Application of protein acetyl donor in ultralow-temperature cryopreservation of poultry semen

Abstract

The invention relates to the technical field of poultry breed conservation biology, and discloses application of a protein acetyl donor in ultralow-temperature cryopreservation of poultry semen. According to the application of the protein acetyl donor in ultralow-temperature cryopreservation of the poultry semen, the protein acetyl donor comprises but is not limited to magnesium acetate, potassium acetate and sodium acetate. Tests prove that the protein acetyl donor can improve the acetylation modification level of poultry sperms and improve the fertilization ability of frozen-remelted poultry sperms; therefore, the protein acetyl donor can be used as an effective component for ultralow-temperature cryopreservation of poultry semen, and plays an important role in improving the ultralow-temperature cryopreservation efficiency of poultry.

Description

Technical Field

[0001] The invention relates to the field of poultry seed preservation biotechnology, and in particular to application of a protein acetyl donor in ultra-low temperature cryopreservation of poultry semen. Background Art

[0002] Semen ultra-low temperature cryopreservation technology is a method of placing sperm under ultra-low temperature conditions such as liquid nitrogen (-196℃) to inhibit sperm metabolism and achieve long-term storage of sperm for subsequent use. Ultra-low temperature cryopreserved semen can restore its fertilization ability after thawing and warming. Therefore, semen ultra-low temperature cryopreservation technology is of great significance in livestock and poultry conservation and breeding. Semen ultra-low temperature cryopreservation technology can also be applied to the construction of sperm banks, which can effectively preserve the genetic information of species, prevent the loss of genetic information due to environmental and selection factors, maintain the genetic diversity of species, and reduce the risk of species extinction. It is an important means of livestock and poultry translocation conservation; in addition, semen ultra-low temperature cryopreservation technology can break through the limitations of time and space, realize the exchange of genetic resources of different generations and regions, and promote livestock and poultry genetic improvement and the cultivation of new varieties.

[0003] The world has abundant chicken genetic resources, which are a valuable asset for the livestock industry. When ultra-low temperature freezing and preservation of chicken semen, the chicken sperm has a small head and a long tail, which leads to poor antifreeze ability. Therefore, establishing a stable and efficient chicken semen freezing and preservation technology has always been a hot spot and difficulty in research in this field. Existing theoretical research on chicken semen freezing and preservation mainly focuses on reducing sperm structural damage caused by ice crystallization and inhibiting sperm chemical damage caused by oxidative stress, but lacks research on the protection and damage repair mechanism of sperm in response to low temperature stimulation. The transcription and translation of mature sperm are stagnant, and protein post-translational modification is an important way for mature sperm to respond to internal and external environmental stimuli, and may play an important role in maintaining sperm structural integrity, mitochondrial functional integrity and DNA structural integrity. Based on this, a poultry semen cryoprotectant based on protein post-translational modification is provided, which can promote the further development of semen ultra-low temperature freezing and preservation technology. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a protein acetyl donor in the ultra-low temperature cryopreservation of poultry semen. The protein acetyl donor is used as an effective ingredient for the ultra-low temperature cryopreservation of poultry semen, which plays an important role in improving the ultra-low temperature cryopreservation efficiency of poultry.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Application of protein acetyl donors in ultra-low temperature cryopreservation of poultry semen.

[0007] Furthermore, the protein acetyl donor includes but is not limited to magnesium acetate, potassium acetate and sodium acetate.

[0008] Furthermore, the application of the protein acetyl donor in ultra-low temperature cryopreservation of poultry semen is specifically: adding the protein acetyl donor to semen freezing diluent, semen cryoprotectant and thawing solution.

[0009] Furthermore, the protein acetyl donor can be used in combination with other cryoprotectant agents.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The application of the protein acetyl donor provided by the present invention in ultra-low temperature cryopreservation of poultry semen has been proved by experiments that the acetylation modification of lysine in poultry sperm protein is involved in the freezing stimulation response of poultry sperm, and the protein acetyl donor can inhibit the deacetylation modification of poultry sperm cells to maintain a high level of acetylation modification, thereby improving the ability of poultry sperm to respond to ultra-low temperature freezing stimulation and improving the fertilization ability of poultry frozen-rethawed sperm; therefore, the protein acetyl donor can be used as an effective ingredient for ultra-low temperature cryopreservation of poultry semen, which plays an important role in improving the ultra-low temperature cryopreservation efficiency of poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0013] Figure 1 This is the result of the test of sperm protein acetylation modification level in the high-freeze resistance rooster group and the low-freeze resistance rooster group;

