Application of protein deacetylase inhibitor NRD167 in ultra-low temperature cryopreservation of poultry semen

Abstract

The present application relates to the field of poultry germplasm preservation biotechnology, and discloses application of protein deacetylase inhibitor NRD167 in poultry semen ultra-low temperature cryopreservation. It is proved by experiments that the protein deacetylase inhibitor NRD167 can improve the acetylation modification level of poultry sperm and the fertilization ability of frozen-thawed poultry sperm; therefore, the protein deacetylase inhibitor NRD167 can be used as an effective component for the ultra-low temperature cryopreservation of poultry semen, and plays an important role in improving the efficiency of poultry ultra-low temperature cryopreservation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of poultry germplasm preservation biotechnology, in particular, to application of protein deacetylase inhibitor NRD167 in poultry semen ultra-low temperature cryopreservation. BACKGROUND

[0002] Semen ultra-low temperature cryopreservation technology is a method of placing sperm in ultra-low temperature conditions such as liquid nitrogen (-196℃) to achieve long-term preservation of sperm by inhibiting sperm metabolism for subsequent use. The semen cryopreserved by ultra-low temperature can restore its fertilization ability after thawing. Therefore, semen ultra-low temperature cryopreservation technology has important significance in livestock and poultry breeding and breeding work. Semen ultra-low temperature cryopreservation technology can also be applied to the construction of sperm bank, which can effectively preserve the genetic information of species, prevent the loss of genetic information due to environmental and selection factors, maintain the genetic diversity of species, and reduce the risk of species extinction. It is an important means of livestock and poultry translocation preservation. In addition, semen ultra-low temperature cryopreservation technology can break through the time and space limitations, realize the exchange of genetic resources of different generations and regions, and promote the genetic improvement and breeding of new breeds of livestock and poultry.

[0003] The world has rich chicken genetic resources, which is a valuable treasure of animal husbandry. When chicken semen is subjected to semen ultra-low temperature cryopreservation, the chicken sperm has poor freezing resistance due to its small head and long tail. Therefore, establishing a stable and efficient chicken semen cryopreservation technology has always been a hot and difficult point in the field. The existing theoretical research on chicken semen cryopreservation mainly focuses on reducing the damage to sperm structure caused by ice crystal and inhibiting the chemical damage to sperm caused by oxidative stress, and lacks research on the protection and damage repair mechanism of sperm in response to low temperature stimulation. The transcription and translation of mature sperm are stalled, and post-translational modification of proteins is an important way for mature sperm to respond to internal and external environmental stimuli, which may play an important role in maintaining the structural integrity of sperm, mitochondrial function integrity and DNA structure 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 cryopreservation technology. SUMMARY

[0004] The purpose of the present application is to provide an application of protein deacetylase inhibitor NRD167 in poultry semen ultra-low temperature cryopreservation, which uses protein deacetylase inhibitor NRD167 as an effective ingredient for the ultra-low temperature cryopreservation of poultry semen, and plays an important role in improving the efficiency of poultry ultra-low temperature cryopreservation.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

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

[0007] The protein deacetylase inhibitor NRD167 provided by the application has the structural formula as shown in the following:

[0008]

[0009] Further, the application of the protein deacetylase inhibitor NRD167 in the ultra-low temperature cryopreservation of poultry semen is as follows: according to the osmotic pressure of 360 mOsm / kg, the protein deacetylase inhibitor NRD167 is added into the semen freezing diluent and the semen cryoprotective solution.

[0010] Further, the protein deacetylase inhibitor NRD167 can be used in combination with other cryoprotective reagents.

