Application of protein deacetylase inhibitor SirReal2 in ultra-low temperature cryopreservation of poultry semen

Abstract

This invention provides the application of the protein deacetylase inhibitor SirReal2 in the cryopreservation of poultry semen, belonging to the field of poultry breeding technology. The protein deacetylase inhibitor SirReal2 described in this invention can inhibit sperm cell deacetylation modification to maintain a high level of acetylation modification, improve the sperm's ability to respond to cryopreservation stimulation, and enhance the fertilization capacity of frozen-thawed sperm. Using SirReal2 as a cryoprotectant, or in combination with other cryoprotectants, as an active ingredient, can improve the efficiency of cryopreservation of poultry.

Description

Technical Field

[0001] This invention relates to the field of poultry conservation technology, and in particular to the application of the protein deacetylase inhibitor SirReal2 in the cryopreservation of poultry semen. Background Technology

[0002] Cryopreservation of semen involves inhibiting sperm metabolism under ultra-low temperature conditions such as liquid nitrogen (-196℃) to achieve long-term effective preservation of sperm. After thawing and warming, the sperm can regain its fertilization capacity. Semen cryopreservation is of great significance in livestock and poultry conservation and breeding. Cryopreservation is an important means of translocation preservation in livestock and poultry. Establishing sperm banks using this technology can effectively preserve the genetic information of a species, prevent the loss of genetic information due to environmental factors and selection, maintain genetic diversity, and reduce the risk of species extinction. Cryopreservation technology can overcome the limitations of time and space, enabling the exchange of genetic resources between different generations and regions, promoting livestock and poultry genetic improvement and the breeding of new breeds. my country possesses the world's richest chicken genetic resources, with 115 local chicken breeds, a valuable asset to animal husbandry. However, the small head and long tail structure of chicken sperm results in poor cryopreservation resistance. Establishing stable and efficient chicken semen cryopreservation technology has always been a hot topic and a challenge in this field.

[0003] Current research on chicken semen cryopreservation primarily focuses on reducing sperm structural damage caused by ice crystallization and inhibiting sperm chemical damage caused by oxidative stress, lacking research on sperm's protective and repair mechanisms against low-temperature stimuli. Transcriptional and translational arrest in mature sperm, along with post-translational protein modifications, are crucial pathways for mature sperm to respond to internal and external environmental stimuli, potentially playing a vital role in maintaining sperm structural integrity, mitochondrial functional integrity, and DNA structural integrity. Therefore, researching sperm's protective and repair mechanisms against low-temperature stimuli and identifying protease inhibitors that can improve the efficiency of ultra-low-temperature cryopreservation of poultry semen is of significant practical importance. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the protein deacetylase inhibitor SirReal2 in the cryopreservation of poultry semen. The lysine acetylation modification of sperm proteins participates in the cryostimulation response of poultry sperm. The protein deacetylase inhibitor SirReal2 can inhibit the deacetylation modification of sperm cells to maintain a high level of acetylation modification, improve the sperm's ability to respond to cryopreservation stimulation, and improve the fertilization capacity 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] This invention provides the application of the protein deacetylase inhibitor SirReal2 in the cryopreservation of poultry semen.

[0007] Furthermore, the protein deacetylase inhibitor SirReal2 can improve the fertilization capacity of frozen-thawed sperm in poultry.

[0008] Furthermore, the protein deacetylase inhibitor SirReal2 improves the fertilization capacity of poultry sperm after cryopreservation by increasing the lysine acetylation modification level of poultry semen proteins.

[0009] Furthermore, the protein deacetylase inhibitor SirReal2 can be used alone or in combination with other poultry semen cryoprotectants.

[0010] Furthermore, this also includes the application of the protein deacetylase inhibitor SirReal2 in the preparation of reagents for poultry conservation.

[0011] The present invention also provides a reagent for cryopreservation of poultry semen, wherein the reagent for cryopreservation of poultry semen contains the protein deacetylase inhibitor SirReal2.

