Goat semen cryopreservation diluent and application thereof

Abstract

The invention relates to the technical field of goat semen cryopreservation, in particular to a goat semen cryopreservation diluent and application thereof. The invention relates to application of vitamin A in cryopreservation of goat semen, and a goat semen cryopreservation diluent contains vitamin A, glucose, lactose, sodium citrate, penicillin, streptomycin, yolk and glycerol. According to the goat semen cryopreservation diluent and the application thereof provided by the invention, the vitamin A is added into the goat semen cryopreservation diluent, so that the motility rate, plasma membrane integrity and acrosome integrity of thawed sperms can be improved, the antioxidant enzyme level in the cryopreservation process is improved, and oxidative stress injury is reduced; therefore, the cryopreservation effect of the goat semen is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cryopreservation of goat semen, and particularly to the application of vitamin A in the cryopreservation diluent of goat semen and the cryopreservation method of goat semen. Background Art

[0002] Semen cryopreservation is one of the key links in the animal biotechnology system, mainly solving the problem of long-term stable preservation of semen. Semen cryopreservation technology can break through the barriers of time and space to preserve semen for a long time, improve the utilization rate of excellent breeding rams, and accelerate the process of breed improvement. It is one of the key links in artificial insemination technology.

[0003] Goat sperm is very sensitive to low temperature and has poor resistance to low temperature shock, and is extremely vulnerable to low temperature damage during the freezing process. First, the content of unsaturated fatty acids in the sperm plasma membrane is very high, while the cholesterol content is relatively low, which cannot coagulate fatty acids together, resulting in a more open structure, lower stability and adhesion of the goat sperm plasma membrane, making it more difficult for goat sperm to resist the structural damage caused by ice crystals during low temperature and freezing. Second, reactive oxygen species will be generated during the cryopreservation of semen. When the reactive oxygen species are excessive, they will disrupt the redox balance of sperm, cause excessive oxidative stress of sperm, resulting in permanent changes in the protein structure, thereby changing the catalytic activities of glycolytic enzymes, Krebs cycle enzymes, electron transfer chain protein complexes and oxidative phosphorylation, reducing the intracellular ATP production and mitochondrial dysfunction, and causing irreversible damage to the stability of sperm structure and genetic material. These damages lead to a decrease in sperm motility, impaired plasma membrane function and reduced fertilization rate. Currently, in the goat semen freezing experiment, the motility of most frozen semen after thawing cannot be stably maintained above 50%. There is still a certain gap between goat frozen semen and commercial production and sales.

[0004] Existing studies have shown that adding antioxidants and cryoprotectants to the cryopreservation diluent of animal semen can improve the oxidation level and membrane integrity of sperm after thawing. However, due to the different absorption, utilization and interaction pathways of each animal for antioxidants and cryoprotectants, the improvement effects of antioxidants and cryoprotectants on the frozen semen of each animal vary greatly. Due to its own species specificity, goat frozen semen has more stringent requirements for antioxidants and cryoprotectants.

[0005] Vitamin A has strong antioxidant capacity. Adding vitamin A-like additives to the diet can significantly increase the semen collection volume, total sperm count, and sperm density of boars, and improve the sperm malformation rate. Supplementing vitamin A to the diet of rams lacking vitamin A can restore the activities of sperm acrosin and fibrinogen activator to normal. In the case of high-temperature oxidative stress, vitamin A has a protective effect on the integrity of the sperm acrosome membrane. Vitamin A can protect human sperm from reactive oxygen species (ROS) damage and enhance the activity of antioxidant enzymes. The diene conjugated bond in the vitamin A molecule plays an important role and can act as a quencher and scavenger of lipid peroxidation free radicals, hydroxyl free radicals, and other various free radicals, effectively reducing the damage of free radicals to the body and playing a positive and effective role in maintaining the normal physiological functions of cells.

[0006] Therefore, a dilution solution for cryopreserving goat semen and its application are designed to provide another technical solution to the above technical problems. Summary of the Invention

[0007] Based on this, it is necessary to provide a dilution solution for cryopreserving goat semen and its application to overcome the defects and deficiencies in the prior art, provide the application of vitamin A in cryopreserving goat semen, the dilution solution for cryopreserving goat semen and its application, and improve the post-thaw survival rate, plasma membrane integrity, and acrosome integrity of goat semen, and reduce oxidative damage.

[0008] To solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The application of vitamin A in cryopreserving goat semen.

[0010] The application of vitamin A as an additive in the preparation of frozen goat semen, and the added concentration of vitamin A is 0.25 - 1 μmol / L.