[0014] Figure 2 The results of the sperm protein acetylation modification level detection in groups Ⅰ, Ⅱ and Ⅲ are shown in the figure;

[0015] Figure 3 This is the result of fertilization rate test of group Ⅰ, group Ⅱ and group Ⅲ. DETAILED DESCRIPTION

[0016] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0017] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0018] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0019] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0020] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0021] In the present invention, unless otherwise specified, the semen freezing diluent, semen cryoprotectant and thawing solution used are the same as those in "Interspecific differences in chicken sperm antifreeze and their correlation with seminal plasma biochemical indices and candidate gene expression" Zong Yunhe et al., China Poultry, 2020: 42(12): 6-13. The protein lysis solution used contains 8M urea, 1% TritonX-100, 10mM dithiothreitol and 1% protease inhibitor mixture.

[0022] 1. Acetylation level of poultry sperm and sperm antifreeze ability

[0023] 1. Sample collection

[0024] According to the "Test Method for Quality of Poultry Semen (NY / T 4047-2021)", sperm motility higher than 60%, semen volume higher than 350 μL, and density higher than 16×10 8 55 healthy adult Beijing oily chickens with a concentration of 100 g / mL were selected, and semen was collected by dorsal and abdominal massage.

[0025] 2. Semen dilution, balancing and freezing

[0026] 200 μL of fresh semen was taken and mixed with 200 μL of semen cryoprotectant preheated to 37°C, and then equilibrated at 4°C for 30 min. Then 400 μL of 4°C semen cryoprotectant was added and divided into semen tubes. After the tubes were sealed, they were placed in a programmed freezer for cooling and freezing. The freezing program was set to 4°C to -44°C at a rate of 12°C / min; -44°C to -120°C at a rate of 40°C / min, and then stored in liquid nitrogen.

[0027] 3. Thawing and sperm motility test

[0028] Take out the semen tube from liquid nitrogen, immerse it in 4℃ water to thaw for 3 minutes, pour it into a centrifuge tube, measure the volume and add 2 times the volume of 4℃ thawing solution to obtain a frozen-rethawed sperm sample;

[0029] The computer-assisted sperm analysis system was used to detect the sperm motility in the frozen-thawed sperm samples, and the change in sperm motility before and after freezing was used as an indicator of sperm antifreeze ability.

[0030] Sperm motility change range = fresh sperm motility - frozen-thawed sperm motility

[0031] After 4 tests, 5 high-freeze-resistance roosters and 5 low-freeze-resistance roosters with stable traits were selected for subsequent experiments. The changes in sperm motility in the high-freeze-resistance rooster group and the low-freeze-resistance rooster group are shown in Table 1;

[0032] Table 1 Variation of sperm motility in high-freeze-resistant rooster group and low-freeze-resistant rooster group

[0033]

[0034] 4. Extraction of sperm protein

[0035] Semen from high-antifreeze rooster group and low-antifreeze rooster group were collected respectively, centrifuged at 4°C and 12000g for 10 min to remove seminal plasma, washed twice with PBS, then added with 4 times volume of protein lysis buffer, ultrasonically lysed, centrifuged at 4°C and 12000g for 10 min to remove cell debris, and the supernatant was transferred to a new centrifuge tube to obtain sperm protein samples from high-antifreeze rooster group and low-antifreeze rooster group; protein concentration was detected using BCA kit;

[0036] 5. Detection of sperm protein acetylation modification level

[0037] 13 μg of sperm protein samples from high antifreeze rooster group and low antifreeze rooster group were taken respectively. After electrophoresis, membrane transfer and blocking, protein acetylation pan-antibody (Ant i-acety l lys i ne Antibody, PTM-101, 12838533L303) was added for primary antibody incubation. After the primary antibody incubation, secondary antibody incubation (Goat anti-Mouse IgG (H+L), Peroxidase Conjugated, 31430) was performed. After the secondary antibody incubation, the samples were rinsed and incubated with chemiluminescent HRP substrate for 2 minutes. Signal capture was performed according to the operating instructions of the chemiluminescent imaging system.

[0038] 6. Experimental results

[0039] The results of sperm protein acetylation modification in the high and low frost resistance rooster groups are as follows Figure 1 As shown, samples 1 to 5 are sperm samples from the high-freeze-resistance rooster group, and samples 6 to 10 are sperm samples from the low-freeze-resistance rooster group;

[0040] Depend on Figure 1 It can be seen that there are certain differences in the acetylation modification levels of sperm proteins in the high-freeze resistance rooster group and the low-freeze resistance rooster group. Among them, the acetylation modification level of protein near 35kD in the low-freeze resistance rooster group is higher, while the acetylation modification level of protein near 30kD is relatively low, which indicates that the acetylation modification level of poultry sperm is related to the antifreeze ability of sperm.

