Application of protein acetyl donor in ultra-low temperature cryopreservation of poultry semen

Abstract

The present application relates to the field of poultry germplasm preservation biotechnology, and discloses application of protein acetyl donor in poultry semen ultra-low temperature cryopreservation.The application of the protein acetyl donor in poultry semen ultra-low temperature cryopreservation, wherein the protein acetyl donor includes but is not limited to magnesium acetate, potassium acetate and sodium acetate.It is proved by experiments that the protein acetyl donor can improve the acetylation modification level of poultry sperm and the fertilization ability of frozen-thawed poultry sperm; therefore, the protein acetyl donor can be used as an effective component for the ultra-low temperature cryopreservation of poultry semen, and plays an important role in improving the efficiency of poultry ultra-low temperature cryopreservation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of poultry germplasm preservation biotechnology, in particular to the application of a protein acetyl donor in the ultra-low temperature cryopreservation of poultry semen. BACKGROUND

[0002] The ultra-low temperature cryopreservation technology of semen is a method of placing sperm in ultra-low temperature conditions such as liquid nitrogen (-196℃) to inhibit the metabolism of sperm and achieve long-term preservation of sperm for subsequent use. The semen cryopreserved by ultra-low temperature can restore its fertilization ability after thawing, so the semen ultra-low temperature cryopreservation technology has important significance in livestock and poultry breeding and breeding work. The semen ultra-low temperature cryopreservation technology can also be applied to the construction of sperm bank, 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 preservation. In addition, the semen ultra-low temperature cryopreservation technology can break through the time and space limitations, realize the exchange of genetic resources of different generations and regions, and promote the genetic improvement and breeding of new breeds of livestock and poultry.

[0003] There are rich chicken genetic resources in the world, which is a valuable wealth of animal husbandry. When the chicken semen is subjected to semen ultra-low temperature cryopreservation, the chicken sperm has poor freezing resistance due to its small head and long tail. Therefore, establishing a stable and efficient chicken semen cryopreservation technology has always been a hot and difficult point in the field. The existing theoretical research on chicken semen cryopreservation mainly focuses on reducing the damage to sperm structure caused by ice crystal and inhibiting the chemical damage to sperm caused by oxidative stress, and 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 arrested, and post-translational modification of protein is an important way for mature sperm to respond to internal and external environmental stimuli, which may play an important role in maintaining the structural integrity of sperm, the functional integrity of mitochondria and the structural integrity of DNA. Based on this, a poultry semen cryoprotectant based on post-translational modification of protein is provided, which can promote the further development of semen ultra-low temperature cryopreservation technology. SUMMARY

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

[0005] To achieve the above purpose, the present application provides the following technical scheme:

[0006] The application of a protein acetyl donor in the ultra-low temperature cryopreservation of poultry semen.

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

[0008] Further, the application of the protein acetyl donor in the ultra-low temperature cryopreservation of poultry semen, specifically, adding the protein acetyl donor into the semen freezing diluent, the semen cryoprotective solution and the thawing solution.

[0009] Further, the protein acetyl donor can be used in combination with other cryoprotective reagents.

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

[0011] The application of the protein acetyl donor in the ultra-low temperature cryopreservation of poultry semen, as proved by tests, the protein lysine acetylation modification of poultry sperm participates in the freezing stimulation response of poultry sperm, the protein acetyl donor can inhibit the deacetylation modification of poultry sperm cells, so as to maintain a high level of acetylation modification, improve the ability of poultry sperm to respond to the ultra-low temperature freezing stimulation, and improve the fertilization ability of the frozen-thawed poultry sperm; therefore, the protein acetyl donor can be used as an effective component for the ultra-low temperature cryopreservation of poultry semen, and plays an important role in improving the efficiency of the ultra-low temperature cryopreservation of poultry. BRIEF DESCRIPTION OF DRAWINGS

[0012] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included to provide a description of the preferred embodiments and are not intended to limit the scope of the application. Furthermore, the drawings are not necessarily drawn to scale. In the drawings:

