An egg yolk antibody-based biological agent for improving survival rate of fish fry

Abstract

This invention discloses a biological agent based on egg yolk antibodies to improve the survival rate of fish fry. By weight, it comprises: 10-15% flour, 2-5% vegetable oil, 2-3% earthworm powder, 0.2-2% modified egg yolk antibody, and the remainder being miscellaneous grain flour. The preparation method of the modified egg yolk antibody is as follows: S1, immunization of laying hens; S2, extraction of the modified egg yolk antibody. This biological agent, prepared by modifying egg yolk antibodies containing *Cyclophila columnaris*, effectively improves the survival rate of grass carp fry. A series of improved methods were employed in the preparation of the modified egg yolk antibody to enhance the survival rate of the immunized laying hens. The resulting modified egg yolk antibody exhibits good immunizing effects and, in conjunction with the application method of this invention, significantly reduces the proportion of grass carp fry infected with *Cyclophila columnaris*, improving the overall immunity of the fry. The cumulative survival rate of grass carp fry within 8 days is approximately 82%.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, specifically to a biological agent based on egg yolk antibodies to improve the survival rate of fish fry. Background Technology

[0002] Fish fry survival rate refers to the proportion of fry that survive the hatching or introduction process. Improving fish fry survival rate is crucial for aquaculture and river ecosystem restoration. Here are some methods to improve fish fry survival rate:

[0003] 1. Optimize water quality: Fish fry have high requirements for water quality, so maintaining clean and stable water quality is key to improving their survival rate. Monitor and maintain suitable water temperature, dissolved oxygen, and pH levels, and avoid the accumulation of harmful substances such as ammonia nitrogen and hydrogen sulfide. Use filtration equipment and change the water regularly to ensure good water quality;

[0004] 2. Provide suitable feed: Providing suitable feed for fish fry is one of the important factors in improving their survival rate. According to the feeding requirements of various fish species, provide them with suitable feed, including live fish, artificial feed, small invertebrates, etc., and ensure the freshness and diversity of the feed to meet the nutritional needs of fish at different growth stages.

[0005] 3. Provide a suitable habitat: Fish have high requirements for their habitat. Providing a suitable habitat can help fish reduce stress and the risk of disease, thereby improving their survival rate. Based on the characteristics of fish, provide suitable water depth, water flow, humidity, vegetation and benthic organisms.

[0006] In addition, there are many other ways to improve the survival rate of fish fry. Using a variety of methods can further improve the survival rate of fish fry. Among them, the research on feed is currently a hot topic. Many people have studied the use of egg yolk antibodies to improve the survival rate of fish fry. Egg yolk antibodies are the main serum antibodies found in amphibians, reptiles and birds. They mainly protect developing embryos from potential pathogens and provide effective passive immune protection for newborn chicks.

[0007] Patent CN109438575B discloses a largemouth bass rhabdovirus yolk antibody, prepared by the following method: (1) isolating and culturing the largemouth bass rhabdovirus from largemouth bass infected with it; (2) inactivating the largemouth bass rhabdovirus; (3) immunizing 180-190 day old laying hens with the inactivated largemouth bass rhabdovirus as an antigen, collecting eggs from the immunized hens, and extracting and purifying the yolk antibody from the egg yolk. This patent effectively neutralizes the largemouth bass rhabdovirus, thereby terminating viral infection, but it has certain limitations. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a biological agent based on egg yolk antibodies to improve the survival rate of fish fry.

[0009] The technical solution of this invention is:

[0010] A biological agent based on egg yolk antibodies to improve the survival rate of fish fry, comprising, by weight: 10-15% flour, 2-5% vegetable oil, 2-3% earthworm powder, 0.2-2% modified egg yolk antibodies, with the remainder being mixed grain flour;

[0011] The method for preparing the modified egg yolk antibody is as follows:

[0012] S1. Immunization of laying hens: Inject inactivated columnar fibrophilic bacteria into laying hens for immunization. A total of four injections are given, each injection being 1-1.6 mL. One week after each injection, feed the laying hens only with Bacillus feed twice a day. After feeding for one week, administer the injection again.

[0013] S2. Extraction of modified egg yolk antibodies: Two weeks after completing the four-injection immunization of laying hens, eggs were collected daily for three consecutive days. The collected eggs were yolk separated to obtain yolk fluid. The yolk fluid was mixed with deionized water at a weight ratio of 1:8, and hydrochloric acid was added to adjust the pH to 4.5-5. The mixture was stored at -18-20℃ for 12 hours, and then thawed at 4-5℃. The supernatant was filtered to obtain an aqueous solution of modified egg yolk antibodies. The aqueous solution of modified egg yolk antibodies was subjected to two salting-out processes, and then concentrated by ultrafiltration to obtain a concentrated modified egg yolk antibody solution. The concentrated modified egg yolk antibody solution was freeze-dried to obtain freeze-dried modified egg yolk antibody powder.

