CN116491498B - Application of quercetin as diluent for goat semen low-temperature preservation - Google Patents
Application of quercetin as diluent for goat semen low-temperature preservation Download PDFInfo
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- CN116491498B CN116491498B CN202310468966.7A CN202310468966A CN116491498B CN 116491498 B CN116491498 B CN 116491498B CN 202310468966 A CN202310468966 A CN 202310468966A CN 116491498 B CN116491498 B CN 116491498B
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- diluent
- semen
- quercetin
- sperm
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- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 title claims abstract description 134
- 210000000582 semen Anatomy 0.000 title claims abstract description 99
- 239000003085 diluting agent Substances 0.000 title claims abstract description 91
- 241000283707 Capra Species 0.000 title claims abstract description 69
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 title claims abstract description 67
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 title claims abstract description 67
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 title claims abstract description 67
- 235000005875 quercetin Nutrition 0.000 title claims abstract description 67
- 229960001285 quercetin Drugs 0.000 title claims abstract description 67
- 238000004321 preservation Methods 0.000 title claims abstract description 43
- 230000019100 sperm motility Effects 0.000 claims abstract description 11
- 239000000126 substance Substances 0.000 claims description 27
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 20
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 12
- 229930182555 Penicillin Natural products 0.000 claims description 10
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 10
- 229940049954 penicillin Drugs 0.000 claims description 10
- 229960005322 streptomycin Drugs 0.000 claims description 10
- 230000000844 anti-bacterial effect Effects 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 9
- 229930091371 Fructose Natural products 0.000 claims description 8
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 8
- 239000005715 Fructose Substances 0.000 claims description 8
- 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 8
- 239000008103 glucose Substances 0.000 claims 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 description 8
- 239000001509 sodium citrate Substances 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 6
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
- 238000003860 storage Methods 0.000 claims description 5
- 230000001681 protective effect Effects 0.000 claims description 4
- 230000004899 motility Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 32
- 230000004083 survival effect Effects 0.000 abstract description 6
- 238000003975 animal breeding Methods 0.000 abstract description 2
- 239000012895 dilution Substances 0.000 description 24
- 238000010790 dilution Methods 0.000 description 24
- 230000000052 comparative effect Effects 0.000 description 23
- 239000000243 solution Substances 0.000 description 20
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- 238000002360 preparation method Methods 0.000 description 18
- 241001494479 Pecora Species 0.000 description 15
- 239000003642 reactive oxygen metabolite Substances 0.000 description 15
- 239000011259 mixed solution Substances 0.000 description 14
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 13
- 102000019197 Superoxide Dismutase Human genes 0.000 description 13
- 108010012715 Superoxide dismutase Proteins 0.000 description 13
- 229940118019 malondialdehyde Drugs 0.000 description 13
- 210000000170 cell membrane Anatomy 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 230000003078 antioxidant effect Effects 0.000 description 10
- 238000005138 cryopreservation Methods 0.000 description 10
- 239000003963 antioxidant agent Substances 0.000 description 9
- 230000035899 viability Effects 0.000 description 9
- 235000006708 antioxidants Nutrition 0.000 description 8
- 238000007865 diluting Methods 0.000 description 8
- 238000001556 precipitation Methods 0.000 description 8
- 238000011160 research Methods 0.000 description 7
- 238000002156 mixing Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 235000013336 milk Nutrition 0.000 description 5
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- 210000004080 milk Anatomy 0.000 description 5
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- 238000001514 detection method Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000012928 buffer substance Substances 0.000 description 3
- 229930003935 flavonoid Natural products 0.000 description 3
- 235000017173 flavonoids Nutrition 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000002577 cryoprotective agent Substances 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 150000002215 flavonoids Chemical class 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000009027 insemination Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000003879 sperm preservation Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 210000001215 vagina Anatomy 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000009470 Theobroma cacao Nutrition 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 235000021028 berry Nutrition 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- HVQAJTFOCKOKIN-UHFFFAOYSA-N flavonol Natural products O1C2=CC=CC=C2C(=O)C(O)=C1C1=CC=CC=C1 HVQAJTFOCKOKIN-UHFFFAOYSA-N 0.000 description 1
- -1 flavonol flavonoid Chemical class 0.000 description 1
- 235000011957 flavonols Nutrition 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 210000003953 foreskin Anatomy 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000015143 herbs and spices Nutrition 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 210000003794 male germ cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 230000003562 morphometric effect Effects 0.000 description 1
- 238000013425 morphometry Methods 0.000 description 1
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- 230000004792 oxidative damage Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 108010071584 oxidized low density lipoprotein Proteins 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 235000017807 phytochemicals Nutrition 0.000 description 1
- 229930000223 plant secondary metabolite Natural products 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
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- 235000020095 red wine Nutrition 0.000 description 1
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- 230000002000 scavenging effect Effects 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
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the technical field of animal breeding, in particular to application of quercetin as a diluent for goat semen low-temperature preservation. The invention provides application of quercetin as a diluent for goat semen low-temperature preservation, and also provides a diluent containing quercetin and application of the diluent in improving goat semen activity and/or sperm survival rate. The quercetin is used as the diluent, so that the quality of goat semen, especially the sperm motility and the sperm survival rate can be improved, and the conception rate is high.
