Preparation and application of low-temperature boar semen diluent based on ferulic acid

Abstract

The invention relates to the field of boar semen preservation, in particular to preparation and application of a ferulic acid-based boar semen low-temperature diluent. The proportion of the ferulic acid in the basic diluent is 0.2 to 0.8 mmol / L; wherein the basic diluent is prepared by dissolving 3.00 g of glucose, 4.00 g of cane sugar, 0.03 g of gentamicin sulfate and 20 mL of yolk in 80 mL of double distilled water, uniformly stirring, centrifuging at 4 DEG C and 7000 * g for 25 min, adjusting the pH value of the solution to 6.80-7.00, and adjusting the osmotic pressure of the solution to 290-300 mOsm / kg. The method has the beneficial effects that ferulic acid is used as a protective agent for preserving the boar semen at 4 DEG C for the first time, the effect is reliable, popularization is easy, the preserved boar semen is suitable for artificial insemination and artificial insemination after long-distance transportation, the motility, motility rate, acrosome integrity rate and plasma membrane integrity rate of boar sperms preserved at 4 DEG C can be remarkably improved, and the fertilization activity of the sperms is kept.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pig semen preservation, in particular to a pig semen diluent with ferulic acid as an additive, and a preparation method and application thereof. BACKGROUND

[0002] With the implementation of sustainable development strategy and the development of modern breeding industry, large-scale and intensive breeding has become the overall trend of the pig industry. Under this trend, pig artificial insemination technology is particularly important, and its development is increasingly mature and perfect. The use rate of artificial insemination technology in China, the United States, New Zealand and other countries has exceeded 90%. Semen preservation technology is an important part of artificial insemination technology. Semen preservation is crucial in artificial insemination technology, directly affecting the utilization rate of boars and subsequent sow mating and pregnancy, thereby affecting the scale and economic benefits of the pig industry. It can prolong the survival time of sperm and maintain its fertilization ability, so as to facilitate long-distance transportation or long-term preservation, thereby expanding the use range of semen, improving the utilization rate of breeding boars, saving costs and not being limited by region and time. The preservation of pig semen is of great significance to the study of boar reproductive medicine, the promotion of pig breeding, the preservation of pig genetic resources, and the popularization of artificial insemination technology.

[0003] At present, in order to meet the different needs of production practice for semen, the preservation methods of semen can be divided into three categories: normal temperature preservation, low temperature preservation and freezing preservation. The temperature of normal temperature preservation is 15-25℃, which uses the acid metabolites produced by pig semen at this temperature to achieve the purpose of preservation. This method does not require special equipment, is simple and easy to operate, is convenient for popularization and application, and is suitable for short-term preservation of various livestock semen, especially for short-term preservation of pig whole semen. The temperature of low temperature preservation is 0-4℃, which inhibits the metabolism of sperm, reduces the activity of sperm, and makes sperm in a dormant state. However, in order to avoid cold shock of sperm, certain protective agents need to be added in the diluent, and egg yolk is commonly used as a protective agent in production. Normal temperature preservation and low temperature preservation are in liquid form, which are used for short-term storage. Freezing preservation refers to the method of preserving semen in a frozen form in an ultra-low temperature environment by using liquid nitrogen as a cold source after special treatment. It is commonly used for the preservation of cow and sheep semen. Due to the specificity of pig sperm cell structure, especially the composition of cell membrane, pig sperm is sensitive to temperature changes, and a large number of pig sperm are damaged during freezing, with significantly lower activity, acrosome integrity and plasma membrane integrity than fresh semen, resulting in a general decrease in pregnancy rate and litter rate of sows in commercial pig production. In addition, the cost of freezing preservation device is high, and most pig farms do not have freezing devices, so freezing preservation is difficult to popularize and apply in actual production. Normal temperature preservation method is simple, but sperm metabolism is vigorous under normal temperature conditions, and the preservation time is relatively short, not more than 5 days, making it difficult to achieve long-distance transportation. Therefore, low temperature preservation is commonly used in production practice.