[0014] Furthermore, the mixed grain powder is a mixture of rice bran and wheat bran in a 1:1 weight ratio.

[0015] Note: By using whole grain powder as the main nutrient component of this biological agent, the absorption of egg yolk antibodies can be ensured.

[0016] Furthermore, the method for preparing the inactivated columnar fibrophilic bacteria in step S1 is as follows:

[0017] Grass carp infected with *Pseudomonas columnaris* were whole-crushed and shaken well. The mixture was centrifuged at 1000–2000 rpm for 15 min, and the supernatant was collected and filtered through a bacterial filter to obtain a crude extract of *Pseudomonas columnaris*. Uninfected grass carp kidney cell line CIK cells were cultured in 2216E medium. When the confluence of CIK cells reached 60–80%, the crude extract of *Pseudomonas columnaris* was added. Once the CIK cell lesion rate was >60%, the mixture was centrifuged at 1000–2000 rpm for 15 min, and the supernatant was collected. The supernatant was stored at -80℃ for 24 h, subjected to two freeze-thaw cycles, and then inactivated for 24 h at 24–28℃ by adding a 0.5% methanol solution and shaking. The cells were then washed three times with PBS solution by centrifugation at 100–200 rpm for 15 min to obtain a 10% concentration. 9 Inactivated columnar fibrophilic bacteria at CFU / mL.

[0018] Note: Successful vaccination of laying hens can be ensured through proper inactivation methods.

[0019] Furthermore, in step S1, during the first two injections, 0.4 mL is injected subcutaneously into the neck and 0.3 mL is injected into each of the left and right pectoral muscles. During the third injection, 0.4 mL is injected subcutaneously into the neck and 0.4 mL is injected into each of the left and right pectoral muscles. During the fourth injection, 0.6 mL is injected subcutaneously into the neck and 0.5 mL is injected into each of the left and right pectoral muscles.

[0020] Note: By selecting the most suitable vaccination method, we can ensure the successful vaccination of laying hens while maintaining their survival rate.

[0021] Further, in step S1, the Bacillus feed, by weight, includes a concentration of 5–6 × 10⁻⁶. 6 The concentration of Lactobacillus spores powder (CFU / g) is 4-8 parts, corn (15-25 parts), soybean meal (10-15 parts), and garlic (1-2 parts), at a concentration of 1-2 × 10⁻⁶. 6 Two to three portions of Lactobacillus powder (CFU / g) and Bacillus feed with a moisture content of 10 to 15%.

[0022] Note: By adjusting the feed formula during the vaccination process, the survival rate of laying hens can be further improved.

[0023] Furthermore, the method of double salting out the modified egg yolk antibody aqueous solution in step S2 is as follows:

[0024] First salting out: Add 40% ammonium sulfate solution to the modified egg yolk antibody aqueous solution to make the volume ratio of modified egg yolk antibody aqueous solution to ammonium sulfate solution 5:3. After stirring evenly, store at 4℃ for 12h. After centrifugation at 10000-12000rpm for 15min, take the precipitate and use deionized water to make up to the original volume of modified egg yolk antibody aqueous solution.

[0025] Second salting out: Add 40% sodium sulfate solution to the modified egg yolk antibody aqueous solution to make the volume ratio of modified egg yolk antibody aqueous solution to sodium sulfate solution 5:1 after the first salting out. After stirring evenly, store at 4℃ for 12h. After centrifugation at 10000-12000rpm for 15min, take the precipitate and make up to the original volume of modified egg yolk antibody aqueous solution with deionized water to obtain modified egg yolk antibody extract.

[0026] Note: By optimizing and adjusting the salting-out method, the properties of the egg yolk antibodies were preserved.

[0027] Furthermore, the ultrafiltration concentration method in step S2 is as follows:

[0028] The modified egg yolk antibody extract was concentrated by ultrafiltration using an ultrafiltration membrane until the solution volume was 1 / 8 to 1 / 6 of the original modified egg yolk antibody extract volume. The ultrafiltration membrane was then washed with a 1-2 mol / L NaOH solution until the original modified egg yolk antibody extract volume was reached, yielding a washing solution. The NaOH solution in the washing solution was then washed and replaced with deionized water. 20-30% of the original modified egg yolk antibody extract volume of a 75% ethanol solution was added, and the solution was stored at 4°C for 12 hours to obtain the modified egg yolk antibody concentrate.