[0011] Compared with the prior art, the application has the beneficial effects that:

[0012] The application of the protein deacetylase inhibitor NRD167 provided by the application in the ultra-low temperature cryopreservation of poultry semen is proved by tests that the protein lysine acetylation modification of poultry sperm participates in the freezing stimulation response of poultry sperm, and the protein deacetylase inhibitor NRD167 can inhibit the deacetylation modification (specifically, inhibit the acetylation enzyme NAD + dependent SIRT family SIRT5 activity) of poultry sperm cells, so as to maintain a high level of acetylation modification and improve the ability of poultry sperm to respond to the ultra-low temperature freezing stimulation and the fertilization ability of the frozen-thawed poultry sperm; therefore, the protein deacetylase inhibitor NRD167 can be used as an effective component for the ultra-low temperature cryopreservation of poultry semen and plays an important role in improving the efficiency of the ultra-low temperature cryopreservation of poultry. BRIEF DESCRIPTION OF DRAWINGS

[0013] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included to provide a description of the preferred embodiments and are not intended to limit the scope of the application. Moreover, the same reference numerals are used throughout the same figures. In the drawings:

[0014] Figure 1 The figure is the detection result graph of the acetylation modification level of sperm protein of the high-freeze-resistant cock group and the low-freeze-resistant cock group;

[0015] Figure 2 The figure is the detection result graph of the acetylation modification level of sperm protein of the TSA group, the NRD167 group and the blank control group;

[0016] Figure 3 The figure is the detection result graph of the fertilization rate of the TSA group, the NRD167 group and the blank control group. DETAILED DESCRIPTION

[0017] The following detailed description of various example embodiments of the application is not to be considered limiting of the scope or spirit of the application as described in the appended claims, but is intended to describe certain aspects, features and embodiments of the application in more detail. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0018] In addition, for numerical ranges recited herein, every intervening value between the upper and lower limits is specifically contemplated. In addition, any other stated or intervening value or intervening range of values in any stated value or range of values is also specifically contemplated. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification prevails.

[0020] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0021] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to mean including, but not limited to.

[0022] In the present application, unless otherwise specified, the semen freezing diluent, semen freezing protective solution and thawing solution used are as described in "Chicken sperm freezing resistance interspecific differences and its correlation with seminal plasma biochemical indicators and candidate gene expression" by Zong Yunhe et al., China Poultry, 2020: 42(12): 6-13. The protein lysis solution used contains 8M urea, 1% Triton X-100, 10 mM dithiothreitol and 1% protease inhibitor cocktail.

[0023] I. The level of acetylation modification of poultry sperm and the freezing resistance ability of sperm

[0024] 1. Collection of samples

[0025] According to the Poultry Semen Quality Detection Method (NY / T 4047-2021), 55 adult healthy Beijing oil cock chickens with sperm motility higher than 60%, semen volume higher than 350 μL, and density higher than 16 × 10 8 The semen of the cock chickens was collected by back and abdomen massage method;

[0026] 2. Semen dilution, balance and freezing

[0027] 200 μL fresh semen was mixed with 200 μL semen freezing diluent preheated to 37℃, balanced at 4℃ for 30 min, then 400 μL 4℃ semen cryoprotective solution was added, and the semen was divided into a thin tube and sealed. The thin tube was placed in a programmed freezer for cooling and freezing, and the freezing program was set as 4℃ to -44℃ at a rate of 12℃ / min, -44℃ to -120℃ at a rate of 40℃ / min, and then put into liquid nitrogen for storage;

[0028] 3. Thawing and sperm motility detection

[0029] The semen thin tube was taken out from the liquid nitrogen and immersed in 4℃ water for 3 min, then poured into a centrifuge tube, and after the volume was measured, 2 times the volume of 4℃ thawing solution was added to obtain the frozen-thawed sperm sample;

[0030] The sperm motility in the frozen-thawed sperm sample was detected by computer-aided sperm analysis system, and the change range of sperm motility before and after freezing was used as the index for measuring the sperm freezing resistance;

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

[0032] After 4 times of detection, 5 high freezing-resistant cock chickens and 5 low freezing-resistant cock chickens with stable traits were selected for subsequent experiments, and the sperm motility change range of the high freezing-resistant cock chicken group and the low freezing-resistant cock chicken group is shown in Table 1;

[0033] Table 1 Sperm motility change range of high freezing-resistant cock chicken group and low freezing-resistant cock chicken group