Description
Technical Field
The invention relates to the technical field of animal breeding, in particular to application of quercetin as a diluent for goat semen low-temperature preservation.
Background
In recent years, the use of plant-derived biomolecules to improve male reproductive performance has become a modern trend. Flavonoids are a widely distributed class of phytochemicals that have a variety of beneficial effects on animal health and production. Many natural flavonoids are associated with male germ cells and tissues, wherein quercetin is of value for further research by researchers. Quercetin is a flavonol flavonoid found in citrus fruits, berries, herbs and spices, tea leaves, cocoa, and red wine and juice. A large number of researches show that the quercetin has the effects of preventing canceration, delaying aging, protecting cardiovascular and nervous systems and the like, and has a strong antioxidant function. Quercetin expresses its antioxidant activity by maintaining oxidative balance, and is mainly characterized by scavenging oxygen free radicals, chelating metal ions, inhibiting oxidative damage caused by ox-LDL and improving antioxidant enzyme activity. Quercetin exerts antioxidant effects in cells and animal models, as well as in humans, by modulating signal pathways and affecting gene expression processes.
Although sperm is now successfully stored at 4 ℃, sub-lethal damage to sperm, such as destruction of lipid peroxidation, enrichment of reactive oxygen species, and deterioration of sperm preservation quality, can still occur, thereby affecting the success of artificial reproduction. In addition, in view of the biosafety issues of egg yolk additives in semen dilutions, further research is critical to evaluate the preservation ability of the formulated dilutions and whether other antioxidants can be added to maximize sperm survival during storage at 4 ℃.
The research shows that the conception rate of the sperms is reduced after recovery at normal temperature and during the freezing preservation period, and the preservation at low temperature has the advantage of longer storage time compared with the normal temperature preservation and the advantage of higher activity compared with the freezing preservation of recovered semen. At present, the semen of cattle or pigs is stored under low temperature, but the semen of sheep is less in sheep, and the semen of sheep in the prior art is stored under low temperature, so that the conception rate is not high, and the storage quality is lower than that of other species.
Disclosure of Invention
In order to solve the problems, the invention provides application of quercetin as a diluent for goat semen low-temperature preservation. The quercetin disclosed by the invention can improve the quality of goat semen, especially the sperm motility and the survival rate, and is high in conception rate.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides application of quercetin as a diluent for goat semen low-temperature preservation.
The invention provides a diluent for preserving goat semen at low temperature, which comprises a low-temperature protective substance and a basic low-temperature diluent;
the cryoprotectant comprises quercetin; the concentration of quercetin in the diluent is 5-50 mu M.
Preferably, the base cryogenic diluent comprises a nutrient, a buffer and an antimicrobial substance.
Preferably, the concentration of the nutrient substances in the basic low-temperature diluent is 19-23 g/L, the concentration of the buffer substances is 1-1.12 g/L, and the concentration of the antibacterial substances is 1-1.1 g/L.
Preferably, the nutrients include fructose and glucose; the buffer substance comprises sodium citrate and sodium bicarbonate; the antibacterial substance comprises penicillin and streptomycin.
The invention provides application of the diluent in improving the semen activity and/or sperm activity rate of goats.
The invention provides the diluent or the application of the technical scheme, and the temperature for low-temperature preservation is 4 ℃.
The invention provides a method for improving the activity of goat semen and/or the sperm motility rate, which comprises the following steps: and mixing goat semen with the diluent and preserving at a low temperature.
Preferably, the volume ratio of the goat semen to the diluent is 1-1.5: 7-12.
Preferably, the temperature of the low temperature storage is 4 ℃.
