Cryopreservation kit for chicken semen and use method of cryopreservation kit

Abstract

The invention relates to the technical field of poultry breed conservation biology, and discloses a cryopreservation kit for chicken semen and a use method of the cryopreservation kit. The invention relates to a cryopreservation kit for chicken semen. The cryopreservation kit comprises a cryopreservation diluent, a cryoprotectant, an insemination diluent, a DMEM (dulbecco's modified eagle medium) and a frozen semen tube. The cryopreservation diluent in the kit provided by the invention can provide a better living environment for chicken sperms, and the cryoprotectant in the kit can successfully perform cryopreservation on the chicken sperms; when the kit provided by the invention is used for freezing and storing chicken semen, the unfrozen chicken semen is used for artificial insemination, the fertilization rate can reach 70% or above, and the requirement for breed conservation of chicken genetic resources is met.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of poultry germplasm preservation biotechnology, in particular, to a freezing preservation kit for chicken semen and a method of use thereof. BACKGROUND

[0002] Semen cryopreservation is a technology for long-term preservation of sperm in a supercooling environment, which is an important technical means for livestock and poultry translocation breeding, and has important application value in the protection of endangered species and the rapid propagation of important economic animals. However, due to the unique morphological structure characteristics of poultry sperm and other factors, the development of poultry semen cryopreservation technology is slow, and a stable and efficient cryopreservation technology system needs to be established.

[0003] The semen freezing and application process is generally divided into semen dilution, balancing, freezing, thawing, glycerol removal and artificial insemination, etc. The main factor affecting the freezing efficiency of poultry semen is the freezing technical parameters, including the type and concentration of cryoprotectant, the composition and concentration of diluent, the cooling speed, the type of semen packaging and the thawing temperature, etc. Since 1951, POLGE et al. (1951), MITCHELL et al. (1977), LAKE et al. (1978), DUPLAIX et al. (1984), SEIGNEURIN (1995), TSELUTIN et al. (1999), Han Xuefeng (2003), LONG et al. (2004), Deng Tuoshun (2013), Yang Xiaohu et al. (2018) and other domestic and foreign scholars have carried out nearly 70 years of research. Some countries have developed methods for freezing and preserving poultry semen, but the maturity and stability of the technology are poor. At present, many diluents are used to make frozen semen, including 5% to 7% glucose solution, 1% NaCl solution and glucose yolk solution, as well as Lake-type semen diluent and BHSV-type semen diluent, etc., but their effects are not ideal. Practical research has proved that the preparation of diluent with appropriate pH, osmotic pressure, energy substance and antioxidant content is crucial for reducing freezing damage and protecting chicken sperm motility and fertility, and affects the effectiveness of chicken frozen semen production and chicken genetic resource protection. There are many types of diluents used for semen freezing, including preliminary balancing diluent, cryoprotective solution, glycerol removal diluent, insemination diluent, etc. The preparation and quality control process is complex, special instruments and consumables are needed, which can easily lead to unstable preparation effect, etc. The sperm motility preservation time and fertility rate of chicken semen after thawing are not ideal, which is a recognized technical problem in this field (MURUGESAN and MAHAPATRA, 2016; Nie Ruixue et al. 2021), therefore, it is urgent to establish a chicken semen freezing preservation kit that can efficiently, high-quality and conveniently realize the freezing preservation of chicken semen and improve the work effectiveness of chicken frozen semen preparation. SUMMARY

[0004] The application aims to provide a freezing preservation kit for chicken semen and a use method thereof, which can effectively freeze and preserve chicken semen.

[0005] To achieve the above-mentioned purpose, the application provides the following technical scheme. A freezing preservation kit for chicken semen comprises: A freezing preservation diluent, a cryoprotectant, an insemination diluent, DMEM and a fine frozen semen tube.

[0006] Further, the freezing preservation diluent contains 5.5-6.0 g of glucose, 14.5-15.5 g of sodium L-glutamate, 1.20-1.30 g of potassium citrate, 0.80-0.88 g of magnesium acetate, 30.5-31.5 g of N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid, 10-40 mL of 200 mmol / L pyrroloquinoline quinone, and the rest is water, and the osmotic pressure is 340-390 mOsm / kg per 1000 mL.