[0034]

[0035] 4. Extraction of sperm protein

[0036] The semen of the high freezing-resistant cock chicken group and the low freezing-resistant cock chicken group was collected, centrifuged at 4℃ and 12000g for 10 min to remove the seminal plasma, washed twice with PBS, then 4 times the volume of protein lysis buffer was added, ultrasonicated, centrifuged at 4℃ and 12000g for 10 min to remove the cell debris, and the supernatant was transferred to a new centrifuge tube to obtain the sperm protein samples of the high freezing-resistant cock chicken group and the low freezing-resistant cock chicken group; the protein concentration was detected by BCA kit;

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

[0038] Take 13 μg sperm protein sample of high cold-resistant cock group and low cold-resistant cock group respectively, after electrophoresis, membrane transfer and blocking, add protein acetylation pan antibody (Anti-acetyl lysine Antibody, PTM-101, 12838533L303) for primary antibody incubation, after primary antibody incubation, carry out secondary antibody incubation (Goat anti-Mouse IgG (H+L), Peroxidase Conjugated, 31430), after secondary antibody incubation, rinse, and add chemical luminescence HRP substrate for incubation for 2 minutes, according to the operation instruction of chemical luminescence imaging system to carry out signal capture;

[0039] 6. Experimental results

[0040] The detection results of acetylation modification level of sperm protein of high cold-resistant cock group and low cold-resistant cock group are shown in Table 1, wherein, the samples 1-5 are sperm samples of high cold-resistant cock group, and the samples 6-10 are sperm samples of low cold-resistant cock group. Figure 1

[0041] It can be known from Table 1 that there is a certain difference in acetylation modification level of sperm protein of high cold-resistant cock group and low cold-resistant cock group, wherein, the acetylation modification level of protein near 35kD of low cold-resistant cock group is relatively high, and the acetylation modification level of protein near 30kD is relatively low, which shows that the acetylation modification level of poultry sperm is related to the anti-freezing ability of sperm. Figure 1

[0042] II. Analysis of deacetylase involved in acetylation modification of sperm protein

[0043] 1. Collection of samples and extraction of protein

[0044] ​​Six adult healthy Beijing-You chickens were randomly selected, and the semen was collected by back and abdomen massage method, centrifuged at 5000g for 10 min at 4℃; the supernatant was transferred to a new centrifuge tube, centrifuged at 12000g for 10 min at 4℃, 2 times the volume of protein lysis buffer was added, and the sperm protein sample was obtained; the precipitate was resuspended with PBS, washed by centrifugation, 4 times the volume of protein lysis buffer was added, ultrasonic lysis, centrifuged at 12000g for 10 min at 4℃ to remove cell debris, and the supernatant was transferred to a new centrifuge tube to obtain the sperm protein sample; the sperm protein sample, testis protein sample and seminal plasma protein sample were detected for protein concentration by BCA kit;

[0045] 2. Qualitative and quantitative analysis of proteins

[0046] The sperm protein sample, testis protein sample and seminal plasma protein sample were subjected to liquid chromatography tandem mass spectrometry analysis, and the expression profiles of sperm proteins, testis proteins and seminal plasma proteins were obtained by database search analysis;

[0047] 3. Experimental results

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

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

[0050]

[0051] As shown in Table 2, high-abundance deacetylases are expressed in sperm proteins and testis proteins, including Zn 2+ dependent histone deacetylase family and NAD + dependent SIRT family.

