Application of protein deacetylase inhibitor NRD167 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 deacetylase inhibitor NRD167 in ultralow-temperature cryopreservation of poultry semen. Tests prove that the protein deacetylase inhibitor NRD167 can improve the acetylation modification level of poultry sperms and improve the fertilization capacity of frozen-remelted poultry sperms; therefore, the protein deacetylase inhibitor NRD167 can be used as an effective component for ultralow-temperature cryopreservation of poultry seminal fluid, 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 deacetylase inhibitor NRD167 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 deacetylase inhibitor NRD167 in ultra-low temperature cryopreservation of poultry semen. The protein deacetylase inhibitor NRD167 is 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.

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

[0006] Application of protein deacetylase inhibitor NRD167 in ultra-low temperature cryopreservation of poultry semen.

[0007] The protein deacetylase inhibitor NRD167 provided by the present invention has a structural formula as follows:

[0008]

[0009] Furthermore, the application of the protein deacetylase inhibitor NRD167 in ultra-low temperature cryopreservation of poultry semen is specifically as follows: adding the protein deacetylase inhibitor NRD167 to semen freezing diluent and semen cryoprotectant at an osmotic pressure of 360mOsm / kg.

[0010] Furthermore, the protein deacetylase inhibitor NRD167 can be used in combination with other cryoprotectant agents.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The application of the protein deacetylase inhibitor NRD167 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 deacetylase inhibitor NRD167 can inhibit the deacetylation modification of poultry sperm cells (specifically inhibiting the acetylase NAD + The activity of SIRT5 of the SIRT family is dependent on the SIRT to maintain a high level of acetylation modification, improve the ability of poultry sperm to respond to ultra-low temperature freezing stimulation, and improve the fertilization ability of poultry frozen-rethawed sperm; therefore, the protein deacetylase inhibitor NRD167 can be used as an effective ingredient for ultra-low temperature cryopreservation of poultry semen, which plays an important role in improving the efficiency of ultra-low temperature cryopreservation of poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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:

[0014] Figure 1 The results of the test of sperm protein acetylation modification level in high-freeze-resistant rooster group and low-freeze-resistant rooster group;

[0015] Figure 2 The results of sperm protein acetylation modification level detection in TSA group, NRD167 group and blank control group are shown in the figure;

[0016] Figure 3 This is a graph showing the fertilization rate test results of the TSA group, NRD167 group and blank control group. DETAILED DESCRIPTION

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] 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.

[0023] 1. Acetylation level of poultry sperm and sperm antifreeze ability

[0024] 1. Sample collection

[0025] 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.

[0026] 2. Semen dilution, balancing and freezing

[0027] 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.

[0028] 3. Thawing and sperm motility test

[0029] 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;

[0030] 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.

[0031] Sperm motility change range = fresh sperm motility - frozen-thawed sperm motility

[0032] 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;

[0033] Table 1 Variation of sperm motility in high-freeze-resistant rooster group and low-freeze-resistant rooster group

[0034]

[0035] 4. Extraction of sperm protein

[0036] 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;

[0037] 5. Detection of sperm protein acetylation modification level

[0038] 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.

[0039] 6. Experimental results

[0040] 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;

[0041] 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.

[0042] 2. Analysis of deacetylases involved in sperm protein acetylation

[0043] 1. Sample collection and protein extraction

[0044] Six adult healthy Beijing oily roosters were randomly selected, and semen was collected by dorsal and abdominal massage method, and centrifuged at 4℃ and 5000g for 10min; the supernatant was aspirated and transferred to a new centrifuge tube, centrifuged at 4℃ and 12000g for 10min, and 2 volumes of protein lysis buffer were added to obtain a seminal plasma protein sample; the precipitate was added to PBS for resuspending, centrifuged and washed, 4 volumes of protein lysis buffer were added, ultrasonic lysis was performed, and cell debris was removed by centrifugation at 4℃ and 12000g for 10min, and the supernatant was transferred to a new centrifuge tube to obtain a sperm protein sample; the roosters were slaughtered, the testicular tissue was separated, and a soybean-sized testicular sample was taken, 4 volumes of lysis buffer were added, homogenized and lysed, centrifuged at 4℃ and 12000g for 10min, cell debris was removed, and the supernatant was transferred to a new centrifuge tube to obtain a testicular protein sample; the protein concentration of sperm protein samples, testicular protein samples and seminal plasma protein samples was detected using a BCA kit;

[0045] 2. Qualitative and quantitative analysis of proteins

[0046] The sperm protein samples, testicular protein samples and seminal plasma protein samples were analyzed by liquid phase tandem mass spectrometry respectively, and the expression profiles of sperm protein, testicular protein and seminal plasma protein were obtained by library search analysis;

[0047] 3. Experimental results

[0048] The deacetylases in sperm protein samples, testis protein samples, and seminal plasma protein samples are shown in Table 2 ;

[0049] Table 2 Deacetylases in sperm protein, testis protein and seminal plasma protein

[0050]

[0051] From the data in Table 2, we can see that both sperm and testis proteins express high abundance of deacetylases, including Zn 2+ NAD-dependent histone deacetylase family + Dependent SIRT family.

