CN115428786A - Normal-temperature storage diluent for improving semen storage quality and preparation and application thereof - Google Patents
Normal-temperature storage diluent for improving semen storage quality and preparation and application thereof Download PDFInfo
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- 238000003860 storage Methods 0.000 title claims abstract description 17
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- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 claims abstract description 39
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims abstract description 39
- 229940114124 ferulic acid Drugs 0.000 claims abstract description 39
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 claims abstract description 39
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- 238000000034 method Methods 0.000 claims abstract description 12
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- 239000007983 Tris buffer Substances 0.000 claims abstract description 8
- 229940049954 penicillin Drugs 0.000 claims abstract description 8
- 239000001509 sodium citrate Substances 0.000 claims abstract description 8
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 8
- 229960005322 streptomycin Drugs 0.000 claims abstract description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 8
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- 241001494479 Pecora Species 0.000 claims description 8
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- 238000002156 mixing Methods 0.000 claims description 3
- 229960002737 fructose Drugs 0.000 claims description 2
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- 238000005138 cryopreservation Methods 0.000 claims 1
- 238000010438 heat treatment Methods 0.000 claims 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 9
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 7
- 210000000170 cell membrane Anatomy 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
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- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
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- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 1
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- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
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- 238000001574 biopsy Methods 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
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- 230000031700 light absorption Effects 0.000 description 1
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- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 235000006109 methionine Nutrition 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
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Abstract
The invention discloses a normal-temperature preservation diluent for improving semen preservation quality, and a preparation method and an application thereof, wherein the diluent comprises a basic diluent and ferulic acid added in the basic diluent, 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. The diluent provided by the invention can obviously improve the semen quality in the normal-temperature semen storage process, prolong the storage time and effectively solve the timeliness problem of fresh semen inseminating.
Description
Technical Field
The invention belongs to the technical field of animal breeding, and particularly relates to a normal-temperature preservation diluent for improving semen preservation quality, and preparation and application thereof.
Background
The semen preservation and the artificial insemination technology are combined, and the method plays an important role in the production of modern animal husbandry. Semen preservation is divided into three methods of normal temperature, low temperature and freezing preservation, wherein the normal temperature preservation refers to the preservation of diluted semen at 15-25 ℃, and has the advantages of simple operation, good preservation effect, low requirement on preservation condition, convenient transportation and good artificial insemination effect. However, when the semen is stored at normal temperature, on one hand, the sperm growth can quickly affect the semen quality, on the other hand, the sperm motility is reduced quickly, and the storage time is only 1 to 3 days generally.
Research shows that during the normal-temperature preservation of semen, because a large amount of unsaturated fatty acid exists in a sperm plasma membrane, excessive ROS (Reactive Oxygen Species) are easily generated under the influence of oxidative stress, and an oxidation and oxidation resistance balance system in the semen is damaged. The generation of ROS by sperm is a normal consequence of oxidative metabolism, and low concentrations of ROS have important roles in sperm function, such as sperm motility, acrosome reaction, sperm-egg binding and related signaling pathways, etc. High concentrations of ROS reduce sperm motility and plasma membrane integrity through lipid peroxidation, increase DNA fragmentation, cause apoptosis, and reduce semen preservation quality.
The antioxidant defense balance present in semen includes SOD, CAT, GPx, GR, and non-enzymatic antioxidants such as methionine, ascorbic acid and alpha-tocopherol, among others. When semen is stored in vitro, this balance is easily disrupted due to the production of ROS, etc., and it is therefore necessary to add antioxidants or other substances to the semen to act as protective agents.
Disclosure of Invention
In view of the above, the present invention provides a diluent for preserving at room temperature, which can effectively improve the quality of semen preservation and prolong the preservation time at room temperature.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a normal temperature preservation diluent for improving semen preservation quality comprises 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 composition of the basic diluent is preferably as follows: 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.
In the above technical solution, the optimal composition of the basic diluent is as follows: 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.
In the above technical scheme, the optimal concentration of ferulic acid is 50 μmol/L.
The preparation method of the normal-temperature storage diluent comprises the following specific steps: weighing Tris, fructose, sodium citrate, penicillin, streptomycin and ferulic acid according to the formula, dissolving in ultrapure water, and mixing uniformly to obtain the product.
The invention also provides an application method of the normal-temperature preservation diluent in semen preservation, which specifically comprises the following steps: respectively preheating the normal-temperature stored diluent and fresh semen, diluting the semen by using the normal-temperature stored diluent, and storing the diluted semen at a constant temperature of 15-20 ℃.
Further, in the above technical scheme, the preheating temperatures of the diluent and the semen are both 33-38 ℃.
Furthermore, in the above technical solution, when the semen is diluted by the diluent stored at normal temperature, a distributed dilution method is adopted, and the dilution ratio may be 8 to 10 times.
Further, in the above technical scheme, the semen is from Hubei black head sheep.
