CN108719272B - Rabbit semen cryopreservation diluent and using method thereof - Google Patents
Rabbit semen cryopreservation diluent and using method thereof Download PDFInfo
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- CN108719272B CN108719272B CN201810320469.1A CN201810320469A CN108719272B CN 108719272 B CN108719272 B CN 108719272B CN 201810320469 A CN201810320469 A CN 201810320469A CN 108719272 B CN108719272 B CN 108719272B
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- 210000000582 semen Anatomy 0.000 title claims abstract description 63
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- 238000005138 cryopreservation Methods 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 16
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- 238000007710 freezing Methods 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 14
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 13
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 13
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 13
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims abstract description 12
- 102000019197 Superoxide Dismutase Human genes 0.000 claims abstract description 12
- 108010012715 Superoxide dismutase Proteins 0.000 claims abstract description 12
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims abstract description 12
- 229960000367 inositol Drugs 0.000 claims abstract description 12
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000001509 sodium citrate Substances 0.000 claims abstract description 9
- 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 abstract description 8
- 239000008103 glucose Substances 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 239000008213 purified water 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
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 7
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 6
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 6
- 235000013345 egg yolk Nutrition 0.000 claims abstract description 6
- 210000002969 egg yolk Anatomy 0.000 claims abstract description 6
- 238000010438 heat treatment Methods 0.000 claims abstract description 4
- 238000003756 stirring Methods 0.000 claims description 3
- 230000035899 viability Effects 0.000 claims description 3
- 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 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 10
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- 210000000170 cell membrane Anatomy 0.000 abstract description 8
- 230000009286 beneficial effect Effects 0.000 abstract description 6
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- 238000004321 preservation Methods 0.000 description 9
- 230000019100 sperm motility Effects 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000002577 cryoprotective agent Substances 0.000 description 6
- 230000006872 improvement Effects 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
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- 239000011521 glass Substances 0.000 description 3
- 230000009027 insemination Effects 0.000 description 3
- 230000003204 osmotic effect Effects 0.000 description 3
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- 230000003078 antioxidant effect Effects 0.000 description 2
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- 108010065337 fluorescein isothiocyanate-peanut agglutinin Proteins 0.000 description 2
- 239000000815 hypotonic solution Substances 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
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- 238000012986 modification Methods 0.000 description 2
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- 240000002930 Alternanthera sessilis Species 0.000 description 1
- 235000015579 Alternanthera sessilis Nutrition 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
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- 238000004820 blood count Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000000959 cryoprotective effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000004899 motility Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003761 preservation solution Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- 239000000725 suspension Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 210000005239 tubule Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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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 discloses a rabbit semen cryopreservation diluent and a use method thereof. Every 100ml of purified water of the rabbit semen cryopreservation diluent comprises the following components: 4-5g of glucose, 0.2-0.4g of sodium citrate, 1-3g of hyaluronic acid, 3500 IU of superoxide dismutase, 4000IU of inositol, 0.4-0.6g and 5-6ml of fresh egg yolk. The specific using method comprises the following steps: (1) heating the frozen and preserved diluent to 37 ℃ in a water bath, then slowly pouring the heated and preserved diluent into the rabbit semen, and slightly shaking the heated and preserved diluent while adding the heated and preserved diluent to fully mix the frozen and preserved diluent with the rabbit semen; (2) subpackaging the diluted rabbit semen by using a thin tube, and balancing for 30-60 min at 5 ℃; (3) and (5) freezing and storing the balanced rabbit semen by liquid nitrogen. The cryopreservation diluent greatly improves the survival rate, plasma membrane integrity rate and acrosome integrity rate of frozen sperms, and is beneficial to improving the breeding efficiency and breeding speed of rabbits.
Description
Technical Field
The invention belongs to the technical field of artificial insemination, and particularly relates to a diluent for preserving frozen rabbit semen and a using method thereof.
Background
The rabbit breeding industry has the advantages of low investment, quick response and small risk, is a good way for farmers to get rid of poverty and become rich, and is called as 'a new industry with bright future' and 'a new economic growth point in rural areas' by experts. In recent years, along with the implementation of the policy of accurate poverty relief in China and the implementation of the western big development strategy, the majority of people, particularly poverty-stricken farmers and western region farmers and herdsmen, take the rabbit farming industry as the first choice, the country invests a large amount of poverty relief funds to help the farmers and herdsmen to develop rabbit farming production every year, the breeding amount of various rabbits in the country exceeds 4 hundred million, the annual rabbit hair yield reaches 8000 tons, the annual rabbit meat yield exceeds 45 million tons, and the whole rabbit farming industry has a value of over one hundred million yuan.
