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

Abstract

The invention provides application of a protein deacetylase inhibitor NAM in ultralow-temperature cryopreservation of poultry semen, and belongs to the technical field of poultry breed conservation. The protein deacetylase inhibitor NAM is used as a cryoprotectant or is combined with other cryoprotectants for use, and the protein deacetylase inhibitor NAM is used as an effective component, so that the ultralow-temperature cryopreservation efficiency of poultry can be improved. Wherein the protein deacetylase inhibitor NAM can inhibit sperm cell deacetylation modification so as to maintain a high acetylation modification level, improve the ability of sperms to respond to ultralow temperature freezing stimulation and improve the fertilization ability of frozen-remelted sperms.

Description

Technical Field

[0001] The 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 is to inhibit the metabolism of sperm under ultra-low temperature conditions such as liquid nitrogen (-196℃) to achieve long-term effective storage of sperm. After thawing and warming, sperm can restore its fertilization ability. Cryopreservation of semen is of great significance in livestock and poultry seed conservation and breeding. Ultra-low temperature cryopreservation of semen is an important means of livestock and poultry translocation seed conservation. The application of this technology to establish a sperm bank can effectively preserve the genetic information of species, prevent the loss of genetic information caused by environmental and selection factors, maintain the genetic diversity of species, and reduce the risk of species extinction; ultra-low temperature freezing technology of semen can break through the limitations of time and space, realize the exchange of genetic resources of different generations and regions, and promote genetic improvement of livestock and poultry and the cultivation of new varieties. my country has the richest chicken genetic resources in the world, with 115 local chicken breeds, which are precious wealth of animal husbandry. However, the structural characteristics of chicken sperm with a small head and a long tail lead to its poor antifreeze ability. Establishing a stable and efficient chicken semen cryopreservation technology has always been a hot spot and difficulty in research in this field.

[0003] Theoretical research on the cryopreservation of chicken semen 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, which may play an important role in maintaining sperm structural integrity, mitochondrial functional integrity and DNA structural integrity. Therefore, it is of great practical significance to study the protection and damage repair mechanism of sperm in response to low temperature stimulation and to find proteinase inhibitors that can improve the efficiency of ultra-low temperature cryopreservation of poultry semen. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a protein deacetylase inhibitor NAM in ultra-low temperature cryopreservation of poultry semen. The acetylation modification of sperm protein lysine is involved in the freezing stimulation response of poultry sperm. 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 improving 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 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 cryoinjury 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 reaction of poultry sperm, and the protein deacetylase inhibitor NAM can inhibit the deacetylation modification of sperm cells to maintain a higher 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 effective ingredient can improve the ultra-low temperature freezing storage efficiency of poultry. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0015] Figure 2A The Western blot results 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 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 activity of the SIRT family.

[0022] In the present invention, the protein deacetylase inhibitor NAM reduces sperm cryoinjury 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 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 products known to those skilled in the art. The 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 indicators and candidate gene expression, China Poultry, 2020: 42 (12): 6-13.) 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, the semen quality was determined according to the "Poultry Semen Quality Test Method (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] The 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 balanced at 4°C for 30 minutes. Then 400 μL of semen cryoprotectant at 4°C was added and 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 tubes were stored in liquid nitrogen.

[0032] 3. Thawing and determination of sperm motility

[0033] Take out the semen tube from 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 change in sperm motility before and after freezing (fresh sperm motility - frozen-rethawed 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 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 of high- and low-freeze-resistant roosters were collected respectively, and centrifuged at 4°C and 12000g 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; centrifuged at 4°C and 12000g for 10 min to remove cell debris, and 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 level

[0038] 13 μg protein samples of rooster semen in the high and low antifreeze groups were taken respectively. After electrophoresis, transfer and blocking, protein acetylated 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. The signal was captured according to the operating instructions of the chemiluminescent imaging system.

[0039] The results are as follows Figure 1 As shown, there are certain differences in acetylation modification between the two groups. In the low antifreeze group, acetylated proteins near 35kD are more abundant, while the acetylation modification level of proteins 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 oily roosters were randomly selected, and semen was collected by dorsal and abdominal massage, 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 times the volume of protein lysis buffer was added to obtain a seminal plasma protein sample; PBS was added to the precipitate for resuspending, centrifugation and washing, 4 times the volume of protein lysis buffer was added, ultrasonic lysis was performed, centrifuged at 4℃ and 12000g for 10min to remove cell debris, 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 times the volume of lysis buffer was 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 concentrations of the testicular protein samples, sperm protein samples, and seminal plasma protein samples obtained above were determined using the BCA kit.