The invention has the beneficial effects that: the normal-temperature preservation diluent provided by the invention can effectively improve the normal-temperature preservation activity of semen, the activity is still more than 80% at the 5 th day of preservation, compared with a basic diluent without ferulic acid, the integrity of a plasma membrane and a acrosome is also obviously improved, the content of ROS and MDA is obviously reduced, the total oxidation resistance is also improved to a certain extent, the operation is simple and effective, and the method is very effective for solving the timeliness problem of fresh semen deposition.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and experimental data. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
In the following examples, unless otherwise specified, all methods are conventional; the reagents and materials are commercially available unless otherwise specified.
Example 1
The composition of the room-temperature-storage diluent provided in this example was: a base diluent and ferulic acid added in the base diluent, wherein the concentration of the ferulic acid is 25 mu mol/L, and the composition of the base diluent is as follows: the solvent is ultrapure water, and the ultrapure water contains Tris 250mmol/L, fructose 100mmol/L, sodium citrate 75mmol/L, penicillin 5 ten thousand IU/100mL, and streptomycin 5 ten thousand IU/100mL.
Example 2
Unlike example 1, the concentration of ferulic acid was 50. Mu. Mol/L.
Example 3
Unlike example 1, the concentration of ferulic acid was 100. Mu. Mol/L.
Comparative example 1
Unlike example 1, the ambient temperature storage diluent in this example contained no ferulic acid, but only the base diluent.
Comparative example 2
Unlike example 1, the concentration of ferulic acid in this example was 200. Mu. Mol/L.
The normal-temperature preservation diluent prepared in each example and comparative example is used for respectively preserving semen of the Hubei black goats, and the method specifically comprises the following steps: respectively preheating the collected fresh semen and the collected diluent at 37 ℃, diluting the semen and the diluent according to a ratio of 1.
The stored semen is detected as follows:
(1) Detecting sperm motility by adopting HT CASA II for detection and analysis, and specifically comprising the following steps:
(1) randomly taking 10 mu L of semen from the sample tubes of each treatment group, slowly shaking uniformly before taking, adding the isothermal basic diluent for diluting to 10 times, and placing the mixture in a water bath kettle at 37 ℃ for incubation for 3min;
(2) dripping 5 mu L of the incubated semen on a glass slide preheated in advance, covering the glass slide with a cover glass, placing the glass slide on a HT CASA II constant-temperature objective table, and then carrying out basic index detection;
(3) the visual field selection requires that more than 5 visual fields are selected at least, and the total number of observed sperms is not less than 800.
The results are shown in table 1:
TABLE 1 Effect of different concentrations of Ferulic acid on the sperm motility of blackhead sheep (%)
Note: the same column of data is shoulder marked with different lower case letters to indicate significant difference (P < 0.05) and the same letters to indicate insignificant difference (P > 0.05).
As can be seen from Table 1, the activity of the diluent containing 25. Mu. Mol/L and 50. Mu. Mol/L ferulic acid during the preservation of semen is significantly higher than that of the control group without ferulic acid (P < 0.05).
(2) The method is characterized in that the integrity rate of the plasma membrane of the sperms is detected by adopting a sperm biopsy kit, and the method comprises the following specific steps:
(1) preheating the sperm hypotonic expansive solution in a water bath for 5min, adding 20 μ L semen into 200 μ L preheated hypotonic solution, stirring gently, and incubating at 37 deg.C for 30min;
(2) and after the incubation is finished, uniformly smearing 10 mu L of the mixture on a glass slide, observing the mixture by using an optical microscope, and randomly selecting at least 3 visual fields, wherein the total number of sperms is not less than 200.
The results are shown in table 2:
TABLE 2 influence of different concentrations of ferulic acid on the plasma membrane integrity of black-headed sheep (in%)
Note: the same column of data is shoulder marked with different lower case letters to indicate significant difference (P < 0.05) and the same letters to indicate insignificant difference (P > 0.05).
As can be seen from Table 2, the plasma membrane integrity during semen preservation was significantly higher in the 50. Mu. Mol/L dilutions than in the control without ferulic acid (P < 0.05).
(3) Detecting the sperm acrosome integrity, specifically adopting fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) fluorescent dye to perform acrosome staining, and comprising the following steps:
(1) adding PBS into the centrifuge tube to resuspend the semen to a concentration of 1X 10 6 Adding 10 mu L of FITC-PNA dye solution, and incubating for 10min at 37 ℃ in a dark place;
(2) centrifuging at 1500rpm for 5min after incubation, discarding supernatant, repeating for 1-2 times, washing off floating color, and adding formaldehyde for fixing for 30min;
(3) centrifuging at 1500rpm for 5min, discarding the supernatant, resuspending the precipitate with PBS, taking 10 μ L of the resuspended semen, uniformly smearing on a glass slide, after drying, dropwise adding 5 μ L of DAPI, and incubating at room temperature for 5min; immediately after the incubation, the cells were mounted and photographed under a fluorescence microscope for at least 3 fields, and the number of sperm was not less than 200.
