Application of protein deacetylase inhibitor 3-TYP 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 3-TYP in poultry semen ultra-low temperature cryopreservation. It is proved by experiments that the protein deacetylase inhibitor 3-TYP can improve the acetylation modification level of poultry sperm and the fertilization ability of frozen-thawed poultry sperm; therefore, the protein deacetylase inhibitor 3-TYP 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 the application of protein deacetylase inhibitor 3-TYP 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 the application of protein deacetylase inhibitor 3-TYP in poultry semen ultra-low temperature cryopreservation, which uses protein deacetylase inhibitor 3-TYP 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.

[0005] To achieve the above purpose, the present application provides the following technical scheme:

[0006] The application of protein deacetylase inhibitor 3-TYP in poultry semen ultra-low temperature cryopreservation.

[0007] The protein deacetylase inhibitor 3-TYP provided by the application has the structural formula as shown in the following:

[0008]

[0009] Further, the application of the protein deacetylase inhibitor 3-TYP in the ultra-low temperature cryopreservation of poultry semen is specifically: according to the osmotic pressure of 360 mOsm / kg, the protein deacetylase inhibitor 3-TYP is added into the semen freezing diluent and the semen cryoprotective solution.

[0010] Further, the protein deacetylase inhibitor 3-TYP 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 3-TYP 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, the protein deacetylase inhibitor 3-TYP can inhibit the deacetylation modification (specifically, inhibit the acetylase 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 3-TYP 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 detailed description is made with reference to the accompanying drawings.

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

[0015] Figure 2 The figure is a graph of the results of the detection of the acetylation modification level of sperm protein for the blank control group, the TSA control group and the 3-TYP group;

[0016] Figure 3 The figure is a graph of the results of the detection of the fertilization rate for the blank control group, the TSA control group and the 3-TYP group. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0018] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0022] Unless otherwise specified, the semen cryopreservation solution, semen cryoprotectant, and thawing solution used in this invention are the same as those used in "Interspecific Differences in Chicken Sperm Antifreeze and Its Correlation with Seminal Plasma Biochemical Indicators and Candidate Gene Expression" by Zong Yunhe et al., 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.

[0023] I. Acetylation Modification Level of Poultry Sperm and Sperm Freeze Resistance

[0024] 1. Sample collection

[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 computer-aided sperm analysis system was used to detect the sperm motility in the frozen-thawed sperm sample, 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-resistance cock chickens and 5 low-freezing-resistance cock chickens with stable traits were selected for subsequent experiments, and the sperm motility change ranges of the high-freezing-resistance cock chicken group and the low-freezing-resistance cock chicken group are shown in Table 1;

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

[0034]

[0035] 4. Extraction of sperm protein

[0036] The semen of the high-freezing-resistance cock chicken group and the low-freezing-resistance 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 solution was added, ultrasonically lysed, 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-resistance cock chicken group and the low-freezing-resistance cock chicken group; the protein concentration was detected by BCA kit;

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

[0038] Sperm protein samples from high-antifreeze roosters and low-antifreeze roosters were taken at 13 μg each. After electrophoresis, transfer to a membrane, and blocking, protein acetylation pan-antibody (Anti-acetylated antibody, PTM-101, 12838533L303) was added for primary antibody incubation. After primary antibody incubation, secondary antibody incubation (Goat anti-mouse IgG (H+L), Peroxidase Conjugated, 31430) was performed. After secondary antibody incubation, the samples were rinsed and incubated with chemiluminescent HRP substrate for 2 minutes. Signal capture was performed according to the operation instructions of the chemiluminescent imaging system.

[0039] 6. Experimental Results

[0040] The results of sperm protein acetylation modification level detection in high-freeze-resistant rooster groups and low-freeze-resistant rooster groups are as follows: Figure 1 As shown, samples 1-5 are sperm samples from the high-freeze-resistant rooster group, and samples 6-10 are sperm samples from the low-freeze-resistant rooster group.

[0041] Depend on Figure 1 It can be seen that there are certain differences in the acetylation modification level of sperm proteins between the high-freeze-resistant rooster group and the low-freeze-resistant rooster group. Among them, the low-freeze-resistant rooster group has a higher level of protein acetylation modification around 35kD, while the level of protein acetylation modification around 30kD is relatively low. This indicates that the acetylation modification level of poultry sperm is related to the sperm's freeze resistance.

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

[0043] 1. Sample collection and protein extraction

[0044] Six adult healthy roosters of the Beijing Oil Chicken were randomly selected, and semen was collected from them using the back and 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 then 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. The roosters were then slaughtered, and testicular tissue was separated. Testicular samples the size of soybeans were collected, and four volumes of lysis buffer were added. The mixture was 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.

