A method for incubating early-stage chicken eggs by opening windows and its application

Abstract

This invention provides a method for window-opening incubation of early-stage chicken embryos and its application, belonging to the field of poultry genetic engineering technology. The method for window-opening incubation of early-stage chicken embryos provided by this invention includes the following steps: Take an embryo that has been incubated for 56-60 hours with its tip facing upwards, open a window at the midpoint between the tip and the equatorial end, observe the embryo by shaking it, add a penicillin-streptomycin solution to the open egg, seal it, and incubate the egg without turning it during the incubation process. The method provided by this invention can significantly improve the hatching rate of chicken embryos and can, to a certain extent, improve the efficiency of preparing genetically modified chicken individuals, saving costs while simplifying the operation process. By combining the method provided by this invention with the technique of microinjecting gene-modifying vectors into chicken embryos, the efficient preparation of gene-edited chickens and transgenic chickens can be promoted, laying a technical foundation for applications such as gene breeding, biomedicine, and gene function research models.

Description

Technical Field

[0001] This invention belongs to the field of poultry genetic engineering technology, specifically relating to a method for incubating early-stage chicken eggs by opening windows and its application. Background Technology

[0002] Chickens are an important source of meat and eggs, and also a commonly used model organism. Through biotechnology, the genetic material of chickens is artificially altered to create genetically modified chickens, including gene-edited chickens and transgenic chickens. These technologies can accelerate the breeding of superior breeds with significantly enhanced traits such as disease resistance and growth rate; simultaneously, genetically modified chickens can serve as bioreactors for high-yield medicinal proteins; and they can also serve as model organisms for developmental biology and genetics research, promoting progress in related basic research. Currently, the method for creating transgenic chickens typically involves injecting viruses packaged with transgenic vectors into the blood vessels of early-stage chicken embryos, while the method for creating gene-edited chickens typically involves injecting in vitro gene-edited chicken germ cells into the blood vessels of early-stage chicken embryos.

[0003] Early embryo windowing is a necessary and crucial technology for producing genetically modified chickens. However, the process can lead to a series of problems, including mechanical damage to the embryo, microbial infection, impaired gas exchange, and moisture loss, ultimately affecting the survival and hatching success of the embryo. Previously, the embryo windowing and incubation process for producing genetically modified chickens involved taking embryos incubated for approximately 2.5 days (HH14-16), making a 0.75-1.5 cm diameter hole at the blunt end of the egg, sealing the egg with plastic wrap, continuing incubation, and turning the egg. After this process, the embryos would gradually stop developing or die during subsequent incubation, with a hatching rate generally not exceeding 20%. For embryos that underwent additional microinjection, the hatching rate would be even lower, severely restricting the efficiency of producing genetically modified chickens and their subsequent application and promotion. Therefore, there is an urgent need to establish a stable and efficient new technology for early embryo windowing and incubation. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a method for incubating early-stage chicken eggs by opening windows, which can significantly improve the hatching rate of chicken eggs.

[0005] The second objective of this invention is to provide the application of the above method in the preparation of genetically modified chickens.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for incubating early-stage chicken eggs by opening a window, comprising the following steps: taking an egg that has been incubated for 56-60 hours with the tip facing upwards, opening a window at the midpoint between the tip and the equatorial end, shaking the egg to observe the chicken embryo, adding a penicillin-streptomycin solution to the egg with the window, sealing it, and incubating the egg without turning it during the incubation process.

[0008] Preferably, the diameter of the window is 0.5 cm.

[0009] Preferably, the concentration of penicillin in the penicillin-streptomycin solution is 10000 U / mL, and the concentration of streptomycin is 10 mg / mL.

[0010] Preferably, the amount of penicillin-streptomycin solution added is 50-100 μL.

[0011] Preferably, the penicillin-streptomycin solution is added directly above the chicken embryo inside the windowed egg.

[0012] Preferably, the sealing material is medical tape.

[0013] Preferably, the incubation temperature is 37.8℃ and the humidity is 65%.

[0014] Preferably, when incubating the eggs after sealing, the pointed end should be facing upwards.

