A goat sperm biofilm protectant and its application method

Abstract

The present invention discloses a goat sperm biofilm protectant and its application method. The goat sperm biofilm protectant includes the following components: 0.036 g / ml Tris, 0.01 g / ml glucose, 0.02 g / ml citric acid, 6% (volume fraction) glycerol, 1% (volume fraction) penicillin-streptomycin mixture, 20 μg / ml peroxidase-6, 10-30 μg / ml glutathione peroxidase 5, and ultrapure water. The present invention can resist the oxidative damage suffered by sperm biofilms during the freezing and recovery process, enabling sperm to exert normal physiological functions. When using the semen preservation method of the present invention compared with using a conventional diluent, under the same conditions, the integrity of the sperm plasma membrane is increased by 27.63%, the integrity of the acrosome membrane is increased by 15.3%, and the mitochondrial membrane potential is increased by 47.2%. The goat sperm biofilm protectant of the present invention can fully improve the utilization rate of breeding rams and is beneficial to the development of livestock improvement and breeding work.

Description

Technical Field

[0001] The present invention relates to the technical field of goat semen preservation solutions, and particularly to a goat sperm biomembrane protectant and its application method. Background Art

[0002] Modern sheep farming is developing towards intensification and large-scale operation, and the lack of excellent breeding males has become an important limitation in sheep production. Semen cryopreservation technology can preserve sperm for a long time and is one of the main measures to improve the utilization rate of excellent germplasm resources, bringing higher economic value to production.

[0003] The integrity of the biomembrane structure is very important for sperm, which is related to sperm intercellular communication, metabolism, sperm-egg fusion, and regulated cell death, etc. During the freezing and thawing process of semen, sperm will be damaged by oxidative stress induced by reactive oxygen species, causing irreversible damage to the sperm biomembrane structure (including plasma membrane, acrosome membrane, and mitochondrial membrane). Sperm biomembranes contain a large amount of polyunsaturated fatty acids and are extremely vulnerable to attack by reactive oxygen species. Since sperm lack cytoplasmic antioxidants and their highly condensed chromatin cannot generate a genomic antioxidant response, it is necessary to study a protectant that can protect the integrity of the sperm biomembrane structure.

[0004] Currently, most of the research on sperm diluents and protectants is related to sperm motility, nutrient requirements, antibacterial properties, and osmotic pressure, etc., while there is no report on protectants for the integrity of the sperm biomembrane structure. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the existing technology and provide a goat sperm biomembrane protectant, aiming to resist oxidative damage to the plasma membrane, acrosome membrane, and mitochondrial membrane of goat sperm during the freezing and thawing process.

[0006] The present invention is achieved by the following technical solutions: A goat sperm biomembrane protectant, comprising the following components: 0.036 g / ml Tris (tris(hydroxymethyl)aminomethane), 0.01 g / ml glucose, 0.02 g / ml citric acid, 6% (v / v) glycerol, 1% (v / v) penicillin-streptomycin mixture, 20 μg / ml peroxidase-6, 10 - 30 μg / ml glutathione peroxidase 5, and ultrapure water.

[0007] The glutathione peroxidase 5 contains 20 μg / ml.

[0008] Another object of the present invention is to provide an application method of a goat sperm biomembrane protectant, comprising the following steps:

[0009] 1) Preparation of the protective agent: 0.036 g / ml Tris, 0.01 g / ml glucose, 0.02 g / ml citric acid, 6% (v / v) glycerol, 1% (v / v) penicillin-streptomycin mixture, 20 μg / ml peroxidase-6, 10 - 30 μg / ml glutathione peroxidase 5 were successively added to ultrapure water for dissolution and then made up to volume to prepare a protective agent for goat sperm biofilm. It was transferred to a reagent bottle through a syringe filter, sealed, and stored at 4°C.

[0010] 2) Semen collection: The artificial vagina collection method was used, and the whole process was carried out under aseptic conditions. After semen collection, the artificial vagina was immediately erected to prevent semen backflow. The freshly collected semen was kept warm at 37°C and sent to the laboratory.

[0011] 3) Semen dilution: Semen collected according to the aseptic operation requirements with a fresh sperm motility of over 75% and a rapid straight-line movement of over 50% was used. The protective agent for goat sperm biofilm was warmed to 37°C, and the semen was added to the protective agent for goat sperm biofilm and diluted at a volume ratio of semen to the protective agent for goat sperm biofilm of 1:10. After dilution, it was mixed evenly.