[0045] The protein concentration of the sperm protein sample, testis protein sample and seminal plasma protein sample was detected by BCA kit;

[0046] 2. Qualitative and quantitative analysis of proteins

[0047] Liquid chromatography tandem mass spectrometry analysis was performed on the sperm protein sample, testis protein sample and seminal plasma protein sample, and the expression profiles of sperm proteins, testis proteins and seminal plasma proteins were obtained by database search analysis;

[0048] 3. Experimental results

[0049] The deacetylases in the sperm protein sample, testis protein sample and seminal plasma protein sample are shown in Table 2;

[0050] Table 2 Deacetylases in sperm proteins, testis proteins and seminal plasma proteins

[0051]

[0052] 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.

[0053] III. Effect of deacetylase inhibitors on increasing the acetylation modification level of poultry sperm

[0054] 1. Screening of protein deacetylase inhibitors

[0055] Through database search, Zn 2+ dependent histone deacetylase family universal inhibitor TSA (C 17 H 22 N2O3), SIRT3 specific inhibitor 3-TYP (C7H6N4) were obtained;

[0056] 2. Collection and grouping of semen

[0057] 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 x 10 8 individual semen was collected by back and abdomen massage method, and the individual semen was mixed and divided into 3 groups, which were blank control group, TSA control group and 3-TYP group;

[0058] 3. Preparation of test samples

[0059] According to the osmotic pressure of 360 mOsm / kg, sodium chloride (NaCl) is added in the semen freezing diluent to obtain the semen freezing diluent with NaCl as the blank control group;

[0060] According to the osmotic pressure of 360 mOsm / kg, sodium chloride (NaCl) is added in the semen freezing diluent to obtain the semen freezing diluent with NaCl as the blank control group;

[0061] According to the osmotic pressure of 360 mOsm / kg, deacetylase inhibitor TSA is added in the semen freezing diluent to obtain the semen freezing diluent added with deacetylase inhibitor TSA;

[0062] According to the osmotic pressure of 360 mOsm / kg, deacetylase inhibitor TSA is added in the semen freezing diluent to obtain the semen freezing diluent added with deacetylase inhibitor TSA;

[0063] According to the osmotic pressure of 360 mOsm / kg, deacetylase inhibitor 3-TYP is added in the semen freezing diluent to obtain the semen freezing diluent added with deacetylase inhibitor 3-TYP;

[0064] According to the osmotic pressure of 360 mOsm / kg, deacetylase inhibitor 3-TYP is added in the semen freezing diluent to obtain the semen freezing diluent added with deacetylase inhibitor 3-TYP;

[0065] 4. Semen dilution, equilibration and freezing

[0066] Blank control group: 200 μL fresh semen is taken and mixed with an equal amount of semen freezing diluent added with NaCl preheated to 37℃, equilibrated at 4℃ for 30 min, then 400 μL of 4℃ semen freezing diluent added with NaCl 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℃ to -44℃ at a rate of 12℃ / min, -44℃ to -120℃ at a rate of 40℃ / min, and then stored in liquid nitrogen;

[0067] TSA control group: 200 μL fresh semen is taken and mixed with an equal amount of semen freezing diluent added with deacetylase inhibitor TSA preheated to 37℃, equilibrated at 4℃ for 30 min, then 400 μL of 4℃ semen freezing diluent added with deacetylase inhibitor TSA 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℃ to -44℃ at a rate of 12℃ / min, -44℃ to -120℃ at a rate of 40℃ / min, and then stored in liquid nitrogen;

[0068] 3-TYP group: 200 μL fresh semen was taken and mixed with an equal amount of preheated to 37°C semen freezing diluent added with deacetylase inhibitor 3-TYP, equilibrated at 4°C for 30 min, then 400 μL of 4°C semen freezing protective solution added with deacetylase inhibitor 3-TYP was added, and the semen was dispensed into a semen thin tube, the thin tube was sealed and placed in a programmed freezer for cooling and freezing, the freezing program was 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, then put into liquid nitrogen for storage;

[0069] 5. Thawing

[0070] The semen thin tubes of the blank control group, the TSA control group and the 3-TYP group were taken out from the liquid nitrogen, immersed in 4°C water for 3 min for thawing, poured into a centrifuge tube, and after the volume was measured, 2 times the volume of 4°C thawing solution was added to obtain the sperm sample of the blank control group, the sperm sample of the TSA control group and the sperm sample of the 3-TYP group;

[0071] 6. Extraction of sperm protein

[0072] 4 times the volume of lysis buffer was added to the sperm sample of the blank control group, the sperm sample of the TSA control group and the sperm sample of the 3-TYP group respectively, and ultrasonic lysis was performed, 4°C, 12000g centrifugation for 10 min, and the cell debris was removed, and the supernatant was transferred to a centrifuge tube to obtain the sperm protein sample of the blank control group, the sperm protein sample of the TSA control group and the sperm protein sample of the 3-TYP group;