[0044] 2. Qualitative and quantitative analysis of proteins

[0045] Testicular protein samples, sperm protein samples and seminal plasma protein samples were respectively taken for liquid phase tandem mass spectrometry analysis, and 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 dependent Sirtuins (SIRT) family is shown in Table 1.

[0047] Example 3

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

[0049] 1. Semen collection and grouping

[0050] 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 Semen was collected from 30 healthy adult Beijing oily chickens with a concentration of 100 g / mL by dorsal and abdominal massage. The semen of the individuals was mixed and divided into three groups: blank control group, TSA control group and NAM group.

[0051] 2. Preparation of semen diluent and protective solution

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

[0053] Sodium chloride (NaCl) was added to the semen cryoprotectant solution at an osmotic pressure of 360 mOsm / kg to obtain a semen cryoprotectant solution with NaCl as a 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 is 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 is 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 containing 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 the blank control group semen freezing diluent preheated at 37°C, and equilibrated at 4°C for 30 min. Subsequently, 400 μL of the blank control group semen freezing protection solution at 4°C 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 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 min. Subsequently, 400 μL of semen freezing protection solution containing the deacetylase inhibitor TSA at 4°C was added and 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 tubes were stored in liquid nitrogen.

[0061] NAM group: 200 μL of fresh semen was mixed with an equal amount of semen cryoprotectant containing deacetylase inhibitor NAM preheated at 37°C, and the mixture was balanced at 4°C for 30 min. Then, 400 μL of semen cryoprotectant containing deacetylase inhibitor NAM of the blank control group at 4°C was added and 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 tubes were stored in liquid nitrogen.

[0062] 4. Thawing

[0063] Take out the semen tube from 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, centrifuge it at 4°C and 12000g for 10 min, remove the supernatant, wash the sperm twice with PBS, add 2 times the volume of lysis solution containing protease inhibitors to obtain a sperm sample; add 4 times the volume of lysis buffer to the sperm sample and perform ultrasonic lysis; centrifuge it at 4°C and 12000g for 10 min to remove cell debris, transfer the supernatant to a new centrifuge tube to obtain a protein sample, and use a BCA kit to determine the protein concentration of the precipitate obtained by centrifugation.

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

[0067] 13 μg of the above protein sample was taken, and after electrophoresis, transfer and blocking, protein acetylation pan-antibody was added for primary antibody incubation. After the primary antibody incubation, secondary antibody incubation was added. After rinsing, chemiluminescent HRP substrate was added for incubation for 2 minutes, and signal capture was performed 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 a protein deacetylase inhibitor can improve the fertilization ability of poultry frozen-thawed sperm.

[0071] 1. Semen collection and grouping

[0072] 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 Semen was collected from 30 healthy adult Beijing oily chickens with a concentration of 100 g / mL by dorsal and abdominal massage. The semen of the individuals was mixed and divided into three groups: blank control group, TSA control group and NAM group.

[0073] 2. Preparation of semen cryoprotectant and diluent

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

[0075] Sodium chloride (NaCl) was added to the semen cryoprotectant solution at an osmotic pressure of 360 mOsm / kg to obtain a semen cryoprotectant solution with NaCl as a 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 is 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 is 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 containing 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 the blank control group semen freezing diluent preheated at 37°C, and equilibrated at 4°C for 30 min. Subsequently, 400 μL of the blank control group semen freezing protection solution at 4°C 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 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 min. Subsequently, 400 μL of semen freezing protection solution containing the deacetylase inhibitor TSA at 4°C was added and 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 tubes were stored in liquid nitrogen.

[0083] NAM group: 200 μL of fresh semen was mixed with an equal amount of semen cryoprotectant containing deacetylase inhibitor NAM preheated at 37°C, and the mixture was balanced at 4°C for 30 min. Then, 400 μL of semen cryoprotectant containing deacetylase inhibitor NAM of the blank control group at 4°C was added and 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 tubes were 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, 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℃ for 8 minutes with a centrifugal force of 600g, remove the supernatant and get the sperm precipitate.

[0085] 5. Determination of fertilization rate

[0086] The blank control group, TSA control group and NAM group sperm precipitates collected in the above steps were resuspended in 4°C DMEM solution of the same volume as the thawed semen. 60 white Leghorn hens were selected and randomly divided into 3 groups. The frozen-reconstituted sperm of the control group, TSA group and NAM group were infused, and 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.

[0087] The results are as follows Figure 3 As shown, it can be seen that the fertilization rates of the groups with added 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 cryoprotectant can improve the fertilization ability of frozen-thawed sperm.

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

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

[0091]

[0092]

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

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 plays the role of reducing sperm freezing damage by inhibiting the activity of deacetylase.

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 NAM, a protein deacetylase inhibitor.

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