[0041] 2. Effect of protein acetyl donors on the acetylation level of poultry sperm

[0042] 1. Semen collection and grouping

[0043] According to the "Test Method for Quality of Poultry Semen (NY / T 4047-2021)", sperm motility higher than 60%, semen volume higher than 350 μL, and density higher than 16×10 8 30 adult healthy Beijing oily chickens with a concentration of 100 g / mL were used to collect semen by dorsal and abdominal massage. The semen of each individual was mixed and divided into three groups, namely group Ⅰ, group Ⅱ and group Ⅲ.

[0044] 2. Preparation of test samples

[0045] Magnesium chloride (MgCl2), potassium chloride (KCl) and sodium chloride (NaCl) in the semen cryodilution liquid were partially replaced by magnesium acetate ((CH3COO)2Mg), potassium acetate (CH3COOK) and sodium acetate (CH3COONa), respectively, to obtain a semen cryodilution liquid with a final acetate concentration of 0, a semen cryodilution liquid with a final acetate concentration of 17 mM and a semen cryodilution liquid with a final acetate concentration of 34 mM;

[0046] Magnesium chloride (MgCl2), potassium chloride (KCl) and sodium chloride (NaCl) in the semen cryoprotectant were partially replaced by magnesium acetate ((CH3COO)2Mg), potassium acetate (CH3COOK) and sodium acetate (CH3COONa), respectively, to obtain semen cryoprotectant with a final acetate concentration of 0, semen cryoprotectant with a final acetate concentration of 17 mM and semen cryoprotectant with a final acetate concentration of 34 mM;

[0047] The magnesium chloride (MgCl2), potassium chloride (KCl) and sodium chloride (NaCl) in the thawing solution were partially replaced by magnesium acetate ((CH3COO)2Mg), potassium acetate (CH3COOK) and sodium acetate (CH3COONa), respectively, to obtain a thawing solution with a final acetate concentration of 0, a thawing solution with a final acetate concentration of 17 mM and a thawing solution with a final acetate concentration of 34 mM;

[0048] 3. Semen dilution, balancing and freezing

[0049] Group I: 200 μL of fresh semen was drawn and mixed with an equal amount of semen cryoprotectant preheated to 37°C with a final acetate concentration of 0, and then equilibrated at 4°C for 30 min. Then 400 μL of semen cryoprotectant preheated to 4°C with a final acetate concentration of 0 was added, and the semen was divided into semen tubes. After the tubes were sealed, they were placed in a programmed freezer for cooling and freezing. The freezing program was set to 4°C to -44°C, with a rate of 12°C / min; -44°C to -120°C, with a rate of 40°C / min, and then stored in liquid nitrogen;

[0050] Group II: 200 μL of fresh semen was drawn and mixed with an equal amount of semen cryoprotectant preheated to 37°C with a final acetate concentration of 17 mM, equilibrated at 4°C for 30 min, then 400 μL of semen cryoprotectant preheated to 4°C with a final acetate concentration of 17 mM was added, divided into semen tubes, and the tubes were sealed and placed in a programmed freezer for cooling and freezing. The freezing program was set to 4°C to -44°C, at a rate of 12°C / min; -44°C to -120°C, at a rate of 40°C / min, and then stored in liquid nitrogen;

[0051] Group III: 200 μL of fresh semen was drawn and mixed with an equal amount of semen cryoprotectant preheated to 37°C with a final acetate concentration of 34 mM, equilibrated at 4°C for 30 min, then 400 μL of semen cryoprotectant preheated to 4°C with a final acetate concentration of 34 mM was added, divided into semen tubes, and the tubes were sealed and placed in a programmed freezer for cooling and freezing. The freezing program was set to 4°C to -44°C, at a rate of 12°C / min; -44°C to -120°C, at a rate of 40°C / min, and then stored in liquid nitrogen;

[0052] 4. Thawing

[0053] Group I: Take out the semen tubes of Group I from liquid nitrogen, thaw them in 4°C water for 3 min, pour them into centrifuge tubes, measure the volume, and add 2 times the volume of 4°C thawing solution with a final acetate concentration of 0 to obtain the sperm samples of Group I;