[0052] 3. Effect of deacetylase inhibitors on the acetylation level of poultry sperm

[0053] 1. Screening of protein deacetylase inhibitors

[0054] Through database search, Zn 2+ TSA (C-dependent histone deacetylase family general inhibitor 17 H 22N2O3), SIRT5 specific inhibitor NRD167 (C 34 H 46 N6O6S);

[0055] 2. Semen collection and grouping

[0056] 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 oil 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 TSA group, NRD167 group and blank control group.

[0057] 3. Preparation of test samples

[0058] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor TSA is added to the semen freezing diluent to obtain the semen freezing diluent added with the deacetylase inhibitor TSA;

[0059] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor TSA is added to the semen cryoprotective solution to obtain the semen cryoprotective solution added with the deacetylase inhibitor TSA;

[0060] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor NRD167 is added to the semen freezing diluent to obtain the semen freezing diluent added with the deacetylase inhibitor NRD167;

[0061] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor NRD167 is added to the semen cryoprotective solution to obtain the semen cryoprotective solution added with the deacetylase inhibitor NRD167;

[0062] According to the osmotic pressure of 360mOsm / kg, sodium chloride (NaCl) was added to the semen freezing diluent to obtain a semen freezing diluent with NaCl as the blank control group;

[0063] Sodium chloride (NaCl) was added to the semen cryoprotective solution at an osmotic pressure of 360 mOsm / kg to obtain a semen cryoprotective solution with NaCl as a blank control group;

[0064] 4. Semen dilution, balancing and freezing

[0065] TSA group: 200 μL fresh semen was drawn and mixed with an equal amount of semen cryoprotectant preheated to 37°C and supplemented with the deacetylase inhibitor TSA, and then equilibrated at 4°C for 30 min. Then 400 μL of 4°C semen cryoprotectant supplemented with the deacetylase inhibitor TSA 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;

[0066] NRD167 group: 200 μL fresh semen was drawn and mixed with an equal amount of semen cryoprotectant preheated to 37°C and supplemented with the deacetylase inhibitor NRD167, and then equilibrated at 4°C for 30 min. Then 400 μL of 4°C semen cryoprotectant supplemented with the deacetylase inhibitor NRD167 was added and divided into semen tubes. 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;

[0067] Blank control group: 200 μL fresh semen was taken and mixed with an equal amount of semen cryoprotectant preheated to 37°C and added with NaCl, and then balanced at 4°C for 30 min. Then 400 μL of 4°C semen cryoprotectant added with NaCl 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, 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;

[0068] 5. Thawing

[0069] Semen tubes of the TSA group, NRD167 group and blank control group were taken out of liquid nitrogen, immersed in 4°C water for 3 min, poured into centrifuge tubes, and after measuring the volume, 2 times the volume of 4°C thawing solution was added to obtain sperm samples of the TSA group, sperm samples of the NRD167 group and sperm samples of the blank control group;

[0070] 6. Extraction of sperm protein

[0071] Four times the volume of lysis buffer was added to the sperm samples of the TSA group, the NRD167 group and the blank control group, respectively, and the samples were ultrasonically lysed, centrifuged at 4°C and 12000g for 10 min, the cell debris was removed, and the supernatant was transferred to a centrifuge tube to obtain the sperm protein samples of the TSA group, the NRD167 group and the blank control group;

[0072] 7. Detection of sperm protein acetylation modification level

[0073] 13 μg sperm protein samples of TSA group, NRD167 group and blank control group were collected respectively. After electrophoresis, membrane transfer and blocking, protein acetylation pan-antibody (Anti-acety l lysineAnti body, PTM-101, 12838533L303) was added for primary antibody incubation. After the primary antibody incubation, secondary antibody incubation (Goat ai-Mouse IgG (H+L), Peroxidase Conjugated, 31430) was carried out. 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.

[0074] 8. Test results

[0075] The results of sperm protein acetylation modification in TSA group, NRD167 group and blank control group are as follows: Figure 2 As shown, A is the sperm protein acetylation modification level map, and B is the gray value of the protein acetylation modification level;

[0076] Depend on Figure 2 It can be seen that the acetylation modification level of sperm protein in the TSA group and NRD167 group was higher than that in the control group.