[0029] Preferably, the freeze-drying method in step S2 is as follows:

[0030] The modified egg yolk antibody concentrate was frozen at -20℃ for 24 hours, then frozen at -70℃ for 2 hours, and then freeze-dried at a vacuum of 15-20 Pa for 36 hours to obtain lyophilized modified egg yolk antibody powder.

[0031] The above-mentioned application of a yolk antibody-based biological agent for improving fish fry survival rate involves applying the biological agent to grass carp farming, specifically as follows:

[0032] During the fry feeding period from April to October, the weight ratio of biological agents to ordinary fish feed was 1:3, and the content of modified egg yolk antibodies in the biological agents was 2wt%.

[0033] During the fry feeding period from November to March of the following year, the weight ratio of biological agents to ordinary fish feed was 1:5, and the content of modified egg yolk antibodies in the biological agents was 0.2 wt%.

[0034] Note: By optimizing and adjusting the specific dosage of the biological agent of the present invention in the grass carp farming process, different dosages are used in different months, thereby maximizing the effect of the biological agent of the present invention for different farming environments.

[0035] The beneficial effects of this invention are:

[0036] (1) The biological agent based on egg yolk antibody for improving the survival rate of fish fry of the present invention is prepared by modifying egg yolk antibody containing columnar fibrophilic bacteria. It can effectively improve the survival rate of grass carp fry. A series of improved methods were adopted in the preparation of modified egg yolk antibody to improve the survival rate of immunized laying hens. The modified egg yolk antibody has good immune effect. In addition, the application method of the present invention can significantly reduce the proportion of grass carp fry infected with columnar fibrophilic bacteria and improve the overall immunity of the fry. The cumulative survival rate of grass carp fry within 8 days is about 82%.

[0037] (2) The present invention provides a biological agent based on egg yolk antibody to improve the survival rate of fish fry. When preparing the modified egg yolk antibody, a related egg-laying hen feed formula was developed simultaneously, thereby reducing the probability of death of egg-laying hens after inoculation with inactivated columnar fibrophilic bacteria, making the preparation of biological agents more efficient and cost-saving.

[0038] (3) The application of the biological agent based on egg yolk antibody to improve the survival rate of fish fry in this invention is achieved by optimizing and adjusting the specific addition dosage of the biological agent of this invention in the grass carp farming process, using different addition amounts in different months, thereby maximizing the effect of the biological agent of this invention for different farming environments. Detailed Implementation

[0039] Example 1

[0040] A biological agent based on egg yolk antibodies to improve the survival rate of fish fry, comprising, by weight 100%, 12% flour, 3% vegetable oil, 2.5% earthworm powder, 0.4% modified egg yolk antibodies, and the remainder being miscellaneous grain powder, which is a mixture of rice bran and wheat bran in a 1:1 weight ratio.

[0041] The preparation method of modified egg yolk antibody is as follows:

[0042] S1. Immunization of laying hens: Inactivated *Bacillus columnaris* was injected into laying hens four times. For the first two injections, 0.4 mL was injected subcutaneously into the neck and 0.3 mL into each pectoral muscle. For the third injection, 0.4 mL was injected subcutaneously into the neck and 0.4 mL into each pectoral muscle. For the fourth injection, 0.6 mL was injected subcutaneously into the neck and 0.5 mL into each pectoral muscle. One week after each injection, the laying hens were fed only *Bacillus columnaris* feed twice daily for one week. After this period, the injections were repeated. The remaining time, the hens were fed commercially available chicken feed. The *Bacillus columnaris* feed, by weight, included a concentration of 5.5 × 10⁻⁶. 6 The concentration of Lactobacillus spores powder (CFU / g) is 1.5 × 10⁻⁶ parts: 6 parts corn, 20 parts soybean meal, 12 parts garlic, and 1.5 parts corn. 6 2.5 parts of Lactobacillus powder with CFU / g, and Bacillus feed with a moisture content of 12%;

[0043] The method for preparing inactivated columnar fibrophilic bacteria is as follows:

[0044] Grass carp infected with *Pseudomonas columnaris* were whole-crushed and shaken well. The mixture was centrifuged at 1500 rpm for 15 min, and the supernatant was collected and filtered through a commercially available 0.25 μm bacterial filter to obtain a crude extract of *Pseudomonas columnaris*. Uninfected grass carp kidney cell line CIK cells were cultured in 2216E medium. When the confluence of CIK cells reached 70%, the crude extract of *Pseudomonas columnaris* was added. After the CIK cell lesion rate exceeded 60%, the mixture was centrifuged at 1500 rpm for 15 min, and the supernatant was collected. The supernatant was stored at -80℃ for 24 h, subjected to two freeze-thaw cycles, and then inactivated for 24 h at 26℃ by adding a 0.5% methanol solution and shaking. The cells were then washed three times with PBS solution after centrifugation at 150 rpm for 15 min to obtain a 10% concentration extract. 9 Inactivated columnar fibrophilic bacteria at CFU / mL;