The beneficial effects are that: the invention provides application of quercetin as a diluent for goat semen low-temperature preservation. In the specific embodiment of the invention, 4 groups of dilutions containing quercetin with different concentrations are selected differently, goat semen diluted by the dilutions is preserved at a low temperature of 4 ℃, and the sperm motility parameters, vitality, activity rate and the like are detected by means of a computer-aided sperm analyzer (CASA), and experimental results show that: the quercetin disclosed by the invention can improve the quality of goat semen, especially the sperm motility, the activity rate, the plasma membrane integrity rate and the acrosome integrity rate, and a treatment group added with the quercetin is found when the goat is bred, the number of sperm subjected to sperm microscopic examination is higher than that of a control group, and the embryo count obtained during embryo flushing is also higher than that of the control group, so that the quercetin adopted as a diluent for the low-temperature preservation of goat semen has the advantage of high conception rate.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 shows the T-AOC content of sperm when quercetin with different concentrations was added on day 3 of sheep semen cryopreservation;
FIG. 2 shows ATP levels of quercetin Pi Sushi sperm added at day 3 of sheep semen cryopreservation;
FIG. 3 shows the ROS levels of quercetin Pi Sushi sperm added at day 3 of low temperature preservation of sheep semen;
FIG. 4 shows MDA levels of quercetin Pi Sushi sperm added at day 3 of sheep semen cryopreservation;
FIG. 5 shows the SOD levels of the sperm of the quercetin Pi Sushi added at the 3 rd day of low temperature preservation of sheep semen.
Detailed Description
If no special requirements exist, the components of the invention are all obtained by routine purchase by a person skilled in the art.
The invention provides application of quercetin as a diluent for goat semen low-temperature preservation. In the present invention, the temperature of the low-temperature preservation is preferably 4 ℃; the goats preferably comprise a Guanzhong milk goat, alps or Hu goats, more preferably a Guanzhong milk goat. The application of quercetin in the diluent for goat semen low-temperature preservation can effectively improve semen quality, especially sperm motility, survival rate and acrosome integrity rate, and maintain higher conception rate. Therefore, the invention provides a reference basis for optimizing the diluent for the low-temperature preservation of the goat semen.
The invention provides a diluent for low-temperature preservation of goat semen, which comprises a low-temperature protective substance and a basic low-temperature diluent; the cryoprotectant comprises quercetin. In the present invention, the temperature for the low-temperature preservation is preferably 4 ℃. The concentration of quercetin in the diluent of the present invention is preferably 5 to 50. Mu.M, more preferably 10 to 45. Mu.M, still more preferably 15 to 40. Mu.M, and most preferably 25 to 35. Mu.M. According to the invention, the quercetin with proper concentration is added into the diluent for low-temperature preservation of goat semen, so that the activity rate, vitality, plasma membrane integrity and acrosome integrity of the preserved goat semen on the 3 rd day can be effectively improved, the ATP content, total antioxidant capacity (T-AOC) level and superoxide dismutase (SOD) content in the semen are effectively improved, and the Reactive Oxygen Species (ROS) and Malondialdehyde (MDA) content in the semen are effectively reduced. The activity reaches 71.74 +/-1.17% in low temperature for 3 days, and the insemination standard is reached.
In the present invention, the basic low temperature diluent preferably includes a nutrient, a buffer substance, and an antibacterial substance. The concentration of the nutrient substances in the basic low-temperature diluent is preferably 19-23 g/L, more preferably 20-22 g/L, the concentration of the buffer substances is preferably 1-1.12 g/L, more preferably 1.05-1.2 g/L, and the preferred concentration of the antibacterial substances is 1-1.1 g/L.
In the present invention, the nutrient preferably includes fructose and glucose; the mass ratio of the fructose to the glucose is preferably 10-12: 9 to 11, more preferably 10:9; the buffer substance preferably comprises sodium citrate and sodium bicarbonate; the mass ratio of the sodium citrate to the sodium bicarbonate is preferably 0.9-1.1: 0.1 to 0.14, more preferably 1:0.12; the antibacterial substance preferably includes penicillin and streptomycin; the mass ratio of penicillin to streptomycin is preferably 1:1, i.e., the amount of the antibacterial substance is preferably 10 ten thousand units per 100mL, and the amounts of penicillin and streptomycin are each preferably 5 ten thousand units per 100mL. According to the invention, fructose, glucose, sodium citrate, sodium bicarbonate, penicillin and streptomycin are matched with quercetin for dilution of goat semen low-temperature preservation, and fructose and glucose are fully utilized to provide energy substrates and sugar for spermatids, so that key substrates are provided for spermatid metabolism; meanwhile, sodium citrate and sodium bicarbonate are used as pH buffer components, the peak respiratory metabolism rate is maintained, and quercetin is used as an antioxidant active component, so that the bacterial growth inhibition effect of antibiotics such as penicillin, streptomycin and the like is effectively exerted, and the sperm preservation quality is comprehensively ensured.