[0007] Further, the cryoprotectant contains 7.4-8.2 g of glucose, 19.0-20.0 g of sodium L-glutamate, 5.0-5.3 g of potassium acetate, 0.75-0.80 g of magnesium acetate, 2.7-3.0 g of polyvinylpyrrolidone, 0.13-0.14 L of glycerol, and the rest is water, and the osmotic pressure is 340-390 mOsm / kg per 1000 mL.

[0008] Further, the insemination diluent contains 5.5-6.0 g of D-fructose, 18.5-19.5 g of sodium L-glutamate, 1.2-1.4 g of potassium citrate, 0.70-0.80 g of magnesium acetate, 5.0-5.5 g of tris-hydroxymethyl aminomethyl ethanesulfonic acid, 5.0-5.5 g of sodium acetate, and the rest is water, and the osmotic pressure is 340-390 mOsm / kg per 1000 mL.

[0009] Further, the specification of the fine frozen semen tube is 0.25 mL or 0.50 mL.

[0010] Further, the volume ratio of the freezing preservation diluent, the cryoprotectant, the insemination diluent and the DMEM is 1:2:8:4.

[0011] A use method of a freezing preservation kit for chicken semen comprises the following steps: Semen was collected from roosters using a dorsal and ventral massage method and placed in a 1.5 mL centrifuge tube. 200 μL of semen was then mixed thoroughly with 200 μL of cryopreservation diluent preheated to 37°C and equilibrated at 4°C for 20–30 min. Subsequently, 400 μL of cryoprotectant was added and mixed thoroughly, and the mixture was equilibrated at 4°C for 10–20 min. The mixture was then transferred to cryopreservation tubes and placed in a programmed freezer for freezing. The freezing program was set to 4°C to -44°C at a rate of 12°C / min, and -44°C to -120°C at a rate of 4... The sperm was heated to 0℃ / min and then stored in liquid nitrogen. During thawing, the frozen sperm tube was removed from the liquid nitrogen and immersed in 4℃ water for 3 minutes within 10 seconds. After the frozen semen was completely thawed, it was poured into a 5mL centrifuge tube, and the volume was measured. 2-3 times the volume of insemination diluent was added in 5-7 portions, with an interval of 2-3 minutes between each addition. The mixture was then centrifuged at 4℃ for 8-10 minutes with a centrifugal force of 600g. The supernatant was removed, and the sperm precipitate was retained. The precipitate was resuspended in DMEM solution with an equal volume of thawed semen. After testing for sperm motility, the mixture could be used for artificial insemination.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: The cryopreservation diluent in the kit provided by this invention can provide a better survival environment for chicken semen, and the cryoprotectant in the kit can successfully cryopreserve chicken semen. The kit provided by this invention allows for the cryopreservation of chicken semen, which, after thawing, can be used for artificial insemination, achieving a fertilization rate of over 70%, thus meeting the needs of preserving chicken genetic resources. Detailed Implementation

[0013] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0014] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0015] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0016] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0017] In the following examples, all raw materials used were commercially available.

[0018] In the following examples, a cryopreservation kit for chicken semen includes: Cryopreservation diluents, cryoprotectants, insemination diluents, DMEM, and frozen sperm tubes.

[0019] The preparation method of the cryopreservation diluent includes the following steps: weighing 5.5-6.0g of glucose, 14.5-15.5g of L-glutamate sodium, 1.2-1.3g of potassium citrate, 0.80-0.88g of magnesium acetate, 30.5-31.5g of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, and 10-40 mL of 200mmol / L pyrroloquinoline quinone, adding ultrapure water to make up to 1000 mL, adjusting the pH to 7.0-7.4 with sodium hydroxide, sterilizing by filtration, and sealing and storing at -20℃ to obtain the cryopreservation diluent; The preparation method of the cryoprotectant includes the following steps: weighing 7.4-8.2g of glucose, 19.0-20.0g of L-glutamate sodium, 5.0-5.3g of potassium acetate, 0.75-0.80g of magnesium acetate, 2.7-3.0g of polyvinylpyrrolidone, and 0.13-0.14L of glycerol (add glycerol immediately before use), adding ultrapure water to a final volume of 1000mL, adjusting the pH to 7.0-7.4 with sodium hydroxide, sterilizing by filtration, and sealing and storing at -20℃ to obtain the cryoprotectant; The preparation method of the insemination diluent includes the following steps: weighing 5.5-6.0g of D-fructose, 18.5-19.5g of L-glutamate sodium, 1.2-1.4g of potassium citrate, 0.70-0.80g of magnesium acetate, 5.0-5.5g of trimethylolpropionic acid, and 5.0-5.5g of sodium acetate, adding ultrapure water to a final volume of 1000mL, adjusting the pH to 7.0-7.4 with sodium hydroxide, sterilizing by filtration, and sealing and storing at -20℃ to obtain the insemination diluent; The frozen seminal tubes are 0.25 mL or 0.50 mL in size. The volume ratio of the cryopreservation diluent, cryoprotectant, insemination diluent, and DMEM is 1:2:8:4.

