CN106857499B - Application of tea polyphenol and chitosan in preparation of diluent for preserving boar semen - Google Patents
Application of tea polyphenol and chitosan in preparation of diluent for preserving boar semen Download PDFInfo
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- 241001122767 Theaceae Species 0.000 title claims abstract description 47
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 46
- 150000008442 polyphenolic compounds Chemical class 0.000 title claims abstract description 42
- 235000013824 polyphenols Nutrition 0.000 title claims abstract description 42
- 239000003085 diluting agent Substances 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 5
- 229920000858 Cyclodextrin Polymers 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 239000001116 FEMA 4028 Substances 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 4
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 4
- 229960004853 betadex Drugs 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- 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 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 2
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- 238000012360 testing method Methods 0.000 description 2
- 102100026041 Acrosin Human genes 0.000 description 1
- 108090000107 Acrosin Proteins 0.000 description 1
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- 238000005502 peroxidation Methods 0.000 description 1
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- 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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- Physiology (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention relates to application of tea polyphenol and chitosan in compatibility as a diluent for preserving boar semen. The antioxidant for preserving the boar semen at the normal temperature adopts the compatibility of the tea polyphenol and the chitosan as the antioxidant, and the tea polyphenol and the chitosan have a dependency synergistic relationship, so that the physiological activity of the boar semen can be well protected in the normal-temperature preserving process of the boar semen by the compatibility of the tea polyphenol and the chitosan with proper concentration.
Description
Technical Field
The invention relates to a formula of a tea polyphenol and chitosan compatible diluent for preparing normal-temperature preservation of porcine semen and an optimal compatible concentration thereof, which can effectively improve the semen preservation effect, effectively reduce the content of bacteria in the semen and improve the activity of antioxidant enzyme in the semen.
Background
The normal-temperature preservation of the boar semen effectively prolongs the in-vitro preservation time of the semen, practically improves the seed value of the improved boar, reduces the occurrence of reproductive system diseases and promotes the development and application of the artificial insemination technology of the boar. Because the formula of the normal-temperature preservation dilution powder is the key for determining the normal-temperature preservation effect of the boar semen, the current domestic boar semen normal-temperature preservation dilution powder formula still cannot completely break the monopoly abroad, and the normal-temperature preservation of the boar semen requires more efficient and stable dilution powder. However, the type and amount of the protective agent used in the formula of the diluent powder are the core of the preservation effect of the diluted semen, and the proportion and compatibility of the protective agent with other components are directly related to the physiological state and activity of the preserved sperm.
Reactive Oxygen Species (ROS) are unstable metabolites produced during the aerobic metabolism of cells (Junling et al 2013). The mammalian semen itself presents a certain ROS defense system (Awda)et al, 2009) mainly enzymatic antioxidant systems including SOD, CAT and GSH-PX. Although the antioxidant enzyme activity varies among species (Strzezek et al 2002), the SOD activity reflects the level of antioxidant enzymes in the whole system for the same animal, the higher the SOD activity, the stronger the antioxidant capacity. Although CAT and GSH-PX play a role in assisting the SOD in scavenging free radicals, CAT and GSH-PX activities also laterally reflect the antioxidant level of the system (Orzolek et al.2013). Thus, laboratory tests for antioxidant levels in the metabolic system of cells have typically employed SOD, CAT and GSH-PX kits to test their activity. H2O2Is one of oxygen free radicals, excess H2O2Can cause damage to cells. In the case of sperm cells, the fertilization ability of the sperm is impaired, and thus H in the body2O2The content of (b) can also be used as an index for detecting the peroxidation damage of the sperm plasma membrane. By measuring the change of the indexes, the survival condition of the sperms in the environment of the diluent is reflected.
The normal temperature preservation of semen is a technology for prolonging the survival time of sperms in vitro by adding diluent under the condition of 17 ℃. Sperm acrosin was able to reach optimal activity at 17 ℃ (Pinart 2013). Because sperm can carry out normal metabolic activity at 17 ℃ and generate harmful substances, corresponding antioxidants or other medicines such as streptomycin and the like are often required to be added into the diluent to keep the relative stability of the microenvironment.
