Application of protein deacetylase inhibitor NAM in ultra-low temperature cryopreservation of poultry semen

Abstract

The application provides application of a protein deacetylase inhibitor NAM in poultry semen ultra-low temperature cryopreservation and belongs to the technical field of poultry breeding preservation. The protein deacetylase inhibitor NAM is used as a cryoprotective agent or is combined with other cryoprotective agents, and the protein deacetylase inhibitor NAM is used as an effective component, so that the efficiency of the poultry ultra-low temperature cryopreservation can be improved. The protein deacetylase inhibitor NAM can inhibit sperm cell deacetylation modification, maintain a high level of acetylation modification level, improve the ability of sperm to respond to ultra-low temperature freezing stimulation, and improve the fertilization ability of frozen-thawed sperm.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry seed preservation, in particular to application of a protein deacetylase inhibitor NAM in ultra-low temperature cryopreservation of poultry semen. Background Art

[0002] Ultra-low temperature cryopreservation of semen involves freezing under ultra-low temperatures, such as liquid nitrogen (-196°C), to inhibit sperm metabolism and achieve long-term effective sperm storage. Upon thawing and warming, the sperm regains its fertilizing capacity. Semen cryopreservation is crucial for livestock and poultry conservation and breeding. Ultra-low temperature cryopreservation of semen is a crucial method for livestock and poultry translocation conservation. Establishing sperm banks using this technology effectively preserves genetic information, preventing loss due to environmental and selective factors, maintaining genetic diversity, and reducing the risk of extinction. Ultra-low temperature freezing of semen can transcend time and space limitations, enabling the exchange of genetic resources across generations and regions, promoting genetic improvement and the development of new breeds in livestock and poultry. my country possesses the world's richest chicken genetic resources, with 115 indigenous chicken breeds, representing a valuable asset for the livestock industry. However, the structural characteristics of chicken sperm, such as a small head and long tail, result in poor freezing resistance. Developing a stable and efficient chicken semen cryopreservation technology has been a hot topic and a challenge in this area.

[0003] Theoretical research on chicken semen cryopreservation has primarily focused on reducing sperm structural damage caused by ice crystallization and inhibiting chemical damage caused by oxidative stress. However, there is a lack of research on the mechanisms by which sperm protect against and repair damage from low temperatures. Transcription and translation arrest in mature sperm, and post-translational protein modification, are crucial pathways by which mature sperm respond to internal and external environmental stimuli and may play a crucial role in maintaining sperm structural integrity, mitochondrial function, and DNA structural integrity. Therefore, research on the mechanisms by which sperm protect against and repair damage from low temperatures, as well as the search for protease inhibitors that could improve the efficiency of poultry semen cryopreservation, is of great practical significance. Summary of the Invention

[0004] The present invention aims to provide an application of a protein deacetylase inhibitor, NAM, in ultra-low temperature cryopreservation of poultry semen. Lysine acetylation modification of sperm proteins is involved in the freezing stimulation response of poultry sperm. The protein deacetylase inhibitor, NAM, can inhibit deacetylation modification of sperm cells to maintain a high level of acetylation modification, thereby improving the ability of sperm to respond to ultra-low temperature freezing stimulation and the fertilization ability of frozen-thawed sperm.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides application of protein deacetylase inhibitor NAM in ultra-low temperature cryopreservation of poultry semen.

[0007] Furthermore, the protein deacetylase inhibitor NAM reduces sperm freezing damage by inhibiting deacetylase activity.

[0008] Furthermore, the protein deacetylase inhibitor NAM can improve the fertilization ability of poultry frozen-thawed sperm.

[0009] Furthermore, the protein deacetylase inhibitor NAM is used alone or in combination with other poultry semen cryoprotectants.

[0010] Furthermore, the invention also includes the use of the protein deacetylase inhibitor NAM in preparing a reagent for poultry seed preservation.

[0011] The present invention also provides a reagent for ultra-low temperature cryopreservation of poultry semen, wherein the reagent for ultra-low temperature cryopreservation of poultry semen comprises a protein deacetylase inhibitor NAM.