Based on the advantages, the invention provides application of the diluent in improving the activity of goat semen and/or sperm motility. In the present invention, the goat is preferably a dairy goat. The temperature of the low-temperature preservation according to the invention is preferably 4 ℃.
The invention provides a method for improving the activity of goat semen and/or the sperm motility rate, which comprises the following steps: and mixing goat semen with the diluent, and preserving at a low temperature. In the present invention, the goat is preferably a dairy goat; the volume ratio of the goat semen to the diluent is preferably 1-1.5: 7 to 12, and preferably 1 to 1.5: 7-11; more preferably 1:7 to 9.
Before the low-temperature preservation, the mixed solution of goat semen and the diluent is preferably diluted after being balanced for 3-5 min, more preferably after being balanced for 5 min. In the invention, the mixed solution and the diluent are preferably diluted according to the volume ratio of 1:4. The liquid obtained by dilution is required to be slightly and evenly shaken after each dilution, so that the influence of sperm precipitation on the activity rate is prevented. The method for preserving goat semen can effectively ensure the sperm viability, the activity and the acrosome integrity, effectively improve the ATP content in sperm, the total antioxidant capacity (T-AOC) level and the superoxide dismutase (SOD) content, and effectively reduce the Reactive Oxygen Species (ROS) and Malondialdehyde (MDA) content in sperm. The activity reaches 71.74 +/-1.17% in low temperature for 3 days, and the insemination standard is reached.
The technical solutions provided by the present invention are described in detail below in conjunction with examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Preparation example 1
Preparation of diluent for goat semen low-temperature preservation:
Preparation of quercetin solution
Purchasing 20mg of quercetin standard substance, dissolving the quercetin standard substance in a sample bottle by using DMSO, transferring the sample bottle into a sterile 10mL centrifuge tube, diluting the sample bottle into 5mL by using DMSO to obtain a quercetin solution with the concentration of 4g/L, filtering the quercetin solution by using a 5 μm filter, and sealing and preserving the quercetin solution at the temperature of 4 ℃. The preparation method of the quercetin solutions with different concentrations comprises the following steps:
(1) Taking 750 mu L of quercetin solution with the concentration of 4g/L, adding 250 mu L of DMSO, and diluting to 1mL to obtain 0.01M quercetin solution for later use;
(2) Taking 500 mu L of the quercetin solution in the step (1), adding 500 mu L of DMSO, and diluting to 1mL to obtain 0.005M quercetin solution for later use;
(3) Taking 400 mu L of quercetin solution in the step (2), adding 600 mu L of DMSO, and diluting to 1M to obtain 0.002M of quercetin solution for later use;
(4) After 500. Mu.L of the quercetin solution in (3) was taken, 500. Mu.L of DMSO was added thereto, and the mixture was diluted to 1mL to obtain a 0.001M quercetin solution for use.
Preparing basic low-temperature diluent
1.2G of glucose, 1g of fructose, 1g of sodium citrate and 0.12g of sodium bicarbonate are accurately weighed by an analytical balance, carefully added into a 100mL wide-mouth bottle, 5 ten thousand units of penicillin and streptomycin are added in combination, the volume of ultrapure water is fixed to 100mL, and the pH value is adjusted to 7 for later use.
The prepared basic diluent is divided into 4 centrifuge tubes, 9950 mu L of basic diluent is added into each tube by a pipetting gun, then 50 mu L of quercetin solutions with different concentrations prepared by (1) to (4) are respectively added into the centrifuge tubes, the total volume of the quercetin solutions in the low-temperature diluent is 0.5%, and the dilutions for goat semen low-temperature preservation with final quercetin concentrations of 50 mu M, 25 mu M, 10 mu M and 5 mu M are sequentially obtained.
Note that: the diluent for preserving the goat semen at low temperature should be prepared at present, and penicillin and streptomycin are added into the diluent before semen collection.