[0013] Figure 1 The figure is the detection result graph of the sperm protein acetylation modification level of the high-freeze-resistant cock group and the low-freeze-resistant cock group;

[0014] Figure 2 The figure is the detection result graph of the sperm protein acetylation modification level of group I, group II and group III;

[0015] Figure 3 The figure is the detection result graph of the fertilization rate of group I, group II and group III. DETAILED DESCRIPTION

[0016] Various example embodiments of the application are described in detail below. The detailed description is presented in terms of specific embodiments which include particular components, devices, or steps. Those skilled in the art will recognize that various substitutions and alterations can be made from this description without departing from the spirit and scope of the application. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0017] Also, for ranges of values, the disclosure herein also contemplates each and every value and sub-range within the range. The disclosure herein also contemplates that, in some embodiments, the ranges should be greater than or equal to the minimum value and less than or equal to the maximum value. In this document, the terms "comprise", "comprising", "include", "including", "contain", "containing", "have", "having", "engage", "engaging", "connect", "connecting" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited only to those features but can include other features not expressly listed or inherent to such process, method, article, or apparatus.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the present document will control.

[0019] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples that follow. The description and examples are illustrative only.

[0020] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0021] In the present application, unless otherwise specified, the semen freezing diluent, the semen freezing protective solution and the thawing solution used are as described in "Chicken Semen Freezing Resistance Interspecific Differences and Its Correlation with Semen Biochemical Indicators and Candidate Gene Expression", Zong Yunhe et al., China Poultry, 2020: 42(12): 6-13. The protein lysis solution contains 8M urea, 1% Triton X-100, 10 mM dithiothreitol and 1% protease inhibitor cocktail.

[0022] I. The level of acetylation modification of poultry sperm and the freezing resistance ability of the sperm

[0023] 1. Collection of samples

[0024] According to the "Poultry Semen Quality Detection Method (NY / T 4047-2021)", 55 adult healthy Beijing oil cock chickens with sperm motility higher than 60%, semen volume higher than 350 μL and density higher than 16 x 10 8 The semen of the cock chickens was collected by back and abdomen massage method;

[0025] 2. Semen dilution, equilibration and freezing

[0026] Take 200 μL fresh semen and mix with 200 μL of semen freezing diluent preheated to 37°C, equilibrate at 4°C for 30 min, then add 400 μL of 4°C semen cryoprotective solution, dispense into semen tubes, seal the tubes and place them in a programmed freezer for cooling and freezing, with a freezing program set at 4°C to -44°C at a rate of 12°C / min; -44°C to -120°C at a rate of 40°C / min, then put into liquid nitrogen for storage;

[0027] 3. Thawing and sperm motility detection

[0028] Take the semen tubes out of the liquid nitrogen and immerse them in 4°C water for 3 min to thaw, pour them into centrifuge tubes, add 2 volumes of 4°C thawing solution after measuring the volume, and obtain the frozen-thawed sperm sample;

[0029] Use the computer-aided sperm analysis system to detect the sperm motility in the frozen-thawed sperm sample, and use the change in sperm motility before and after freezing as an indicator of sperm freeze resistance;

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

[0031] After 4 tests, 5 high freeze-resistant roosters and 5 low freeze-resistant roosters with stable traits were selected for subsequent experiments, and the sperm motility change of the high freeze-resistant rooster group and the low freeze-resistant rooster group is shown in Table 1;

[0032] Table 1 Sperm motility change of high freeze-resistant rooster group and low freeze-resistant rooster group

[0033]

[0034] 4. Sperm protein extraction

[0035] Collect the semen of high freeze-resistant rooster group and low freeze-resistant rooster group, centrifuge at 4°C, 12000g for 10 min to remove seminal plasma, wash twice with PBS, then add 4 volumes of protein lysis buffer, ultrasonic lysis, centrifuge at 4°C, 12000g for 10 min to remove cell debris, transfer the supernatant to a new centrifuge tube, and obtain the sperm protein samples of high freeze-resistant rooster group and low freeze-resistant rooster group; use BCA kit for protein concentration detection;