[0054] Group II: Take out the semen tubes from liquid nitrogen, thaw them in 4°C water for 3 min, pour them into centrifuge tubes, measure the volume, and add 2 times the volume of 4°C thawing solution with a final acetate concentration of 17 mM to obtain sperm samples of Group II;

[0055] Group III: Take out the semen tubes of Group III from liquid nitrogen, thaw them in 4°C water for 3 min, pour them into centrifuge tubes, measure the volume, and add 2 times the volume of 4°C thawing solution with a final acetate concentration of 34 mM to obtain sperm samples of Group III;

[0056] 5. Extraction of sperm protein

[0057] Four times the volume of lysis buffer was added to the sperm samples of group I, group II and group III, respectively, and the samples were ultrasonically lysed, centrifuged at 4°C and 12000 g for 10 min, cell debris were removed, and the supernatant was transferred to a centrifuge tube to obtain sperm protein samples of group I, group II and group III;

[0058] 6. Detection of sperm protein acetylation modification level

[0059] 13 μg of sperm protein samples from group I, group II and group III were taken respectively. After electrophoresis, membrane transfer and blocking, protein acetylation pan-antibody (Ant i-acety l lys i ne Antibody, PTM-101, 12838533L303) was added for primary antibody incubation. After the primary antibody incubation, secondary antibody incubation (Goat anti-Mouse IgG (H+L), Peroxidase Conjugated, 31430) was performed. After the secondary antibody incubation, the samples were rinsed and incubated with chemiluminescent HRP substrate for 2 minutes. Signal capture was performed according to the operating instructions of the chemiluminescent imaging system.

[0060] 7. Test results

[0061] The results of sperm protein acetylation modification in groups Ⅰ, Ⅱ and Ⅲ are as follows Figure 2 As shown;

[0062] Depend on Figure 2It can be seen that the acetylation modification level of sperm protein in group II and group III was significantly higher than that in group I, which indicates that adding protein acetyl donors to semen freezing diluent, semen cryoprotectant and thawing solution can increase the acetylation modification level of frozen-thawed sperm protein.

[0063] 3. Effect of protein acetyl donors on the fertilization ability of poultry frozen-thawed sperm

[0064] 1. Semen collection and grouping

[0065] According to the "Test Method for Quality of Poultry Semen (NY / T 4047-2021)", sperm motility higher than 60%, semen volume higher than 350 μL, and density higher than 16×10 8 30 adult healthy Beijing oily chickens with a concentration of 100 g / mL were used to collect semen by dorsal and abdominal massage. The semen of each individual was mixed and divided into three groups, namely group Ⅰ, group Ⅱ and group Ⅲ.

[0066] 2. Preparation of test samples

[0067] Magnesium chloride (MgCl2), potassium chloride (KCl) and sodium chloride (NaCl) in the semen cryodilution liquid were partially replaced by magnesium acetate ((CH3COO)2Mg), potassium acetate (CH3COOK) and sodium acetate (CH3COONa), respectively, to obtain a semen cryodilution liquid with a final acetate concentration of 0, a semen cryodilution liquid with a final acetate concentration of 17 mM and a semen cryodilution liquid with a final acetate concentration of 34 mM;

[0068] Magnesium chloride (MgCl2), potassium chloride (KCl) and sodium chloride (NaCl) in the semen cryoprotectant were partially replaced by magnesium acetate ((CH3COO)2Mg), potassium acetate (CH3COOK) and sodium acetate (CH3COONa), respectively, to obtain semen cryoprotectant with a final acetate concentration of 0, semen cryoprotectant with a final acetate concentration of 17 mM and semen cryoprotectant with a final acetate concentration of 34 mM;

[0069] The magnesium chloride (MgCl2), potassium chloride (KCl) and sodium chloride (NaCl) in the thawing solution were partially replaced by magnesium acetate ((CH3COO)2Mg), potassium acetate (CH3COOK) and sodium acetate (CH3COONa), respectively, to obtain a thawing solution with a final acetate concentration of 0, a thawing solution with a final acetate concentration of 17 mM and a thawing solution with a final acetate concentration of 34 mM;

[0070] 3. Semen dilution, balancing and freezing

[0071] Group I: 200 μL of fresh semen was drawn and mixed with an equal amount of semen cryoprotectant preheated to 37°C with a final acetate concentration of 0, and then equilibrated at 4°C for 30 min. Then 400 μL of semen cryoprotectant preheated to 4°C with a final acetate concentration of 0 was added, and the semen was divided into semen tubes. After the tubes were sealed, they were placed in a programmed freezer for cooling and freezing. The freezing program was set to 4°C to -44°C, with a rate of 12°C / min; -44°C to -120°C, with a rate of 40°C / min, and then stored in liquid nitrogen;