[0015] This invention also provides the application of the above method in the preparation of genetically modified chickens.

[0016] This invention also provides the application of the above method in chicken embryo development research.

[0017] The beneficial effects of this invention are:

[0018] The method for window-opening incubation of early-stage chicken eggs provided by this invention can significantly improve the hatching rate of chicken eggs and, to a certain extent, increase the efficiency of preparing genetically modified chicken individuals, while simplifying the operation process and saving costs. Furthermore, the method provided by this invention can be extended to research on chicken embryo development, such as inoculation with viruses and the addition of drugs. By combining the method provided by this invention with the technique of microinjecting gene-modifying vectors into chicken embryos, the efficient preparation of gene-edited chickens and transgenic chickens can be promoted, laying a technical foundation for applications in gene breeding, biomedicine, and gene function research models. Attached Figure Description

[0019] Figure 1 The images show the opening and sealing of the embryos in Example 1 and Comparative Example 1, where A is the opening and sealing of the embryos in Comparative Example 1 and B is the opening and sealing of the embryos in Example 1.

[0020] Figure 2 The chart shows the hatching rate of eggs for the methods in Example 1 and Comparative Example 1, where Comparative Example 1 is shown before optimization and Example 1 is shown after optimization. *** indicates P<0.001.

[0021] Figure 3This is a statistical chart comparing the hatching rates of eggs under different single-factor comparisons during windowed incubation. A represents the hatching rate with and without the addition of double antibiotics; B represents the hatching rate with window diameters of 0.75cm and 0.5cm; C represents the hatching rate with windows opened at the blunt end and the equatorial end; D represents the hatching rate with eggs sealed using plastic wrap and medical tape; E represents the hatching rate with and without turning the eggs when they are re-inserted after windowing; and F represents the hatching rate with windows opened at the blunt end, the pointed end, and the middle position of the equatorial end. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. Detailed Implementation

[0022] This invention provides a method for incubating early-stage chicken eggs by opening a window, comprising the following steps: taking an egg that has been incubated for 56-60 hours with the tip facing upwards, opening a window at the midpoint between the tip and the equatorial end, shaking the egg to observe the chicken embryo, adding a penicillin-streptomycin solution to the egg with the window, sealing it, and incubating the egg without turning it during the incubation process.

[0023] In this invention, when incubating fresh eggs, it is preferable to first soak them in a benzalkonium chloride solution for 5 minutes before incubation. The preferred incubation temperature is 37.8°C, and the preferred incubation humidity is 65%. The blunt end of the egg is preferably facing upwards during incubation. In some embodiments of this invention, eggs incubated for 56, 57, 58, 59, or 60 hours can be windowed. In this invention, before windowing, the pointed end of the egg is facing upwards, and a window is opened at the midpoint between the pointed end and the equatorial end to observe the embryo. If the embryo is not visible at the window opening, the egg is shaken until it is visible at the window opening.

[0024] In this invention, the diameter of the window opening is preferably 0.5 cm. After opening the window, a penicillin-streptomycin solution is preferably added directly above the chicken embryo inside the windowed egg. The concentration of penicillin in the penicillin-streptomycin solution is preferably 10000 U / mL, the concentration of streptomycin is preferably 10 mg / mL, the solvent of the penicillin-streptomycin solution is preferably 0.85% physiological saline, and the amount of penicillin-streptomycin solution added is preferably 50-100 μL, more preferably 60-90 μL, and even more preferably 70-80 μL.

[0025] In this invention, the sealing material is preferably medical tape, and the length of the medical tape is preferably 1.5 cm. After sealing, the embryo is preferably incubated with the pointed end facing upwards. The incubation temperature is preferably 37.8°C, and the incubation humidity is preferably 65%.