[0011] A dilution solution for cryopreserving goat semen, containing vitamin A, as well as glucose, lactose, sodium citrate, penicillin, streptomycin, egg yolk, and glycerol.

[0012] As a preferred embodiment of the dilution solution for cryopreserving goat semen provided by the present invention, it includes the following components: 156.43 mmol / L glucose, 127.66 mmol / L lactose, 51.00 mmol / L sodium citrate, 100,000 IU penicillin, 100,000 IU streptomycin, 100 mL of double-distilled water, 15% egg yolk, and 4% glycerol.

[0013] The application of the dilution solution for cryopreserving semen in improving the quality of frozen goat semen.

[0014] A method for preparing a cryopreservation diluent for goat semen comprises the following steps:

[0015] Mix glucose, lactose, sodium citrate, penicillin, and streptomycin to form Solution A.

[0016] Vibrate and mix Solution A with egg yolk, and then vibrate and mix the resulting liquid with glycerol to form a cryopreservation diluent for goat semen.

[0017] A method for cryopreserving goat semen comprises the following steps:

[0018] Mix the cryopreservation diluent for goat semen, vitamin A, and goat semen to obtain diluted semen.

[0019] Fill 0.25 ml veterinary freezing straws, seal them, wrap them with 18 layers of gauze, place them in an environment at 4°C for 1.5 h to cool down, balance for 2 h, and then place them in a programmable freezer for cooling and freezing. After completion, quickly take them out of the programmable freezer and store them in liquid nitrogen.

[0020] It can be clearly seen that through the above technical solution of the present application, the technical problems to be solved by the present application can surely be solved.

[0021] Meanwhile, through the above technical solution, the present invention has at least the following beneficial effects:

[0022] The present invention provides a cryopreservation diluent for goat semen and its application. Using vitamin A for cryopreserving goat semen can improve the post-thaw survival rate, plasma membrane integrity, acrosome integrity, and antioxidant capacity of goat semen. Detailed Embodiments

[0023] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the following further elaborates on the present invention in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described.

[0025] It should be noted that, without conflict, the embodiments in the present invention and the features and technical solutions in the embodiments can be combined with each other.

[0026] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0027] I. Experimental Animals

[0028] The goat semen used in this invention is all from the agricultural and animal husbandry experimental base of Yulin University.

[0029] II. Solution Preparation

[0030] Solution A: Weigh 3.1 g of glucose, 4.6 g of lactose, 1.5 g of sodium citrate, 0.1 g of streptomycin, and 0.0625 g of penicillin, dissolve them in 50 ml of double-distilled water, make up the volume to 100 ml, filter with a 0.22 μm filter, and store in a 4°C refrigerator for later use.

[0031] Solution B: Vigorously shake and mix 8.5 ml of Solution A with 1.5 ml of egg yolk. Take 9.6 ml of the mixed liquid and mix it with 0.4 ml of glycerol again by shaking. Prepare it for immediate use.

[0032] Hypotonic solution preparation: Weigh 0.9 g of fructose and 0.49 g of sodium citrate, add double-distilled water to make up the volume to 100 ml, filter with a 0.22 μm filter, and store at 4°C for later use.

[0033] III. Specific Operations for Semen Freezing

[0034] Semen collection and sperm motility detection: Use the artificial vagina method to collect semen from Shaanbei white cashmere goats. Immediately after semen collection, use an ordinary optical microscope to detect the quality of fresh semen. Select semen with sperm motility above 75% for mixed use to eliminate the error caused by the difference in sperm motility among individual breeding rams.

[0035] Dilution: Mix the qualified semen with Solution B at a ratio of 1:9 isothermally. Immediately after dilution, use an ordinary optical microscope to detect the quality after dilution.

[0036] Filling: Fill the diluted semen into 0.25 ml veterinary semen freezing straws. There should be a gap in the straw and it should not be filled completely to prevent the straw from bursting during thawing. Seal the filled straws with a sealer.

[0037] Equilibration: Wrap the filled semen freezing straws with 18 layers of gauze and place them in a 4°C environment to cool down for 1.5 h and equilibrate for 2 h. Immediately after equilibration, use an ordinary optical microscope to detect the quality after equilibration.

[0038] Freezing: Place the equilibrated semen freezing straws in a freezing program instrument for cooling and freezing; place the completed semen freezing straws in liquid nitrogen for cryopreservation.

