CN115428786A - Normal-temperature storage diluent for improving semen storage quality and preparation and application thereof - Google Patents

Abstract

The invention discloses a normal temperature storage diluent for improving semen storage quality and its preparation and application. The diluent includes a basic diluent and ferulic acid added to the basic diluent, wherein the concentration of ferulic acid is 25 ～100μmol/L, the composition of the basic diluent is: using ultrapure water as the solvent, containing 200～300mmol/L Tris, 50～150mmol/L fructose, 50～100mmol/L sodium citrate, 50,000 IU/100mL penicillin and 50,000 IU/100mL streptomycin. The diluent provided by the invention can significantly improve the quality of the semen in the process of storing the semen at room temperature, prolong the storage time, and effectively solve the timeliness problem of fresh semen insemination.

Description

A normal-temperature storage diluent for improving the quality of semen storage and its preparation and application

technical field

The invention belongs to the technical field of animal reproduction, and in particular relates to a diluent for storage at room temperature for improving the quality of semen storage and its preparation and application.

Background technique

The combination of semen preservation and artificial insemination technology plays an important role in modern animal husbandry production. Semen storage is divided into three methods: normal temperature, low temperature and cryopreservation. Among them, normal temperature storage means that the semen is diluted and stored at 15-25°C. Normal temperature storage has the advantages of simple operation, good storage effect, low storage condition requirements, and convenient transportation. Advantages of artificial insemination effect. However, when stored at room temperature, on the one hand, the growth of microorganisms will quickly affect the quality of semen, and on the other hand, the sperm motility will decline rapidly, and the storage time is usually only 1 to 3 days.

Studies have shown that during the storage of semen at room temperature, due to the presence of a large number of unsaturated fatty acids in the sperm plasma membrane, they are easily affected by oxidative stress and produce excessive ROS (Reactive Oxygen Species, active oxygen), which leads to the oxidation and anti-oxidation in semen. Oxidation balance system is destroyed. The production of ROS by sperm is a normal result of oxidative metabolism. Low concentration of ROS plays an important role in the function of sperm, such as sperm energy, acrosome reaction, sperm-egg combination and related signaling pathways. High concentrations of ROS reduce sperm motility and plasma membrane integrity through lipid peroxidation, increase DNA fragmentation, lead to apoptosis, and reduce the quality of semen preservation.

The antioxidant defense balance system in semen includes SOD, CAT, GPx, GR, and non-enzymatic antioxidants such as methionine, ascorbic acid, and α-tocopherol. When semen is stored in vitro, this balance is easily broken due to the production of ROS and other reasons, so it is necessary to add antioxidants or other substances to semen as protective agents.

Contents of the invention

In view of this, the object of the present invention is to provide a diluent for storage at room temperature, which can effectively improve the quality of semen storage and prolong the storage time at room temperature.

In order to achieve the above object, the technical solution of the present invention is specifically as follows:

A normal-temperature storage diluent for improving the quality of semen preservation, comprising a basic diluent and ferulic acid added to the basic diluent, wherein the concentration of ferulic acid is 25-100 μmol/L, and the composition of the basic diluent is preferably: Using ultrapure water as a solvent, it contains 200-300mmol/L Tris, 50-150mmol/L fructose, 50-100mmol/L sodium citrate, 50,000 IU/100mL penicillin and 50,000 IU/100mL streptomycin.

In the above technical scheme, the optimal composition of the basic diluent is: using ultrapure water as a solvent, containing Tris250mmol/L, fructose 100mmol/L, sodium citrate 75mmol/L, penicillin 50,000 IU/100mL, streptomycin 5 Ten thousand IU/100mL.

In the above technical scheme, the optimum concentration of ferulic acid is 50 μmol/L.

The preparation method of the above normal temperature preservation diluent is as follows: according to the formula, take Tris, fructose, sodium citrate, penicillin, streptomycin and ferulic acid, dissolve them in ultrapure water, and mix them evenly.

