A method for cryopreservation of equine semen using an antifreeze polypeptide

Abstract

This invention discloses a method for cryopreservation of donkey semen using antifreeze peptides, belonging to the field of animal reproductive technology. This invention involves adding 8-polyproline (L-Pro) to the donkey semen cryopreservation solution. 8 5mM and 8-polyarginine (L-Arg) 8 5mM, and reduce the glycerol concentration to 1%. L-Pro 8 and L-Arg 8 Possessing a PPII double helix structure, it can effectively inhibit ice crystal growth and protect cell membrane structure. This invention can significantly improve the motility of frozen donkey sperm, protect intracellular structures such as mitochondria and DNA, and increase the conception rate of frozen sperm insemination, thus having significant application value.

Description

Technical Field

[0001] This invention belongs to the field of animal reproduction technology, specifically relating to a method for cryopreservation of donkey semen using antifreeze polypeptides. Background Technology

[0002] Sperm inevitably suffer damage from low temperatures during freezing. Adding cryoprotectants to the freezing solution can protect sperm; commonly used cryoprotectants are permeable cryoprotectants, meaning they can penetrate the sperm and cell membrane structures. For donkey sperm freezing, 4% glycerol (Gly) is commonly used as a permeable cryoprotectant. Although permeable cryoprotectants are commonly used, their penetration and exudation processes can cause severe damage to the sperm membrane structure, reducing cell viability and damaging intracellular structures such as mitochondria and DNA.

[0003] Antifreeze proteins (AFPs) are a class of non-permeable cryoprotectants widely found in bacteria, fungi, crustaceans, microalgae, insects, and fish living in cryogenic environments. The main functions of AFPs are to inhibit ice crystal formation and to interact with biofilms. Different types of AFPs exhibit different activities and can bind to different surfaces of ice crystals. Polyamino acids formed by the polymerization of several amino acids possess the same functions as AFPs.

[0004] However, there are currently no reports of applying antifreeze peptides to the cryopreservation of donkey semen, nor are there any technical solutions that utilize antifreeze peptides to reduce the concentration of conventional permeable cryoprotectants to reduce their permeability toxicity. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems and provide a method for cryopreservation of donkey semen using antifreeze peptides.

[0006] To achieve the above objectives, the present invention provides a method for cryopreservation of donkey semen using antifreeze peptides, comprising the following steps: S1. Prepare the centrifugal solution; S2. Prepare the coolant; S3. Perform cryopreservation of donkey semen; S1, the preparation of centrifugal solution includes: To prepare 100 mL of solution, take distilled water, add 2.5 g of glucose, 0.15 g of lactose, 0.15 g of raffinose, 0.03 g of sodium citrate, 0.041 g of potassium citrate, 0.47 g of HEPES, and 4 g of skim milk powder. Stir until completely dissolved, adjust the osmotic pressure to 280-320 mOsm / kg, and make up to 100 mL with distilled water. After sterilization, cool to room temperature, and add 15 mg of penicillin sodium and 10 mg of streptomycin.

[0007] S2. Preparation of the freezing solution includes: taking 94 mL of the centrifuged liquid from step S1, adding 5 mL of egg yolk, 1 mL of glycerol, 5 mM of 8-polyproline and 5 mM of 8-polyarginine.

[0008] Furthermore, when used for donkey semen preservation, the collected fresh donkey semen is mixed with the centrifuged liquid described in step S1 at a ratio of 1:1 and then centrifuged to remove the supernatant and retain the sperm precipitate. The freezing solution was added to the obtained sperm precipitate to make the final sperm concentration 100 million effective sperm counts / mL; After mixing sperm and cryoprotectant, the mixture was dispensed into frozen sperm tubes, equilibrated at 4°C for 2 hours, then frozen according to a programmed cooling curve, and finally stored in liquid nitrogen.