[0012] 4) Semen freezing: It was cooled in a constant temperature refrigerator at 4°C for 2 h. After cooling, the semen was filled into 0.25 ml straws. After equilibration for 1 h, it was moved to a position 4 cm above the liquid nitrogen surface, fumigated for 7 min, and then quickly immersed in liquid nitrogen for cryopreservation.

[0013] 5) Semen thawing: After taking out the frozen straw from liquid nitrogen, it was immersed in a water bath at 37°C for 30 s for use.

[0014] The beneficial effects of the present invention are as follows: The present invention can resist the oxidative damage suffered by sperm biofilm during the freezing and recovery process, enabling sperm to exert normal physiological functions. When using the present invention to preserve semen compared with using a conventional diluent, under the same conditions, the integrity of sperm plasma membrane is increased by 27.6%, the integrity of acrosome membrane is increased by 15.3%, and the mitochondrial membrane potential is increased by 47.2%. The protective agent for goat sperm biofilm of the present invention can fully improve the utilization rate of breeding rams and is conducive to carrying out livestock improvement and breeding work. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 shows the effect of the protective agent for goat sperm biofilm on the integrity of sperm plasma membrane;

[0016] Figure 2 shows the effect of the protective agent for goat sperm biofilm on the integrity of acrosome membrane;

[0017] Figure 3 shows the effect of the protective agent for goat sperm biofilm on mitochondrial membrane potential. EMBODIMENTS

[0018] The present invention will be described in detail below in conjunction with specific embodiments, which are for understanding rather than limiting the present invention.

[0019] Example: Influence of a Goat Sperm Biofilm Protective Agent on the Structure of Sperm Biofilm during the Freezing and Recovery Process of Cashmere Goat Semen

[0020] Experimental Animals

[0021] The cashmere goat semen used in this experiment was from Inner Mongolia Jinlai Animal Husbandry Technology Co., Ltd. Four 2-year-old adult rams were selected, requiring good health, no diseases, and unified feeding and management.

[0022] Experimental Methods

[0023] 1) Preparation of the protective agent:

[0024] Control group: Take 1.8 g of Tris, 0.5 g of glucose, 1 g of citric acid, 3 ml of glycerol, and 0.5 ml of penicillin-streptomycin mixture. Dissolve them separately with ultrapure water and make up the volume to 50 ml. Transfer it to a reagent bottle through a 0.22 μm filter, seal it, and store it in a 4°C refrigerator for use within one week.

[0025] Experimental group 1: Take 1.8 g of Tris, 0.5 g of glucose, 1 g of citric acid, 3 ml of glycerol, 0.5 ml of penicillin-streptomycin mixture, 0.5 mg of peroxidase-6, and 0.5 mg of glutathione peroxidase. Dissolve them separately with ultrapure water and make up the volume to 50 ml. Transfer it to a reagent bottle through a 0.22 μm filter, seal it, and store it in a 4°C refrigerator for use within one week.

[0026] Experimental group 2: Take 1.8 g of Tris, 0.5 g of glucose, 1 g of citric acid, 3 ml of glycerol, 0.5 ml of penicillin-streptomycin mixture, 1 mg of peroxidase-6, and 0.5 mg of glutathione peroxidase. Dissolve them separately with ultrapure water and make up the volume to 50 ml. Transfer it to a reagent bottle through a 0.22 μm filter, seal it, and store it in a 4°C refrigerator for use within one week.

[0027] Experimental group 3: Take 1.8 g of Tris, 0.5 g of glucose, 1 g of citric acid, 3 ml of glycerol, 0.5 ml of penicillin-streptomycin mixture, 1.5 mg of peroxidase-6, and 0.5 mg of glutathione peroxidase. Dissolve them separately with ultrapure water and make up the volume to 50 ml. Transfer it to a reagent bottle through a 0.22 μm filter, seal it, and store it in a 4°C refrigerator for use within one week.

