Sperm diluting powder for maintaining sperm motility

Abstract

The invention provides sperm diluting powder for maintaining sperm motility, and belongs to the technical field of animal breeding. Heat shock proteins HSP 90-alpha and HSPB1 are expressed through genetic engineering escherichia coli, are purified and are compounded with inorganic salt and the like to form a sperm diluent, and the sperm diluent is freeze-dried to prepare the sperm freeze-dried powder. The sperm dilution powder containing the heat shock protein HSP 90-alpha or the heat shock protein HSPB1 can reduce the injury of chicken sperms and pig sperms and maintain the sperm activity, and meanwhile, the sperm dilution powder containing the heat shock protein HSP 90-alpha and the heat shock protein HSPB1 has the advantages that the sperm activity maintaining effect is better than that of the sperm dilution powder containing the heat shock protein HSP 90-alpha and the heat shock protein HSPB1 under the synergistic effect compared with that of the sperm dilution powder containing the heat shock protein HSP 90-alpha and the heat shock protein HSPB1 containing the heat shock protein HSP 90-alpha and the heat shock protein HSPB1 containing the heat shock protein HSP 90-alpha.

Description

Technical Field

[0001] The invention belongs to the technical field of animal breeding, and in particular relates to a sperm diluting powder for maintaining sperm motility. Background Art

[0002] Artificial insemination technology is widely used in animal husbandry and reproduction, enabling efficient cross-regional transfer of superior genes and rapid advancement of population genetics. In vitro manipulation of semen is a key support technology for the development of high-quality animal seed industries, improved breeding technology systems, and the efficient development of genetic resources. Sperm extenders are widely used in artificial insemination, in vitro fertilization, and semen analysis. Their composition and physicochemical properties directly influence sperm motility, which in turn determines reproductive efficiency and genetic gain. Due to the unique lipid composition of their biomembranes and the characteristics of their seminal plasma, sperm are susceptible to multiple stresses during in vitro manipulation, including oxidative stress caused by a surge in reactive oxygen species and abnormal energy metabolism due to sperm cell damage. These stresses can cause irreversible damage to sperm, a key factor limiting the success rate of artificial insemination. Studies have found that adding appropriate amounts of exogenous substances, such as vitamins, amino acids, hormones, plant extracts, and other exogenous additives, to semen extenders can reduce sperm stress and damage to energy metabolism.

[0003] Common sperm dilution products currently on the market suffer from numerous deficiencies, leading to decreased sperm motility after dilution. Conventional sperm dilution products fail to fully meet the nutritional and environmental stability requirements of sperm during in vitro dilution, resulting in a rapid decline in sperm motility. This decline in sperm motility prior to fertilization leads to low fertilization efficiency. Therefore, there is an urgent need to develop a high-performance, easy-to-use sperm dilution product that can maintain high sperm motility during dilution and insemination procedures, thereby ensuring fertilization rates. Summary of the Invention

[0004] The invention aims to provide a sperm diluent powder for maintaining sperm motility, belonging to the technical field of animal breeding.

[0005] To achieve the above object, the present invention provides the following technical solutions: Firstly, the present invention provides a sperm diluent powder for maintaining sperm motility, wherein the sperm diluent powder contains one or both of heat shock protein HSP 90-alpha and heat shock protein HSPB1.

[0006] Furthermore, the DNA sequence of the heat shock protein HSP 90-alpha is SEQ ID NO. 2, and the DNA sequence of the heat shock protein HSPB1 is SEQ ID NO. 4.

[0007] Furthermore, the formula of the sperm diluent powder is NaCl 101.6mM / L, KCl 4.69mM / L, CaCl2·2H2O 1.71mM / L, MgSO4·7H2O 1.19mM / L, KH2PO4 1.19mM / L, HEPES 20mM / L, glucose 2.00mM / L, sodium pyruvate 0.30mM / L, sodium lactate 21.4mM / L, 5% w / v heat shock protein HSP 90-alpha, penicillin 100 IU / mL, and streptomycin 100μg / mL.

[0008] Furthermore, the formula of the sperm diluent powder is NaCl 101.6mM / L, KCl 4.69mM / L, CaCl2·2H2O 1.71mM / L, MgSO4·7H2O 1.19mM / L, KH2PO4 1.19mM / L, HEPES 20mM / L, glucose 2.00mM / L, sodium pyruvate 0.30mM / L, sodium lactate 21.4mM / L, 5% w / v heat shock protein HSPB1, penicillin 100IU / mL, and streptomycin 100μg / mL.