[0052] III. Effect of deacetylase inhibitors on increasing the acetylation modification level of poultry sperm

[0053] 1. Screening of protein deacetylase inhibitors

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

[0055] 2. Collection and grouping of semen

[0056] According to the Poultry Semen Quality Detection Method (NY / T 4047-2021), 30 adult healthy Beijing oil cock chickens with sperm motility higher than 60%, semen volume higher than 350 μL, and density higher than 16 × 10 8 individual semen was mixed and divided into 3 groups, namely, the TSA group, the NRD167 group, and the blank control group;

[0057] 3. Preparation of test samples

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

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

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

[0063] According to the osmotic pressure of 360 mOsm / kg, sodium chloride (NaCl) was added to the semen freezing diluent to obtain the semen freezing diluent with NaCl as the blank control group;

[0064] 4. Semen dilution, equilibration, and freezing

[0065] TSA group: 200 μL fresh semen was taken and mixed with an equal amount of preheated to 37℃ semen freezing diluent added with deacetylase inhibitor TSA, equilibrated at 4℃ for 30 min, then 400 μL of 4℃ semen cryoprotective solution added with deacetylase inhibitor TSA was added, and then the semen was divided into a semen tube, the tube was sealed and placed in a programmed freezer for cooling and freezing, the freezing program was set at 4℃ to-44℃ at a rate of 12℃ / min, then-44℃ to-120℃ at a rate of 40℃ / min, and then placed in liquid nitrogen for storage;

[0066] NRD167 group: 200 μL fresh semen was taken and mixed with an equal amount of preheated to 37℃ semen freezing diluent added with deacetylase inhibitor NRD167, equilibrated at 4℃ for 30 min, then 400 μL of 4℃ semen cryoprotective solution added with deacetylase inhibitor NRD167 was added, and then the semen was divided into a semen tube, the tube was sealed and placed in a programmed freezer for cooling and freezing, the freezing program was set at 4℃ to-44℃ at a rate of 12℃ / min, then-44℃ to-120℃ at a rate of 40℃ / min, and then placed in liquid nitrogen for storage;

[0067] Blank control group: 200 μL fresh semen was taken and mixed with an equal amount of preheated to 37℃ semen freezing diluent added with NaCl, equilibrated at 4℃ for 30 min, then 400 μL of 4℃ semen cryoprotective solution added with NaCl was added, and then the semen was divided into a semen tube, the tube was sealed and placed in a programmed freezer for cooling and freezing, the freezing program was set at 4℃ to-44℃ at a rate of 12℃ / min, then-44℃ to-120℃ at a rate of 40℃ / min, and then placed in liquid nitrogen for storage;

[0068] 5. Thawing

[0069] The semen tubes of the TSA group, the NRD167 group and the blank control group were taken out from the liquid nitrogen, immersed in 4℃ water for 3 min, poured into a centrifuge tube, and then 2 times the volume of 4℃ thawing solution was added to obtain the TSA group sperm sample, the NRD167 group sperm sample and the blank control group sperm sample;

[0070] 6. Sperm protein extraction

[0071] 4 times the volume of lysis buffer was added to the TSA group sperm sample, the NRD167 group sperm sample and the blank control group sperm sample, respectively, and then ultrasonic lysis was performed, 4℃, 12000g centrifugation for 10 min, cell debris was removed, and the supernatant was transferred to a centrifuge tube to obtain the TSA group sperm protein sample, the NRD167 group sperm protein sample and the blank control group sperm protein sample;

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

[0073] Take 13 μg of sperm protein sample from the TSA group, 13 μg of sperm protein sample from the NRD167 group and 13 μg of sperm protein sample from the blank control group, respectively, after electrophoresis, membrane transfer and blocking, add protein acetylation general antibody (Anti-acetyl lysine antibody, PTM-101, 12838533L303) for primary antibody incubation, after primary antibody incubation, secondary antibody incubation (Goat anti-Mouse IgG (H+L), Peroxidase Conjugated, 31430) is carried out, after secondary antibody incubation, rinse, and add chemical luminescence HRP substrate for incubation for 2 minutes, according to the operation instructions of the chemical luminescence imaging system, signal capture is carried out;

[0074] 8. Detection results

[0075] The detection results of the acetylation modification level of sperm protein in the TSA group, the NRD167 group and the blank control group are shown in Figure 2 , wherein A is the acetylation modification level diagram of sperm protein, and B is the gray value of the acetylation modification level of protein;

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

[0077] Four, the effect of deacetylase inhibitor on the improvement of the fertilization ability of poultry remelted sperm

[0078] 1. Semen collection and grouping

[0079] According to the Poultry Semen Quality Detection Method (NY / T 4047-2021), 30 adult healthy Beijing broiler roosters with sperm motility higher than 60%, semen volume higher than 350 μL, and density higher than 16×10 8 / mL were selected, and semen was collected by back and abdomen massage method. The individual semen was mixed and divided into 3 groups, namely TSA group, NRD167 group and blank control group.