[0004] During the cryopreservation of pig semen, reactive oxygen species (ROS) will be enriched and react with the plasma membrane, proteins and even DNA in sperm, causing oxidative stress in pig sperm, affecting the structure of pig sperm and the quality of semen. Therefore, by adding an appropriate amount of antioxidant to the pig sperm diluent, the oxidative stress of ROS on pig sperm can be reduced, the quality of pig semen during cryopreservation can be improved, and better service can be provided for pig production. SUMMARY

[0005] The purpose of the application is to solve the above technical problems. Adding antioxidant components to the diluent during the cryopreservation of pig semen can play a protective role. In order to improve the quality of pig semen after cryopreservation and improve the success rate of artificial insemination of semen after preservation, it is necessary to find an antioxidant protective agent or semen diluent suitable for 4℃.

[0006] As an antioxidant, ferulic acid can reduce the damage of free radicals to cell membranes and genetic material.

[0007] So far, there is no relevant report on the use of ferulic acid as a protective agent for the 4℃ preservation of pig semen at home and abroad.

[0008] In order to achieve the above purpose, the application adopts the following technical solutions: The protective agent for the 4℃ preservation of pig semen, characterized in that the proportion of ferulic acid in the basic diluent is 0.02-0.08mmol / L; wherein the ratio of the basic diluent is: glucose 3.00g, sucrose 4.00g, gentamicin sulfate 0.03g, yolk 20mL dissolved in 80mL double distilled water, stirred uniformly, centrifuged at 4℃, 7000xg for 25min.

[0009] The further technical solution of the application is that the pH of the solution is 6.80-7.00, and the osmotic pressure is 290-300mOsm / kg.

[0010] The further technical solution of the application is that the preservation temperature is 4℃.

[0011] The preparation method of the protective agent for the 4℃ preservation of pig semen as described above, characterized in that it comprises the following steps: 1) Glucose 3.00g, sucrose 4.00g, gentamicin sulfate 0.03g, yolk 20mL dissolved in 80mL double distilled water, stirred uniformly, centrifuged at 4℃, 7000xg for 25min, prepared into a basic diluent; 2) Add 0.02-0.08 mmol ferulic acid to the above basic solution, adjust the pH of the solution to 6.80-7.00, and the osmotic pressure to 290-300 mOsm / kg; filter and sterilize, cool to room temperature and store in a refrigerator at 4℃.

[0012] A method for preserving boar semen at 4°C, characterized by the following steps: 1) According to the volume ratio of 1:1 of the protective agent described in claim 1, the treated boar semen is added to the pre-warmed preservation diluent, wrapped in gauze, and equilibrated in a constant temperature incubator at 4°C for 1-2 hours. 2) Store the semen after the equilibration treatment in step 1) at 4°C.

[0013] A further technical solution of the present invention is that the low temperature is 4°C.

[0014] Ferulic acid is used as a preservative for preserving bovine semen at 4°C.

[0015] The present invention, which adopts the above technical solution, has the following beneficial effects compared with the prior art: The present invention is the first to use ferulic acid as a protective agent for the preservation of boar semen at 4°C. The effect is reliable and easy to promote. The preserved boar semen is suitable for artificial insemination and artificial insemination after long-distance transportation. It can significantly improve the motility, viability, acrosome integrity rate and plasma membrane integrity rate of boar sperm after preservation at 4°C, and maintain the fertilization vitality of sperm. Detailed Implementation

[0016] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0017] Example 1 Accurately measure 3.00g of glucose, 4.00g of sucrose, 0.03g of gentamicin sulfate, and 20mL of egg yolk, dissolve them in 80mL of double-distilled water, stir well, and centrifuge at 7000×g for 25min at 4℃ to prepare a basic dilution. Accurately measure 0.2mmol of ferulic acid, mix it with the basic dilution to prepare a dilution. Filter and sterilize, cool to room temperature, and store in a 4℃ refrigerator for later use.