[0009] Furthermore, the programmed cooling curves are as follows: the cooling rate from 4℃ to -10℃ is 6℃ / min, the cooling rate from -10℃ to -100℃ is 36℃ / min, and the cooling rate from -100℃ to -140℃ is 20℃ / min.

[0010] The beneficial effects of this invention are as follows: The 8-polyproline and 8-polyarginine used in this invention are non-permeable cryoprotectants, non-toxic, and biocompatible, making them suitable for cryopreservation of donkey sperm. The present invention adds appropriate amounts of 8-polyproline and 8-polyarginine to the donkey semen cryopreservation fluid, which can reduce the glycerol concentration in the semen cryopreservation fluid from the conventional 4% to 1%, thereby significantly reducing the osmotic toxicity damage to sperm caused by high concentrations of permeable cryoprotectants. 8-Polyproline and 8-polyarginine have a PPII helical structure, which can effectively inhibit the formation of ice crystals during the cooling process, protect sperm from freezing damage caused by ice crystals, protect intracellular structures such as mitochondria and DNA, and effectively improve sperm motility after freezing. Attached Figure Description

[0011] Figure 1 This diagram illustrates the acrosome integrity rate of sperm after freezing and thawing according to one embodiment of the present invention. Figure 2 This diagram illustrates the mitochondrial membrane potential of sperm after freezing and thawing according to one embodiment of the present invention. Figure 3 This diagram illustrates the sperm DNA integrity rate after semen freezing and thawing according to one embodiment of the present invention. Detailed Implementation

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0013] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The embodiments cannot be described in detail here, but the embodiments of the present invention are not limited to the following embodiments.

[0014] This invention provides a method for cryopreservation of donkey semen using antifreeze peptides, comprising the following steps: S1, preparing centrifuge buffer; S2, preparing cryopreservation solution; and S3, cryopreservation of donkey semen. Specific examples are provided below for illustration: Example 1: Cryopreservation of Donkey Semen 1. Preparation of semen freezing solution S1. Preparation of centrifugal solution (taking 100mL as an example): Accurately weigh 2.5g glucose, 0.15g lactose, 0.15g raffinose, 0.03g sodium citrate, 0.041g potassium citrate, 0.47g HEPES, and 4g skim milk powder. Add an appropriate amount of ultrapure water and stir until completely dissolved. Measure and adjust the osmotic pressure to 280-320 mOsm / kg using an osmometer, and bring the volume to 100mL with distilled water. Autoclave at 110-115℃ for 5 minutes. After cooling to room temperature, add 15mg sodium penicillin and 10mg streptomycin, and shake well before use.

[0015] S2. Preparation of the cryogenic solution (taking 100mL as an example): Take 94 mL of the centrifuged liquid cooled to room temperature, add 5 mL of egg yolk (final concentration 5%), 1 mL of glycerol (final concentration 1%), and L-Pro. 8 5mM, L-Arg 8 5mM, after the above medicine is fully dissolved, refrigerate at 4℃ for later use.

[0016] 2. Semen freezing procedure (1) After semen collection, sperm motility, volume and density were tested.

[0017] (2) Fresh semen and centrifuged liquid were diluted at a ratio of 1:1 and centrifuged at 1800 r / min for 12 min. After centrifugation, the upper part of the semen and centrifuged liquid were removed, and the sperm precipitate at the bottom of the centrifuge tube was retained.

[0018] (3) Calculate the volume of cryosol to be added based on sperm motility, volume and density, so that the final sperm concentration is 100 million effective sperm / mL. Effective sperm count is calculated according to the following formula: Effective sperm count = volume × density × motility × 80% (20% is centrifugation loss).

[0019] (4) Mix the sperm and the freezing solution thoroughly, and use a fine tube dispenser to dispense the semen into 0.5 mL frozen sperm tubes. Wrap the tubes with absorbent cotton and equilibrate them in a 4°C freezer for 2 hours.