[0039] IV. Thawing of Semen and Subsequent Detection

[0040] After 3 days of liquid nitrogen storage, use pre-cooled forceps to take out the semen freezing straws from the liquid nitrogen tank, quickly put them into a 37°C constant temperature water bath, thaw for 30 s, and transfer the semen to a 1.5 ml centrifuge tube for semen quality evaluation after thawing.

[0041] 1. Sperm motility test: Use a pipette to draw 10 μL of semen, drop it on a slide preheated at 37°C, cover it with a cover slip preheated at 37°C, and test it under an ordinary optical microscope. Use the ten-level scoring method to evaluate. Repeat each dilution three times and take the average value.

[0042] 2. Detection of sperm deformity rate: Take 5 μL of semen, dilute it 200 times with 0.9% NaCl solution, take 10 μL and smear it on one end of the slide, air-dry it, fix it in 95% alcohol for 5-6 minutes, stain it with blue ink for 3-5 minutes after air-drying, rinse it with running water, observe 300 sperms under a microscope after air-drying, calculate the proportion of abnormal sperms, repeat 3 times and take the average value.

[0043] Sperm abnormality rate (%) = number of abnormal sperm / total number of sperm × 100%

[0044] 3. Detection of sperm plasma membrane integrity: The hypoosmotic swelling method (HOST) was used to detect the integrity of sperm plasma membrane. 10 μL of semen was added to a preheated centrifuge tube, and then 400 μL of preheated hypotonic solution was added and mixed. Incubate in a 37°C constant temperature water bath for 30 minutes. 15 μL was taken and the number of sperm with unbent or large bent tails was recorded under a microscope. A total of 300 sperm were counted, repeated 3 times, and the average value was taken.

[0045] Sperm plasma membrane integrity rate (%) = (number of sperm with unbent tails + number of sperm with large curved tails) / total number of sperm × 100%

[0046] 4. Sperm acrosome integrity detection: Use fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) fluorescent dye for staining, and observe and record data under a fluorescence microscope. Use a pipette to absorb 30μI of semen and evenly apply it on a clean glass slide to air dry naturally; after the sample is air-dried, use anhydrous methanol to fix it for 10 minutes and air-dry again. After air-drying, take 30μL FITC-PNA dye solution to cover the air-dried sample and place it in a 37℃ incubator for 30 minutes, and keep it away from light throughout the process. At the same time, use DAPI fluorescent dye to stain the sperm nucleus to exclude the effect of false positives caused by non-cellular substances such as impurities on sperm count. After the incubation is completed, take 30μL DAPI dye solution and evenly add it to the sample slide to ensure that the sample is covered, and stain it at room temperature for 5-10 minutes. After DAPI staining, the sperm nucleus emits blue fluorescence under the fluorescence microscope. Rinse the slide 2-3 times with PBS to wash off the floating color and place it in a ventilated place to air dry. Then add an appropriate amount of glycerol to seal the slide and place it under an upright fluorescence microscope for observation and counting. The sperm with a complete and bright green fluorescence at the front of the acrosome is a sperm with a complete acrosome, and the sperm with a damaged acrosome is a sperm with a defective or no fluorescence at the front. When taking pictures and counting, at least 5 fields of view should be selected, and the number of sperm in each field of view should not be less than 200. Keep the operation away from light throughout the process.

[0047] Acrosome integrity rate of sperm (%) = (Number of sperm with intact acrosome / Total number of sperm) × 100%

[0048] 5. Detection of SOD enzyme activity: Use the SOD assay kit of Shanghai Beyotime Biotechnology Co., Ltd. to detect the SOD content. The detection steps are shown in the kit instruction manual.

[0049] 6. Detection of CAT enzyme activity: Use the CAT assay kit of Shanghai Beyotime Biotechnology Co., Ltd. to detect the CAT content. The detection steps are shown in the kit instruction manual.

[0050] 7. Detection of GSH-PX enzyme activity: Use the GSH-PX assay kit of Shanghai Beyotime Biotechnology Co., Ltd. to detect the GSH-PX content. The detection steps are shown in the kit instruction manual.

[0051] Example 1

[0052] Prepare the basic semen cryopreservation diluent solution I (solution A) and the basic semen cryopreservation diluent solution II (solution B) according to the above method. Add the pre-prepared vitamin A to solution B to make the final concentration of vitamin A 0.25 μmol / L, and perform semen cryopreservation according to the above method.

[0053] Example 2

[0054] Refer to Example 1, the only difference is that the pre-prepared vitamin A is added to solution B to make the final concentration of vitamin A 0.5 μmol / L.