Collection of semen from milk goats
Semen used in the test is obtained from the stock farm of the Guanzhong milk goats in Fu Ping county of Shaanxi, and the selected sheep is Guanzhong milk goats. Sheep were treated prior to semen collection. The ewe wipes the vagina with warm water and the ram wipes the foreskin with warm water. Allowing ram to climb and exercise, and collecting semen in pseudo vagina when sexual desire is vigorous.
Semen inspection
Semen with normal color, smell, was selected for testing, observed under an optical microscope, 10 μl was pipetted onto a slide glass with a pipette, the activation rate was examined under a microscope at 37 ℃, and semen with a sperm activation rate above 0.8 was selected for testing.
Preparation example 2
Preparation of diluent for goat semen low-temperature preservation:
Accurately weighing 1.2g of glucose, 1g of fructose, 1g of sodium citrate, 0.12g of sodium bicarbonate, 0.05g of penicillin and streptomycin respectively, and fixing the volume of sterile water to 100mL.
Example 1-1
Semen dilution of goats
In this example, a diluent for low-temperature preservation of goat sperm prepared in preparation example 1 and having a final quercetin concentration of 5. Mu.M was used, and the diluent will be hereinafter referred to as "diluent".
The diluent is added into the semen obtained in preparation example 1, and the diluent is added by slowly rotating a centrifuge tube to adhere to the wall, so that the excessive impact on sperm dilution is prevented. The method comprises the following specific steps: firstly, the volume ratio of semen to diluent is 1:1, mixing and diluting, after diluting, shaking gently and uniformly to prevent sperm precipitation from affecting the activity rate, balancing for 5min to obtain a mixed solution, and then mixing the mixed solution and the diluted solution according to the volume ratio of 1:4, the volume ratio of the semen to the diluent in the obtained liquid is 1:9; after dilution, the mixture is gently shaken to prevent sperm precipitation from affecting the activity.
Examples 1 to 2
In this example, a diluent for low-temperature preservation of goat sperm prepared in preparation example 1 and having a final quercetin concentration of 5. Mu.M was used, and the diluent will be hereinafter referred to as "diluent".
The diluent is added into the semen obtained in preparation example 1, and the diluent is added by slowly rotating a centrifuge tube to adhere to the wall, so that the excessive impact on sperm dilution is prevented. The method comprises the following specific steps: firstly, the volume ratio of semen to diluent is 1:1, mixing and diluting, after diluting, shaking gently and uniformly to prevent sperm precipitation from affecting the activity rate, balancing for 5min to obtain a mixed solution, and then mixing the mixed solution and the diluted solution according to the volume ratio of 1:3, and the volume ratio of the semen to the diluent in the obtained liquid is 1:7.
Comparative example 1
Semen dilution of goats
The diluent prepared in preparation example 2 is added into the semen prepared in preparation example 1, and the diluent is added by slowly rotating a centrifuge tube to adhere to the wall, so that excessive impact is prevented when the sperm is diluted. The specific steps are that firstly, according to the volume ratio of semen to diluent of 1:1, after balancing for 5min, obtaining a mixed solution, and then according to the volume ratio of the mixed solution to the diluent of 1:4, after each dilution, gently shaking the mixture to prevent sperm precipitation from affecting the activity.
Example 2
Semen dilution of goats
In this example, a diluent for low-temperature preservation of goat sperm prepared in preparation example 1 and having a final quercetin concentration of 10. Mu.M was used, and the diluent will be hereinafter referred to as "diluent".
The diluent is added into the semen obtained in preparation example 1, and the diluent is added by slowly rotating a centrifuge tube to adhere to the wall, so that the excessive impact on sperm dilution is prevented. The method comprises the following specific steps: firstly, the volume ratio of semen to diluent is 1:1, after balancing for 5min, obtaining a mixed solution, and then according to the volume ratio of the mixed solution to the diluent of 1:4, after each dilution, gently shaking the mixture to prevent sperm precipitation from affecting the activity.
Example 3
Semen dilution of goats
In this example, a diluent for low-temperature preservation of goat sperm prepared in preparation example 1 and having a final quercetin concentration of 25. Mu.M was used, and the diluent will be hereinafter referred to as "diluent".
