52g, VE 72g, VD3 0.8g, VK3 2g, VB1 3.2g, VB2 9g, VB6 4g, VB12 0.08g, niacin 16g, folic acid 1g, inositol 64g, biotin 0.2g, calcium pantothenate 14g, and zeolite powder 777.02g.

[0044] The mineral premix formula (g / kg): copper sulfate pentahydrate CuSO4·5H2O 1.95g, zinc sulfate heptahydrate ZnSO4·7H2O 30.91g, manganese sulfate monohydrate MnSO4·H2O 5.23g, zinc sulfate heptahydrate FeSO4·7H2O 24.83g, calcium iodate Ca(IO3)2 0.46g, sodium selenite Na2SeO3 0.09g, cobalt chloride hexahydrate CoCl2·6H2O 0.32g, magnesium sulfate heptahydrate MgSO4·7H2O 244.9g.

[0045] Fish meal, dissolved fish paste powder and wheat gluten powder were crushed and passed through a 60-mesh sieve. Calcium dihydrogen phosphate and trace elements were passed through an 80-mesh sieve and mixed by a step-by-step expansion method. Fat source and water were added and mixed thoroughly. The mixture was extruded through a 1.5 mm die using an F-26 twin-screw extruder (Institute of Chemical Engineering, South China University of Technology, Guangzhou, China) to granulate. The granules were then dried in an oven at 40 °C. The ground feed was passed through 20, 40, 60 and 80 mesh sieves, packaged and labeled, and stored in a -20 °C refrigerator until use.

[0046] Nutrient composition determination: We analyzed the proximate chemical composition of three replicates of each experimental feed according to the regulations of the Association of Official Analytical Chemists (AOAC, 2005). The feeds were dried at 105 °C until their weight remained constant for the measurement of dry matter. Crude lipid was extracted and determined using a Soxhlet extraction system. Ash content was determined by combustion at 550 °C for 6 h in a muffle furnace. Crude protein content was determined using the Dumas nitrogen analyzer combustion method. The fatty acid composition of the feeds was determined by gas chromatography-mass spectrometry (GC-MS, 8890B-7693A, Agilent Technologies, USA) as described by Liang et al. (2022).

[0047] Example 2

[0048] Management of zoeae breeding and feeding:

[0049] Larvae: Healthy Macrobrachium rosenbergii zoeae (average weight 0.25 mg) provided by Zhejiang Zhongyi Aquatic Seed Co., Ltd. were selected.

[0050] Grouping: Set up 5 groups - FO group with fish oil as fat source, SO group with soybean oil as fat source, RO group with rapeseed oil as fat source, CO group with coconut oil as fat source, and SL group with soybean lecithin as fat source. Feed them with corresponding feeds respectively, with 6 parallel barrels for each group and 5,000 larvae in each barrel.

[0051] Breeding environment: The capacity of the plastic breeding tank is about 200L, and the breeding water is artificially prepared freshwater: salinity 12‰, water temperature 30-32℃, pH 7.6-8.1, dissolved oxygen>6.5mg / L, ammonia and nitrate content <0.1mg / L. During the breeding period, the feeding status of the experimental shrimp was observed in time and the residual feed was collected. 1 / 3 of the water in the breeding tank was replaced every day to maintain good water quality.

[0052] Feeding strategy: In the early stage of the experiment, Artemia nauplii were fed with Artemia hatched on the same day at 7:00 and 16:00 every day. One week later, feeding began, using 80-mesh sieve feed and Artemia nauplii to alternate feeding (specific feeding time: 7:00, 7:30, 13:00, 13:30, 16:00, 16:30), and the daily feeding amount of feed was approximately consistent with the total weight of the larvae. In the third week, 60-mesh feed was replaced, in the fourth week, 40-mesh feed was replaced, and in the fifth week, 20-mesh sieved feed was replaced, and the feeding frequency and amount of feed were gradually increased until Artemia was completely replaced. The experiment lasted for 6 weeks.

[0053] The preparation method of the artemia feed is as follows: taking out frozen Artemia (Artemia) eggs from a refrigerator in advance, incubating them in artificial seawater (salinity 30‰, 28°C, pH 8-9) for 24 hours, covering them with black cloth for 20 minutes to separate the Artemia from the egg shell, taking out the Artemia from the bottom of a conical bottle, flushing them with fresh water, and feeding them.

[0054] Example 3

[0055] Growth performance assay:

[0056] After the breeding experiment, the number of surviving shrimp fry and metamorphosed shrimp fry in each breeding bucket was counted, and all shrimps were taken out of the breeding bucket and rinsed with ultrapure water, and weighed after draining. 1.5 ml of completely metamorphosed juvenile shrimps in each bucket were randomly selected and placed in a centrifuge tube, quickly frozen in liquid nitrogen, and then stored in a -80℃ refrigerator for biochemical analysis. At the same time, 30 completely metamorphosed juveniles were randomly selected from each parallel for subsequent stress resistance tests.

[0057] Survival rate (SR, %) = 100 × N2 / N1

[0058] Weight gain (WG, %) = 100 × (W t -W0) / W0

[0059] Specific growth rate (% / d) = 100 × (lnW t -lnW0) / D

[0060] Perverted rate (PR, %) = 100 × Np / N2

[0061] Where, W0 is the initial body weight (g); W t is the final body weight (g); D is the number of breeding days; N1 is the initial number, N2 is the final number, N p The number of abnormalities.