[0018] Example 2 Accurately measure 3.00g of glucose, 4.00g of sucrose, 0.03g of gentamicin sulfate, and 20mL of egg yolk, dissolve them in 80mL of double-distilled water, stir well, and centrifuge at 7000×g for 25min at 4℃ to prepare a basic dilution. Accurately measure 0.4mmol of ferulic acid, mix it with the basic dilution to prepare a dilution. Filter and sterilize, cool to room temperature, and store in a 4℃ refrigerator for later use.

[0019] Example 2 Accurately measure 3.00g of glucose, 4.00g of sucrose, 0.03g of gentamicin sulfate, and 20mL of egg yolk, dissolve them in 80mL of double-distilled water, stir well, and centrifuge at 7000×g for 25min at 4℃ to prepare a basic dilution. Accurately measure 0.8mmol of ferulic acid, mix it with the basic dilution to prepare a dilution. Filter and sterilize, cool to room temperature, and store in a 4℃ refrigerator for later use.

[0020] Comparison Example Accurately measure 3.00g of glucose, 4.00g of sucrose, 0.03g of gentamicin sulfate, and 20mL of egg yolk, dissolve them in 80mL of double-distilled water, stir well, centrifuge at 7000×g for 25min at 4℃ to prepare a basic dilution; adjust the pH of the solution to 6.80~7.00, filter and sterilize, cool to room temperature, and store in a 4℃ refrigerator for later use.

[0021] The following describes the steps for cryopreservation of boar semen and the method for assessing sperm quality.

[0022] (I) Semen Collection Semen was collected by hand, focusing on the midstream sperm-rich portion. The collected semen was filtered through four layers of sterile gauze to remove any gelatinous residue. After being diluted 1:1 with a basic diluent, routine quality checks were performed under a microscope at 37°C. Semen that was milky white in color, had a uniform, cloudy, colloidal consistency, a slightly fishy odor, normal sperm morphology, a motility of 0.8 or higher, and a density of "dense" was selected for the test.

[0023] (II) Semen Processing and Preservation Centrifuge fresh semen (800×g, 10min), discard the supernatant, add pre-warmed storage diluent at a semen:diluent ratio of 1:1, wrap with 4 layers of gauze, and equilibrate in a 4℃ incubator for 1–2 hours. Store the equilibrated semen at 4℃.

[0024] (III) Post-sperm preservation sperm quality assessment 1. Sperm motility and viability Take 10 mL of semen sample and incubate at 37℃ for 30 min. After mixing, take 10 μL and drop it onto a glass slide for microscopic examination. Use a computer-assisted semen analysis system (CASA) to detect sperm motility and viability. Randomly select 3 fields of view for each measurement, with each field of view requiring a sperm count greater than 200. The results are averaged.

[0025] 2. Sperm acrosome integrity rate After mixing the semen sample thoroughly, take 50 μL and place it in a centrifuge tube. Add 1 mL of 4% paraformaldehyde, mix well, and fix for 10 min. Centrifuge (800×g) for 5 min, discard the supernatant, and take 10 μL of the lower semen layer to prepare a smear. Air dry for 5 min, stain with FITC-PNA fluorescent dye, incubate in the dark for 30 min, and then fluoresce with DAPI dye for 10 min. Wash off excess dye with water and quickly observe and photograph under a fluorescence microscope. Sperm with intact acrosomes exhibiting complete bright green fluorescence at the anterior acrosome are considered intact, while sperm with incomplete or no fluorescence at the anterior acrosome are considered damaged. When photographing, select at least 3 clear fields of view, each containing at least 200 sperm. The entire experiment must be conducted in the dark.

[0026] 3. Sperm plasma membrane integrity rate Take 100 μL of semen sample and place it in a 1.5 mL centrifuge tube. Incubate at 37°C for 10 min. Add 0.2 μL of SYBR-14 working solution, mix gently, and incubate at 37°C for 10 min. Then add 1 μL of PI working solution, mix gently, and incubate at 37°C for another 10 min. After incubation, drop 10 μL of semen onto a special poly-L-lysine-treated adhesive slide, cover with a coverslip, and quickly place under a fluorescence microscope to take a picture. Sperm nuclei fluorescing green indicate sperm with intact plasma membranes, while those fluorescing red-orange indicate sperm with damaged plasma membranes. When taking pictures, select at least three clear fields of view, each containing at least 200 sperm.