[0045] S2. Extraction of modified egg yolk antibodies: Two weeks after completing the four injections of immunization for laying hens, eggs were collected daily for three consecutive days. The collected eggs were separated into egg yolks to obtain egg yolk fluid. The egg yolk fluid was mixed with deionized water at a weight ratio of 1:8, and hydrochloric acid was added to adjust the pH to 4.8. The mixture was stored at -19℃ for 12 hours and then thawed at 4.5℃. The supernatant was filtered to obtain an aqueous solution of modified egg yolk antibodies. The aqueous solution of modified egg yolk antibodies was subjected to two salting-out processes and then concentrated by ultrafiltration to obtain a concentrated solution of modified egg yolk antibodies. The concentrated solution of modified egg yolk antibodies was freeze-dried to obtain freeze-dried modified egg yolk antibody powder.

[0046] The method of two salting-out steps for modified egg yolk antibody aqueous solution is as follows:

[0047] First salting out: Add 40% ammonium sulfate solution to the modified egg yolk antibody aqueous solution to make the volume ratio of modified egg yolk antibody aqueous solution to ammonium sulfate solution 5:3. After stirring evenly, store at 4℃ for 12h. After centrifugation at 11000rpm for 15min, take the precipitate and use deionized water to make up to the original volume of modified egg yolk antibody aqueous solution.

[0048] Second salting out: Add 40% sodium sulfate solution to the modified egg yolk antibody aqueous solution to make the volume ratio of modified egg yolk antibody aqueous solution to sodium sulfate solution after the first salting out 5:1. After stirring evenly, store at 4℃ for 12h. After centrifugation at 11000rpm for 15min, take the precipitate and make up to the original volume of modified egg yolk antibody aqueous solution with deionized water to obtain modified egg yolk antibody extract.

[0049] The method of ultrafiltration concentration is as follows:

[0050] The modified egg yolk antibody extract was concentrated by ultrafiltration using an ultrafiltration membrane until the solution volume was 1 / 7 of the original modified egg yolk antibody extract volume. The ultrafiltration membrane was then washed with a 1.5 mol / L NaOH solution until the original modified egg yolk antibody extract volume was reached, resulting in a washing solution. The NaOH solution in the washing solution was then washed and replaced with deionized water. A 75% ethanol solution with a mass concentration of 25% of the original modified egg yolk antibody extract volume was added, and the solution was stored at 4°C for 12 hours to obtain the modified egg yolk antibody concentrate.

[0051] The freeze-drying method is as follows:

[0052] The modified egg yolk antibody concentrate was frozen at -20℃ for 24 hours, then frozen at -70℃ for 2 hours, and finally freeze-dried at a vacuum of 17 Pa for 36 hours to obtain lyophilized modified egg yolk antibody powder.

[0053] Example 2

[0054] The difference between this embodiment and Embodiment 1 is that the specific parameters of the biological agent are different.

[0055] A biological agent based on egg yolk antibodies to improve the survival rate of fish fry, comprising, by weight: 10% flour, 2% vegetable oil, 2% earthworm powder, 0.2% modified egg yolk antibodies, with the remainder being miscellaneous grain powder, which is a mixture of rice bran and wheat bran in a 1:1 weight ratio.

[0056] Example 3

[0057] The difference between this embodiment and Embodiment 1 is that the specific parameters of the biological agent are different.

[0058] A biological agent based on egg yolk antibodies to improve the survival rate of fish fry, comprising, by weight: 15% flour, 5% vegetable oil, 3% earthworm powder, 2% modified egg yolk antibodies, and the remainder being mixed grain powder, which is a 1:1 weight mixture of rice bran and wheat bran.

[0059] Example 4

[0060] The difference between this embodiment and Example 1 is that the specific parameters of the preparation method for the modified egg yolk antibody are different.