The present invention also provides a method for applying the above diluent for storage at room temperature in semen storage, specifically: after preheating the diluent for storage at room temperature and fresh semen respectively, dilute the semen with the diluent for storage at room temperature, and place the diluted semen in Store at constant temperature at 15-20°C.

Further, in the above technical solution, the preheating temperatures of the diluent and the semen are both 33-38°C.

Further, in the above technical solution, when diluting the semen with the normal temperature preservation diluent, the method of distribution dilution is adopted, and the dilution factor can be 8-10 times.

Further, in the above technical solution, the semen comes from Hubei black-headed sheep.

The beneficial effects of the present invention are: the normal temperature storage diluent provided by the present invention can effectively improve the viability of semen stored at room temperature, and the viability is still above 80% on the 5th day of storage. Compared with the basic diluent without ferulic acid, the plasma membrane is intact The integrity of sex and acrosome is also significantly improved, the content of ROS and MDA is significantly reduced, the total antioxidant capacity is also improved to a certain extent, and the operation is simple and effective, which is very effective in solving the timeliness problem of fresh sperm insemination.

detailed description

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in combination with examples and experimental data. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

In the following examples, unless otherwise specified, all are conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial sources.

Example 1

The composition of the diluent stored at room temperature provided in this example is: basic diluent and ferulic acid added in the basic diluent, wherein the concentration of ferulic acid is 25 μmol/L, and the composition of the basic diluent is: ultrapure water As a solvent, it contains Tris 250mmol/L, fructose 100mmol/L, sodium citrate 75mmol/L, penicillin 50,000 IU/100mL, and streptomycin 50,000 IU/100mL.

Example 2

The difference from Example 1 is that the concentration of ferulic acid is 50 μmol/L.

Example 3

The difference from Example 1 is that the concentration of ferulic acid is 100 μmol/L.

Comparative example 1

The difference from Example 1 is that the normal temperature storage diluent in this example does not contain ferulic acid, only the basic diluent.

Comparative example 2

Different from Example 1, the concentration of ferulic acid in this example is 200 μmol/L.

Use the normal temperature preservation diluents prepared in each example and comparative example to preserve the semen of Hubei black goat respectively. The method is as follows: preheat the collected fresh semen and the diluent at 37°C respectively, and use the stepwise dilution method to dilute the semen Dilute it with the diluent at a ratio of 1:8, wrap the diluted semen with gauze, put it in a constant temperature refrigerator at 17°C, and shake it every 12 hours.

The preserved semen is tested as follows:

(1) Sperm motility detection, using HT CASA Ⅱ for detection and analysis, the specific steps are as follows:

① Randomly take 10 μL of semen from the sample tubes of each treatment group, shake slowly before taking, add isothermal basic diluent to dilute to 10 times, and place it in a 37°C water bath for 3 minutes;

② Take 5 μL of the incubated semen and drop it on the preheated glass slide, cover it with a cover glass and place it on the HT CASAⅡ constant temperature stage, and then perform the basic index detection;

③ Field of view selection requires at least 5 fields of view to be selected, and the total number of observed spermatozoa should not be less than 800.

The test results are shown in Table 1:

Table 1 Effect of different concentrations of ferulic acid on motility of black-headed sheep sperm (%)

Note: Different lowercase letters on the shoulders of data in the same column indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

It can be seen from Table 1 that during the semen preservation process, the ferulic acid content of 25 μmol/L and 50 μmol/L dilutions had significantly higher activity than the control group without ferulic acid during semen preservation (P<0.05) .

(2) The integrity rate of sperm plasma membrane is detected by using sperm biopsy detection kit, and the specific steps are as follows:

①Preheat the sperm hypotonic expansion solution in a water bath for 5 minutes, take 20 μL of semen and add it to 200 μL of the preheated hypotonic solution, stir gently, and incubate at 37°C for 30 minutes;

②After the incubation, take 10 μL and evenly smear it on the glass slide, observe with an optical microscope, randomly select at least 3 fields of view, and the total number of spermatozoa should not be less than 200.

