Boar semen diluent at 15-20 DEG C with ferulic acid as additive as well as preparation method and application of boar semen diluent

Abstract

The invention relates to the field of pig semen preservation, in particular to a 15-20 DEG C diluent of pig semen with ferulic acid as an additive as well as a preparation method and application of the 15-20 DEG C 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 3.715 g of glucose, 0.125 g of sodium citrate, 0.125 g of sodium ethylene diamine tetracetate, 0.075 g of potassium chloride and 0.03 g of gentamicin sulfate into 100 mL of double distilled water, and after the materials are fully dissolved, the pH value of the solution is adjusted to 6.80-7.00, and the osmotic pressure of the solution is adjusted to 290-300 mOsm / kg. The method has the advantages that ferulic acid is used as a protective agent for preservation of the boar semen at 15-20 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 vitality, motility, acrosome integrity and plasma membrane integrity of boar sperms preserved at 17 DEG C can be remarkably improved, and the fertilization vitality of the sperms is kept.

Description

Technical Field

[0001] This invention relates to the field of boar semen preservation, and more particularly to a room-temperature dilution of boar semen with ferulic acid as an additive, its preparation method, and its application. Background Technology

[0002] With the implementation of sustainable development strategies and the development of modern animal husbandry, large-scale and intensive farming has become the overall trend in the pig industry. Under this trend, artificial insemination technology for pigs has become particularly important. Semen preservation technology is an important component of artificial insemination. Semen preservation can extend the survival time of sperm and maintain its fertilization capacity, facilitating long-distance transportation or long-term storage, thereby expanding the scope of semen use, improving the utilization rate of breeding boars, saving costs, and not being limited by location or time. The preservation of pig semen is of great significance for research on boar reproductive medicine, promoting pig genetic breeding, preserving pig genetic resources, and promoting artificial insemination technology.

[0003] Semen preservation methods can be categorized into three types: room temperature preservation, cryopreservation, and cryopreservation. Room temperature preservation (17℃) requires no special equipment, is simple and easy to implement, and is suitable for short-term preservation of semen from various livestock, especially whole pig semen. Cryopreservation (4℃) inhibits sperm metabolism, weakens sperm motility, and induces a dormant state. However, to prevent cold shock, a cryoprotectant must be added to the diluent; egg yolk is commonly used in production. Cryopreservation involves specially treating the semen and then using liquid nitrogen as a cold source to freeze it in an ultra-low temperature environment for long-term preservation. This method is commonly used for preserving bovine and ovine semen. Due to the specificity of the structure of pig sperm cells, especially the cell membrane components, they are sensitive to temperature changes. A large number of pig sperm are damaged during the freezing process, and their viability, acrosome integrity rate, and plasma membrane integrity rate are significantly lower than those of fresh sperm. This leads to a general decrease in the pregnancy rate and farrowing rate of sows in commercial pig production. In addition, cryopreservation equipment is expensive, and most pig farms are not equipped with cryopreservation equipment. Therefore, cryopreservation is difficult to promote and apply in actual production. The commonly used methods for preserving pig semen are low-temperature preservation and room temperature preservation.

[0004] Studies have shown that during the storage of porcine semen at room temperature and low temperature, porcine sperm metabolism produces a large amount of reactive oxygen species (ROS), which carry oxygen free radicals (O2). 2- H2O2 and -OH can cause lipid peroxidation of unsaturated fatty acids in sperm cell membranes, resulting in damage to the sperm cell membranes and ultimately reducing the fertilization capacity of pig sperm. Summary of the Invention

[0005] Purpose of the invention: To solve the above-mentioned technical problems. Adding antioxidants to the diluent during the room temperature preservation of boar semen can provide protection. To improve the preservation efficiency of boar semen and increase the success rate of artificial insemination with preserved semen, it is necessary to find an antioxidant or semen diluent suitable for 15–20℃.

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

[0007] To date, there have been no reports, either domestically or internationally, on the use of ferulic acid as a preservative for the preservation of boar semen at 15–20°C.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A preservative for preserving bovine semen at 15–20°C, characterized in that the proportion of ferulic acid in the basal diluent is 0.02–0.08 mmol / L; wherein the basal diluent is prepared by dissolving 3.715 g glucose, 0.125 g sodium citrate, 0.125 g sodium EDTA, 0.075 g potassium chloride, and 0.03 g gentamicin sulfate in 100 mL double-distilled water.

[0009] A further technical solution of the present invention is that the solution pH is 6.80-7.00 and the osmotic pressure is 290-300 mOsm / kg.

[0010] A further technical solution of the present invention is that the storage temperature is 15-20℃.