[0036] 5. Sperm protein acetylation modification level detection

[0037] Sperm protein samples from high-antifreeze roosters and low-antifreeze roosters were taken at 13 μg each. After electrophoresis, transfer to a membrane, and blocking, protein acetylation pan-antibody (Anti-acetylated antibody, PTM-101, 12838533L303) was added for primary antibody incubation. After primary antibody incubation, secondary antibody incubation (Goat anti-mouse IgG (H+L), Peroxidase Conjugated, 31430) was performed. After secondary antibody incubation, the samples were rinsed and incubated with chemiluminescent HRP substrate for 2 minutes. Signal capture was performed according to the operation instructions of the chemiluminescent imaging system.

[0038] 6. Experimental Results

[0039] The results of sperm protein acetylation modification level detection in high-freeze-resistant rooster groups and low-freeze-resistant rooster groups are as follows: Figure 1 As shown, samples 1-5 are sperm samples from the high-freeze-resistant rooster group, and samples 6-10 are sperm samples from the low-freeze-resistant rooster group.

[0040] Depend on Figure 1 It can be seen that there are certain differences in the acetylation modification level of sperm proteins between the high-freeze-resistant rooster group and the low-freeze-resistant rooster group. Among them, the low-freeze-resistant rooster group has a higher level of protein acetylation modification around 35kD, while the level of protein acetylation modification around 30kD is relatively low. This indicates that the acetylation modification level of poultry sperm is related to the sperm's freeze resistance.

[0041] II. The effect of protein acetyl donors on increasing the acetylation level of poultry sperm

[0042] 1. Semen collection and grouping

[0043] Referring to the "Test Methods for Poultry Semen Quality (NY / T 4047-2021)," samples with sperm motility higher than 60%, semen volume higher than 350 μL, and semen density higher than 16 × 10⁻⁶ were selected. 8 Thirty healthy adult Beijing Oil Chicken roosters with a semen volume of 1 / mL were used to collect semen using the back and abdomen massage method. The individual semen was mixed and divided into three groups, designated as Group I, Group II and Group III.

[0044] 2. Preparation of the test sample

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

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

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

[0048] 3. Semen dilution, equilibration and freezing

[0049] Group I: 200μL fresh semen was mixed with an equal amount of preheated 37℃ semen freezing diluent with an acetate final concentration of 0, equilibrated at 4℃ for 30min, then 400μL 4℃ semen freezing protective solution with an acetate final concentration of 0 was added, and the semen was aliquoted into a semen straw, the straw was sealed and placed in a programmed freezer for cooling and freezing, the freezing program was set as 4℃ to -44℃ at a rate of 12℃ / min, -44℃ to -120℃ at a rate of 40℃ / min, and then placed in liquid nitrogen for storage;

[0050] Group II: 200μL fresh semen was mixed with an equal amount of preheated 37℃ semen freezing diluent with an acetate final concentration of 17mM, equilibrated at 4℃ for 30min, then 400μL 4℃ semen freezing protective solution with an acetate final concentration of 17mM was added, and the semen was aliquoted into a semen straw, the straw was sealed and placed in a programmed freezer for cooling and freezing, the freezing program was set as 4℃ to -44℃ at a rate of 12℃ / min, -44℃ to -120℃ at a rate of 40℃ / min, and then placed in liquid nitrogen for storage;

[0051] Group III: 200μL fresh semen was mixed with an equal amount of preheated 37℃ semen freezing diluent with an acetate final concentration of 34mM, equilibrated at 4℃ for 30min, then 400μL 4℃ semen freezing protective solution with an acetate final concentration of 34mM was added, and the semen was aliquoted into a semen straw, the straw was sealed and placed in a programmed freezer for cooling and freezing, the freezing program was set as 4℃ to -44℃ at a rate of 12℃ / min, -44℃ to -120℃ at a rate of 40℃ / min, and then placed in liquid nitrogen for storage;