[0020] The following examples illustrate a method for using a cryopreservation kit for chicken semen, comprising the following steps: Semen was collected from roosters using a dorsal and ventral massage method and placed in a 1.5 mL centrifuge tube. 200 μL of semen was then mixed thoroughly with 200 μL of cryopreservation diluent preheated to 37°C and equilibrated at 4°C for 20–30 min. Subsequently, 400 μL of cryoprotectant was added and mixed thoroughly, and the mixture was equilibrated at 4°C for 10–20 min. The mixture was then transferred to cryopreservation tubes and placed in a programmed freezer for freezing. The freezing program was set to 4°C to -44°C at a rate of 12°C / min, and -44°C to -120°C at a rate of 4... The sperm was heated to 0℃ / min and then stored in liquid nitrogen. During thawing, the frozen sperm tube was removed from the liquid nitrogen and immersed in 4℃ water for 3 minutes within 10 seconds. After the frozen semen was completely thawed, it was poured into a 5mL centrifuge tube, and the volume was measured. 2-3 times the volume of insemination diluent was added in 5-7 portions, with an interval of 2-3 minutes between each addition. The mixture was then centrifuged at 4℃ for 8-10 minutes with a centrifugal force of 600g. The supernatant was removed, and the sperm precipitate was retained. The precipitate was resuspended in DMEM solution with an equal volume of thawed semen. After testing for sperm motility, the mixture could be used for artificial insemination.

[0021] Example 1 Prepare diluent, cryoprotectant and insemination diluent separately. (1) Diluent 100 mL of diluent contains: 0.60 g glucose, 1.52 g L-glutamate, 0.13 g potassium citrate, 0.08 g magnesium acetate, 3.05 g N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 1 mL of 20 mmol / L PQQ (pyrroloquinoline quinone), and the remainder is water. Adjust the pH to 7.0–7.4 with 1 mol / L–3 mol / L sodium hydroxide.

[0022] (2) Cryoprotectant 100 mL of cryopreservation solution contains: 0.80 g glucose, 1.92 g L-glutamate, 0.08 g magnesium acetate, 0.50 g potassium acetate, 0.30 g polyvinylpyrrolidone, and the balance is water. Adjust the pH to 7.0-7.4 with 1 mol / L-3 mol / L sodium hydroxide, and add 12 mL of glycerol before use.

[0023] (3) Insemination diluent 100 mL of thawing solution contains: 1.92 g L-glutamate, 0.12 g potassium citrate, 0.08 g magnesium acetate, 0.51 g sodium acetate, 0.60 g D-fructose, and 0.50 g tris(hydroxymethyl)ethanesulfonic acid. Adjust the pH to 7.0–7.4 with 1 mol / L–3 mol / L sodium hydroxide. Examples 2-4 Everything else is the same as in Example 1, except that in Examples 2-4, the amount of 20 mmol / L PQQ (pyrroloquinoline quinone) added per 100 mL of diluent is 2 mL, 3 mL and 4 mL respectively, and the corresponding final concentrations of PQQ are 400 μmol / L, 600 μmol / L and 800 μmol / L respectively.

[0024] Comparative Example 1 Everything else is the same as in Example 1, except that Comparative Example 1 does not contain PQQ, i.e., pyrroloquinoline quinone.