[0080] 2. Preparation of test samples

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

[0082] According to the osmotic pressure of 360 mOsm / kg, deacetylase inhibitor TSA was added to the semen freezing diluent to obtain semen freezing diluent added with deacetylase inhibitor TSA;

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

[0084] The deacetylase inhibitor NRD167 is added to the semen freezing protective solution with an osmotic pressure of 360 mOsm / kg to obtain the semen freezing protective solution added with the deacetylase inhibitor NRD167;

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

[0086] The sodium chloride (NaCl) is added to the semen freezing protective solution with an osmotic pressure of 360 mOsm / kg to obtain the semen freezing protective solution with NaCl as a blank control group;3. Semen dilution, equilibration and freezing

[0087] The TSA group: 200 μL of fresh semen is taken and mixed with an equal amount of the semen freezing diluent added with the deacetylase inhibitor TSA preheated to 37°C, equilibrated at 4°C for 30 min, and then 400 μL of the semen freezing protective solution added with the deacetylase inhibitor TSA at 4°C is added, and the semen is aliquoted into a semen straw, the straw is sealed and placed in a programmed freezer for cooling and freezing, the freezing program is set as 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 placed in liquid nitrogen for storage;

[0088] The NRD167 group: 200 μL of fresh semen is taken and mixed with an equal amount of the semen freezing diluent added with the deacetylase inhibitor NRD167 preheated to 37°C, equilibrated at 4°C for 30 min, and then 400 μL of the semen freezing protective solution added with the deacetylase inhibitor NRD167 at 4°C is added, and the semen is aliquoted into a semen straw, the straw is sealed and placed in a programmed freezer for cooling and freezing, the freezing program is set as 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 placed in liquid nitrogen for storage;

[0089] The blank control group: 200 μL of fresh semen is taken and mixed with an equal amount of the semen freezing diluent added with NaCl preheated to 37°C, equilibrated at 4°C for 30 min, and then 400 μL of the semen freezing protective solution added with NaCl at 4°C is added, and the semen is aliquoted into a semen straw, the straw is sealed and placed in a programmed freezer for cooling and freezing, the freezing program is set as 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 placed in liquid nitrogen for storage;

[0090] 4. Thaw and remove glycerin

[0091] Semen capillaries from the TSA group, NRD167 group, and blank control 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. Then, the tubes were centrifuged at 4°C and 600g for 8-10 minutes, and the supernatant was removed to obtain sperm precipitates from the TSA group, NRD167 group, and blank control group.

[0092] 5. Fertilization rate detection

[0093] Sperm precipitates from the TSA group, NRD167 group, and blank control group were resuspended in 4℃ DMEM solution with an equal volume of thawed semen. Sixty healthy Beijing Oil Chicken hens were randomly divided into three groups and infused with frozen-thawed sperm from the TSA group, NRD167 group, and blank control group, respectively. Each hen was inseminated with approximately 100 million sperm. Insemination was performed continuously for 2 days, and eggs were collected after 4 days of incubation. The fertilization rate was then calculated.

[0094] 6 Experimental Results

[0095] The fertilization rate results of the 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 both the TSA group and the NRD167 group were higher than those of the blank control group, and the NRD167 group was higher than that of the TSA group. This indicates that adding the protein deacetylase inhibitor NRD167 to the semen cryopreservation solution and the semen cryoprotectant solution can improve the fertilization capacity 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 and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. Application of protein deacetylase inhibitor NRD167 in the ultra-low temperature cryopreservation of poultry semen.

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

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

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