[0012] Compared with the prior art, the sperm protein lysine acetylation described in the present invention participates in the freezing stimulation response of poultry sperm, and the protein deacetylase inhibitor NAM can inhibit the deacetylation modification of sperm cells to maintain a high level of acetylation modification, thereby improving the ability of sperm to respond to ultra-low temperature freezing stimulation and the fertilization ability of frozen-thawed sperm. Using the protein deacetylase inhibitor NAM as a cryoprotectant, or using it in combination with other cryoprotectants, and using the protein deacetylase inhibitor NAM as an active ingredient will improve the ultra-low temperature freezing preservation efficiency of poultry. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a comparison of the protein acetylation patterns of the antifreeze-resistant sperm and the low-antifreeze-resistant sperm obtained in Example 1;

[0014] Among them, samples 1-5 are frozen sperm samples of the antifreeze group, and samples 6-10 are frozen sperm samples of the low-antifreeze group;

[0015] Figure 2A This is a Western blot result of protein acetylation of sperm with and without the addition of protein deacetylase inhibitors in Example 3;

[0016] Figure 2B This is a grayscale value result diagram of the protein acetylation modification level of sperm with and without the addition of protein deacetylase inhibitors in Example 3;

[0017] Figure 3This is a graph showing the fertilization rate results of frozen-thawed sperm with and without the addition of a protein deacetylase inhibitor in Example 4. DETAILED DESCRIPTION

[0018] The present invention provides application of protein deacetylase inhibitor NAM in ultra-low temperature cryopreservation of poultry semen.

[0019] The protein deacetylase inhibitor NAM provided by the present invention is a compound with the structural formula shown below:

[0020]

[0021] In the present invention, the protein deacetylase inhibitor NAM inhibits the acetylase NAD + dependent on the activity of the SIRT family.

[0022] In the present invention, the protein deacetylase inhibitor NAM reduces sperm freezing damage by inhibiting deacetylase activity.

[0023] In the invention, the protein deacetylase inhibitor NAM can improve the fertilization ability of poultry frozen-thawed sperm.

[0024] In the present invention, the protein deacetylase inhibitor NAM is used alone or in combination with other poultry semen cryoprotectants.

[0025] The invention also includes the use of protein deacetylase inhibitor NAM in the preparation of poultry seed preservation reagents.

[0026] In the present invention, unless otherwise specified, the raw materials required for preparation are commercially available commodities well known to those skilled in the art. Semen diluent, semen cryoprotectant, and thawing solution are selected from (Zong Yunhe et al., Interspecific differences in chicken sperm antifreeze and its correlation with seminal plasma biochemical indices and candidate gene expression, China Poultry, 2020: 42 (12): 6-13.) described in the semen diluent, semen cryoprotectant, and thawing solution; the protein lysate used contains 8M urea, 1% TritonX-100, 10mM dithiothreitol, and 1% protease inhibitor mixture.

[0027] Example 1

[0028] This example is used to verify that the acetylation level of poultry sperm is related to the antifreeze ability of sperm. 1. Semen collection and quality testing

[0029] Among 160 Beijing oily chicken roosters, semen quality was determined according to the "Test Method for Poultry Semen Quality (NY / T4047-2021)", and the semen with sperm motility higher than 60%, semen volume higher than 350 μL, and density higher than 16×10 8 55 adult roosters per mL.

[0030] 2. Semen dilution, balancing and freezing

[0031] Rooster semen was collected by abdominal massage. 200 μL of fresh semen was mixed with 200 μL of semen diluent preheated at 37°C, and the mixture was balanced at 4°C for 30 minutes. Subsequently, 400 μL of semen cryoprotectant at 4°C was added and the mixture was evenly divided into semen tubes. The semen tubes were sealed and placed in a programmed freezer for cooling and freezing. The freezing program was set at a cooling rate of 12°C / min at a temperature of 4°C to -44°C and a cooling rate of 40°C / min at a temperature of -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0032] 3. Thawing and determination of sperm motility

[0033] Take the semen tube out of liquid nitrogen and 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.

[0034] Sperm motility was measured using a computer-assisted sperm analysis system. The magnitude of change in sperm motility before and after freezing (fresh sperm motility - frozen-thawed sperm motility) was used as a measure of sperm antifreeze ability. After four measurements, five high-freeze-resistant roosters and five low-freeze-resistant roosters with stable traits were finally selected for subsequent experiments. The magnitude of change in sperm motility in the high-freeze-resistant group was significantly smaller than that in the low-freeze-resistant group.

[0035] 4. Extraction of Sperm Protein

[0036] Semen from high- and low-freeze-resistant roosters were collected respectively, and centrifuged at 4°C and 12,000 g for 10 min. The seminal plasma was removed, and the sperm was washed twice with PBS. Four times the volume of protein lysis buffer was added and ultrasonic lysis was performed. The sperm was centrifuged at 4°C and 12,000 g for 10 min to remove cell debris. The supernatant was transferred to a new centrifuge tube, and the protein concentration was determined using a BCA kit.