[0025] The chicken semen was cryopreserved using the cryopreservation kits for chicken semen obtained in Examples 1-4 and Comparative Example 1. The specific procedures were as follows: 1. Trim the feathers around the cloaca of a 28-week-old healthy rooster. Collect fresh semen from the rooster using the back and abdomen massage method and place it in a 1.5mL centrifuge tube. Evaluate the semen quality using a phase contrast microscope or a computer-aided sperm analysis system. Qualified semen with no contamination, sperm motility greater than 50%, and sperm density greater than 1.6 billion / mL will be used for subsequent operations. II. Semen Freezing and Thawing Procedures (1) Collect rooster semen using the back and abdomen massage method and place it in a centrifuge tube. Take 200 μL of semen and mix it evenly with 200 μL of diluent preheated to 37°C. Equilibrate at 4°C for 20-30 min. (2) Then add 400 μL of cryoprotectant and mix well. Equilibrate at 4℃ for 10-20 min. Then put it into a cryo-sperm tube and put it into a programmed freezer for cooling and freezing. The freezing program is set to 4℃ to -44℃ at a rate of 12℃ / min and -44℃ to -120℃ at a rate of 40℃ / min. Then put it into liquid nitrogen for storage. (3) When thawing, take the frozen semen tube out of liquid nitrogen and immerse it in water at 4°C for 3 minutes within 10 seconds. After the frozen semen is completely thawed, pour it into a centrifuge tube, measure the volume, add 2 to 3 times the volume of insemination diluent in 5 to 7 portions, with an interval of 2 to 3 minutes each time, and then centrifuge at 4°C for 8 to 10 minutes with a centrifugal force of 600g. Remove the supernatant, retain the sperm precipitate, add DMEM equal to the volume of thawed semen to resuspend the precipitate, and after testing the motility, it can be used for artificial insemination.

[0026] III. Determination of Frozen Sperm Motility Add an equal volume of 4°C DMEM to the sperm precipitate collected in step two to resuspend the precipitate, and then detect sperm motility using a phase contrast microscope or a computer-aided sperm analysis system.

[0027] IV. Structural Damage Measurement During Freezing Step 3: Using the same samples, determine sperm viability, sperm abnormality rate, sperm plasma membrane integrity, and sperm acrosome integrity.

[0028] V. Determination of fertilization rate 150 white-feathered aircraft carrier hens (5 groups of 30 each) were selected for artificial insemination. Each white-feathered aircraft carrier hen was inseminated with approximately 100 million sperm. After two consecutive days of insemination, the eggs were collected and labeled for incubation, and the fertilization rate was calculated.

[0029] VI. Test Results (1) The results of the comparison of sperm motility and sperm motility in different embodiments and comparative examples are shown in Table 1. Table 1. Comparison of sperm motility and performance between different embodiments and comparative examples.

[0030] (2) The results of the comparison of sperm structure and functional integrity between different embodiments and comparative examples are shown in Table 2. Table 2. Comparison of sperm structure and functional integrity between different embodiments and comparative examples.

[0031] Based on Tables 1-2, it can be seen that the PQQ added to the kit described in this invention can significantly improve the sperm motility and the integrity of its structure and function in frozen semen, and the protective effect of PQQ on sperm during freezing has a dose-dependent effect.

[0032] (3) The sperm fertilization rate results of different embodiments and comparative examples are shown in Table 3; Table 3. Sperm fertilization rate results of different embodiments and comparative examples.

[0033] As shown in Table 3, the addition of PQQ can significantly improve the fertilization capacity of frozen semen. The protective effect of PQQ on sperm and the maintenance of fertilization capacity during freezing have a dose-dependent effect, and the effect is best when the final concentration of PQQ in the diluent is 400 μmol / L.

[0034] (4) Comparison Example 1 and Example 2 are compared, specifically: ① Collection and quality testing of chicken semen Feathers around the cloaca of roosters were clipped. Fresh semen was collected from healthy roosters aged 40 weeks using a back-abdomen massage method and placed in a 1.5 mL centrifuge tube. Semen quality was assessed using a phase contrast microscope or a computer-aided sperm analysis system. Semen that was uncontaminated, had sperm motility greater than 50%, and sperm density greater than 1.6 billion / mL was used for further processing. ② Semen dilution and cryopreservation ③ Thawing ④ Determination of fertilization rate Two hundred white-feathered aircraft carrier hens (two groups of 100 each) were selected for artificial insemination. Each white-feathered aircraft carrier hen was inseminated with approximately 100 million sperm. After two consecutive days of insemination, the eggs were collected, labeled, and incubated for three days, and the fertilization rate was calculated.