[0028] 2) Semen collection: The artificial vagina method was used for semen collection, and semen was collected once every two days. A long-handled forceps was used to pick up a 75% alcohol cotton ball to wipe and disinfect the inner tube of the artificial vagina, and then it was wiped with sterilized physiological saline 2-3 times. The inner tube of the artificial vagina was rinsed with a sterilized Tris-citrate-glucose basal solution, and the remaining liquid in the artificial vagina was allowed to air dry naturally. Hot water accounting for about 2 / 3 of the volume of the sandwich cavity of the artificial vagina was injected into the water injection hole of the artificial vagina shell. The water temperature was generally 45-50 °C, and the water injection port was plugged with a rubber stopper. The amount of water injection should vary according to the livestock species and individual. The temperature of the inner tube was measured with a thermometer and maintained at 38-40 °C. A sterilized glass rod was dipped in sterilized lubricant (medical vaseline) and applied to the front 1 / 3-1 / 2 of the artificial vagina to lubricate its inner cavity. The sperm collection cup that had been sterilized by high-pressure drying was inserted into the other end without vaseline applied, and the pressure in the inner cavity of the artificial vagina was adjusted according to the insertion depth of the sperm collection cup. During semen collection, the collector was at the right rear of the semen collection platform. When the male animal straddled, the penis was quickly guided into the artificial vagina. After semen collection was completed, the artificial vagina was immediately erected to prevent semen backflow. The freshly collected semen was kept warm at 37 °C and sent to the laboratory for processing. Fresh semen quality detection and processing. 5 μl of fresh semen was diluted with Tris-citrate-glucose basal solution, 5 μl of the diluted semen was placed on a glass slide, covered with a coverslip and pressed, and at least 5 fields of view were detected using a computer-assisted semen analysis system. The detection system captured at least 1000 sperm cells. The motility of fresh semen reached more than 75%, and semen samples with more than 50% of the sperm cells moving rapidly in a straight line were used for subsequent experiments.

[0029] 3) Sperm freezing and thawing: Fresh semen of cashmere goats and the cryoprotectant were diluted at 37 °C, and the sperm density was adjusted to 2×10 8 cells / ml. The diluted semen was placed in a 37 °C water bath and then placed in a 4 °C freezer for 3 h of temperature equilibration. After the water bath temperature dropped to 5 °C, 200 μl was aspirated with a pipette into a 0.25 ml cryotube, sealed with sealing powder, placed equidistantly on a freezing rack, and fumigated with liquid nitrogen 4 cm above the liquid nitrogen surface for 7 min, and then put into liquid nitrogen for storage. The frozen semen was thawed in a water bath three days later, and thawed in a 37 °C water bath for 30 s.

[0030] 4) Plasma membrane integrity detection: The low-osmotic swelling test was used to detect the plasma membrane integrity rate. First, 20 μl of semen was drawn from each experimental group and mixed with 200 μl of low-osmotic solution (0.49 g of sodium citrate and 0.9 g of fructose made up to 100 ml with distilled water) respectively, shaken well, incubated in an incubator at 37 °C for 30 min, observed using an optical microscope, 5 fields of view were randomly selected, and the number of sperm cells in each field of view was more than 200. The proportion of sperm cells with coiled tails in the total number of sperm cells was counted.

[0031] As shown by the results of the low-osmotic swelling test for detecting the plasma membrane integrity rate (see Figure 1), the sperm plasma membrane integrity rates of experimental groups 1, 2, and 3 were all significantly higher than those of the control group (P < 0.05). The sperm plasma membrane integrity rate of experimental group 2 was the highest, at 72.05%, while that of the control group was 44.42%, an increase of 27.6%.

[0032] 5) Detection of acrosome membrane integrity: Using a peanut agglutinin fluorescence labeling (PNA-FITC) detection kit, detect with a flow cytometer. Incubate the thawed semen at 37 °C for 30 min, centrifuge at 600 g for 5 min to remove the supernatant, add GENMED preservation solution to adjust the sperm concentration to 2×10 7 sperm / ml. Draw 100 μl of the suspension, add 500 μl of GENMED cleaning solution to mix the sperm, centrifuge and take the supernatant (using the same centrifugation method as above), then add 200 μl of GENMED staining solution B, mix the sperm population, incubate in the dark at room temperature for 20 min, then centrifuge to remove the supernatant (using the same centrifugation method as above), add 200 μl of self-prepared propidium iodide (0.4 μl / mg), incubate in the dark at room temperature for 5 min, centrifuge to take the supernatant (using the same centrifugation method as above), then add 1 ml of GENMED cleaning solution, and finally detect with a flow cytometer.