[0012] Compared with the prior art, the lysine acetylation of sperm protein described in this invention participates in the cryostimulation response of poultry sperm. The protein deacetylase inhibitor SirReal2 can inhibit sperm cell deacetylation modification to maintain a high level of acetylation modification, improve the sperm's ability to respond to cryogenic freezing stimulation, and improve the fertilization ability of frozen-thawed sperm. Using the protein deacetylase inhibitor SirReal2 as a cryoprotectant, or using it in combination with other cryoprotectants, or using the protein deacetylase inhibitor SirReal2 as an active ingredient, can improve the efficiency of cryogenic cryopreservation of poultry. Attached Figure Description

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

[0014] Samples 1-5 were frozen sperm samples from the antifreeze group, and samples 6-10 were frozen sperm samples from the low-antifreeze group.

[0015] Figure 2A Western blot results of protein acetylation in sperm from Example 3 with and without protein deacetylase inhibitors.

[0016] Figure 2BThe image shows the grayscale results of the protein acetylation modification levels of sperm in Example 3 with and without the addition of a protein deacetylase inhibitor.

[0017] Figure 3 The graph shows the fertilization rate results of frozen-thawed sperm in Example 4 with and without the addition of a protein deacetylase inhibitor. Detailed Implementation

[0018] This invention provides the application of the protein deacetylase inhibitor SirReal2 in the cryopreservation of poultry semen.

[0019] The protein deacetylase inhibitor SirReal2 provided by this invention is a compound with the following structural formula:

[0020]

[0021] The protein deacetylase inhibitor SirReal2 provided by this invention protects sperm and reduces cryopreservation damage by inhibiting the activity of deacetylase.

[0022] The protein deacetylase inhibitor SirReal2 provided by this invention inhibits the deacetylase NAD. + The activity of SIRT2, a member of the SIRT family, is dependent.

[0023] The protein deacetylase inhibitor SirReal2 provided by this invention can be used to prepare cryoprotective solutions that protect sperm from damage caused by changes in protein acetylation modification levels.

[0024] In this invention, the protein deacetylase inhibitor SirReal2 improves the fertilization capacity of poultry sperm after cryopreservation by increasing the lysine acetylation modification level of poultry semen proteins.

[0025] In this invention, the protein deacetylase inhibitor SirReal2 is able to improve the fertilization capacity of frozen-thawed sperm in poultry.

[0026] In this invention, the protein deacetylase inhibitor SirReal2 is used alone or in combination with other poultry semen cryoprotectants.

[0027] The invention also includes the use of the protein deacetylase inhibitor SirReal2 in the preparation of reagents for poultry conservation.

[0028] The invention includes the protein deacetylase inhibitor SirReal2.

[0029] In this invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well known to those skilled in the art. Among them, the semen diluent, semen cryoprotectant, and thawing solution are selected from those used in (Zong Yunhe et al., Interspecies Differences in Chicken Semen Antifreeze and Its Correlation with Seminal Plasma Biochemical Indicators and Candidate Gene Expression, Chinese Poultry, 2020:42(12):6-13.); the protein lysis buffer used contains a mixture of 8M urea, 1% Triton X-100, 10mM dithiothreitol, and 1% protease inhibitor.

[0030] Example 1

[0031] This embodiment is used to verify the relationship between the acetylation modification level of poultry sperm and the sperm's antifreeze ability. I. Semen collection and quality testing

[0032] Semen quality was determined in 160 Beijing Oil Chicken roosters according to the "Poultry Semen Quality Test Method (NY / T4047-2021)". Chickens with sperm motility above 60%, semen volume above 350 μL, and semen density above 16 × 10⁻⁶ were selected. 8 55 adult roosters per mL.