The diluent is added into the semen obtained in preparation example 1, and the diluent is added by slowly rotating a centrifuge tube to adhere to the wall, so that the excessive impact on sperm dilution is prevented. The method comprises the following specific steps: firstly, the volume ratio of semen to diluent is 1:1, after balancing for 5min, obtaining a mixed solution, and then according to the volume ratio of the mixed solution to the diluent of 1:4, after each dilution, gently shaking the mixture to prevent sperm precipitation from affecting the activity.
Example 4
Semen dilution of goats
In this example, a diluent for low-temperature preservation of goat sperm prepared in preparation example 1 and having a final quercetin concentration of 50. Mu.M was used, and the diluent will be hereinafter referred to as "diluent".
The diluent is added into the semen obtained in preparation example 1, and the diluent is added by slowly rotating a centrifuge tube to adhere to the wall, so that the excessive impact on sperm dilution is prevented. The method comprises the following specific steps: firstly, the volume ratio of semen to diluent is 1:1, after balancing for 5min, obtaining a mixed solution, and then according to the volume ratio of the mixed solution to the diluent of 1:4, after each dilution, gently shaking the mixture to prevent sperm precipitation from affecting the activity.
After the dilution of examples 1-1 to 4 and comparative example 1 is completed, the room temperature is balanced for 1.5 hours, then 18 layers of gauze are covered outside a centrifuge tube, the centrifuge tube is put into a refrigerator at 4 ℃, semen is gently shaken to prevent semen from precipitating every 12 hours, and the activation rate, the vitality and the acrosome integrity of the sperm are detected every 24 hours, wherein the activation rate of the sperm is the percentage of the number of the viable sperm to the total number of the sperm in one picture, and the vitality of the sperm is the percentage of the total number of the sperm which moves forward in one picture of a microscope.
At 72h of semen preservation (day 3), a portion of cryopreserved semen was resuscitated and slides were simultaneously pre-heated at 37 ℃ and the sperm viability, motility and morphometric rate were measured by means of a Computer Aided Sperm Analyzer (CASA).
The results of the investigation of examples 1-1 to 5 and comparative example 1 are shown in Table 1.
Table 1 results of investigation of sperm motility, viability and acrosome integrity at day 3
| Activity (%) | Vitality (%) | Plasma membrane integrity (%) | Top body integrity (%) | |
| Comparative example 1 | 60.76±1.53b | 58.23±1.35c | 69.71±0.31b | 75.29±1.10b |
| Example 1-1 | 62.69±1.53b | 61..21±2.15bc | 70.99±0.37b | 75.56±0.39b |
| Example 2 | 73.25±0.63a | 65.98±0.59b | 71.46±0.77b | 78.32±0.37ac |
| Example 3 | 72.85±1.06a | 71.74±1.17a | 78.18±0.21a | 80.47±0.68a |
| Example 4 | 62.51±0.22b | 58.49±2.26c | 75.72±0.88c | 76.77±0.91c |
As is clear from Table 1, the sperm of comparative example 1 had a viability of 60.76% + -1.53% at day 3 of the preservation of goat semen at low temperature; the vitality is 58.23% +/-1.35%; the plasma membrane integrity rate is 69.71% +/-0.31%; the top body integrity rate is 75.29 percent plus or minus 1.10 percent.
On day 3 of cryopreservation of goat semen, the sperm (5. Mu. Mol/L) activity of example 1-1 was not significantly different (p > 0.05) from comparative example 1, the viability was not significantly different (p > 0.05) from comparative example 1, the plasma membrane integrity was not significantly different (p > 0.05) from comparative example, and the acrosome integrity was not significantly different (p > 0.05) from comparative example 1. Although the data of example 1-1 is not significantly different from that of comparative example 1, from a numerical point of view, example 1-1 is improved in viability, viability and plasma membrane integrity as compared to comparative example 1.
On day 3 of cryopreservation of goat semen, the sperm (10. Mu. Mol/L) of example 2 had significantly different viability (p < 0.05) from comparative example 1, no significantly different viability (p > 0.05) from comparative example 1, no significantly different plasma membrane integrity rate (p > 0.05) from comparative example 1, and no significantly different acrosome integrity rate (p > 0.05) from comparative example 1.
On day 3 of cryopreservation of goat semen, the sperm (25. Mu. Mol/L) activity of example 3 was significantly different from that of comparative example 1 (p < 0.05), the activity was significantly different from that of comparative example 1 (p < 0.05), the plasma membrane integrity was significantly different from that of comparative example 1 (p < 0.05), and the acrosome integrity was significantly different from that of comparative example 1 (p < 0.05).