[0033] As shown by the results of flow cytometer detection of sperm acrosome membrane integrity rate (see Figure 2 ), the sperm acrosome membrane integrity rates of experimental groups 1 and 2 were significantly higher than those of experimental group 3 and the control group (P < 0.05). The acrosome integrity rate of experimental group 1 was 77.30%, the sperm acrosome membrane integrity rate of experimental group 2 was 80.27%, while that of the control group was 64.97%. The acrosome membrane integrity of experimental group 2 increased by 15.3% compared with the control group.

[0034] 6) Detection of mitochondrial membrane potential: Using a mitochondrial membrane potential detection kit (JC-1), detect with a flow cytometer. After thawing the semen, take 1×10 6 sperm, add 0.5 ml of PBS, then add 0.5 ml of JC-1 staining working solution, invert several times to mix evenly, incubate at 37 °C for 20 min. After incubation, centrifuge at 600 g for 5 minutes to discard the supernatant, then add 1 ml of JC-1 staining buffer (1x) to resuspend the sperm, centrifuge and discard the supernatant (using the same centrifugation method as above), repeat the above operation once, and finally add 500 μl of JC-1 staining buffer (1x), resuspend the sperm, and then detect with a flow cytometer.

[0035] As shown by the results of flow cytometer detection of mitochondrial membrane potential (see Figure 3), the mitochondrial membrane potentials of experimental group 1, experimental group 2 and experimental group 3 were all significantly higher than those of the control group (P < 0.05). The mitochondrial membrane potential of experimental group 2 was the highest, being 1.84 ΔΨm, while that of the control group was 1.25 ΔΨm, and the mitochondrial membrane potential of experimental group 2 increased by 47.2% compared with the control group.

[0036] The above results indicate that the combination of 0.036 g / ml Tris, 0.01 g / ml glucose, 0.02 g / ml citric acid, 6% (v / v) glycerol, 1% (v / v) penicillin-streptomycin mixture, 20 μg / ml peroxidase-6, 10 - 30 μg / ml glutathione peroxidase 5 and ultrapure water in experimental group 2 can significantly and effectively protect goat sperm from oxidative damage during the freezing-thawing process, improve the integrity of the sperm biomembrane structure, and enable the sperm to perform normal physiological functions.

[0037] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A goat sperm biofilm protectant, characterized in that It includes the following components: 0.036 g / ml Tris, 0.01 g / ml glucose, 0.02 g / ml citric acid, 6% (volume fraction) glycerol, 1% (volume fraction) penicillin-streptomycin mixture, 20 μg / ml peroxidase-6, 20 μg / ml glutathione peroxidase 5 and ultrapure water.

2. The application method of a goat sperm biofilm protectant according to claim 1, characterized in that It includes the following steps: 1) Prepare the protective agent: 0.036 g / ml Tris, 0.01 g / ml glucose, 0.02 g / ml citric acid, 6% (volume fraction) glycerol, 1% (volume fraction) penicillin-streptomycin mixture, 20 μg / ml peroxidase-6, 10 - 30 μg / ml glutathione peroxidase 5. Add them to ultrapure water in sequence for dissolution, make up the volume, prepare the biological membrane protective agent for goat sperm, transfer it to a reagent bottle through a syringe filter, seal it, and store it at 4°C. 2) Semen collection: Use the artificial vagina collection method and perform aseptic operation throughout the process. After semen collection, immediately erect the artificial vagina to avoid semen backflow. Keep the freshly collected semen at 37°C and send it to the laboratory. 3) Semen dilution: Select semen collected according to the requirements of aseptic operation, with the motility of fresh semen reaching over 75% and the rapid straight-line movement rate over 50% for use. Take the biological membrane protective agent for goat sperm, warm it up to 37°C, add the semen to the biological membrane protective agent for goat sperm, and dilute it according to the volume ratio of semen to the biological membrane protective agent for goat sperm of 1:

10. Mix well after dilution. 4) Semen freezing: Cool it in a constant temperature refrigerator at 4°C for 2 h. After cooling, fill the semen into 0.25 ml thin tubes. After equilibration for 1 h, move it to a position 4 cm above the liquid nitrogen surface, fumigate for 7 min, and then quickly immerse it in liquid nitrogen for cryopreservation. 5) Semen thawing: Take out the frozen thin tubes from liquid nitrogen and immerse them in a water bath at 37°C for thawing, waiting for use.

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