[0033] II. Semen dilution, equilibration, and freezing

[0034] Rooster semen was collected using the abdominal massage method. 200 μL of fresh semen was mixed with 200 μL of semen diluent preheated at 37°C and equilibrated at 4°C for 30 min. Then, 400 μL of semen cryoprotectant at 4°C was added and the mixture was evenly distributed into semen capillary tubes. The capillary tubes were sealed and placed in a programmed cryostat for freezing. The freezing program was set to a cooling rate of 12°C / min from 4°C to -44°C and a cooling rate of 40°C / min from -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0035] III. Thawing and Sperm Motility Measurement

[0036] Remove the semen capillary from liquid nitrogen and immerse it in 4°C water for 3 minutes to thaw. Pour the semen into a centrifuge tube, measure the volume, and then add twice the volume of 4°C thawing solution.

[0037] Sperm motility was measured using a computer-aided sperm analysis system: the change in sperm motility before and after freezing (fresh sperm motility - frozen-thawed sperm motility) was used as an indicator of sperm resistance to freezing. After four measurements, five roosters with stable traits and high freezing resistance and five roosters with low freezing resistance were finally selected for subsequent experiments. The change in sperm motility in the high freezing resistance group was significantly smaller than that in the low freezing resistance group.

[0038] IV. Extraction of Sperm Protein

[0039] Semen from high- and low-freeze roosters was collected and centrifuged at 12000g for 10 min at 4℃ to remove seminal plasma. The sperm were washed twice with PBS, and then lysed by sonication with 4 times the volume of protein lysis buffer. The lysate was then centrifuged at 12000g for 10 min at 4℃ to remove cell debris. The supernatant was transferred to a new centrifuge tube, and the protein concentration was determined using a BCA kit.

[0040] V. Detection of sperm protein acetylation modification level

[0041] 13 μg protein samples were taken from the semen of roosters in the high and low antifreeze groups, respectively. After electrophoresis, membrane transfer, and blocking, protein acetylation pan-antibody (Anti-acetyllysine Antibody, PTM-101, 12838533L303) was added for primary antibody incubation. After primary antibody incubation, secondary antibody (Goat anti-Mouse IgG (H+L), Peroxidase Conjugated, 31430) was added for incubation. After secondary antibody incubation, the samples were rinsed and then incubated with chemiluminescent HRP substrate for 2 minutes. Signal capture was performed according to the operation instructions of the chemiluminescent imaging system.

[0042] The results are as follows Figure 1 As shown, there are certain differences in acetylation modification between the two groups. The low-freezing-resistant group has a higher level of acetylated protein near 35kD, while the level of acetylation modification of protein near 30kD is relatively low. It can be seen that the level of acetylation modification in poultry sperm is related to the sperm's freezing resistance.

[0043] Example 2

[0044] This embodiment analyzes key deacetylases involved in sperm acetylation modification.

[0045] I. Sample Collection

[0046] Six adult healthy Beijing Oil Chicken roosters were randomly selected, and semen was collected using the back-abdomen massage method. The semen was centrifuged at 5000g for 10 min at 4℃. The supernatant was collected, transferred to a new centrifuge tube, and centrifuged at 12000g for 10 min at 4℃. Two volumes of protein lysis buffer were added to obtain seminal plasma protein samples. The precipitate was resuspended in PBS, washed by centrifugation, and four volumes of protein lysis buffer were added. The mixture was sonicated and centrifuged at 12000g for 10 min at 4℃ to remove cell debris. The supernatant was transferred to a new centrifuge tube to obtain sperm protein samples. Roosters were slaughtered, and testicular tissue was separated. Testicular samples the size of soybeans were collected, and four volumes of lysis buffer were added. The samples were homogenized and lysed, and centrifuged at 12000g for 10 min at 4℃ to remove cell debris. The supernatant was transferred to a new centrifuge tube to obtain testicular protein samples.

[0047] The protein concentrations of the testicular protein samples, sperm protein samples, and seminal plasma protein samples obtained above were determined using a BCA kit.