[0052] 4. Thawing

[0053] Group I: The Group I sperm fine tube was taken out from liquid nitrogen, immersed in 4°C water for 3 min, poured into a centrifuge tube, the volume was measured, and then 2 times the volume of 4°C acetic acid was added to obtain a final concentration of 0 mM of the thawing solution, to obtain the Group I sperm sample;

[0054] Group II: The Group II sperm fine tube was taken out from liquid nitrogen, immersed in 4°C water for 3 min, poured into a centrifuge tube, the volume was measured, and then 2 times the volume of 4°C acetic acid was added to obtain a final concentration of 17 mM of the thawing solution, to obtain the Group II sperm sample;

[0055] Group III: The Group III sperm fine tube was taken out from liquid nitrogen, immersed in 4°C water for 3 min, poured into a centrifuge tube, the volume was measured, and then 2 times the volume of 4°C acetic acid was added to obtain a final concentration of 34 mM of the thawing solution, to obtain the Group III sperm sample;

[0056] 5. Sperm protein extraction

[0057] 4 times the volume of lysis buffer was added to the Group I sperm sample, the Group II sperm sample and the Group III sperm sample respectively, and ultrasonic lysis was performed. After centrifugation at 4°C, 12000g for 10 min, the cell debris was removed, and the supernatant was transferred to a centrifuge tube to obtain the Group I sperm protein sample, the Group II sperm protein sample and the Group III sperm protein sample;

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

[0059] 13 μg of the Group I sperm protein sample, the Group II sperm protein sample and the Group III sperm protein sample were taken respectively, and after electrophoresis, membrane transfer and blocking, protein acetylation pan antibody (Anti-acetyl lysine Antibody, PTM-101, 12838533L303) was added for primary antibody incubation. After the primary antibody incubation, secondary antibody incubation was performed (Goat anti-Mouse IgG (H+L), Peroxidase Conjugated, 31430). After the secondary antibody incubation, rinsing was performed, and chemical luminescence HRP substrate was added for incubation for 2 min. According to the operation instructions of the chemical luminescence imaging system, signal capture was performed;

[0060] 7. Detection results

[0061] The detection results of the Group I, the Group II and the Group III sperm protein acetylation modification level are shown in Table 1. Figure 2

[0062] Figure 2 ​​It can be seen that the acetylation modification level of sperm protein in group II and group III is significantly higher than that in group I, which shows that adding protein acetyl donor into semen freezing diluent, semen cryoprotective solution and thawing solution can improve the acetylation modification level of frozen-thawed sperm protein.

[0063] III. Effect of protein acetyl donor on the improvement of the fertility of poultry frozen-thawed sperm

[0064] 1. Semen collection and grouping

[0065] According to the Poultry Semen Quality Detection Method (NY / T 4047-2021), 30 adult healthy Beijing oil cockerels with sperm motility higher than 60%, semen volume higher than 350 μL, and density higher than 16 × 10 8 individual semen was mixed and divided into 3 groups, denoted as group I, group II and group III;

[0066] 2. Preparation of test samples

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

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

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

[0070] 3. Semen dilution, equilibration and freezing

[0071] Group I: 200 μL fresh semen was taken and mixed with equal amount of semen freezing diluent with 0 mM final concentration of acetate preheated to 37℃, equilibrated at 4℃ for 30 min, then 400 μL of semen freezing protective solution with 0 mM final concentration of acetate at 4℃ was added, and the semen was aliquoted into the semen thin tube, the thin tube was sealed and put into the programmed freezer for cooling and freezing, the freezing program was set as 4℃ to -44℃ with a rate of 12℃ / min, -44℃ to -120℃ with a rate of 40℃ / min, then put into liquid nitrogen for storage;