[0077] 4. Effect of Sirtuin Inhibitors on the Fertilization Capacity of Poultry Reconstituted Sperm

[0078] 1. Semen collection and grouping

[0079] 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 oil 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 TSA group, NRD167 group and blank control group.

[0080] 2. Preparation of test samples

[0081] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor TSA is added to the semen freezing diluent to obtain the semen freezing diluent added with the deacetylase inhibitor TSA;

[0082] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor TSA is added to the semen cryoprotective solution to obtain the semen cryoprotective solution added with the deacetylase inhibitor TSA;

[0083] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor NRD167 is added to the semen freezing diluent to obtain the semen freezing diluent added with the deacetylase inhibitor NRD167;

[0084] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor NRD167 is added to the semen cryoprotective solution to obtain the semen cryoprotective solution added with the deacetylase inhibitor NRD167;

[0085] According to the osmotic pressure of 360mOsm / kg, sodium chloride (NaCl) was added to the semen freezing diluent to obtain a semen freezing diluent with NaCl as the blank control group;

[0086] Sodium chloride (NaCl) was added to the semen cryoprotective solution at an osmotic pressure of 360 mOsm / kg to obtain a semen cryoprotective solution with NaCl as the blank control group; 3. Semen dilution, balance and freezing

[0087] TSA group: 200 μL fresh semen was drawn and mixed with an equal amount of semen cryoprotectant preheated to 37°C and supplemented with the deacetylase inhibitor TSA, and then equilibrated at 4°C for 30 min. Then 400 μL of 4°C semen cryoprotectant supplemented with the deacetylase inhibitor TSA 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;

[0088] NRD167 group: 200 μL fresh semen was drawn and mixed with an equal amount of semen cryoprotectant preheated to 37°C and supplemented with the deacetylase inhibitor NRD167, and then equilibrated at 4°C for 30 min. Then 400 μL of 4°C semen cryoprotectant supplemented with the deacetylase inhibitor NRD167 was added and divided into semen tubes. 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;

[0089] Blank control group: 200 μL fresh semen was taken and mixed with an equal amount of semen cryoprotectant preheated to 37°C and added with NaCl, and then balanced at 4°C for 30 min. Then 400 μL of 4°C semen cryoprotectant added with NaCl 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, 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;

[0090] 4. Thawing and removing glycerol

[0091] Semen tubes of the TSA group, NRD167 group and blank control group were taken out of liquid nitrogen, immersed in 4°C water for 3 min, poured into centrifuge tubes, and the volume was measured and 2 times the volume of 4°C thawing solution was added. Then, the tubes were centrifuged at 4°C and 600g for 8-10 min, and the supernatant was removed to obtain sperm pellets of the TSA group, NRD167 group and blank control group.

[0092] 5. Fertilization rate detection

[0093] The sperm sediments of the TSA group, NRD167 group and blank control group were resuspended with 4℃ DMEM solution of the same volume as the thawed semen. 60 healthy Beijing oil chicken hens were selected and randomly divided into 3 groups. They were infused with frozen-rethawed sperm in the TSA group, NRD167 group and blank control group, respectively. The insemination volume of each hen was about 100 million sperms. The insemination was continuous for 2 days, and the breeding eggs were collected for 4 days, incubated and the fertilization rate was calculated.

[0094] 6 Experimental results

[0095] The results of fertilization rate test in TSA group, NRD167 group and blank control group are as follows Figure 3 As shown;

[0096] Depend on Figure 3 It can be seen that the fertilization rates of the TSA group and the NRD167 group were higher than those of the blank control group, and that of the NRD167 group was higher than that of the TSA group. This indicates that adding the protein deacetylase inhibitor NRD167 to semen cryo-diluent and semen cryoprotectant can improve the fertilization ability of frozen-thawed sperm.

[0097] 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 deacetylase inhibitor NRD167 in ultra-low temperature cryopreservation of poultry semen.

2. The use of the protein deacetylase inhibitor NRD167 according to claim 1 in ultra-low temperature cryopreservation of poultry semen, characterized in that: The application of the protein deacetylase inhibitor NRD167 in ultra-low temperature cryopreservation of poultry semen specifically comprises: adding the protein deacetylase inhibitor NRD167 into semen freezing diluent and semen freezing protection solution.

3. The use of the protein deacetylase inhibitor NRD167 according to claim 1 in ultra-low temperature cryopreservation of poultry semen, characterized in that: The protein deacetylase inhibitor NRD167 can be used in combination with other cryoprotectant agents.

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