[0048] II. Qualitative and quantitative analysis of proteins

[0049] Testicular protein samples, sperm protein samples, and seminal plasma protein samples were collected and subjected to liquid chromatography-tandem mass spectrometry analysis. Through library search analysis, the protein expression profiles of the testicular protein samples, sperm protein samples, and seminal plasma protein samples were obtained.

[0050] The results showed that high abundance of deacetylases, including Zn, were expressed in both the testes and sperm. 2+ Histone deacetylase (HDAC) family and NAD + The dependent Sirtuins (SIRT) family is shown in Table 1.

[0051] Example 3

[0052] This embodiment is used to verify that adding a protein deacetylase inhibitor can increase the acetylation modification level of poultry sperm.

[0053] I. Semen Collection and Grouping

[0054] 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 samples were mixed and divided into three groups: blank control group, TSA control group, and SirReal2 group.

[0055] II. Preparation of Semen Diluent and Protective Solution

[0056] Sodium chloride (NaCl) was added to the semen cryopreservation diluent with an osmotic pressure of 360 mOsm / kg to obtain a semen cryopreservation diluent with NaCl as the blank control group.

[0057] Sodium chloride (NaCl) was added to the semen cryopreservation solution with an osmotic pressure of 360 mOsm / kg to obtain a semen cryopreservation solution with NaCl as the blank control group.

[0058] With an osmotic pressure of 360 mOsm / kg, the deacetylase inhibitor TSA was added to the semen cryopreservation diluent to obtain the semen cryopreservation diluent with added deacetylase inhibitor TSA.

[0059] With an osmotic pressure of 360 mOsm / kg, the deacetylase inhibitor TSA was added to the semen cryopreservation solution to obtain the semen cryopreservation solution with added deacetylase inhibitor TSA.

[0060] With an osmotic pressure of 360 mOsm / kg, SirReal2, a deacetylase inhibitor, was added to the semen cryopreservation diluent to obtain a semen cryopreservation diluent with added SirReal2.

[0061] With an osmotic pressure of 360 mOsm / kg, SirReal2, a deacetylase inhibitor, was added to the semen cryopreservation solution to obtain a semen cryopreservation solution with added SirReal2.

[0062] III. Semen Balance and Freezing

[0063] Blank control group: 200 μL of fresh semen was mixed with an equal volume of preheated (37℃) semen cryopreservation diluent from the blank control group and equilibrated at 4℃ for 30 min. Then, 400 μL of cryoprotectant solution from the blank control group at 4℃ was added, and the mixture was dispensed into semen capillary tubes. The capillary tubes were sealed and placed in a programmed cryostat for cooling and freezing. The freezing program was set to a cooling rate of 12℃ / min from 4℃ to -44℃ and a cooling rate of 40℃ / min from -44℃ to -120℃. After the program was completed, the semen was stored in liquid nitrogen.

[0064] TSA control group: 200 μL of fresh semen was mixed with an equal volume of semen cryopreservation diluent containing the deacetylase inhibitor TSA preheated at 37°C. The mixture was equilibrated at 4°C for 30 min. Then, 400 μL of semen cryopreservation solution containing the deacetylase inhibitor TSA at 4°C was added. The mixture was dispensed into semen caps, sealed, and placed in a programmed cryostat for freezing. The freezing program was set to a cooling rate of 12°C / min from 4°C to -44°C and a cooling rate of 40°C / min from -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0065] SirReal2 group: 200 μL of fresh semen was mixed with an equal volume of preheated (37°C) semen cryopreservation buffer containing the deacetylase inhibitor SirReal2. The mixture was equilibrated at 4°C for 30 min. Then, 400 μL of the blank control group's semen cryopreservation buffer containing the deacetylase inhibitor SirReal2 was added at 4°C. The mixture was dispensed into semen capillary tubes, sealed, and placed in a programmed cryostat for freezing. The freezing program was set to a cooling rate of 12°C / min from 4°C to -44°C and a cooling rate of 40°C / min from -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0066] IV. Thawing

[0067] Remove the semen capillary from liquid nitrogen and immerse it in 4°C water for 3 minutes to thaw. Pour the semen into a centrifuge tube, measure the volume, and then add twice the volume of 4°C thawing solution.