[0026] In this field, when fertilized chicken eggs are incubated, the blunt end, that is, the end containing the air cell, is typically facing upwards. At this point, the less dense yolk rises, causing the embryo on the yolk surface to float and approach the air cell, facilitating respiration. This field employs a method of opening a window at the blunt end. First, the embryo is located as much as possible using a candler, then the eggshell is broken open. Based on the embryo's location, the shell membrane covering the embryo is peeled away from the outer edge to expose it. If the embryo is completely obscured by the air cell, it cannot be located using a candler; in this case, a window must be blindly opened at the blunt end and gradually enlarged until the embryo beneath the shell membrane is visible. Opening a window at the blunt end provides a buffer zone within the air cell, reducing the risk of accidentally damaging the yolk sac when breaking the shell membrane. It also simplifies embryo handling and egg sealing, and prevents egg liquid from spilling out. However, this invention is the first to propose opening a window midway between the pointed end and the equatorial end of the egg, which significantly improves the hatching rate.

[0027] This invention proposes adding a penicillin-streptomycin solution above the chicken embryo inside the egg before sealing the windowed egg. This inhibits bacterial contamination and prevents the embryo surface from drying out. Existing windowing methods in the art involve using tweezers to create windows with diameters ranging from 0.75 to 1.5 cm to ensure accessibility of the blunt-end embryo (meaning exposure and micromanipulation of the blunt-end embryo). Larger apertures facilitate micromanipulation. This invention controls the window on the eggshell to a diameter of 0.5 cm, reducing moisture evaporation and the risk of contamination, and also providing favorable conditions for sealing the windowed egg. Previously, sealing eggs typically involved applying egg white to the outer shell of the window and sealing it with a piece of cling film. This invention optimizes this process by using sterile, breathable medical tape to directly seal the egg window, offering advantages such as a tight seal, breathability, ease of operation, and reduced risk of contamination. Conventionally, in the subsequent incubation of windowed eggs, a turning step is performed to prevent the embryo from adhering to the shell and affecting development. This invention is the first to propose that open-window eggs, which do not require turning, have a higher hatching rate.

[0028] In summary, this invention significantly improves the hatching rate of chicken eggs by optimizing conditions such as antibiotic addition, window aperture, window location, egg sealing material, and incubation turning.

[0029] This invention also provides the application of the above method in the preparation of genetically modified chickens or in chicken embryo development research. In this invention, the genetically modified chicken preferably includes gene-edited chickens or transgenic chickens, and the preferred breed of chicken includes the White Leghorn.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Unless otherwise specified, the following embodiments are all conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] In the following examples, the data analysis software used for statistical analysis was GraphPadPrism. The results are presented in the form of mean ± standard error; the significance of the difference between the two groups of data was determined using Ttests. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0034] Example 1

[0035] A method for incubating early-stage chicken eggs by opening a window, the steps of which are as follows:

[0036] Fresh eggs were soaked in a benzalkonium chloride solution for 5 minutes and then placed in an incubator at 37.8°C and 65% humidity with the blunt end facing upwards for incubation.

[0037] Take an egg that has been incubated for 56 hours, place it with the pointed end facing upwards, and use tweezers to make a window (shell-breaking hole) 0.5 cm in diameter between the pointed end and the equatorial end. After gently shaking the egg and observing the embryo at the window, add 50 μL of penicillin-streptomycin solution (purchased from Thermo Fisher, #15140122) directly above the embryo. The solution is 0.85% physiological saline, with a penicillin concentration of 10000 U / mL and a streptomycin concentration of 10 mg / mL. Then, cut a 1.5 cm length of medical tape and stick it to the window to seal the egg.

[0038] After opening the window and sealing the embryos, place them in the incubator with the pointed end facing up, and incubate them at a temperature of 37.8℃ and a humidity of 65% without turning them over.

[0039] Example 2

[0040] A method for incubating early-stage chicken eggs by opening a window, the steps of which are as follows:

[0041] Fresh eggs were soaked in a benzalkonium chloride solution for 5 minutes and then placed in an incubator at 37.8°C and 65% humidity with the blunt end facing upwards for incubation.