[0055] Example 3

[0056] Refer to Example 1, the only difference is that the pre-prepared vitamin A is added to solution B to make the final concentration of vitamin A 0.75 μmol / L.

[0057] Example 4

[0058] Refer to Example 1, the only difference is that the pre-prepared vitamin A is added to solution B to make the final concentration of vitamin A 1 μmol / L.

[0059] Comparative Example 1

[0060] Refer to Example 1, the only difference is that vitamin A is not added to solution B.

[0061] Using the goat semen cryopreservation diluent of the present invention, the evaluation results of the preservation effect are as follows:

[0062] Table 1 Detection results of the motility rate of thawed goat semen under different treatment methods

[0063]

[0064] Note: The same capital letters in the same column indicate no significant difference between groups (P > 0.05), and different capital letters indicate significant difference between groups (P < 0.05).

[0065] As can be seen from Table 1, cryopreserving goat semen with 0.25 - 1 μmol / L vitamin A can improve the sperm motility rate of thawed goat semen.

[0066] Table 2 Detection results of sperm malformation rate of thawed goat semen under different treatment methods

[0067]

[0068] Note: The same capital letters in the same column indicate no significant difference between groups (P > 0.05), and different capital letters indicate significant difference between groups (P < 0.05).

[0069] As can be seen from Table 2, using 0.25 - 1 μmol / L vitamin A to cryopreserve goat semen slightly increases the sperm malformation rate of thawed goat semen, but the difference is not significant.

[0070] Table 3 Detection results of plasma membrane integrity rate of thawed goat semen under different treatment methods

[0071]

[0072] Note: Different lowercase letters indicate significant difference (P < 0.05), and different capital letters indicate extremely significant difference (P < 0.01).

[0073] As can be seen from Table 3, cryopreserving goat semen with 0.25 - 1 μmol / L vitamin A can improve the plasma membrane integrity rate of thawed goat semen.

[0074] Table 4 Detection results of acrosome integrity rate of thawed goat semen under different treatment methods

[0075]

[0076] Note: Different lowercase letters indicate significant difference (P < 0.05), and different capital letters indicate extremely significant difference (P < 0.01).

[0077] As can be seen from Table 4, cryopreserving goat semen with 0.25 - 1 μmol / L vitamin A can improve the acrosome integrity rate of thawed goat semen.

[0078] Table 5 Detection results of antioxidant indexes of thawed goat semen under different treatment methods

[0079]

[0080] Note: Different lowercase letters indicate significant differences (P < 0.05), and different uppercase letters indicate extremely significant differences (P < 0.01).

[0081] It can be seen from Table 5 that cryopreserving goat semen with 0.25 - 1 μmol / L vitamin A can improve the activities of SOD, CAT, and DSH-PX enzymes in thawed goat semen, reduce oxidative stress damage, and enhance the antioxidant capacity of sperm in thawed goat semen.

[0082] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principles and practical applications of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. Application of vitamin A in cryopreserving goat semen.

2. Application of vitamin A as an additive in preparing cryopreserved goat semen, wherein the added concentration of vitamin A is 0.25 - 1 μmol / L.

3. A dilution solution for cryopreservation of goat semen, characterized in that, Containing vitamin A, as well as glucose, lactose, sodium citrate, penicillin, streptomycin, egg yolk, and glycerol.

4. The cryopreservation diluent for goat semen according to claim 3, wherein Comprising the following components: 156.43 mmol / L glucose, 127.66 mmol / L lactose, 51.00 mmol / L sodium citrate, 100000 IU penicillin, 100000 IU streptomycin, 100 mL double-distilled water, 15% egg yolk, 4% glycerol.

5. Application of the semen cryopreservation diluent according to claim 3 or 4 in improving the quality of cryopreserved goat semen.

6. A preparation method of a cryopreservation diluent for goat semen, characterized in that, The steps are as follows: Mix glucose, lactose, sodium citrate, penicillin, and streptomycin to form solution A; Vibrate and mix solution A with egg yolk, and then vibrate and mix the mixed liquid with glycerol again to form a cryopreservation diluent for goat semen.

7. A method for cryopreserving goat semen, characterized in that, The steps are as follows: Mix the cryopreservation diluent for goat semen according to claim 3 or 4, vitamin A, and goat semen to obtain diluted semen; Fill it into a 0.25 ml veterinary semen freezing straw, seal it, wrap it with 18 layers of gauze, place it in an environment at 4°C to cool for 1.5 h, equilibrate for 2 h, then place it in a programmable freezer to cool and freeze. After completion, quickly take it out from the programmable freezer and store it in liquid nitrogen.

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