[0009] Furthermore, the formula of the sperm diluent powder is NaCl 101.6mM / L, KCl 4.69mM / L, CaCl2·2H2O 1.71mM / L, MgSO4·7H2O 1.19mM / L, KH2PO4 1.19mM / L, HEPES 20mM / L, glucose 2.00mM / L, sodium pyruvate 0.30mM / L, sodium lactate 21.4mM / L, 2.5% w / v heat shock protein HSP 90-alpha, 2.5% w / v heat shock protein HSPB1, penicillin 100 IU / mL, and streptomycin 100μg / mL.

[0010] Secondly, the present invention provides a complex for maintaining sperm motility, wherein the complex comprises heat shock protein HSP 90-alpha and heat shock protein HSPB1.

[0011] Furthermore, the DNA sequence of the heat shock protein HSP 90-alpha in the complex is SEQ ID NO. 2, and the DNA sequence of the heat shock protein HSPB1 is SEQ ID NO. 4.

[0012] The beneficial effects of the present invention are that heat shock proteins HSP 90-alpha and HSPB1 are expressed and purified by genetically engineered Escherichia coli, compounded with inorganic salts and the like to form a sperm diluent, and freeze-dried to produce sperm freeze-dried powder. Chicken and porcine sperm diluted with sperm diluent powder containing bovine serum albumin as the protein component show a rapid decline in activity, while sperm diluted with sperm diluent powder containing heat shock protein HSP 90-alpha or heat shock protein HSPB1 show a slower decline in activity. Sperm diluted with sperm diluent powder containing both heat shock protein HSP 90-alpha and heat shock protein HSPB1 show the slowest decline in activity. Fifteen hours after dilution, the linear motility rate of chicken and porcine sperm remains at nearly 50%. The sperm diluent powders of the present invention containing either or both heat shock protein HSP 90-alpha and heat shock protein HSPB1 can reduce sperm damage in chicken and porcine, maintain sperm activity, and effectively address the problem of rapid decline in sperm motility during sperm dilution. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : SDS electrophoresis detection of heat shock protein HSP 90-alpha and HSPB1.

[0014] Figure 1 Label numbers: M: Protein ladder, 1: HSPB1 band, 2: HSP 90-alpha band.

[0015] Figure 2 : Detection of the effect of sperm extender powder on chicken sperm motility.

[0016] Figure 3 : Detection of the effect of sperm extender powder on pig sperm motility. DETAILED DESCRIPTION

[0017] Example 1: Expression of Heat Shock Protein HSP 90-alpha (1) Construction of expression plasmid The amino acid sequence of human heat shock protein HSP 90-alpha is NP_001017963.2 in GenBank, and the amino acid sequence is SEQ ID NO. 1. Based on the codon-preferred expression of E. coli, the DNA sequence for expressing HSP 90-alpha protein was optimized. SEQ ID NO. 2 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and cloned into the BamHI and XhoI sites of the pET28a plasmid, named pET28a-HSP 90.

[0018] (2) Construction of recombinant expression strain The pET28a-HSP 90 plasmid was transformed into E. coli BL21 competent cells, and a single colony was picked for PCR identification of the positive transformant, named E. coli BL21 / pET28a-HSP 90. The single colony was cultured in 10 mL of LB culture medium containing 50 μg / mL kanamycin sulfate at 37°C and 250 rpm for 12 h to serve as the seed solution.

[0019] (3) Expression and identification of target protein: 4 mL of seed solution was pipetted into 400 mL of LB medium supplemented with 50 μg / mL kanamycin. The culture was shaken at 37°C until the OD600nm reached 0.6-0.8. 100 mM / L α-lactose was added to a final concentration of 0.5 mM / L and the cells were cultured at 37°C for another 6 h. After removal, the cells were centrifuged at 12,000 rpm for 10 min to collect the cells. PBS (1 / 10 volume of the original culture medium) was added to the cells for suspension and ultrasonic disruption. The cells were centrifuged at 12,000 rpm for 10 min at 4°C and the supernatant and precipitate were collected.

[0020] (4) Purification of recombinant protein Before loading, use 10 column volumes of Binding Buffer to equilibrate the nickel column at a flow rate of 5 mL / min. After centrifugation and filtration of the supernatant, the protein solution is loaded onto the equilibrated Ni column at a flow rate of 1 mL / min through a constant flow pump. The permeate is collected and the sample can be loaded repeatedly to improve the efficiency of the sample hanging on the column. The Ni column that has bound the target protein is re-equilibrated with 10 column volumes of Binding Buffer at a flow rate of 5 mL / min to remove unbound proteins. Elute the Ni column with a mixture of equal volumes of Elution Buffer and Binding Buffer at a flow rate of 5 mL / min, measure the OD280 value, and collect the eluate corresponding to the protein absorption peak. Take an appropriate amount of eluate for SDS-PAGE electrophoresis detection. The protein band size is about 101.7 kD. The electrophoresis results are as follows Figure 1 .