[0011] A method for preparing the preservative for preserving bovine semen at 15-20°C as described above, characterized by comprising the following steps: 1) Dissolve 3.715g of glucose, 0.125g of sodium citrate, 0.125g of sodium EDTA, 0.075g of potassium chloride, and 0.03g of gentamicin sulfate in 100mL of double-distilled water to prepare a basic dilution solution; 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 17℃.

[0012] A method for preserving boar semen at 15–20°C, characterized by the following steps: 1) According to the volume ratio of the preservative 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 17°C for 1-2 hours. 2) Store the semen after equilibration in step 1) at 17°C.

[0013] A further technical solution of the present invention is that the room temperature is 15-20°C.

[0014] Ferulic acid is used as a preservative for preserving bovine semen at 15–20°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 15-20℃. 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 15-20℃, 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 3.715g of glucose, 0.125g of sodium citrate, 0.125g of sodium EDTA, 0.075g of potassium chloride, and 0.03g of gentamicin sulfate, and dissolve them in 100mL of double-distilled water to prepare a basic dilution. Accurately measure 0.2mmol of ferulic acid and mix it with the basic dilution to prepare a dilution. Filter and sterilize, cool to room temperature, and store in a 17℃ refrigerator for later use.

[0018] Example 2 Accurately measure 3.715g of glucose, 0.125g of sodium citrate, 0.125g of sodium EDTA, 0.075g of potassium chloride, and 0.03g of gentamicin sulfate, and dissolve them in 100mL of double-distilled water to prepare a basic dilution. Accurately measure 0.4mmol of ferulic acid and mix it with the basic dilution to prepare a dilution. Filter and sterilize, cool to room temperature, and store in a 17℃ refrigerator for later use.

[0019] Comparison Example Accurately measure 3.715g of glucose, 0.125g of sodium citrate, 0.125g of sodium EDTA, 0.075g of potassium chloride, and 0.03g of gentamicin sulfate, dissolve them in 100mL of double-distilled water to prepare a basic dilution solution. Adjust the pH of the solution to 7.00–7.20, filter and sterilize, cool to room temperature, and store in a 17°C refrigerator for later use.

[0020] The present invention is described below with reference to the steps of cryopreservation of boar semen and the method of sperm quality assessment.

[0021] (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.

[0022] (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 17℃ incubator for 1–2 hours. Store the equilibrated semen at 17℃.

[0023] (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.

[0024] 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.

[0025] 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 are those with intact plasma membranes, while those with nuclei fluorescing red-orange are those with damaged plasma membranes. When taking pictures, select at least 3 clear fields of view, and each field of view should contain at least 200 sperm.

[0026] 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.

[0027] (iv) Sperm quality assessment results The evaluation results of the porcine semen preservation agent and method at 15-20°C according to the present invention are as follows: When ferulic acid was added at 0.2 mmol, after storage at 17°C for 5 days, sperm motility reached 77%, sperm viability reached 69%, acrosome integrity reached 76%, and plasma membrane integrity reached 70%. When ferulic acid was added at 0.4 mmol, after storage at 17°C, sperm motility reached 75%, sperm viability reached 64%, acrosome integrity reached 69%, and plasma membrane integrity reached 66%. The above describes 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 without departing from the spirit and scope of the invention, and all such changes and modifications fall within the scope of the claims.

Claims

1. A preservative for storing bovine semen at 15-20°C, characterized in that, The proportion of ferulic acid in the basal diluent is 0.2~0.8 mmol / L; the basal diluent is prepared as follows: 3.715 g glucose, 0.125 g sodium citrate, 0.125 g sodium EDTA, 0.075 g potassium chloride, and 0.03 g gentamicin sulfate are dissolved in 100 mL double-distilled water. After complete dissolution, the pH of the solution is adjusted to 6.80-7.00, and the osmotic pressure is adjusted to 290-300 mOsm / kg.

2. The preservative for preserving bovine semen at 15-20°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 15-20°C as described in claim 1, characterized in that, Includes the following steps: 1) Dissolve 3.715g of glucose, 0.125g of sodium citrate, 0.125g of sodium EDTA, 0.075g of potassium chloride, and 0.03g of gentamicin sulfate in 100 mL of double-distilled water to prepare a basic dilution solution; 2) Add 0.02~0.08 mmol ferulic acid to the above basic solution, the pH of the solution is 6.80-7.00, the osmotic pressure is 290-300 mOsm / kg, filter and sterilize, cool to room temperature and store in a 17℃ constant temperature incubator.

4. A method for preserving boar semen at 15-20℃, 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 17°C for 1-2 hours. 2) Store the semen after equilibration in step 1) at 17°C.

5. The method for preserving boar semen at 15-20°C as described in claim 4, characterized in that, The room temperature is 15~20℃.

6. Use of ferulic acid as a preservative for preserving bovine semen at 15-20℃.