[0061] The preparation method of modified egg yolk antibody is as follows:

[0062] S1. Immunization of laying hens: Inactivated *Bacillus columnaris* was injected into laying hens four times. For the first two injections, 0.4 mL was injected subcutaneously into the neck and 0.3 mL into each pectoral muscle. For the third injection, 0.4 mL was injected subcutaneously into the neck and 0.4 mL into each pectoral muscle. For the fourth injection, 0.6 mL was injected subcutaneously into the neck and 0.5 mL into each pectoral muscle. One week after each injection, the laying hens were fed a *Bacillus* feed twice daily for one week. The *Bacillus* feed, by weight, included a concentration of 5×10... 6 Four parts of Bacillus spores powder (CFU / g), 15 parts of corn, 10 parts of soybean meal, and 1 part of garlic were mixed to a concentration of 1×10⁻⁶. 6 Two portions of Lactobacillus powder (CFU / g) and Bacillus feed with a moisture content of 10%;

[0063] The method for preparing inactivated columnar fibrophilic bacteria is as follows:

[0064] Grass carp infected with *Pseudomonas columnaris* were whole-crushed and shaken well. The mixture was centrifuged at 1000 rpm for 15 min, and the supernatant was collected and filtered through a bacterial filter to obtain a crude extract of *Pseudomonas columnaris*. Uninfected grass carp kidney cell line CIK cells were cultured in 2216E medium. When the confluence of CIK cells reached 60%, the crude extract of *Pseudomonas columnaris* was added. When the CIK cell lesion rate reached 70%, the mixture was centrifuged at 1000 rpm for 15 min, and the supernatant was collected. The supernatant was stored at -80℃ for 24 h, subjected to two freeze-thaw cycles, and then inactivated for 24 h at 24℃ by adding a 0.5% methanol solution and shaking. The cells were then washed three times with PBS solution by centrifugation at 100 rpm for 15 min to obtain a 10% concentration extract. 9 Inactivated columnar fibrophilic bacteria at CFU / mL;

[0065] S2. Extraction of modified egg yolk antibodies: Two weeks after completing the four injections of immunization for laying hens, eggs were collected daily for three consecutive days. The collected eggs were separated into egg yolks to obtain egg yolk fluid. The egg yolk fluid was mixed with deionized water at a weight ratio of 1:8, and hydrochloric acid was added to adjust the pH to 4.5. The mixture was stored at -18℃ for 12 hours and then thawed at 4℃. The supernatant was filtered to obtain an aqueous solution of modified egg yolk antibodies. The aqueous solution of modified egg yolk antibodies was subjected to two salting-out processes and then concentrated by ultrafiltration to obtain a concentrated solution of modified egg yolk antibodies. The concentrated solution of modified egg yolk antibodies was freeze-dried to obtain freeze-dried modified egg yolk antibody powder.

[0066] The method of two salting-out steps for modified egg yolk antibody aqueous solution is as follows:

[0067] First salting out: Add 40% ammonium sulfate solution to the modified egg yolk antibody aqueous solution to make the volume ratio of modified egg yolk antibody aqueous solution to ammonium sulfate solution 5:3. After stirring evenly, store at 4℃ for 12h. After centrifugation at 10000rpm for 15min, take the precipitate and use deionized water to make up to the original volume of modified egg yolk antibody aqueous solution.

[0068] Second salting out: Add 40% sodium sulfate solution to the modified egg yolk antibody aqueous solution to make the volume ratio of modified egg yolk antibody aqueous solution to sodium sulfate solution after the first salting out 5:1. After stirring evenly, store at 4℃ for 12h. After centrifugation at 10000rpm for 15min, take the precipitate and make up to the original volume of modified egg yolk antibody aqueous solution with deionized water to obtain modified egg yolk antibody extract.

[0069] The method of ultrafiltration concentration is as follows:

[0070] The modified egg yolk antibody extract was concentrated by ultrafiltration using an ultrafiltration membrane until the solution volume was 1 / 8 of the original modified egg yolk antibody extract volume. The ultrafiltration membrane was then washed with a 1 mol / L NaOH solution until the original modified egg yolk antibody extract volume was reached, resulting in a washing solution. The NaOH solution in the washing solution was then washed and replaced with deionized water. A 75% ethanol solution with a mass concentration of 20% of the original modified egg yolk antibody extract volume was added, and the solution was stored at 4°C for 12 hours to obtain the modified egg yolk antibody concentrate.

[0071] The freeze-drying method is as follows:

[0072] The modified egg yolk antibody concentrate was frozen at -20℃ for 24 hours, then frozen at -70℃ for 2 hours, and then freeze-dried at a vacuum of 15 Pa for 36 hours to obtain lyophilized modified egg yolk antibody powder.

[0073] Example 5

[0074] The difference between this embodiment and Example 1 is that the specific parameters of the preparation method for the modified egg yolk antibody are different.