[0035] ⑤ Test Results Comparative Example 1 yielded 250 hatching eggs, 190 fertilized eggs, and a fertilization rate of 76.0%; Example 2 yielded 270 hatching eggs, 230 fertilized eggs, and a fertilization rate of 85.2%.

[0036] ⑥ Conclusion After expanding the population size, the fertilization rate of Example 2 was significantly higher than that of Comparative Example 1 by χ² test, indicating that the effect of adding PQQ was best when the final concentration of PQQ in the diluent was 400 μmol / L.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A kit for cryopreservation of chicken semen, characterized in that, The cryopreservation kit for chicken semen includes: cryopreservation diluent, cryoprotectant, insemination diluent, DMEM, and frozen sperm tubes.

2. The cryopreservation kit for chicken semen according to claim 1, characterized in that, Each 1L of the cryopreservation diluent contains: 5.5–6.0g glucose, 14.5–15.5g L-glutamate, 1.20–1.30g potassium citrate, 0.80–0.88g magnesium acetate, 30.5–31.5g N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 10–40mL 200mmol / L pyrroloquinoline quinone, with the balance being water, and an osmotic pressure of 340–390 mOsm / kg.

3. The cryopreservation kit for chicken semen according to claim 1, characterized in that, Each 1L of the cryoprotectant contains: 7.4–8.2g glucose, 19.0–20.0g L-glutamate, 5.0–5.3g potassium acetate, 0.75–0.80g magnesium acetate, 2.7–3.0g polyvinylpyrrolidone, 0.12–0.14L glycerol, with the balance being water, and an osmotic pressure of 340–390 mOsm / kg.

4. The cryopreservation kit for chicken semen according to claim 1, characterized in that, Each 1L of the insemination diluent contains: 5.5–6.0g of D-fructose, 18.5–19.5g of L-glutamate, 1.2–1.4g of potassium citrate, 0.70–0.80g of magnesium acetate, 5.0–5.5g of trimethylolpropionic acid, 5.0–5.5g of sodium acetate, with the balance being water, and an osmotic pressure of 340–390 mOsm / kg.

5. The cryopreservation kit for chicken semen according to claim 1, characterized in that, The frozen microtubes are available in 0.25 mL or 0.50 mL sizes.

6. The cryopreservation kit for chicken semen according to claim 1, characterized in that, The volume ratio of the cryopreservation diluent, cryoprotectant, insemination diluent, and DMEM is 1:2:8:

4.

7. A method of using a cryopreservation kit for chicken semen as described in any one of claims 1 to 6, characterized in that, Includes the following steps: Semen was collected from roosters using a dorsal and ventral massage method and placed in centrifuge tubes. 200 μL of semen was mixed thoroughly with 200 μL of cryopreservation diluent preheated to 37°C and equilibrated at 4°C for 20–30 min. Then, 400 μL of cryoprotectant was added and mixed thoroughly, equilibrated at 4°C for 10–20 min. The mixture was then transferred to frozen semen tubes and placed in a programmed freezer for freezing. The freezing program was set to 4°C to -44°C at a rate of 12°C / min, and -44°C to -120°C at a rate of 40°C / min. The volume of the frozen semen was measured at a rate of 1000 g / min, and then stored in liquid nitrogen. During thawing, the frozen semen tube was removed from the liquid nitrogen and immersed in water at 4°C for 3 minutes within 10 seconds. After the frozen semen was completely thawed, it was poured into a centrifuge tube, and the volume was measured. Two to three times the volume of insemination diluent was added in 5 to 7 portions, with an interval of 2 to 3 minutes between each addition. The tubes were then centrifuged at 4°C for 8 to 10 minutes at a centrifugal force of 600g. The supernatant was removed, and the sperm precipitate was retained. The precipitate was resuspended in DMEM solution with an equal volume of thawed semen. After testing for sperm motility, the semen could be used for artificial insemination.

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