DAPI makes sperm nucleus appear blue light under fluorescence microscope, and is used for cell counting and eliminating influence of false positive caused by non-cell substance such as impurity on sperm counting. The FITC-PNA staining criteria were: sperm with damaged acrosome are stained with green fluorescence, and intact acrosome sperm without green fluorescence. The results are shown in table 3:
TABLE 3 influence of different concentrations of ferulic acid on the acrosome integrity of blackhead sheep sperm (%)
Note: the data in the same column are marked with different lower case letters to indicate significant difference (P < 0.05), and the same letters to indicate insignificant difference (P > 0.05).
As can be seen from Table 3, the acrosome integrity was slightly higher when the ferulic acid content was 25. Mu. Mol/L and 100. Mu. Mol/L than that of the control group without ferulic acid (P > 0.05), and the acrosome integrity was significantly higher when the ferulic acid content was 50. Mu. Mol/L than that of the control group without ferulic acid (P < 0.05).
(4) The method for detecting the total antioxidant capacity of the sperms adopts a total antioxidant capacity determination kit to operate and comprises the following steps:
(1) taking 100 mu L of semen, centrifuging for 10min at 800g, discarding supernatant, collecting sperm cell precipitate, and washing twice with precooled PBS;
(2) 100 μ L of a precooled lysate was added, the sperm cells were disrupted using an ultrasonic disruptor (ice-on procedure), and thereafter centrifuged at 12,000g, and the supernatant was taken as a sample to be tested, while the supernatant protein concentration was measured using BCA protein concentration kit (Takara).
(3) After adding corresponding reagents according to the kit operation instructions, adjusting the optical path to be 1cm, adjusting the double distilled water to zero, detecting the light absorption value at 520nm, and calculating according to the following formula through the detection value:
total antioxidant capacity (U/mgprot) = (A determination-A control)/0.01/T × V Inverse assembly V Sample (II) /Cpr
Wherein T is the reaction time, V Inverse assembly Is the total volume of the reaction system, V Sample (II) For sample measurements, cpr is the homogenate protein concentration.
The calculation results are shown in table 4:
TABLE 4 Effect of different concentrations of Ferulic acid on ROS content of sperm in black-headed sheep at day 3
Note: different lower case letters indicate significant difference (P < 0.05) and the same letters indicate insignificant difference (P > 0.05).
As can be seen from the above table, the ROS content was significantly lower than the control group without ferulic acid (P < 0.05) at ferulic acid contents of 25. Mu. Mol/L, 50. Mu. Mol/L and 100. Mu. Mol/L on day 3 of storage. In addition, on day 5 of storage, at a ferulic acid content of 50 μmol/L, the ROS content was significantly lower than the control without ferulic acid (P < 0.05).
(5) Detecting the content of MDA (malondialdehyde) in semen, and operating by adopting an MDA determination kit:
configuring a detection system according to the kit operation instruction, binding a small hole on a centrifuge tube cover after mixing uniformly, carrying out water bath for 40min at 95 ℃, cooling and centrifuging (4000 rmp and 10 min), taking 200 mu L of supernatant, adding the supernatant into an ELISA plate, measuring the OD value at 530nm, and calculating by the following formula:
the results are shown in table 5:
TABLE 5 influence of different concentrations of ferulic acid on the MDA content of sperm of black-headed sheep
Note: the data in the same column are marked with different lower case letters to indicate significant difference (P < 0.05), and the same letters to indicate insignificant difference (P > 0.05).
As can be seen from Table 5, on days 3 and 5 of storage, when the ferulic acid content was 50. Mu. Mol/L, the MDA content was significantly lower than that of the control group containing no ferulic acid (P < 0.05).
(6) Detecting the content of the ROS in the sperm by adopting an ROS detection kit:
(1) DCFH-DA was diluted with PBS at a ratio of 1;
(2) taking 100 mu L of semen into a centrifuge tube, centrifuging for 10min at 1000rmp, removing supernatant, adding 500 mu L of diluted DCFH-DA for resuspension and precipitation, and carrying out water bath at 37 ℃ in a dark place for 30min;
(3) centrifuging at 1000rmp for 10min, removing supernatant, washing the precipitate with PBS for 2 times, centrifuging to collect cell precipitate, adding 600 μ LPBS for suspension, and adding sample to 96-well plate; fluorescence was measured using a multifunctional microplate reader with an excitation wavelength of 488nm and an emission wavelength of 525 nm.
The results are shown in Table 6:
TABLE 6 influence of different concentrations of ferulic acid on the T-AOC content of sperm of black-headed sheep
Note: the data in the same column are marked with different lower case letters to indicate significant difference (P < 0.05), and the same letters to indicate insignificant difference (P > 0.05).
As can be seen from Table 6, the T-AOC content was significantly higher than that of the control group containing no ferulic acid (P < 0.05) when the ferulic acid content was 50. Mu. Mol/L on days 3 and 5 of storage.
In conclusion, the addition of ferulic acid in the basic diluent can significantly improve the plasma membrane integrity and acrosome integrity of the sperms, significantly reduce the ROS content and MDA content, and further prolong the storage time of the semen at normal temperature.
The above description is for the purpose of illustrating the preferred embodiments of the present invention and is not intended to limit the scope of the present invention, and it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
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.
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