[0072] Group II: 200 μL fresh semen was taken and mixed with equal amount of semen freezing diluent with 17 mM final concentration of acetate preheated to 37℃, equilibrated at 4℃ for 30 min, then 400 μL of semen freezing protective solution with 17 mM final concentration of acetate at 4℃ was added, and the semen was aliquoted into the semen thin tube, the thin tube was sealed and put into the programmed freezer for cooling and freezing, the freezing program was set as 4℃ to -44℃ with a rate of 12℃ / min, -44℃ to -120℃ with a rate of 40℃ / min, then put into liquid nitrogen for storage;

[0073] Group III: 200 μL fresh semen was taken and mixed with equal amount of semen freezing diluent with 34 mM final concentration of acetate preheated to 37℃, equilibrated at 4℃ for 30 min, then 400 μL of semen freezing protective solution with 34 mM final concentration of acetate at 4℃ was added, and the semen was aliquoted into the semen thin tube, the thin tube was sealed and put into the programmed freezer for cooling and freezing, the freezing program was set as 4℃ to -44℃ with a rate of 12℃ / min, -44℃ to -120℃ with a rate of 40℃ / min, then put into liquid nitrogen for storage;

[0074] 4. Thawing and removing glycerol

[0075] Group I: The semen thin tube of Group I was taken out from liquid nitrogen and immersed in 4℃ water for 3 min for thawing, then poured into a centrifuge tube, after measuring the volume, 2 times volume of thawing solution with 0 mM final concentration of acetate at 4℃ was added, then centrifuged at 4℃ and 600g for 8-10 min, the supernatant was removed to obtain the sperm precipitate of Group I;

[0076] Group II: The semen thin tube of Group II was taken out from liquid nitrogen and immersed in 4℃ water for 3 min for thawing, then poured into a centrifuge tube, after measuring the volume, 2 times volume of thawing solution with 17 mM final concentration of acetate at 4℃ was added, then centrifuged at 4℃ and 600g for 8-10 min, the supernatant was removed to obtain the sperm precipitate of Group II;

[0077] Group III: The semen thin tube of Group III was taken out from liquid nitrogen and immersed in 4℃ water for 3 min for thawing, then poured into a centrifuge tube, after measuring the volume, 2 times volume of thawing solution with 34 mM final concentration of acetate at 4℃ was added, then centrifuged at 4℃ and 600g for 8-10 min, the supernatant was removed to obtain the sperm precipitate of Group III;

[0078] 5. Fertilization rate detection

[0079] The sperm precipitates from Group I, Group II, and Group III were resuspended in 4°C DMEM solution with an equal volume of thawed semen. Sixty healthy Beijing Oil Chicken hens were randomly divided into three groups and infused with frozen-thawed sperm from Group I, Group II, and Group III, respectively. Each hen was inseminated with approximately 100 million sperm. Insemination was performed continuously for 2 days, and eggs were collected after 6 days of incubation. The fertilization rate was then calculated.

[0080] 6 Experimental Results

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

[0082] Depend on Figure 3 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 those of Group I. This indicates that adding protein acetyl donors to the semen cryopreservation solution, semen cryoprotectant, and thawing solution can improve the fertilization capacity of frozen-thawed sperm.

[0083] 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. Use of a protein acetyl donor in the ultra-low temperature cryopreservation of poultry semen, characterized in that, The protein acetyl donor includes magnesium acetate, potassium acetate and sodium acetate; The application of the protein acetyl donor in the ultra-low temperature cryopreservation of poultry semen, specifically, adding the protein acetyl donor into semen freezing diluent, semen cryoprotective solution and thawing solution; The final concentration of acetate in the semen freezing diluent is 34 mM, and the final concentration of acetate in the thawing solution is 34 mM.

2. Use of the protein acetyl donor according to claim 1 in the ultra-low temperature cryopreservation of poultry semen, characterized in that, The protein acetyl donor can be used in combination with other cryoprotective reagents.

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