[0068] V. Extraction of Sperm Protein

[0069] After thawing, the sperm were centrifuged at 12000g for 10 min at 4℃, the supernatant was removed, and the sperm were washed twice with PBS. Two volumes of lysis buffer containing protease inhibitors were added to obtain sperm samples. Four volumes of lysis buffer were added to the sperm samples and the samples were lysed by sonication. The samples were then centrifuged at 12000g for 10 min at 4℃ to remove cell debris. The supernatant was transferred to a new centrifuge tube to obtain protein samples. The protein concentration of the precipitate obtained by centrifugation was determined using a BCA kit.

[0070] VI. Detection of sperm protein acetylation modification level

[0071] Take 13 μg of the above protein sample, and after electrophoresis, transfer to a membrane, and blocking, add protein acetylation pan-antibody for primary antibody incubation. After the primary antibody incubation, add secondary antibody incubation. After rinsing, add chemiluminescent HRP substrate and incubate for 2 minutes. Perform signal capture according to the operation instructions of the chemiluminescent imaging system.

[0072] As shown in Figure 2, the acetylation modification level of frozen-thawed sperm was higher than that of the control group after the addition of protease inhibitors TSA and SirReal2.

[0073] Example 4

[0074] This embodiment is used to verify that adding the protein deacetylase inhibitor SirReal2 can improve the fertilization ability of frozen-thawed sperm in poultry.

[0075] I. Semen Collection and Grouping

[0076] 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 samples were mixed and divided into three groups: blank control group, TSA control group, and SirReal2 group.

[0077] II. Preparation of Semen Freezing Diluent and Preservative Solution

[0078] Sodium chloride (NaCl) was added to the semen cryopreservation diluent with an osmotic pressure of 360 mOsm / kg to obtain a semen cryopreservation diluent with NaCl as the blank control group.

[0079] Sodium chloride (NaCl) was added to the semen cryopreservation solution with an osmotic pressure of 360 mOsm / kg to obtain a semen cryopreservation solution with NaCl as the blank control group.

[0080] With an osmotic pressure of 360 mOsm / kg, the deacetylase inhibitor TSA was added to the semen cryopreservation diluent to obtain the semen cryopreservation diluent with added deacetylase inhibitor TSA.

[0081] With an osmotic pressure of 360 mOsm / kg, the deacetylase inhibitor TSA was added to the semen cryopreservation solution to obtain the semen cryopreservation solution with added deacetylase inhibitor TSA.

[0082] With an osmotic pressure of 360 mOsm / kg, SirReal2, a deacetylase inhibitor, was added to the semen cryopreservation diluent to obtain a semen cryopreservation diluent with added SirReal2.

[0083] With an osmotic pressure of 360 mOsm / kg, SirReal2, a deacetylase inhibitor, was added to the semen cryopreservation solution to obtain a semen cryopreservation solution with added SirReal2.

[0084] III. Semen dilution, equilibration, and freezing

[0085] Blank control group: 200 μL of fresh semen was mixed with an equal volume of preheated (37℃) semen cryopreservation diluent from the blank control group and equilibrated at 4℃ for 30 min. Then, 400 μL of cryoprotectant solution from the blank control group at 4℃ was added, and the mixture was dispensed into semen capillary tubes. The capillary tubes were sealed and placed in a programmed cryostat for cooling and freezing. The freezing program was set to a cooling rate of 12℃ / min from 4℃ to -44℃ and a cooling rate of 40℃ / min from -44℃ to -120℃. After the program was completed, the semen was stored in liquid nitrogen.