[0020] (5) After equilibration, place the frozen semi-tissue tubes in a programmed freezer and freeze them according to the following cooling curves: 6℃ / min for cooling from 4℃ to -10℃, 36℃ / min for cooling from -10℃ to -100℃, and 20℃ / min for cooling from -100℃ to -140℃. When the temperature drops to -140℃, transfer the frozen semi-tissue tubes to a liquid nitrogen tank for storage.

[0021] 3. Quality testing of frozen semen after thawing (1) Detection of sperm motility parameters: Frozen sperm tubes were removed from the liquid nitrogen tank, left to stand in the air for 3 seconds, and then quickly immersed in a 37°C water bath to thaw for 30 seconds. Sperm motility parameters were measured using the CASA system, and the total motility rate (TM), forward linear motility rate (PM), varagic motility rate (VCL), varagic motility rate (VSL), and average motility rate (VAP) were recorded.

[0022] (2) Sperm acrosome integrity test (FITC-PNA method): After thawing, 100 μL of semen was mixed with 1 mL of preheated DMEM / F-12 solution and centrifuged at 600 g for 4 min. The precipitate was washed twice. FITC-PNA working solution (1 μL FITC-PNA + 1 mL DMEM / F-12) was added to the precipitate and incubated at 37 °C in the dark for 15 min. After incubation, the mixture was centrifuged at 600 g for 4 min in the dark, and the precipitate was washed twice. The precipitate was then resuspended in 1 mL of DMEM / F-12. The morphology of the sperm acrosome was observed under a laser confocal microscope: sperm with intact acrosomes emitted bright green fluorescence in the anterior region of the head, while sperm with damaged acrosomes showed incomplete or no fluorescence in the anterior region of the head.

[0023] (3) Detection of sperm mitochondrial membrane potential (JC-1 method): Add 100 μL of semen to 1 mL of DMEM solution and mix by inversion. Centrifuge at 600 g for 4 min at room temperature and discard the supernatant. Add 1 mL of JC-1 working solution (50 μL of JC-1 (200X) mixed with 8 mL of ultrapure water), gently pipet, incubate at 37°C for 20 min in the dark, centrifuge at 600 g for 4 min, and discard the supernatant. Add 1 mL of JC-1 buffer (1X) and mix gently by pipet. Centrifuge at 650 g for 4 min at room temperature and discard the supernatant. Add 600 μL of JC-1 buffer (1X) and mix gently. Observe under a laser confocal microscope: normal sperm mitochondria show bright red fluorescence, and the fluorescence color gradually changes from red to green as the mitochondrial membrane potential decreases.

[0024] (4) Sperm DNA integrity testing (acidine orange method): Prepare a 0.01% acridine orange staining solution (dissolve 0.1g of acridine orange powder in 10mL of PBS (1X) to obtain a 0.1% stock solution; dilute with PBS 10 times before use). Observe the DNA integrity of the sperm head under a laser confocal microscope: acridine orange shows green fluorescence when bound to double-stranded DNA and red fluorescence when bound to single-stranded DNA.

[0025] 4. Conception rate experiment after thawing frozen semen One hundred healthy female donkeys aged 3-7 years were randomly divided into two groups: a control group (Con, n=41) using frozen semen containing 4% glycerol, which was then directly inseminated after thawing; and an experimental group (MSm, n=43) using semen containing 1% glycerol and 5mM L-Pro. 8 and 5mM L-Arg 8 The freezing fluid is used to freeze semen.

[0026] Every morning at 8:00 AM, ultrasound was used to examine the ovaries and uterus of the female donkey. When follicles larger than 30mm appeared on the ovaries, 0.22mg of GnRH analogue was injected intramuscularly. Insemination was performed on the female donkey 30 hours after injection, and ovulation was monitored 12 hours after insemination. Female donkeys that ovulated within 30 hours of GnRH analogue injection or did not ovulate within 24 hours of insemination were not included in the statistics. Pregnancy was checked on the female donkey 15 and 30 days after ovulation; the presence of blastocysts in the uterus indicated pregnancy.