Disclosure of Invention
The invention takes tea polyphenol (TPP) and Chitosan (CS) as main components of an antioxidant, and selects the optimal compatible concentration of the tea polyphenol and the chitosan by jointly adding the tea polyphenol and the chitosan with different concentrations and taking the motility rate of diluted sperms, the plasma membrane integrity rate, the MDA content, CAT activity, SOD activity, GSH-Px activity and the bacterial content in semen as evaluation indexes so as to ensure that the boar semen maintains good biological activity in the normal-temperature storage process.
The invention provides application of tea polyphenol and chitosan in compatibility as a diluent for preserving boar semen at normal temperature.
The invention also provides a diluent for preserving the boar semen at normal temperature, which comprises tea polyphenol and chitosan.
Furthermore, the diluent for normal-temperature preservation of the boar semen comprises, per 1L of the diluent for normal-temperature preservation of the boar semen, 0.02g of tea polyphenol, 0.05g of chitosan, 38g of glucose, 6.5g of sodium citrate, 1.2g of EDTA, 1g of sodium bicarbonate, 0.25g of citric acid, 0.6g of potassium chloride, 0.9g of β -cyclodextrin and the balance of ultrapure water.
The antioxidant for preserving the boar semen at the normal temperature adopts the compatibility of the tea polyphenol and the chitosan as the antioxidant, and the tea polyphenol and the chitosan have a dependency synergistic relationship, so that the compatibility of the tea polyphenol with proper concentration and the chitosan can well protect the physiological activity of the boar semen in the process of preserving the boar semen at the normal temperature.
Drawings
FIG. 1 shows the effect of tea polyphenol-chitosan combinations of different concentrations on the SOD activity of boar semen preserved at normal temperature (17 ℃);
FIG. 2 shows the effect of tea polyphenol-chitosan combinations of different concentrations on CAT activity in the preservation of boar semen at normal temperature (17 ℃);
FIG. 3 shows the effect of tea polyphenol-chitosan combinations of different concentrations on the GSH-PX activity of preserved porcine semen at room temperature (17 deg.C);
FIG. 4 shows the effect of tea polyphenol-chitosan combinations of different concentrations on the bacterial content of the pig semen preserved at normal temperature (17 ℃).
Detailed Description
The present invention will be described in further detail with reference to specific examples.
According to the technical scheme of the invention, the formula of the compatible diluent of the tea polyphenol and the chitosan for preserving the boar semen at normal temperature is provided in the embodiment. Wherein:
the formula of the diluent comprises 0.02g/L of tea polyphenol, 0.05g/L of chitosan, 38g/L of glucose, 6.5g/L of sodium citrate, 1.2g/L of EDTA, 1g/L of sodium bicarbonate, 0.25g/L of citric acid, 0.6g/L of potassium chloride and 0.9g/L of β -cyclodextrin (Table 1).
TABLE 1 formulation of dilution of tea polyphenols and chitosan
The specific process of the test is as follows:
according to the formula of the diluent shown in the table 1, 38g of glucose, 6.5g of sodium citrate, 1.2g of EDTA, 1g of sodium bicarbonate, 0.25g of citric acid, 0.6g of potassium chloride and 0.9g of β -cyclodextrin are weighed, 0g of tea polyphenol, 0.01g of sodium bicarbonate, 0.02g of citric acid, 0.04g of tea polyphenol, 0g of chitosan and 0.05g of chitosan are respectively weighed, the tea polyphenol and the chitosan are added according to the compatibility scheme shown in the table 2, the mixture is dissolved in a 250mL sterilizing beaker and then fixed in a 1000mL volumetric flask, after 1h, a sterile filter is matched with a 0.22 mu m filter membrane in a sterile operating platform to filter the solution, the mass concentration of the tea polyphenol and the chitosan is obtained, and the prepared diluent is ready for use.
TABLE 2 formulation of tea polyphenols and chitosan compatible diluent
The porcine semen used in this experiment was from a 20 month old duroc breed boar. The semen collected by the hand-held method is filtered and put into a heat preservation box to be quickly taken back to a laboratory. Selecting semen with normal color and smell and sperm motility rate above 85%.