[0072] Group II: 200 μL of fresh semen was drawn and mixed with an equal amount of semen cryoprotectant preheated to 37°C with a final acetate concentration of 17 mM, equilibrated at 4°C for 30 min, then 400 μL of semen cryoprotectant preheated to 4°C with a final acetate concentration of 17 mM was added, divided into semen tubes, and the tubes were sealed and placed in a programmed freezer for cooling and freezing. The freezing program was set to 4°C to -44°C, at a rate of 12°C / min; -44°C to -120°C, at a rate of 40°C / min, and then stored in liquid nitrogen;

[0073] Group III: 200 μL of fresh semen was drawn and mixed with an equal amount of semen cryoprotectant preheated to 37°C with a final acetate concentration of 34 mM, equilibrated at 4°C for 30 min, then 400 μL of semen cryoprotectant preheated to 4°C with a final acetate concentration of 34 mM was added, divided into semen tubes, and the tubes were sealed and placed in a programmed freezer for cooling and freezing. The freezing program was set to 4°C to -44°C, at a rate of 12°C / min; -44°C to -120°C, at a rate of 40°C / min, and then stored in liquid nitrogen;

[0074] 4. Thawing and removing glycerol

[0075] Group I: Take out the semen tubes of Group I from liquid nitrogen, thaw them in 4℃ water for 3 min, pour them into centrifuge tubes, measure the volume, add 2 times the volume of 4℃ thawing solution with a final acetate concentration of 0, then centrifuge at 4℃ and 600g for 8-10 min, remove the supernatant, and obtain the sperm pellet of Group I;

[0076] Group II: Take the semen tube of Group II out of liquid nitrogen, thaw it in 4℃ water for 3 min, pour it into a centrifuge tube, measure the volume, add 2 times the volume of 4℃ thawing solution with a final acetate concentration of 17mM, then centrifuge at 4℃ and 600g for 8-10 min, remove the supernatant, and obtain the sperm pellet of Group II;

[0077] Group III: Take the semen tube of Group III out of liquid nitrogen, thaw it in 4℃ water for 3 min, pour it into a centrifuge tube, measure the volume, add 2 times the volume of 4℃ thawing solution with a final acetate concentration of 34mM, then centrifuge at 4℃ and 600g for 8-10 min, remove the supernatant, and obtain the sperm pellet of Group III;

[0078] 5. Fertilization rate detection

[0079] The same volume of thawed semen was added to the sperm precipitates of group Ⅰ, group Ⅱ and group Ⅲ, and the precipitates were resuspended. 60 healthy Beijing oil chicken hens were selected and randomly divided into 3 groups. The frozen-thawed sperm of group Ⅰ, group Ⅱ and group Ⅲ were infused, and the insemination volume of each hen was about 100 million sperms. The insemination was continuous for 2 days, and the 6-day breeding eggs were collected, incubated and the fertilization rate was calculated.

[0080] 6 Experimental results

[0081] The results of fertilization rate test of group Ⅰ, group Ⅱ and group Ⅲ are as follows Figure 3 As shown;

[0082] Depend on Figure 3 It can be seen that the fertilization rates of group I, group II and group III were 29.53%, 36.48% and 52.12% respectively. The fertilization rates of group II and group III were significantly higher than that of group I, which indicates that adding protein acetyl donors to semen freezing diluent, semen cryoprotectant and thawing solution can improve the fertilization ability of frozen-rethawed sperm.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does 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. Application of protein acetyl donors in ultra-low temperature cryopreservation of poultry semen.

2. The use of the protein acetyl donor according to claim 1 in ultra-low temperature cryopreservation of poultry semen, characterized in that: The protein acetyl donors include, but are not limited to, magnesium acetate, potassium acetate and sodium acetate.

3. The use of the protein acetyl donor according to claim 1 in ultra-low temperature cryopreservation of poultry semen, characterized in that: The application of the protein acetyl donor in the ultra-low temperature cryopreservation of poultry semen specifically comprises: adding the protein acetyl donor into semen freezing diluent, semen freezing protection liquid and thawing liquid.

4. The use of the protein acetyl donor according to claim 1 in ultra-low temperature cryopreservation of poultry semen, characterized in that: The protein acetyl donor can be used in combination with other cryoprotectant agents.

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