[0062] The statistical results of the growth performance of Macrobrachium rosenbergii larvae in different groups are shown in Figure 1 , the results are mean ± SD, n = 3, different superscripts indicate significant differences (P < 0.05). At the end of the experiment, there was no significant difference in weight gain rate and specific growth rate among larvae with different dietary fat sources (P> 0.05). The survival rate of the SL group was 26.85 ± 2.64%, which was significantly higher than that of the FO group (20.24 ± 2.98%), the SO group (21.63 ± 1.74%), and the RO group (20.93 ± 3.48%). The survival rate of the CO group (13.63 ± 1.39%) was the lowest (P < 0.05). At the same time, the metamorphosis rate of the SL group was 93.48 ± 1.86%, which was also significantly higher than that of the other treatment groups (P < 0.05).

[0063] Example 4

[0064] Immune and antioxidant index detection:

[0065] The frozen larvae samples were homogenized in an ice bath with 0.86% physiological saline at a (mass to volume ratio) of 1:9, and then centrifuged at 3500 rpm for 15 min at 4°C. The supernatant was used to detect the contents of alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) using kits purchased from Nanjing Jiancheng. The reagent preparation, sample pretreatment and operation methods were carried out according to the instructions.

[0066] The immune performance of Macrobrachium rosenbergii larvae in different groups Figure 2 , n=3, different superscripts indicate significant differences (P<0.05).

[0067] The activities of acidic AKP and ACP were significantly different between larvae with different dietary fat sources (P<0.05). At the end of the experiment, the ACP activity of the SL group was 0.412±0.018U / g prot, and the AKP activity was 0.407±0.019U / g prot, which were significantly higher than those of the FO group (0.296±0.013U / g prot), the SO group (0.206±0.018U / g prot), the RO group (0.139±0.013U / g prot), and the CO group (0.283±0.023U / g prot).

[0068] The antioxidant capacity and malondialdehyde content of Macrobrachium rosenbergii larvae in different groups Figure 3 , n=3, different superscripts indicate significant differences (P<0.05). At the end of the experiment, the total antioxidant capacity of the SL group was not significantly different from that of the FO group, SO group, and CO group, but was significantly higher than that of the RO group (P<0.05). The activities of superoxide dismutase and glutathione peroxidase in the SL group were not significantly different from those of the FO group and SO group, but were significantly higher than those of the RO group and CO group (P<0.05). The content of malondialdehyde in the CO group was significantly higher than that in the other treatment groups (P<0.05).

[0069] Embodiment 5:

[0070] Test of larval stress resistance

[0071] The air exposure test was used to test the stress resistance of the larvae: 30 larvae from each parallel were placed on a sieve of the same specifications, excess water was removed with a paper towel, and the larvae were exposed to the air for 30 minutes. After the timing was over, the larvae were quickly placed in water, and the survival was observed again after 24 hours. The stress resistance of the post-larvae was determined by the final survival rate.

[0072] The antioxidant capacity and malondialdehyde content of larvae with different dietary fat sources Figure 4, n=3, different superscripts indicate significant differences (P<0.05). Different fat sources significantly affected the survival rate of Macrobrachium rosenbergii larvae after rehydration for 24 hours. The stress resistance of the SL group was not significantly different from that of the FO group, but was significantly higher than that of the SO, RO, and CO groups (P<0.05).

[0073] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A fat source for promoting the survival and metamorphosis of Macrobrachium rosenbergii larvae, characterized in that: The fat source is soybean lecithin, and the added amount thereof is 5-10% of the total mass of the feed. The crude fat content in the feed is 10-12%.

2. The fat source according to claim 1, characterized in that Adding it to feed can significantly increase the survival and metamorphosis rate of Macrobrachium rosenbergii zoeae, enhance their immune capacity and improve their anti-oxidative stress performance.

3. A feed for Macrobrachium rosenbergii zoeae comprising the fat source according to claim 1 or 2, characterized in that: The formula includes the following components in percentage by mass: 30-40% of fish meal; 8-12% of dissolved fish slurry powder; 5-10% of wheat enzymatic protein casein; 3-7% of sodium caseinate; 20-30% of alpha-starch; 5-10% of soybean lecithin; 1-3% of monocalcium phosphate; and 2-5% of trace element premix.

4. The feed according to claim 3, characterized in that The preparation process is as follows: fish meal, dissolved fish paste powder and wheat protein powder are crushed and passed through a 60-mesh sieve, calcium dihydrogen phosphate and trace elements are passed through an 80-mesh sieve, and then a fat source and water are added to be fully mixed, granulated, dried, and ground feed is passed through 20, 40, 60 and 80 mesh sieves for packaging.

5. A method for feeding Macrobrachium rosenbergii zoeae by switching from biological feed to artificial feed, characterized in that: The taming process lasts for 6 weeks. The taming steps are: (1) Feed Artemia nauplii twice a day for the first 7 days; (2) Subsequently, the feed particle size is adjusted gradually every week: on the 8th to 14th day, the 80-mesh artificial feed described in claim 4 is fed alternately with Artemia, and on the 15th to 28th day, it is gradually replaced with the 60-mesh, 40-mesh and 20-mesh feeds described in claim 4, and fed 6 times a day, and the feeding frequency and amount of feed are gradually increased until Artemia is completely replaced. (3) After the 29th day, the rats were fed with 20-mesh feed 6 times a day.

6. The feeding method according to claim 5, characterized in that: During the taming period, the water temperature was maintained at 28-32℃, pH 7.5-8.2, dissolved oxygen content ≥6.0mg / L, and the daily water change volume was ≥30%.

7. The feeding method according to claim 5, characterized in that: After 6 weeks, the survival rate of zoeae is ≥25%, the metamorphosis rate is ≥90%, and the 24-hour survival rate after 30 minutes of air exposure is ≥15%.

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