On day 3 of cryopreservation of goat semen, the sperm (50. Mu. Mol/L) activity of example 4 was not significantly different (p < 0.05) from comparative example 1, the activity was not significantly different (p < 0.05) from comparative example 1, the plasma membrane integrity was significantly different (p < 0.05) from comparative example 1, and the acrosome integrity was significantly different (p < 0.05) from comparative example 1. From the above data we can see that the addition of quercetin at a concentration in goat low temperature base diluent has a significant positive effect on semen preservation, with an optimal addition concentration of 25 μmol/L.
At 72h (day 3) of semen preservation, a portion of the cryopreserved semen was recovered and the total antioxidant capacity (T-AOC), reactive Oxygen Species (ROS) content, ATP content, malondialdehyde content (MDA) superoxide dismutase (SOD) content of the recovered sperm was determined as follows:
The T-AOC level detection is carried out by adopting a total antioxidant capacity (T-AOC) measuring kit (Nanjing established biological institute; product number: A015-1 colorimetric method) and an enzyme-labeled instrument; the activity of superoxide dismutase (SOD) is detected by using a total superoxide dismutase (T-SOD) detection kit (Nanjing institute of biological research; product number: A001-1 hydroxylamine method) enzyme-labeled instrument; the content of malondialdehyde is measured by an enzyme-labeled instrument of Malondialdehyde (MDA) test box instruction book (Nanjing institute of biological research; product number: A003-1 TBA method); the ATP content in semen is measured by using an ATP content detection kit (Solarbio; ultraviolet spectrophotometry; product number: BC 0300) enzyme-labeled instrument; the content of Reactive Oxygen Species (ROS) in semen is determined by a multifunctional enzyme-labeled instrument of a Reactive Oxygen Species (ROS) determination kit (Nanjing institute of biological research; product number: E004-1-1 chemical fluorescence method 100T-500T). The detection results are shown in fig. 1 to 5.
As can be seen from FIG. 1, the addition of 25. Mu.M quercetin at day 3 of cryopreservation of sheep semen significantly increased sperm T-AOC levels (p < 0.05). As can be seen from FIG. 2, the addition of 25. Mu.M quercetin at day 3 of low temperature preservation of sheep semen significantly increased sperm ATP content (p < 0.05). From FIG. 3, it can be seen that addition of 10. Mu.M quercetin at day 3 of cryopreservation of sheep semen significantly reduced sperm ROS levels (p < 0.05). From FIG. 4, it can be seen that addition of 25. Mu.M quercetin at day 3 of sheep semen cryopreservation significantly reduced sperm MDA levels (p < 0.05). As can be seen from FIG. 5, the addition of 25. Mu.M quercetin at day 3 of low temperature preservation of sheep semen significantly increased the SOD level of sperm (p < 0.05).
In conclusion, quercetin (25 mu M) with proper concentration is added into the goat semen low-temperature preservation diluent, so that the sperm survival rate, vitality, plasma membrane integrity and acrosome integrity of the goat can be effectively improved, the ATP content in the sperm, the total antioxidant capacity (T-AOC) level and the superoxide dismutase (SOD) content of the sperm are effectively improved, and the Reactive Oxygen Species (ROS) and Malondialdehyde (MDA) content in the sperm are effectively reduced.
From the above, the quercetin disclosed by the invention can improve the quality of goat semen, especially the sperm motility, the activity rate, the plasma membrane integrity rate and the acrosome integrity rate, and has high conception rate.
While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.
Claims (3)
1. The diluent for the low-temperature preservation of the goat semen is characterized by comprising a low-temperature protective substance and a basic low-temperature diluent;
The low-temperature protective substance is quercetin; the concentration of quercetin in the diluent is 25 mu M;
the basic low-temperature diluent consists of nutrient substances, buffer substances and antibacterial substances;
the concentration of nutrient substances in the basic low-temperature diluent is 19-23 g/L, the concentration of buffer substances is 1-1.12 g/L, and the concentration of antibacterial substances is 1-1.1 g/L;
The nutrient substances are fructose and glucose;
The buffer substances are sodium citrate and sodium bicarbonate;
The antibacterial substances are penicillin and streptomycin.
2. The diluent of claim 1, wherein the low temperature storage temperature is 4 DEG C
3. Use of a diluent according to claim 1 or claim 2 to increase goat semen motility and/or sperm motility.
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