[0021] Example 2: Expression of heat shock protein HSPB1 (1) Construction of expression plasmid The amino acid sequence of human heat shock protein HSPB1 is GenBank accession number: CAG38728.1, and the amino acid sequence is SEQ ID NO. 3. Based on the codon-biased optimization of E. coli, the DNA sequence for expressing HSPB1 protein was commissioned to Sangon Biotech (Shanghai) Co., Ltd. to synthesize SEQ ID NO. 4 and cloned into the BamHI and XhoI sites of the pET28a plasmid, named pET28a-HSPB1.

[0022] (2) Construction of recombinant expression strain pET28a-HSPB1 plasmid was transformed into E. coli BL21 competent cells, and a single colony was picked for PCR identification of positive transformants, named E. coli BL21 / pET28a-HSPB1. The single colony was inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin, and incubated at 37°C, 250 r / min for 12 h as a seed liquid.

[0023] (3) Expression and identification of target protein 4 mL of seed liquid was inoculated into 400 mL of LB medium containing 50 μg / mL kanamycin, and incubated at 37°C with shaking until OD600nm was 0.6-0.8. 100 mM / L of α-lactose was added to a final concentration of 0.5 mM / L, and incubated at 37°C for another 6 h. After centrifugation at 12000 r / min for 10 min, the bacterial cells were collected. PBS was added to the original culture medium at a volume of 1 / 10 to suspend the bacterial cells, and ultrasonic wave was used for crushing. Centrifugation was performed at 4°C, 12000 r / min for 10 min, and the supernatant and precipitate were collected.

[0024] (4) Purification of recombinant protein Before loading, the nickel column was equilibrated with 10 times the column volume of Binding Buffer at a flow rate of 5 mL / min. The protein solution was loaded onto the equilibrated Ni column at a flow rate of 1 mL / min after centrifugation and filtration of the supernatant. The breakthrough liquid was collected, and repeated loading was performed to improve the column hanging efficiency of the sample. The Ni column with bound target protein was equilibrated again with 10 times the column volume of Binding Buffer at a flow rate of 5 mL / min to remove the unbound protein. The Ni column was eluted with an equal volume mixture of Elution Buffer and Binding Buffer at a flow rate of 5 mL / min, and the OD280 value was measured. The eluate corresponding to the protein absorption peak was collected. An appropriate amount of eluate was subjected to SDS-PAGE electrophoresis detection, and the protein band size was about 26.3 kD. The electrophoresis result is shown in Figure 2. Figure 1 .

[0025] Example 3: Preparation of sperm dilution powder I (1) Formula of dilution powder I NaCl 101.6 mM / L, KCl 4.69 mM / L, CaCl2·2H2O 1.71 mM / L, MgSO4·7H2O 1.19 mM / L, KH2PO4 1.19 mM / L, HEPES 20 mM / L, glucose 2.00 mM / L, sodium pyruvate 0.30 mM / L, sodium lactate 21.4 mM / L, 5% w / v bovine serum albumin, penicillin 100 IU / mL, streptomycin 100 μg / mL.

[0026] (2) Preparation method: Take 800 mL of ultrapure water and dissolve NaCl, KCl, CaCl2·2H2O, MgSO4·7H2O, KH2PO4, HEPES, glucose, sodium pyruvate, sodium lactate, bovine serum albumin, penicillin, and streptomycin in sequence. After sufficient dissolution, add HCl / NaOH to adjust the pH to 7.3-7.5, make up to 1000 mL, and calibrate the osmotic pressure to 290-310 mOsm / kg.

[0027] (3) Sterilize by filtering through a 0.22 μm filter, dispense into sterilized vials, 2.0 mL / vial, freeze-dry in a lyophilizer, cap, and store at 4°C for a long term.

[0028] Example 4: Preparation of Sperm Diluent Powder II (1) Dilution powder formula II: NaCl 101.6mM / L, KCl 4.69mM / L, CaCl2·2H2O 1.71mM / L, MgSO4·7H2O 1.19mM / L, KH2PO4 1.19mM / L, HEPES 20mM / L, glucose 2.00mM / L, sodium pyruvate 0.30mM / L, sodium lactate 21.4mM / L, 5% w / v heat shock protein HSP 90-alpha, penicillin 100 IU / mL, streptomycin 100μg / mL.