[0075] The preparation method of modified egg yolk antibody is as follows:

[0076] S1. Immunization of laying hens: Inactivated *Bacillus columnaris* was injected into laying hens four times. For the first two injections, 0.4 mL was injected subcutaneously into the neck and 0.3 mL into each pectoral muscle. For the third injection, 0.4 mL was injected subcutaneously into the neck and 0.4 mL into each pectoral muscle. For the fourth injection, 0.6 mL was injected subcutaneously into the neck and 0.5 mL into each pectoral muscle. One week after each injection, the laying hens were fed a *Bacillus* feed twice daily for one week. The *Bacillus* feed, by weight, included a concentration of 6 × 10⁻⁶. 6 Eight parts of Bacillus spores powder (CFU / g), 25 parts of corn, 15 parts of soybean meal, and 2 parts of garlic, with a concentration of 2×10⁻⁶. 6 Three portions of Lactobacillus powder (CFU / g) and Bacillus feed with a moisture content of 15%;

[0077] The method for preparing inactivated columnar fibrophilic bacteria is as follows:

[0078] Grass carp infected with *Pseudomonas columnaris* were whole-crushed and shaken well. The mixture was centrifuged at 2000 rpm for 15 min, and the supernatant was collected and filtered through a bacterial filter to obtain a crude extract of *Pseudomonas columnaris*. Uninfected grass carp kidney cell line CIK cells were cultured in 2216E medium. When the confluence of CIK cells reached 80%, the crude extract of *Pseudomonas columnaris* was added. When the CIK cell lesion rate reached 65%, the mixture was centrifuged at 2000 rpm for 15 min, and the supernatant was collected. The supernatant was stored at -80℃ for 24 h, subjected to two freeze-thaw cycles, and then inactivated for 24 h at 28℃ by adding a 0.5% methanol solution and shaking. The cells were then washed three times with PBS solution by centrifugation at 200 rpm for 15 min to obtain a 10% concentration. 9 Inactivated columnar fibrophilic bacteria at CFU / mL;

[0079] S2. Extraction of modified egg yolk antibodies: Two weeks after completing the four injections of immunization for laying hens, eggs were collected daily for three consecutive days. The collected eggs were separated into egg yolks to obtain egg yolk fluid. The egg yolk fluid was mixed with deionized water at a weight ratio of 1:8, and hydrochloric acid was added to adjust the pH to 5. The mixture was stored at -20℃ for 12 hours and then thawed at 5℃. The supernatant was filtered to obtain an aqueous solution of modified egg yolk antibodies. The aqueous solution of modified egg yolk antibodies was subjected to two salting-out processes and then concentrated by ultrafiltration to obtain a concentrated solution of modified egg yolk antibodies. The concentrated solution of modified egg yolk antibodies was freeze-dried to obtain freeze-dried modified egg yolk antibody powder.

[0080] The method of two salting-out steps for modified egg yolk antibody aqueous solution is as follows:

[0081] First salting out: Add 40% ammonium sulfate solution to the modified egg yolk antibody aqueous solution to make the volume ratio of modified egg yolk antibody aqueous solution to ammonium sulfate solution 5:3. After stirring evenly, store at 4℃ for 12h. After centrifugation at 12000rpm for 15min, take the precipitate and use deionized water to make up to the original volume of modified egg yolk antibody aqueous solution.

[0082] Second salting out: Add 40% sodium sulfate solution to the modified egg yolk antibody aqueous solution to make the volume ratio of modified egg yolk antibody aqueous solution to sodium sulfate solution after the first salting out 5:1. After stirring evenly, store at 4℃ for 12h. After centrifugation at 12000rpm for 15min, take the precipitate and make up to the original volume of modified egg yolk antibody aqueous solution with deionized water to obtain modified egg yolk antibody extract.

[0083] The method of ultrafiltration concentration is as follows:

[0084] The modified egg yolk antibody extract was concentrated by ultrafiltration using an ultrafiltration membrane until the solution volume was 1 / 6 of the original modified egg yolk antibody extract volume. The ultrafiltration membrane was then washed with a 2 mol / L NaOH solution until the original modified egg yolk antibody extract volume was reached, resulting in a washing solution. The NaOH solution in the washing solution was then washed and replaced with deionized water. A 75% ethanol solution with a mass concentration of 30% of the original modified egg yolk antibody extract volume was added, and the solution was stored at 4°C for 12 hours to obtain the modified egg yolk antibody concentrate.

[0085] The freeze-drying method is as follows:

[0086] The modified egg yolk antibody concentrate was frozen at -20℃ for 24 hours, then frozen at -70℃ for 2 hours, and finally freeze-dried at a vacuum of 20 Pa for 36 hours to obtain lyophilized modified egg yolk antibody powder.