[0073] 7. Detection of acetylation modification level of sperm protein

[0074] 13 μg of the sperm protein sample of the blank control group, the sperm protein sample of the TSA control group and the sperm protein sample of the 3-TYP group were taken respectively, after electrophoresis, membrane transfer and blocking, protein acetylation pan antibody (Anti-acetyl lysine Antibody, PTM-101, 12838533L303) was added for primary antibody incubation, after the primary antibody incubation, secondary antibody incubation was performed (Goat anti-Mouse IgG (H+L), Peroxidase Conjugated, 31430), after the secondary antibody incubation, rinsing was performed, and chemical luminescence HRP substrate was added for incubation for 2 min, and signal capture was performed according to the operation instructions of the chemical luminescence imaging system;

[0075] 8. Detection results

[0076] The detection results of the acetylation modification level of sperm protein of the blank control group, the TSA control group and the 3-TYP group are shown in Table 1. Figure 2The results are shown in Figure 1, wherein A is the sperm protein acetylation modification level map, and B is the gray value of the protein acetylation modification level;

[0077] By Figure 2 It can be seen that the sperm protein acetylation modification level of the TSA control group and the 3-TYP group is higher than that of the blank control group.

[0078] Four, the effect of deacetylase inhibitor on the improvement of the fertility of poultry remelted sperm

[0079] 1. Collection and grouping of semen

[0080] 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 individuals / mL were selected, and semen was collected by back and abdominal massage method. The individual semen was mixed and divided into 3 groups, namely blank control group, TSA control group and 3-TYP group.

[0081] 2. Preparation of test samples

[0082] 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;

[0083] 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;

[0084] According to the osmotic pressure of 360 mOsm / kg, deacetylase inhibitor TSA was added to the semen freezing diluent to obtain the semen freezing diluent added with deacetylase inhibitor TSA;

[0085] According to the osmotic pressure of 360 mOsm / kg, deacetylase inhibitor TSA was added to the semen freezing diluent to obtain the semen freezing diluent added with deacetylase inhibitor TSA;

[0086] According to the osmotic pressure of 360 mOsm / kg, deacetylase inhibitor 3-TYP was added to the semen freezing diluent to obtain the semen freezing diluent added with deacetylase inhibitor 3-TYP;

[0087] According to the osmotic pressure of 360 mOsm / kg, deacetylase inhibitor 3-TYP was added to the semen freezing diluent to obtain the semen freezing diluent added with deacetylase inhibitor 3-TYP;

[0088] 3. Semen dilution, equilibration and freezing

[0089] 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; -44℃ to -120℃ at a rate of 40℃ / min, and then stored in liquid nitrogen;

[0090] TSA control 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; -44℃ to -120℃ at a rate of 40℃ / min, and then stored in liquid nitrogen;

[0091] 3-TYP group: 200 μL fresh semen was taken and mixed with an equal amount of preheated to 37℃ semen freezing diluent added with deacetylase inhibitor 3-TYP, equilibrated at 4℃ for 30 min, then 400 μL of 4℃ semen cryoprotective solution added with deacetylase inhibitor 3-TYP 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; -44℃ to -120℃ at a rate of 40℃ / min, and then stored in liquid nitrogen;

[0092] 4, thawing and removing glycerol

[0093] The semen tubes of the blank control group, TSA control group and 3-TYP group were taken out from the liquid nitrogen, immersed in 4℃ water for 3 min, poured into a centrifuge tube, the volume was measured, 2 times the volume of 4℃ thawing solution was added, then centrifuged at 4℃, 600g for 8-10 min, the supernatant was removed, and the blank control group sperm precipitate, TSA control group sperm precipitate and 3-TYP group sperm precipitate were obtained;

[0094] 5, fertilization rate detection

[0095] The sperm precipitates from the blank control group, the TSA control group, and the 3-TYP 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 blank control group, the TSA control group, and the 3-TYP 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.

[0096] 6 Experimental Results

[0097] The fertilization rate test results of the blank control group, TSA control group, and 3-TYP group are as follows: Figure 3 As shown;

[0098] Depend on Figure 3 It can be seen that the fertilization rates of the TSA control group and the 3-TYP group were higher than those of the blank control group, and the 3-TYP group was higher than that of the TSA group. This indicates that adding the protein deacetylase inhibitor 3-TYP to the semen cryopreservation solution and the semen cryoprotectant solution can improve the fertilization capacity of frozen-thawed sperm.

[0099] 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. The application of protein deacetylase inhibitor 3-TYP in the ultra-low temperature cryopreservation of poultry semen.

2. The use of the protein deacetylase inhibitor 3-TYP according to claim 1 for the ultra- low temperature cryopreservation of poultry semen, characterized in that, The application of protein deacetylase inhibitor 3-TYP in the ultra-low temperature cryopreservation of poultry semen, specifically, adding protein deacetylase inhibitor 3-TYP into semen freezing diluent and semen cryoprotective solution.

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

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