[0029] (2) Preparation method: Take 800 mL of ultrapure water and dissolve NaCl, KCl, CaCl2·2H2O, MgSO4·7H2O, KH2PO4, HEPES, glucose, sodium pyruvate, sodium lactate, heat shock protein HSP 90-alpha, penicillin, and streptomycin in sequence. After sufficient dissolution, add HCl / NaOH to adjust the pH to 7.3-7.5, make up to 1000 mL, and calibrate the osmotic pressure to 290-310 mOsm / kg.

[0030] (3) Sterilize by filtering through a 0.22 μm filter, dispense into sterilized vials, 2.0 mL / vial, freeze-dry in a lyophilizer, cap, and store at 4°C for a long term.

[0031] Example 5: Preparation of Sperm Diluent Powder III (1) Dilution powder formula III: NaCl 101.6mM / L, KCl 4.69mM / L, CaCl2·2H2O 1.71mM / L, MgSO4·7H2O 1.19mM / L, KH2PO4 1.19mM / L, HEPES 20mM / L, glucose 2.00mM / L, sodium pyruvate 0.30mM / L, sodium lactate 21.4mM / L, 5% w / v heat shock protein HSPB1, penicillin 100 IU / mL, streptomycin 100μg / mL.

[0032] (2) Preparation method: Take 800 mL of ultrapure water, dissolve NaCl, KCl, CaCl2.2H2O, MgSO4.7H2O, KH2PO4, HEPES, glucose, sodium pyruvate, sodium lactate, heat shock protein HSPB1, penicillin, streptomycin in turn, after fully dissolving, adjust pH to 7.3-7.5 with HCl / NaOH, constant volume to 1000 mL, calibrate the osmotic pressure to 290-310 mOsm / kg.

[0033] (3) Filtered through a 0.22 μm filter to remove bacteria, divided into sterile test tubes, 2.0 mL / tube, vacuum freeze-dried in a freeze dryer and capped, stored at 4°C for long-term preservation.

[0034] Example 6: Preparation of sperm dilution powder four (1) Dilution powder four formula: NaCl 101.6 mM / L, KCl 4.69 mM / L, CaCl2.2H2O 1.71 mM / L, MgSO4.7H2O 1.19 mM / L, KH2PO4 1.19 mM / L, HEPES 20 mM / L, glucose 2.00 mM / L, sodium pyruvate 0.30 mM / L, sodium lactate 21.4 mM / L, 2.5% w / v heat shock protein HSP 90-alpha, 2.5% w / v heat shock protein HSPB1, penicillin 100 IU / mL, streptomycin 100 μg / mL.

[0035] (2) Preparation method: Take 800 mL of ultrapure water, dissolve NaCl, KCl, CaCl2.2H2O, MgSO4.7H2O, KH2PO4, HEPES, glucose, sodium pyruvate, sodium lactate, heat shock protein HSP 90-alpha, HSPB1, penicillin, streptomycin in turn, after fully dissolving, adjust pH to 7.3-7.5 with HCl / NaOH, constant volume to 1000 mL, calibrate the osmotic pressure to 290-310 mOsm / kg.

[0036] (3) Filtered through a 0.22 μm filter to remove bacteria, divided into sterile test tubes, 2.0 mL / tube, vacuum freeze-dried in a freeze dryer and capped, stored at 4°C for long-term preservation.

[0037] Example 7: Effect of sperm dilution powder on chicken sperm motility (1) Take 3 bottles of sperm dilution powder one, two, three, and four prepared in Examples 3, 4, 5, and 6, respectively, and add 2 mL of sterile ultrapure water to each bottle to dissolve, and incubate at 37°C.

[0038] (2) Semen of healthy adult Hy-Line Brown roosters was collected and mixed with four sperm diluents in a volume ratio of 1:1. Sperm motility was assessed every 3 hours using a computer-assisted semen analysis system (CASA). The geometric mean of the linear motility of sperm after dilution with three bottles of sperm diluent powder was taken from each group. The test was terminated when the linear motility of sperm dropped below 30%.

[0039] Depend on Figure 2 As can be seen, the linear motility rate of freshly diluted chicken sperm was 81.58%. Sperm diluted with diluent powder 1 showed a rapid decline in motility, falling below 30% 12 hours after dilution. Sperm diluted with diluent powders 2 and 3 showed a slower decline in motility, falling below 30% 15 hours after dilution. Sperm diluted with diluent powder 4 showed the slowest decline in motility, remaining near 50% 15 hours after dilution and falling below 30% 21 hours after dilution. This indicates that adding either heat shock protein 90-alpha or heat shock protein HSPB1 to sperm diluent powder can reduce chicken sperm damage and maintain sperm motility. The synergistic effect of the large-molecule heat shock protein HSP 90-alpha and the small-molecule heat shock protein HSPB1 is more effective in maintaining sperm motility.