Taking 2mL of semen into a sterile 10mL centrifuge tube, slowly adding diluent to the tube opening, wrapping the centrifuge tube with 4 layers of towels, storing the centrifuge tube in a 17 ℃ thermostat for 6 days when the temperature of the semen is close to the room temperature, slowly turning the semen for 1 time every 12 hours, and checking the sperm motility rate (taking the number of days with the sperm motility rate of 60% as effective storage time) and the plasma membrane integrity rate every 24 hours; and detecting the bacterial content in the semen when the semen is stored for 0, 2, 4 and 6 days respectively. Each treatment was repeated 3 times.
The results of the measurement of sperm motility, plasma membrane integrity, SOD activity, CAT activity, GSH-Px activity and bacterial content are shown in the form of a graph.
TABLE 3 pig sperm motility (%) -based on the combination of tea polyphenols and chitosan at different dosages
TABLE 4 porcine sperm plasma membrane integrity (%) -after different dosages of tea polyphenols and chitosan
As can be seen from Table 3, except for T3C2And T3C0In addition, the effective preservation time of the sperms of other compatibility treatment groups can reach 6 days, and the difference of the sperm motility rate when the sperms are preserved for 6 days and the sperm motility rate of a control group is obvious (P is less than 0.05); t is2C1The sperm survival rate of the group is the highest in the whole preservation process and is obviously higher than that of the group treated by separately adding tea polyphenol or chitosan; the sperm motility rate is 67.40% when the sperm is preserved for 6 days, which is obviously higher than that of other groups (P is less than 0.05). High concentration group T with increasing combination concentration3C2Sperm motility was the lowest at 6 days of storage, so this combination of concentrations was not conducive to semen preservation.
As can be seen from Table 4, the integrity of the plasma membrane of the sperm decreases with the preservation time, and the difference of the integrity of the plasma membrane of each treatment group is not significant (P is more than 0.05) when the sperm is preserved for 1d at normal temperature; from the preservation 2d, the sperm plasma membrane integrity rate of the treatment group added with tea polyphenol and chitosan is obviously higher than that of the treatment group added with tea polyphenol alone; storing for 2-5 days at normal temperature, removing T3C2The plasma membrane integrity rate of the other treatment groups is obviously higher than that of the control group (P < 0.05), but most of the differences among the treatment groups are not obvious (P > 0.05); after 6 days of storage, T2C1The integrity rate of the plasma membrane of the composition is the highest and is obviously higher than that of the group treated by the tea polyphenol alone (P is less than 0.05), but the difference with the chitosan alone is not obvious (P is more than 0.05), and T is not obvious3C2The plasma membrane integrity of the treated group was the lowest, significantly lower than that of the other treated groups (P < 0.05).
As can be seen from FIG. 1, the SOD activity of each treatment group decreased with the storage time and T was observed after 2 days of storage1C2、T2C1、T2C2The group is higher than the T of separately adding tea polyphenol and chitosan0C2And T3C0Group, but not significantly different (P > 0.05); after 4 days of storageT2C1The group still maintained the highest SOD activity, but the difference was not significant (P > 0.05); the two groups with the highest SOD activity after 6 days of storage are T2C1And T2C2And significantly higher than the other treatment groups (P < 0.05), where T2C1Higher than T2C2But the difference between the two groups was not significant (P > 0.05).
As can be seen from FIG. 2, T2C1The treatment group is helpful to improve CAT activity in normal temperature preservation of the pig semen. The data show that T is measured for 3 times2C1The CAT activity of the treated group is obviously higher than that of T of the treated group which is added with tea polyphenol and chitosan separately0C2And T3C0Group (P < 0.05), slightly higher than T1C2、T2C2Two groups, but the difference was not significant (P > 0.05). The CAT activity of the tea polyphenol group added alone is higher than that of the chitosan group added alone, but the difference is not significant (P is more than 0.05). In general, the effect of the combined addition of the tea polyphenol and the chitosan is better than that of the single addition. T is2C1The best effect on improving the CAT activity is achieved, and the optimal concentration combination is 0.02g/L TPP +0.05g/L CS.