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

[0038] 13 μg protein samples of rooster semen from the high and low antifreeze groups were collected, respectively. After electrophoresis, transfer to a membrane, and blocking, protein acetylation pan-antibody (Anti-acetyllysine Antibody, PTM-101, 12838533L303) was added for primary antibody incubation. After the primary antibody incubation, the secondary antibody (Goat anti-Mouse IgG (H+L), Peroxidase Conjugated, 31430) was incubated. After the secondary antibody incubation, the cells were rinsed and incubated with chemiluminescent HRP substrate for 2 minutes. Signals were captured according to the operating instructions of the chemiluminescent imaging system.

[0039] The results are as follows Figure 1 As shown in the figure, there are certain differences in acetylation modification between the two groups. In the low antifreeze group, the acetylated protein near 35kD is more abundant, while the acetylation modification level of protein near 30kD is relatively low. It can be seen that the acetylation modification level of poultry sperm is related to the antifreeze ability of sperm.

[0040] Example 2

[0041] This example analyzes the key deacetylases involved in sperm acetylation modification.

[0042] 1. Sample Collection

[0043] Six healthy adult Beijing roosters were randomly selected. Semen was collected using dorsal and abdominal massage and centrifuged at 5000 g for 10 min at 4°C. The supernatant was aspirated and transferred to a fresh centrifuge tube and centrifuged at 12000 g for 10 min at 4°C. Two volumes of protein lysis buffer were added to obtain a seminal plasma protein sample. The pellet was resuspended in PBS, washed by centrifugation, and then sonicated with four volumes of protein lysis buffer. Cell debris was removed by centrifugation at 12000 g for 10 min at 4°C. The supernatant was transferred to a fresh centrifuge tube to obtain a sperm protein sample. Roosters were slaughtered, testicular tissue was isolated, and a soybean-sized testicular sample was obtained. Four volumes of lysis buffer were added and homogenized. Cell debris was removed by centrifugation at 12000 g for 10 min at 4°C. The supernatant was transferred to a fresh centrifuge tube to obtain a testicular protein sample. The protein concentrations of the testicular, sperm, and seminal plasma protein samples obtained above were determined using a BCA assay.

[0044] 2. Qualitative and quantitative analysis of proteins

[0045] Testicular protein samples, sperm protein samples and seminal plasma protein samples were taken respectively for liquid phase tandem mass spectrometry analysis. The protein expression profiles of the testicular protein samples, sperm protein samples and seminal plasma protein samples were obtained through library search analysis.

[0046] The results showed that both testis and sperm expressed high abundance of deacetylases, including Zn 2+ The histone deacetylase (HDAC) family and NAD + The sirtuins (SIRT) family is dependent on thymidine, as shown in Table 1.

[0047] Example 3

[0048] This example is used to verify that the addition of protein deacetylase inhibitors can increase the acetylation modification level of poultry sperm.

[0049] 1. Semen collection and grouping

[0050] According to the "Test Method for Poultry Semen Quality (NY / T 4047-2021)", sperm motility higher than 60%, semen volume higher than 350 μL, and density higher than 16×10 8 Semen was collected from 30 healthy adult Beijing oily chickens with a concentration of 100 μg / mL by dorsal and abdominal massage. The semen of each individual was mixed and divided into three groups: blank control group, TSA control group and NAM group.

[0051] 2. Preparation of Semen Diluent and Protective Fluid

[0052] Sodium chloride (NaCl) was added to the semen cryodiluent at an osmotic pressure of 360 mOsm / kg to obtain a semen cryodiluent with NaCl as the blank control group;

[0053] 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;

[0054] 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;

[0055] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor TSA was added to the semen cryoprotective solution to obtain the semen cryoprotective solution added with the deacetylase inhibitor TSA;

[0056] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor NAM was added to the semen freezing diluent to obtain the semen freezing diluent added with the deacetylase inhibitor NAM;

[0057] The deacetylase inhibitor NAM was added to the semen cryoprotectant at an osmotic pressure of 360 mOsm / kg to obtain a semen cryoprotectant supplemented with the deacetylase inhibitor NAM.