[0042] Take an egg that has been incubated for 60 hours, place it with the pointed end facing upwards, and use tweezers to make a window (shell-breaking hole) 0.5 cm in diameter between the pointed end and the equatorial end. Observe the embryo. If the embryo is not observed, gently shake the egg until it can be observed at the window. Then, add 100 μL of penicillin-streptomycin solution (purchased from Thermofisher, #15140122) directly above the embryo in the egg with the window. The solution is made of 0.85% physiological saline, and the concentration of penicillin in the solution is 10000 U / mL, and the concentration of streptomycin is 10 mg / mL. Then, cut a 1.5 cm long piece of medical tape and stick it to the window to seal the egg.

[0043] After opening the window and sealing the embryos, place them in the incubator with the pointed end facing up, and incubate them at a temperature of 37.8℃ and a humidity of 65% without turning them over.

[0044] Example 3

[0045] A method for incubating early-stage chicken eggs by opening a window, the steps of which are as follows:

[0046] Fresh eggs were soaked in a benzalkonium chloride solution for 5 minutes and then placed in an incubator at 37.8°C and 65% humidity with the blunt end facing upwards for incubation.

[0047] Take an egg that has been incubated for 58 hours, place it with the pointed end facing upwards, and use tweezers to make a window (shell-breaking hole) 0.5 cm in diameter between the pointed end and the equatorial end. Observe the embryo. If the embryo is not observed, gently shake the egg until it can be observed at the window. Then, add 80 μL of penicillin-streptomycin solution (purchased from Thermofisher, #15140122) directly above the embryo in the egg with the window. The solution is 0.85% physiological saline, and the concentration of penicillin in the solution is 10000 U / mL, and the concentration of streptomycin is 10 mg / mL. Then, cut a 1.5 cm length of medical tape and stick it to the window to seal the egg.

[0048] After opening the window and sealing the embryos, place them in the incubator with the pointed end facing up, and incubate them at a temperature of 37.8℃ and a humidity of 65% without turning them over.

[0049] Comparative Example 1

[0050] The difference from Example 1 is that a window is made at the blunt end, with a window diameter of 0.75 cm. It is sealed with egg white and plastic wrap, and the blunt end faces upwards during incubation, with an egg-turning step. All other aspects are the same as in Example 1. The specific method of this comparative example 1 is as follows:

[0051] Fresh eggs were soaked in a benzalkonium chloride solution for 5 minutes, then placed in an incubator at 37.8℃ and 65% humidity with the blunt end facing upwards for incubation. After 56 hours of incubation, the eggs were removed and placed at room temperature with the blunt end facing upwards. The position of the embryo at the blunt end was roughly predicted using an egg candler, and marked on the eggshell with a marker. Sterilized tweezers were used to gently tap the marked area to create a crack, and then shell fragments were removed until the window diameter was 0.75cm. The embryo inside the window was observed visually, and the white inner shell membrane was broken open and removed with tweezers. Egg white was applied to the outer edge of the window using a sterile cotton swab, and a 2cm square piece of cling film was cut and sealed. The sealed egg was returned to the incubator at 37.8℃ and 65% humidity, blunt end facing upwards. After 2 days of incubation, the eggs were turned, rotating 45° to the left or right every 90 minutes.

[0052] The window opening and sealing diagrams of Example 1 and Comparative Example 1 are shown below. Figure 1 As shown. After cleaning the eggs, incubation began, and 24 hours was counted as one day of embryonic development, embryonic age ED1. After the window opening operation, incubation continued, and eggs were transferred to hatching trays at ED19. The hatching rate of Example 1 and Comparative Example 1 was calculated between ED21 and ED22. Hatching rate (%) = (Total hatched eggs / Total number of eggs placed in incubation after window opening) × 100%. The method of Example 1 was marked as optimized, and the method of Comparative Example 1 was marked as unoptimized. The results are shown below. Figure 2 As shown, the hatching rate of the early-stage chicken egg hatching method of the present invention is significantly higher than that of the conventional egg hatching method used in the art (Comparative Example 1).