As can be seen from FIG. 3, the GSH-PX activity varied greatly with the storage time, and T was observed during the storage3C0The group effect is better than T0C2And control group, but the difference was not significant (P > 0.05). T is2C1The treatment group was able to increase GSH-PX activity with the best results, but not significantly different from the other groups (P > 0.05).
As can be seen from the graph 4, the bacterial content in the semen changes along with the preservation time of the semen, the bacterial content in the semen is slowly increased by 0-2 d, the bacterial content in the semen is fastest by 2-4 d, the bacterial content in the semen is relatively slowed by 4-6 d, and the antibacterial effect of the complexing agent is superior to that of the two substances which are separately added during the preservation time of the semen for 0-4 d, but the difference is not significant (P is more than 0.05). After semen is preserved for 6 days, the semen bacterial content of the control group is far higher than that of other treatment groups (P is less than 0.05), and the compatible group T1C1The bacteria content is higher than that of a group treated by separately adding tea polyphenol and chitosan (P is less than 0.05), and T is higher than that of a group treated by separately adding tea polyphenol and chitosan2C1Is significantly lower than the treatment group with the two substances added separately (P < 0.05), and the semen contains more semen with the increase of the combined concentrationThe bacterial content gradually decreased, and the high concentration combined treatment group T3C2The bacteria content is minimal.
Compared with the existing diluent for preserving the boar semen at normal temperature, the diluent used in the research simultaneously contains tea polyphenol and chitosan, which is an innovative point of the research. The research result shows that the conventional parameters (sperm motility rate and plasma membrane integrity rate) of the pig sperm can be remarkably improved by jointly adding a proper amount of tea polyphenol and chitosan (for example, 0.02g/L tea polyphenol and 0.05g/L chitosan in the above embodiment), and the effective storage time is as long as 6 d. In addition, the research of the invention finds that the compatibility of tea polyphenol with proper concentration and chitosan can not only improve the activity of antioxidant enzyme (SOD, CAT, GSH-PX) of sperms so as to protect the sperms from oxidative damage, but also reduce the proliferation speed of bacteria in the process of preserving the boar semen at normal temperature, and provide guarantee for a good microenvironment for the boar semen in the process of preserving the boar semen at normal temperature.
It will be understood that modifications and variations can be effected by a person skilled in the art in light of the above description, and all such modifications and variations are intended to be within the scope of the appended claims, and to be construed as T2C1The preservation effect of the formula of 0.02g/L tea polyphenol and 0.05g/L chitosan is unexpected compared with the prior art.
Claims (1)
1. The diluent for preserving the boar semen at the normal temperature is characterized in that the diluent for preserving the boar semen at the normal temperature is prepared by mixing tea polyphenol and chitosan, and comprises the following components:
the formula of the diluent for preserving the boar semen at normal temperature per 1L comprises 0.02g of tea polyphenol, 0.05g of chitosan, 38g of glucose, 6.5g of sodium citrate, 1.2g of EDTA, 1g of sodium bicarbonate, 0.25g of citric acid, 0.6g of potassium chloride, 0.9g of β -cyclodextrin and the balance of ultrapure water.
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| CN107637589A (en) * | 2017-11-21 | 2018-01-30 | 广东中农联生物制药有限公司 | Diluent of Pig's Spermatic Fluid and its production method |
| CN113317312B (en) * | 2021-06-01 | 2022-07-12 | 福建农林大学 | Diluent preparation capable of prolonging normal-temperature survival period of boar semen |
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| CN1817158A (en) * | 2006-03-14 | 2006-08-16 | 中国农业科学院茶叶研究所 | Green-tea biological antistaling agent at normal temperature and use thereof |
| CN102106588A (en) * | 2009-12-25 | 2011-06-29 | 南通远大生物科技发展有限公司 | Preservative for water-swollen squid and using method thereof |
| CN104996543A (en) * | 2015-07-31 | 2015-10-28 | 广东海洋大学 | Improved biological preservation agent for aquatic products, as well as preparation method and application thereof |
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| Title |
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