[0086] TSA control group: 200 μL of fresh semen was mixed with an equal volume of semen cryopreservation diluent containing the deacetylase inhibitor TSA preheated at 37°C. The mixture was equilibrated at 4°C for 30 min. Then, 400 μL of semen cryopreservation solution containing the deacetylase inhibitor TSA at 4°C was added. The mixture was dispensed into semen caps, sealed, and placed in a programmed cryostat for freezing. The freezing program was set to a cooling rate of 12°C / min from 4°C to -44°C and a cooling rate of 40°C / min from -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0087] SirReal2 group: 200 μL of fresh semen was mixed with an equal volume of preheated (37°C) semen cryopreservation buffer containing the deacetylase inhibitor SirReal2. The mixture was equilibrated at 4°C for 30 min. Then, 400 μL of the blank control group's semen cryopreservation buffer containing the deacetylase inhibitor SirReal2 was added at 4°C. The mixture was dispensed into semen capillary tubes, sealed, and placed in a programmed cryostat for freezing. The freezing program was set to a cooling rate of 12°C / min from 4°C to -44°C and a cooling rate of 40°C / min from -44°C to -120°C. After the program was completed, the semen was stored in liquid nitrogen.

[0088] IV. Thawing

[0089] Semen capillaries from the blank control group, TSA control group, and SirReal2 group were removed from liquid nitrogen, immersed in 4°C water for 3 minutes to thaw, poured into centrifuge tubes, and after measuring the volume, 2 times the volume of 4°C thawing solution was added in 5 portions, 2 minutes apart, and gently mixed. Then, the mixture was centrifuged at 4°C for 8 minutes with a centrifugal force of 600g, and the supernatant was removed to obtain sperm precipitate.

[0090] V. Determination of fertilization rate

[0091] The sperm precipitates collected in the blank control group, TSA control group, and SirReal2 group were resuspended in 4℃ DMEM solution with an equal volume of thawed semen. Sixty white-feathered aircraft carrier hens were randomly divided into three groups, and frozen-thawed sperm from the control group, TSA group, and SirReal2 group were introduced into each hen. The insemination volume was approximately 100 million sperm per hen. Insemination was performed continuously for 2 days, and eggs were collected for 5 days. The eggs were then incubated and the fertilization rate was calculated.

[0092] The results are as follows Figure 3 As shown, the fertilization rates of the groups with added protease inhibitor SirReal2 and protease inhibitor TSA were higher than those of the control group, and the SirReal2 group had a higher fertilization rate than the TSA group.

[0093] The above results confirm that adding the protein deacetylase inhibitor SirReal2 to semen cryopreservation diluent, semen cryoprotectant, and saturates can improve the fertilization capacity of frozen-thawed sperm.

[0094] The operations described in the above embodiments are not limited and can be performed using techniques well known to those skilled in the art.

[0095] Table 1. Deacetylases in the testes, sperm, and seminal plasma of Beijing oil chickens.

[0096]

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of SirReal2, a protein deacetylase inhibitor, in the cryopreservation of poultry semen.

2. The application of SirReal2, a protein deacetylase inhibitor according to claim 1, in the cryopreservation of poultry semen, characterized in that, The protein deacetylase inhibitor SirReal2 can improve the fertilization capacity of frozen-thawed sperm in poultry.

3. The application of SirReal2, a protein deacetylase inhibitor according to claim 1, in the cryopreservation of poultry semen, characterized in that... The protein deacetylase inhibitor SirReal2 improves the fertilization capacity of poultry sperm after cryopreservation by increasing the lysine acetylation modification level of poultry semen proteins.

4. The application of SirReal2, a protein deacetylase inhibitor according to claim 1, in the cryopreservation of poultry semen, characterized in that... The protein deacetylase inhibitor SirReal2 can be used alone or in combination with other poultry semen cryoprotectants.

5. The application of SirReal2, a protein deacetylase inhibitor according to claim 1, in the cryopreservation of poultry semen, characterized in that... It also includes the application of the protein deacetylase inhibitor SirReal2 in the preparation of poultry breeding reagents.

6. A reagent for cryopreservation of poultry semen, characterized in that, It contains SirReal2, a protein deacetylase inhibitor.

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