[0053] Example 4

[0054] Single-factor optimization of the chick hatching method based on hatching rate was conducted, involving six single-factor experiments. The fertilized eggs in each group came from the same batch, and the operations and tests were performed simultaneously. However, the fertilized eggs in different single-factor experimental groups came from different batches, and the experiments were not conducted simultaneously, resulting in slight differences in hatching rates. The steps are as follows:

[0055] (1) Cleaning and incubation of hatching eggs

[0056] Collect a sufficient number of fresh Leghorn chicken eggs, soak them in a benzalkonium chloride solution for 5 minutes, wipe them clean, and then mark them into groups of at least 25 eggs each. At least two replicates should be conducted. Place the eggs evenly on a tray with the blunt end facing up, and incubate them uniformly in an incubator at 37.8℃ and 65% humidity. Timing begins after incubation; 24 hours is considered one day of embryonic development, or ED1.

[0057] (2) Opening the window of early-stage embryos and continuing incubation

[0058] Prepare an egg candling device, tweezers, fresh egg whites, sterile cotton swabs, plastic wrap, scissors, medical tape, etc. in advance. Remove the eggs that have been incubated for 56 hours.

[0059] ① Comparison of adding double antibiotics and not adding antibiotics: The embryos were placed at room temperature with the blunt end facing up; the position of the embryo on the blunt end was roughly predicted using an egg candler; the marked area of ​​the embryo was gently tapped with sterilized tweezers to create a crack, and then the shell fragments were removed until the window diameter was 0.5cm; the embryo inside the window was observed with the naked eye, and the white inner shell membrane was broken and removed with tweezers; 50μL of penicillin-streptomycin solution (the penicillin-streptomycin solution is the same as in Example 1) was added to the embryo through the window in one group, and no solution was added to the other group; 1.5cm of medical tape was cut and sealed; the sealed eggs with the window were placed back in the incubator with the blunt end facing up, and the eggs were turned over after 2 days of incubation, turning them 45° to the left or right every 90 minutes.

[0060] ② Comparison of window opening size: The embryos were placed at room temperature with the blunt end facing up; the position of the embryo on the blunt end was roughly predicted using an egg candler; the marked area of ​​the embryo was gently tapped with sterilized tweezers to create a crack, and one group removed eggshell fragments until the window opening diameter was 0.75cm, and the other group removed eggshell fragments until the window opening diameter was 0.5cm; the embryo inside the window was observed with the naked eye, and the white inner shell membrane was broken and removed with tweezers; 50μL of penicillin-streptomycin solution (the penicillin-streptomycin solution is the same as in Example 1) was added above the embryo through the window; 1.5cm of medical tape was cut and sealed; the sealed eggs with the window opened were placed back in the incubator with the blunt end facing up, and the eggs were turned over after 2 days of incubation, turning them 45° to the left or right every 90 minutes.

[0061] ③ Comparison of the window openings at the blunt end and the equatorial end: One group of eggs was placed at room temperature with the blunt end facing upwards. The position of the embryo at the blunt end was roughly predicted using an egg candling device. The marked area of ​​the embryo was gently tapped with sterilized tweezers to form a crack. Eggshell fragments were removed until the window opening diameter was 0.5cm. The embryo inside the window was observed with the naked eye. The white inner shell membrane was then broken open and removed with tweezers. Another group of eggs was placed horizontally at room temperature. The position of the embryo at the equatorial end was roughly predicted using an egg candling device. The marked area was gently tapped with sterilized tweezers to form a crack. Eggshell fragments were removed until the window opening diameter was 0.5cm. 50μL of penicillin-streptomycin solution (the same penicillin-streptomycin solution as in Example 1) was added above the embryo through the window in both groups. 1.5cm of medical tape was cut and sealed. The sealed eggs with the window opening were placed back in the incubator with the window facing upwards. The equatorial end group was adjusted to have the blunt end facing upwards after 12 hours. The eggs were turned after 2 days of incubation, turning them 45° to the left or right every 90 minutes.