[0027] 5. Experimental Results (1) Sperm motility parameters after thawing frozen sperm The experimental results are shown in Table 1. The total rate of motion (TM), forward linear rate of motion (PM), and average velocity of motion (VAP) of the experimental group (MSm) were significantly higher than those of the control group (Con) (P < 0.05).

[0028] Table 1. Effects of antifreeze peptides on exercise parameters of donkey frozen semen after thawing.

[0029] Note: TM: Total motile sperm; PM: Forward linear motile sperm; VCL: Curvilinear motility; VSL: Linear motility; VAP: Average motility; 'a' indicates significant differences in values ​​within the same column (P < 0.05).

[0030] (2) Sperm acrosome integrity, mitochondrial membrane potential and DNA integrity Based on the acrosome integrity rate of sperm after semen freezing and thawing ( Figure 1 ), mitochondrial membrane potential ( Figure 2 ) and DNA integrity rate ( Figure 3 The results showed that the acrosome integrity rate of the MSm group was significantly higher than that of the control group (P<0.05), the red / green fluorescence ratio of the MSm group was significantly higher than that of the control group (P<0.05), and the DNA integrity rate of the MSm group was significantly higher than that of the control group (P<0.05).

[0031] (3) Conception rate The conception rate of female donkeys 30 days after ovulation is shown in Table 2. The conception rate of female donkeys after frozen semen insemination in the control group was 29.27%, while that in the experimental group was 48.84%, which was significantly higher than that in the control group (P<0.05).

[0032] Table 2. Effects of different cryoprotectants on conception rate in female donkeys

[0033] Conclusion: Adding antifreeze peptide (L-Pro) to donkey sperm cryopreservation dilution solution 8 and L-Arg 8 This product can effectively reduce glycerol content, improve sperm motility after thawing, protect the integrity of sperm acrosomes, mitochondria, and DNA, and increase the conception rate of female donkeys after frozen semen insemination.

[0034] The above description is merely one embodiment of the present invention and is not intended to limit the invention. Those skilled in the art will recognize that the present invention can be modified and varied in various ways. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for cryopreservation of donkey semen using antifreeze peptides, characterized in that, Includes the following steps: S1. Prepare the centrifugal solution; S2. Prepare the refrigerant; S3. Perform cryopreservation of donkey semen; S1, preparing the centrifugal solution includes: taking distilled water, adding 2.5g glucose, 0.15g lactose, 0.15g raffinose, 0.03g sodium citrate, 0.041g potassium citrate, 0.47g HEPES, and 4g skim milk powder, stirring until completely dissolved, adjusting the osmotic pressure to 280-320mOsm / kg, making up to 100mL with distilled water, sterilizing and cooling to room temperature, and adding 15mg of penicillin sodium and 10mg of streptomycin; S2. Preparation of the freezing solution includes: taking 94 mL of the centrifuged liquid from step S1, adding 5 mL of egg yolk, 1 mL of glycerol, 5 mM of 8-polyproline and 5 mM of 8-polyarginine.

2. The method for cryopreservation of donkey semen using antifreeze peptides according to claim 1, characterized in that, When used for donkey semen preservation, the collected fresh donkey semen is mixed with the centrifuged liquid described in step S1 at a ratio of 1:1 and then centrifuged to remove the supernatant and retain the sperm precipitate. The freezing solution was added to the obtained sperm precipitate to make the final sperm concentration 100 million effective sperm counts / mL; After mixing sperm and cryoprotectant, the mixture was dispensed into frozen sperm tubes, equilibrated at 4°C for 2 hours, then frozen according to a programmed cooling curve, and finally stored in liquid nitrogen.

3. The method for cryopreservation of donkey semen using antifreeze peptides according to claim 2, characterized in that, The programmed cooling curves are as follows: the cooling rate is 6℃ / min from 4℃ to -10℃, the cooling rate is 36℃ / min from -10℃ to -100℃, and the cooling rate is 20℃ / min from -100℃ to -140℃.

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