The test results are shown in Table 2:

Table 2 Effects of different concentrations of ferulic acid on the integrity of the plasma membrane of black-headed sheep sperm (%)

Note: Different lowercase letters on the shoulders of data in the same column indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

It can be seen from Table 2 that the plasma membrane integrity of the dilution with ferulic acid content of 50 μmol/L was significantly higher than that of the control group without ferulic acid during semen preservation (P<0.05).

(3) Detection of sperm acrosome integrity, specifically using fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) fluorescent dye for acrosome staining, the steps are as follows:

① Add PBS to the centrifuge tube to resuspend the semen to a concentration of 1×10 ⁶ , add 10 μL FITC-PNA dye solution and incubate at 37°C in the dark for 10 min;

② After the incubation, centrifuge at 1500rpm for 5min, discard the supernatant, repeat 1-2 times, wash away the floating color, add formaldehyde to fix for 30min;

③Centrifuge at 1500rpm for 5min, discard the supernatant, resuspend the pellet with PBS, take 10μL of the resuspended semen and smear it evenly on the glass slide, after it dries, add 5μL of DAPI dropwise and incubate at room temperature for 5min; seal the slide immediately after the incubation, and observe under a fluorescent microscope Take pictures, observe at least 3 fields of view, and the number of sperm is not less than 200.

DAPI makes sperm nuclei appear blue light under a fluorescent microscope, which is used for cell counting and excludes false positives caused by impurities and other non-cellular substances that affect sperm counting. The criteria for FITC-PNA staining were as follows: sperm with damaged acrosomes were stained with green fluorescence, and sperm with intact acrosomes were without green fluorescence. The test results are shown in Table 3:

Table 3 Effects of different concentrations of ferulic acid on the acrosome integrity of black-headed sheep sperm (%)

Note: Different lowercase letters on the shoulders of data in the same column indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

It can be seen from Table 3 that when the content of ferulic acid was 25 μmol/L and 100 μmol/L, the acrosome integrity rate was slightly higher than that of the control group without adding ferulic acid (P>0.05). The body integrity rate was significantly higher than that of the control group without ferulic acid (P<0.05).

(4) The detection of the total antioxidant capacity of sperm is carried out using the total antioxidant capacity assay kit, and the steps are as follows:

① Take 100 μL of semen, centrifuge at 800g for 10 minutes, discard the supernatant, collect the sperm cell pellet, and wash twice with pre-cooled PBS;

② Add 100 μL of pre-cooled lysate, use an ultrasonic breaker to crush the sperm cells (operate on ice), and then centrifuge at 12,000 g, take the supernatant as the sample to be tested, and use the BCA protein concentration kit (Takara) to detect the supernatant protein concentration.

③After adding the corresponding reagents according to the operation instructions of the kit, adjust the optical path to 1cm, double-distilled water to zero, and detect the absorbance value at 520nm, and calculate the detected value according to the following formula:

Total antioxidant capacity (U/mgprot)=(A determination-A control)/0.01/T×V _anti-total V _sample /Cpr

In the formula, T is the reaction time, V _total is the total volume of the reaction system, V _sample is the sampling volume, Cpr is the protein concentration of tissue homogenate.

The calculation results are shown in Table 4:

Table 4 Effect of different concentrations of ferulic acid on the ROS content of black-headed sheep sperm on the third day

Note: Different lowercase letters indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

It can be seen from the above table that on the third day of storage, when the ferulic acid content was 25 μmol/L, 50 μmol/L and 100 μmol/L, the ROS content was significantly lower than that of the control group without ferulic acid (P<0.05). In addition, on the fifth day of storage, when the ferulic acid content was 50 μmol/L, the ROS content was significantly lower than that of the control group without ferulic acid (P<0.05).