[0027] 4. Sperm deformity rate Take 10 mL of semen sample and incubate at 37℃ for 30 min. After mixing, take 10 μL and drop it onto a glass slide. Spread the sample drop into a smear and air dry for 5 min. Stain with 0.5% gentian violet alcohol solution or blue ink for 5 min, air dry for 10 min, wash off excess stain with water, and air dry again before microscopic examination. Randomly record at least 200 sperm cells from different fields of view, count the number of abnormal sperm cells, and calculate the abnormality rate.

[0028] (iv) Sperm quality assessment results The results of the evaluation of the porcine semen preservation agent and method at 4°C according to the present invention are as follows: When the amount of ferulic acid added is 0.2 mmol, after 7 days of preservation at 4°C, the sperm motility reaches 77%, the sperm viability reaches 62%, the acrosome integrity rate reaches 75%, the plasma membrane integrity rate reaches 58%, and the abnormality rate is 5%; when the amount of ferulic acid added is 0.4 mmol, after 4°C preservation, the sperm motility reaches 72%, the sperm viability rate reaches 64%, the acrosome integrity rate reaches 68%, the plasma membrane integrity rate reaches 54%, and the abnormality rate is 7%.

[0029] The results of the example are shown in the chart: Table 1. Effects of ferulic acid on sperm motility and viability of porcine semen stored at 4℃

[0030] A patent application entitled "Preparation and Application of a Low-Temperature Diluent for Porcine Semen Based on Ferulic Acid" includes two supplementary figures in its specification. The figures are described below: Figure 1 This is a graph showing the effect of ferulic acid on the acrosome integrity of porcine semen stored at 4°C. Figure 2 This is a graph showing the effect of ferulic acid on the integrity of the plasma membrane of porcine semen stored at 4°C. The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claims.

Claims

1. A preservative for storing bovine semen at 4°C, characterized in that, The proportion of ferulic acid in the basal dilution is 0.2~0.8 mmol / L; the basal dilution is prepared as follows: 3.00 g glucose, 4.00 g sucrose, 0.03 g gentamicin sulfate, 20 mL egg yolk dissolved in 80 mL double-distilled water, stirred evenly, centrifuged at 7000×g for 25 min at 4°C, adjusted the pH of the solution to 6.80-7.00, and adjusted the osmotic pressure of the solution to 290-300 mOsm / kg.

2. The preservative for preserving bovine semen at 4°C as described in claim 1, characterized in that, After complete dissolution, the pH of the basic diluent is 6.80-7.00, and the osmotic pressure is 290-300 mOsm / kg.

3. A method for preparing the preservative for preserving bovine semen at 4°C as described in claim 1, characterized in that, Includes the following steps: 1) Dissolve 3.00 g glucose, 4.00 g sucrose, 0.03 g gentamicin sulfate, and 20 mL egg yolk in 80 mL double-distilled water, stir well, and centrifuge at 7000×g for 25 min at 4℃ to prepare a basic dilution solution; 2) Add 0.02~0.08 mmol ferulic acid to the above basic solution, so that the solution pH is 6.80-7.00 and the osmotic pressure is 290-300 mOsm / kg. Filter and sterilize, cool to room temperature and store in a 4℃ constant temperature incubator.

4. A method for preserving boar semen at 4°C, characterized in that, The steps are as follows: 1) According to the volume ratio of 1:1 of the protective agent described in claim 1, the treated boar semen is added to the pre-warmed preservation diluent, wrapped in gauze, and equilibrated in a constant temperature incubator at 4°C for 1-2 hours. 2) Store the semen after equilibration in step 1) at 4°C.

5. The method for preserving boar semen at 4°C as described in claim 4, characterized in that, The low temperature mentioned is 4°C.

6. Use of ferulic acid as a preservative for preserving bovine semen at 4°C.

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