[0040] Example 8: Effect of sperm diluent on pig sperm motility (1) Take 3 bottles each of sperm diluent powder 1, 2, 3, and 4 prepared in Examples 3, 4, 5, and 6, add 2 mL of sterile ultrapure water to each bottle to dissolve, and keep warm at 37°C.

[0041] (2) Semen of healthy adult Large White boars was collected and mixed with four sperm diluents in a volume ratio of 1:1. Sperm motility was assessed every 3 hours using a computer-assisted semen analysis system (CASA). The geometric mean of the linear motility of sperm after dilution with three bottles of sperm diluent powder was taken from each group. The test was terminated when the linear motility of sperm dropped below 30%.

[0042] Depend on Figure 3It can be seen that the linear movement sperm rate of freshly diluted pig sperm is 84.48%, the activity of sperm diluted by dilution powder one decreases rapidly, and the linear movement sperm rate decreases to less than 30% after 15 hours of dilution; the activity of sperm diluted by dilution powder two and dilution powder three decreases slowly, and the linear movement sperm rate decreases to less than 30% after 18 hours of dilution; the activity of sperm diluted by dilution powder four decreases most slowly, and the linear movement sperm rate still remains close to 50% after 15 hours of dilution, and the linear movement sperm rate decreases to less than 30% after 21 hours of dilution. It can be seen that adding heat shock protein HSP 90-alpha or heat shock protein HSPB1 in the sperm dilution powder can reduce the damage of pig sperm and maintain the activity of sperm, and the synergistic effect of large molecular heat shock protein HSP 90-alpha and small molecular heat shock protein HSPB1 is better in maintaining the activity of sperm.

Claims

1. A sperm diluent powder for maintaining sperm motility, characterized in that: The sperm diluent powder contains one or both of heat shock protein HSP 90-alpha and heat shock protein HSPB1.

2. The sperm diluent powder according to claim 1, characterized in that: The DNA sequence of the heat shock protein HSP 90-alpha is SEQ ID NO. 2, and the DNA sequence of the heat shock protein HSPB1 is SEQ ID NO.

4.

3. The sperm diluent powder according to claim 1, characterized in that: The formula of the sperm diluent powder is NaCl 101.6mM / L, KCl 4.69mM / L, CaCl2·2H2O 1.71mM / L, MgSO4·7H2O 1.19mM / L, KH2PO4 1.19mM / L, HEPES 20mM / L, glucose 2.00mM / L, sodium pyruvate 0.30mM / L, sodium lactate 21.4mM / L, 5% w / v heat shock protein HSP 90-alpha, penicillin 100IU / mL, and streptomycin 100μg / mL.

4. The sperm diluent powder according to claim 1, characterized in that: The formula of the sperm diluent powder is NaCl 101.6mM / L, KCl 4.69mM / L, CaCl2·2H2O 1.71mM / L, MgSO4·7H2O 1.19mM / L, KH2PO4 1.19mM / L, HEPES 20mM / L, glucose 2.00mM / L, sodium pyruvate 0.30mM / L, sodium lactate 21.4mM / L, 5% w / v heat shock protein HSPB1, penicillin 100IU / mL, and streptomycin 100μg / mL.

5. The sperm diluent powder according to claim 1, characterized in that: The formula of the sperm diluent powder is NaCl 101.6mM / L, KCl 4.69mM / L, CaCl2·2H2O 1.71mM / L, MgSO4·7H2O 1.19mM / L, KH2PO4 1.19mM / L, HEPES 20mM / L, glucose 2.00mM / L, sodium pyruvate 0.30mM / L, sodium lactate 21.4mM / L, 2.5% w / v heat shock protein HSP 90-alpha, 2.5% w / v heat shock protein HSPB1, penicillin 100IU / mL, and streptomycin 100μg / mL.

6. A compound for maintaining sperm motility, characterized in that: The complex comprises heat shock protein HSP 90-alpha and heat shock protein HSPB1.

7. The composite according to claim 6, characterized in that The DNA sequence of the heat shock protein HSP 90-alpha in the complex is SEQ ID NO. 2, and the DNA sequence of the heat shock protein HSPB1 is SEQ ID NO. 4.

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