[0087] Example 6

[0088] This embodiment describes the application of a yolk antibody-based biological agent to improve the survival rate of fish fry. The biological agent is applied to grass carp farming, and the specific method is as follows:

[0089] During the fry feeding from April to October, the weight ratio of biological agent to ordinary fish feed is 1:3, and the content of modified egg yolk antibody in the biological agent is 2wt%. The biological agent in Example 3 can be used.

[0090] During the fry feeding period from November to March of the following year, the weight ratio of biological agent to ordinary fish feed is 1:5, and the content of modified egg yolk antibody in the biological agent is 0.2wt%. The biological agent in Example 2 can be used.

[0091] Experimental Example 1

[0092] The effects of the biological agent of the present invention were tracked and monitored, and corresponding comparative examples were established for comparative analysis. In Comparative Example 1, the biological agent used did not contain modified egg yolk antibodies, and the other components were the same as those of the biological agent of the present invention. Comparative Example 2 showed the effects of the biological agent in Example 1, but did not differentiate according to the month. The results are shown in Table 1.

[0093] Table 1. Survival rate of fish fry in each case.

[0094]

[0095] As can be seen from the data in Table 1, after applying the biological agent of the present invention in Comparative Example 2 and Example 6, the survival rate of fish fry was greatly improved in every month. This indicates that the biological agent of the present invention can have an important impact on the survival of grass carp fry. This is mainly because the biological agent of the present invention contains a special modified egg yolk antibody, which can effectively reduce the incidence of columnar fibrophilic bacteria, and at the same time reduce the occurrence of infectious enteritis, hemorrhagic disease and red spot disease, thus greatly improving the overall survival rate.

[0096] Let's look at Comparative Example 2 and Example 6. They are not much different. However, April to October is the peak season for infectious diseases in grass carp. Therefore, it is necessary to appropriately increase the content of modified egg yolk antibodies in the biological agent. At the same time, from November to March of the following year, considering the cost, the content of modified egg yolk antibodies in the biological agent can be appropriately reduced. This can also achieve a high survival rate. Taking all factors into consideration, the application method in Example 6 is selected.

[0097] Experiment Example 2

[0098] The immune status of laying hens during the preparation of the biological agent of the present invention was monitored and a corresponding comparative example was developed for comparative analysis. In Comparative Example 3, the special Bacillus feed of the present invention was not fed after each injection, and the same application method as in Example 6 was applied. The results are shown in Table 2.

[0099] Table 2. Survival rate of fish fry in each case.

[0100]

[0101] As can be seen from the data in Table 2, in Comparative Example 3, the survival rate of laying hens not fed with the special Bacillus feed of this invention decreased, and the effect of the prepared biological agent was also not ideal. Compared with the fish fry survival rate in Example 6, it was slightly lower. This indicates that the special Bacillus feed of this invention can produce a good auxiliary effect in the process of vaccinating laying hens, which can not only improve the survival rate of laying hens, but also ensure the overall activity of the biological agent, thus greatly improving the overall survival rate.

Claims

1. A biological agent based on egg yolk antibodies to improve the survival rate of fish fry, characterized in that, By weight, it includes: 10-15% flour, 2-5% vegetable oil, 2-3% earthworm powder, 0.2-2% modified egg yolk antibody, and the remainder is mixed grain flour; The method for preparing the modified egg yolk antibody is as follows: S1. Immunization of laying hens: Inject inactivated columnar fibrophilic bacteria into laying hens for immunization. A total of four injections are given, each injection being 1-1.6 mL. One week after each injection, feed the laying hens only with Bacillus feed twice a day. After feeding for one week, administer the injection again. The method for preparing the inactivated columnar fibrophilic bacteria in step S1 is as follows: Grass carp infected with *Pseudomonas columnaris* were whole-crushed and shaken well. The mixture was centrifuged at 1000-2000 rpm for 15 min, and the supernatant was collected and filtered through a bacterial filter to obtain a crude extract of *Pseudomonas columnaris*. Uninfected grass carp kidney cell line CIK cells were cultured in 2216E medium. When the confluence of CIK cells reached 60-80%, the crude extract of *Pseudomonas columnaris* was added. Once the CIK cell lesion rate was >60%, the mixture was centrifuged at 1000-2000 rpm for 15 min, and the supernatant was collected. The supernatant was stored at -80℃ for 24 h, subjected to two freeze-thaw cycles, and then inactivated for 24 h at 24-28℃ by adding a 0.5% methanol solution and shaking. The cells were then washed three times with PBS solution by centrifugation at 100-200 rpm for 15 min to obtain a 10% concentration. 9 Inactivated columnar fibrophilic bacteria at CFU / mL; In step S1, the Bacillus feed, by weight, includes a concentration of 5~6×10⁻⁶. 6 4-8 parts of Bacillus spores powder (CFU / g), 15-25 parts of corn, 10-15 parts of soybean meal, and 1-2 parts of garlic, at a concentration of 1-2 × 10⁻⁶. 6 2-3 parts of Lactobacillus powder (CFU / g) and Bacillus feed with a moisture content of 10-15%; S2. Extraction of modified egg yolk antibodies: Two weeks after completing the four-injection immunization of laying hens, eggs were collected daily for three consecutive days. The collected eggs were separated into egg yolk fluid. The egg yolk fluid was mixed with deionized water at a weight ratio of 1:8, and hydrochloric acid was added to adjust the pH to 4.5-5. The mixture was stored at -18 to -20°C for 12 hours, and then thawed at 4-5°C. The supernatant was filtered to obtain an aqueous solution of modified egg yolk antibodies. The aqueous solution of modified egg yolk antibodies was subjected to two salting-out processes and then concentrated by ultrafiltration to obtain a concentrated modified egg yolk antibody solution. The concentrated modified egg yolk antibody solution was freeze-dried to obtain freeze-dried modified egg yolk antibody powder.