[0058] 3. Semen balance and freezing

[0059] Blank control group: 200 μL of fresh semen was mixed with an equal amount of semen freezing diluent of the blank control group preheated at 37°C, and the mixture was balanced at 4°C for 30 min. Subsequently, 400 μL of semen freezing protection solution of the blank control group at 4°C was added, and the semen was divided into semen straws. After the semen straws were sealed, they were placed in a programmed freezer for cooling and freezing. The freezing program was set at a cooling rate of 12°C / min at a temperature of 4°C to -44°C; and a cooling rate of 40°C / min at a temperature of -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0060] TSA control group: 200 μL of fresh semen was mixed with an equal amount of semen freezing diluent containing the deacetylase inhibitor TSA preheated at 37°C, and the mixture was balanced at 4°C for 30 minutes. Subsequently, 400 μL of semen cryoprotectant containing the deacetylase inhibitor TSA at 4°C was added and the mixture was divided into semen tubes. The semen tubes were sealed and placed in a programmed freezer for cooling and freezing. The freezing program was set at a cooling rate of 12°C / min at a temperature of 4°C to -44°C and a cooling rate of 40°C / min at a temperature of -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0061] NAM group: 200 μL of fresh semen was mixed with an equal amount of semen cryoprotectant containing the deacetylase inhibitor NAM preheated at 37°C, and the mixture was balanced at 4°C for 30 min. Subsequently, 400 μL of semen cryoprotectant containing the deacetylase inhibitor NAM of the blank control group at 4°C was added and the semen was divided into semen tubes. The semen tubes were sealed and placed in a programmed freezer for cooling and freezing. The freezing program was set at a cooling rate of 12°C / min at a temperature of 4°C to -44°C and a cooling rate of 40°C / min at a temperature of -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0062] 4. Thawing

[0063] Take the semen tube out of liquid nitrogen and 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.

[0064] 5. Extraction of Sperm Protein

[0065] After thawing, the sperm was centrifuged at 4°C and 12,000 g for 10 min, the supernatant was removed, and the sperm was washed twice with PBS. Two volumes of lysis buffer containing protease inhibitors were added to obtain a sperm sample. Four volumes of lysis buffer were added to the sperm sample and ultrasonically lysed. The sperm was centrifuged at 4°C and 12,000 g for 10 min to remove cell debris. The supernatant was transferred to a new centrifuge tube to obtain a protein sample, and the protein concentration of the precipitate obtained by centrifugation was determined using a BCA kit.

[0066] 6. Detection of sperm protein acetylation modification levels

[0067] 13 μg of the above protein sample was taken, subjected to electrophoresis, membrane transfer, and blocking, and then protein acetylated pan-antibody was added for primary antibody incubation. After the primary antibody incubation, secondary antibody was added for incubation. After rinsing, chemiluminescent HRP substrate was added and incubated for 2 minutes. Signal was captured according to the operating instructions of the chemiluminescent imaging system.

[0068] The results are shown in Figure 2. It can be seen that the acetylation modification level of frozen-thawed sperm was higher than that of the control group after adding protease inhibitors TSA and NAM.

[0069] Example 4

[0070] This example is used to verify that the addition of protein deacetylase inhibitors can improve the fertilizing ability of poultry frozen-thawed sperm.

[0071] 1. Semen collection and grouping

[0072] According to the "Test Method for Poultry Semen Quality (NY / T 4047-2021)", sperm motility higher than 60%, semen volume higher than 350 μL, and density higher than 16×10 8 Semen was collected from 30 healthy adult Beijing oily chickens with a concentration of 100 μg / mL by dorsal and abdominal massage. The semen of each individual was mixed and divided into three groups: blank control group, TSA control group and NAM group.

[0073] 2. Preparation of Semen Freezing Diluent and Protective Fluid

[0074] Sodium chloride (NaCl) was added to the semen cryodiluent at an osmotic pressure of 360 mOsm / kg to obtain a semen cryodiluent with NaCl as the blank control group;

[0075] 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;

[0076] 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;

[0077] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor TSA was added to the semen cryoprotective solution to obtain the semen cryoprotective solution added with the deacetylase inhibitor TSA;

[0078] According to the osmotic pressure of 360mOsm / kg, the deacetylase inhibitor NAM was added to the semen freezing diluent to obtain the semen freezing diluent added with the deacetylase inhibitor NAM;

[0079] The deacetylase inhibitor NAM was added to the semen cryoprotectant at an osmotic pressure of 360 mOsm / kg to obtain a semen cryoprotectant supplemented with the deacetylase inhibitor NAM.