(5) MDA (malondialdehyde) content detection in semen, using MDA assay kit for operation:

Configure the detection system according to the operation instructions of the kit. After mixing, pierce a small hole on the cap of the centrifuge tube, bathe in water at 95°C for 40 minutes, and centrifuge after cooling (4000rmp, 10min). The OD value was measured and calculated by the following formula:

The test results are shown in Table 5:

Table 5 Effect of different concentrations of ferulic acid on MDA content of black-headed sheep sperm

Note: Different lowercase letters on the shoulders of data in the same column indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

It can be seen from Table 5 that on the 3rd and 5th day of storage, when the ferulic acid content was 50 μmol/L, the MDA content was significantly lower than that of the control group without ferulic acid (P<0.05).

(6) Sperm ROS content detection, using the ROS assay kit for operation:

① Dilute DCFH-DA with PBS at a ratio of 1:1000 to make the final concentration 10 μmol/L;

② Take 100 μL of semen in a centrifuge tube, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, add 500 μL of diluted DCFH-DA to resuspend the pellet, and bathe in water at 37°C for 30 minutes in the dark;

③Centrifuge at 1000rmp for 10min, discard the supernatant, wash the pellet twice with PBS, collect the cell pellet by centrifugation, add 600μL PBS to suspend, add the sample to a 96-well plate; use a multi-functional microplate reader, set the excitation wavelength at 488nm, the emission wavelength at 525nm, and measure the fluorescence value .

The test results are shown in Table 6:

Table 6 Effect of different concentrations of ferulic acid on T-AOC content of black-headed sheep sperm

Note: Different lowercase letters on the shoulders of data in the same column indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05).

It can be seen from Table 6 that on the 3rd and 5th day of storage, when the ferulic acid content was 50 μmol/L, the T-AOC content was significantly higher than that of the control group without ferulic acid (P<0.05).

In summary, adding ferulic acid to the basic diluent can significantly improve the integrity of sperm plasma membrane and acrosome, and significantly reduce the content of ROS and MDA, thereby prolonging the storage time of semen at room temperature.

The above description is a preferred embodiment of the present invention, which cannot limit the scope of rights of the present invention. It should be pointed out that for those of ordinary skill in the art, any modifications made within the spirit and principles of the present invention , equivalent replacements and improvements, etc., should all be included within the protection scope of the present invention.

Claims (10)

1. A normal temperature preservation diluent for improving semen preservation quality is characterized by comprising a basic diluent and ferulic acid added in the basic diluent, wherein the concentration of the ferulic acid is 25-100 mu mol/L, and the basic diluent comprises the following components: using ultrapure water as a solvent, and containing 200-300 mmol/L Tris, 50-150 mmol/L fructose, 50-100 mmol/L sodium citrate, 5 ten thousand IU/100mL penicillin and 5 ten thousand IU/100mL streptomycin.

2. The ambient storage diluent as claimed in claim 1, wherein the composition of the base diluent is: the ultrapure water is used as a solvent and contains 250mmol/L Tris, 100mmol/L fructose, 75mmol/L sodium citrate, 5 ten thousand IU/100mL penicillin and 5 ten thousand IU/100mL streptomycin.

3. The cryopreservation diluent of claim 1 or 2, wherein the concentration of ferulic acid is 50 μmol/L.

4. The use of the diluent for preserving at normal temperature according to any one of claims 1 to 3 in preserving semen, wherein the diluent for preserving at normal temperature and fresh semen are preheated respectively, the diluted semen is diluted by the diluent for preserving at normal temperature, and the diluted semen is preserved at constant temperature of 15 to 20 ℃.

5. Use according to claim 4, wherein the temperature of the preheating is 33-38 ℃.

6. Use according to claim 5, wherein the pre-heating temperature is 37 ℃.

7. Use according to claim 4, wherein the dilution is carried out by stepwise dilution.

8. Use according to claim 4, wherein the dilution factor is between 8 and 10.

9. The use according to claim 4, wherein the semen is from Hubei Black sheep.

10. A method for preparing the diluent for ordinary-temp storage as claimed in any one of claims 1-3, which is characterized by dissolving Tris, fructose, sodium citrate, penicillin, streptomycin and ferulic acid in ultrapure water and mixing them uniformly.