2. The biological agent based on egg yolk antibodies for improving the survival rate of fish fry according to claim 1, characterized in that, The mixed grain powder is made by mixing rice bran and wheat bran in a 1:1 weight ratio.

3. The biological agent based on egg yolk antibodies for improving the survival rate of fish fry according to claim 1, characterized in that, In step S1, during the first two injections, 0.4 mL is injected subcutaneously into the neck and 0.3 mL is injected into each of the left and right pectoral muscles. During the third injection, 0.4 mL is injected subcutaneously into the neck and 0.4 mL is injected into each of the left and right pectoral muscles. During the fourth injection, 0.6 mL is injected subcutaneously into the neck and 0.5 mL is injected into each of the left and right pectoral muscles.

4. The biological agent based on egg yolk antibodies for improving the survival rate of fish fry according to claim 1, characterized in that, The method for two salting-out processes of the modified egg yolk antibody aqueous solution in step S2 is as follows: First salting out: Add 40% ammonium sulfate solution to the modified egg yolk antibody aqueous solution to make the volume ratio of modified egg yolk antibody aqueous solution to ammonium sulfate solution 5:

3. After stirring evenly, store at 4℃ for 12h. After centrifugation at 10000~12000rpm for 15min, take the precipitate and use deionized water to make up to the original volume of modified egg yolk antibody aqueous solution. Second salting out: Add 40% sodium sulfate solution to the modified egg yolk antibody aqueous solution to make the volume ratio of modified egg yolk antibody aqueous solution to sodium sulfate solution 5:1 after the first salting out. After stirring evenly, store at 4℃ for 12h. After centrifugation at 10000~12000rpm for 15min, take the precipitate and make up to the original volume of modified egg yolk antibody aqueous solution with deionized water to obtain modified egg yolk antibody extract.

5. A biological agent based on egg yolk antibodies for improving the survival rate of fish fry according to claim 4, characterized in that, The ultrafiltration concentration method in step S2 is as follows: The modified egg yolk antibody extract was concentrated by ultrafiltration using an ultrafiltration membrane until the solution volume was 1 / 8 to 1 / 6 of the original modified egg yolk antibody extract volume. The ultrafiltration membrane was then washed with a 1-2 mol / L NaOH solution until the original modified egg yolk antibody extract volume was reached, yielding a washing solution. The NaOH solution in the washing solution was then washed and replaced with deionized water. 20-30% of the original modified egg yolk antibody extract volume of a 75% ethanol solution was added, and the solution was stored at 4°C for 12 hours to obtain the modified egg yolk antibody concentrate.

6. A biological agent based on egg yolk antibodies for improving the survival rate of fish fry according to claim 5, characterized in that, The freeze-drying method in step S2 is as follows: The modified egg yolk antibody concentrate was frozen at -20℃ for 24 hours, then frozen at -70℃ for 2 hours, and then freeze-dried at a vacuum of 15~20Pa for 36 hours to obtain freeze-dried modified egg yolk antibody powder.

7. The application of a yolk antibody-based biological agent for improving fish fry survival rate according to any one of claims 1 to 6, characterized in that, The biological agent is applied to grass carp farming, specifically through the following method: During the fry feeding period from April to October, the weight ratio of biological agents to ordinary fish feed was 1:3, and the content of modified egg yolk antibodies in the biological agents was 2 wt%. During the fry feeding period from November to March of the following year, the weight ratio of biological agents to ordinary fish feed was 1:5, and the content of modified egg yolk antibodies in the biological agents was 0.2wt%.

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