[0080] 3. Semen dilution, balancing and freezing

[0081] Blank control group: 200 μL of fresh semen was mixed with an equal amount of semen freezing diluent of the blank control group preheated at 37°C, and the mixture was balanced at 4°C for 30 min. Subsequently, 400 μL of semen freezing protection solution of the blank control group at 4°C was added, and the semen was divided into semen straws. After the semen straws were sealed, they were placed in a programmed freezer for cooling and freezing. The freezing program was set at a cooling rate of 12°C / min at a temperature of 4°C to -44°C; and a cooling rate of 40°C / min at a temperature of -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0082] TSA control group: 200 μL of fresh semen was mixed with an equal amount of semen freezing diluent containing the deacetylase inhibitor TSA preheated at 37°C, and the mixture was balanced at 4°C for 30 minutes. Subsequently, 400 μL of semen cryoprotectant containing the deacetylase inhibitor TSA at 4°C was added and the mixture was divided into semen tubes. The semen tubes were sealed and placed in a programmed freezer for cooling and freezing. The freezing program was set at a cooling rate of 12°C / min at a temperature of 4°C to -44°C and a cooling rate of 40°C / min at a temperature of -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0083] NAM group: 200 μL of fresh semen was mixed with an equal amount of semen cryoprotectant containing the deacetylase inhibitor NAM preheated at 37°C, and the mixture was balanced at 4°C for 30 min. Subsequently, 400 μL of semen cryoprotectant containing the deacetylase inhibitor NAM of the blank control group at 4°C was added and the semen was divided into semen tubes. The semen tubes were sealed and placed in a programmed freezer for cooling and freezing. The freezing program was set at a cooling rate of 12°C / min at a temperature of 4°C to -44°C and a cooling rate of 40°C / min at a temperature of -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0084] 4. Thawing: Take out the semen tubes of the blank control group, TSA control group and NAM group from liquid nitrogen respectively, immerse them in 4℃ water to thaw for 3 minutes, pour them into centrifuge tubes, measure the volume and add 2 times the volume of 4℃ thawing solution, add it in 5 times, each time with an interval of 2 minutes, mix gently, then centrifuge at 4℃ temperature for 8 minutes, with a centrifugal force of 600g, remove the supernatant and obtain the sperm pellet.

[0085] 5. Determination of fertilization rate

[0086] To the sperm pellets collected in the blank control, TSA control, and NAM groups, an equal volume of thawed semen was added to the precipitates in 4°C DMEM to resuspend the pellets. Sixty White Leghorn hens were randomly divided into three groups and infused with frozen-thawed sperm from the control, TSA, and NAM groups, respectively. Each hen received approximately 100 million sperm for two consecutive days. Hatching eggs were collected and incubated for four days, and the fertilization rate was calculated.

[0087] The results are as follows Figure 3 As shown, the fertilization rates of the groups supplemented with protease inhibitors NAM and TSA were higher than those of the control group, and the fertilization rates of the NAM group were higher than those of the TSA group.

[0088] The above results confirm that adding the protein deacetylase inhibitor NAM to semen cryoprotectant and semen cryodiluent can improve the fertilizing ability of frozen-thawed sperm.

[0089] The operations described in the above embodiments are not limited to those described in the above embodiments and can be performed using technical means well known to those skilled in the art.

[0090] Table 1 Deacetylases in testicles, sperm and seminal plasma of Beijing oily chicken

[0091]

[0092]

[0093] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. Application of protein deacetylase inhibitor NAM in ultra-low temperature cryopreservation of poultry semen.

2. The use of the protein deacetylase inhibitor NAM in ultra-low temperature cryopreservation of poultry semen according to claim 1, characterized in that: The protein deacetylase inhibitor NAM reduces sperm freezing damage by inhibiting deacetylase activity.

3. The use of the protein deacetylase inhibitor NAM in ultra-low temperature cryopreservation of poultry semen according to claim 1, characterized in that: The protein deacetylase inhibitor NAM can improve the fertilization ability of poultry frozen-thawed sperm.

4. The use of the protein deacetylase inhibitor NAM in ultra-low temperature cryopreservation of poultry semen according to claim 1, characterized in that: The protein deacetylase inhibitor NAM is used alone or in combination with other poultry semen cryoprotectants.

5. The use of the protein deacetylase inhibitor NAM in ultra-low temperature cryopreservation of poultry semen according to claim 1, characterized in that: The invention also includes the use of the protein deacetylase inhibitor NAM in preparing a reagent for poultry seed preservation.

6. A reagent for ultra-low temperature cryopreservation of poultry semen, characterized in that: Contains the protein deacetylase inhibitor NAM.

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