[0062] ④ Comparison of sealing materials: The embryos were placed horizontally at room temperature. The position of the chicken embryo at the equatorial end was roughly predicted using an egg candler. After gently tapping with sterilized tweezers to create a crack, the eggshell fragments were removed until the window diameter was 0.5cm. 50μL of penicillin-streptomycin solution (the same penicillin-streptomycin solution as in Example 1) was added above the chicken embryo through the window. One group used sterile cotton swabs to apply the prepared egg white to the outer edge of the eggshell window and then cut 2cm square pieces of plastic wrap to seal it. Another group used 1.5cm long pieces of medical tape to seal it. The sealed eggs with the window opened were placed back in the incubator with the window facing upwards and placed horizontally for 12 hours, then adjusted so that the blunt end was facing upwards. After 2 days of incubation, the eggs were turned over, 45° to the left or right every 90 minutes.

[0063] ⑤ Comparison of whether or not the eggs were turned again after re-incubation: The embryos were placed horizontally at room temperature. The position of the chicken embryo at the equatorial end was roughly predicted using an egg candling device. After gently tapping with sterilized tweezers to create a crack, the eggshell fragments were removed until the window diameter was 0.5cm. 50μL of penicillin-streptomycin solution (the same penicillin-streptomycin solution as in Example 1) was added above the chicken embryo through the window. 1.5cm of medical tape was cut and sealed. The sealed egg with the window facing up was placed back in the incubator and placed horizontally for 12 hours, then adjusted so that the blunt end faced up. After 2 days of incubation, one group of eggs was turned, turning 45° to the left or right every 90 minutes, while the other group was not turned.

[0064] ⑥ Comparison of the window openings at the midpoint between the blunt end and the pointed end and the equatorial end: One group of eggs was placed at room temperature with the blunt end facing upwards. The position of the embryo at the blunt end was roughly predicted using an egg candler. The marked area of ​​the embryo was gently tapped with sterilized tweezers to form a crack. Eggshell fragments were removed until the window opening diameter was 0.5 cm. The embryo inside the window was observed with the naked eye. The white inner shell membrane was then broken open and removed with tweezers. Another group of eggs was placed at room temperature with the pointed end facing upwards. The midpoint between the pointed end and the equatorial end was gently tapped with sterilized tweezers to form a crack. Eggshell fragments were removed until the window opening diameter was 0.5 cm. The embryo was observed. For both groups, 50 μL of penicillin-streptomycin solution (the same penicillin-streptomycin solution as in Example 1) was added above the embryo through the window. A 1.5 cm long piece of medical tape was cut and sealed. The sealed eggs with the window opened were placed back in the incubator with the pointed end facing upwards, without turning the eggs.

[0065] (3) Hatching statistics of open-window embryos

[0066] Eggs were transferred to hatching trays on day 19 of embryonic development, and turning of all eggs was stopped. On days 21-22 of embryonic development, the number of successfully hatched chicks was observed and recorded. Hatching rate (%) = (Total hatched chicks / Total number of eggs placed in incubation after opening the hatching window) × 100%. Results are as follows: Figure 3As shown, in the early stage of chicken embryo incubation, adding antibiotics, opening the window with a diameter of 0.5 cm, opening the window at the midpoint between the tip and the equatorial end, sealing with medical tape, and not turning the egg after opening the window are significantly better than not adding antibiotics, opening the window with a diameter of 0.75 cm, opening the window at the blunt end or the equatorial end, sealing with egg white and plastic wrap, and turning the egg after opening the window.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for incubating early-stage chicken eggs by opening a window, characterized in that, The steps include: Take an egg that has been incubated for 56-60 hours with the tip facing up, make a window at the midpoint between the tip and the equatorial end, shake it to observe the chicken embryo, add penicillin and streptomycin solution to the egg with the window, seal it and incubate the egg without turning it during the incubation process. The diameter of the window is 0.5cm; The concentration of penicillin in the penicillin-streptomycin solution is 10000 U / mL, and the concentration of streptomycin is 10 mg / mL. The amount of penicillin-streptomycin solution added is 50-100 μL; Add the penicillin-streptomycin solution directly above the chicken embryo inside the windowed egg; The sealing material is medical tape; The incubation temperature was 37.8℃, and the humidity was 65%. When incubating the eggs after sealing, the pointed end should be facing upwards.

2. The application of the method of claim 1 in the preparation of genetically modified chickens.

3. The application of the method described in claim 1 in chicken embryo development research.

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