At present, the artificial insemination technology is applied to most of domestic animals, frozen semen of cows, sheep, pigs and the like is produced in a large-scale commercial mode in China, but the artificial insemination technology of rabbits is not widely applied, and an important factor is the semen preservation problem. The preservation of the semen can be divided into normal temperature, low temperature and freezing preservation, wherein the normal temperature and the low temperature preservation are limited by regions and time, so that the problem of the variety improvement of the rabbits cannot be fundamentally solved. The freezing preservation of the semen can solve the phenomenon of 'summer sterility' of the male rabbits and also can avoid the problem of poor quality of the temporary semen of the male rabbits due to other reasons. Therefore, the freezing preservation of the rabbit semen has very important significance for the quality improvement and large-scale commercial production of rabbits.
The cryoprotectant is an important factor influencing the quality of frozen semen of the rabbit. Since the discovery of the cryoprotective properties of glycerol, the development of frozen livestock semen has been rapidly promoted. However, glycerol, as an osmotic cryoprotectant, can penetrate into the interior of sperm cells, concentrate or bind intracellular water, and reduce the concentration of salts in solution and the osmotic pressure of the diluent. On one hand, the sperm has a protection effect, on the other hand, the sperm has a certain poisoning effect, partial sperm has better freezing effect after being dehydrated, but has no motility and fertilization capability after being thawed. In recent years, efforts have been made to investigate non-osmotic cryoprotectants in combination with the addition of other components to the dilution to improve the frozen sperm quality. Hyaluronic acid has attracted considerable attention from researchers as an impermeable cryoprotectant, primarily because it is capable of forming a protective film on the outside of cell membranes in the event of dehydration of bioactive cells, thereby acting to protect the active cells during the freezing process. Furthermore, semen is vulnerable to free radicals during equilibration and freezing processes, and therefore, the addition of antioxidants to the frozen dilution is beneficial for sperm protection.
On the basis of the traditional diluent, the frozen sperm motility rate, plasma membrane integrity, acrosome integrity and the like are taken as quality evaluation indexes, and the protection effect of non-permeable cryoprotectants, namely hyaluronic acid, antioxidant superoxide dismutase and inositol, on rabbit semen freezing is explored.
Disclosure of Invention
The invention aims to provide a rabbit semen cryopreservation diluent and a use method thereof. The invention can effectively solve the problems of low sperm motility, plasma membrane integrity and acrosome integrity after the rabbit semen is frozen, is beneficial to the popularization of the semen freezing preservation technology in the rabbit industry, and is beneficial to the improvement of the breeding efficiency and the breeding speed of rabbits.
In order to achieve the purpose, the invention adopts the following technical scheme:
a rabbit semen cryopreservation diluent, wherein each 100ml of purified water of the cryopreservation diluent comprises the following components: 4-5g of glucose, 0.2-0.4g of sodium citrate, 1-3g of hyaluronic acid, 3500 IU of superoxide dismutase, 4000IU of inositol, 0.4-0.6g and 5-6ml of fresh egg yolk.
Preferably, the rabbit semen cryopreservation diluent is prepared by adding glucose, sodium citrate, hyaluronic acid, superoxide dismutase and inositol into purified water, fully stirring and uniformly mixing.
Preferably, the cryopreservation diluent comprises the following components per 100ml of purified water: 4.54g of glucose, 0.38g of sodium citrate, 2g of hyaluronic acid, 4000IU of superoxide dismutase, 0.5g of inositol and 5mL of fresh egg yolk.
The invention also provides a use method of the rabbit semen cryopreservation diluent, which comprises the following steps:
(1) heating the frozen and preserved diluent to 37 ℃ in a water bath, slowly pouring the frozen and preserved diluent into the rabbit semen, and slightly shaking while adding to fully mix the frozen and preserved diluent with the rabbit semen;
(2) subpackaging the diluted rabbit semen obtained in the step (1) by using a thin tube, and balancing at 5 ℃ for 30-60 min;
(3) and (3) freezing and storing the rabbit semen balanced in the step (2) by liquid nitrogen.
Preferably, the volume ratio of the cryopreservation diluent to the rabbit semen is 1: 1.
Preferably, the rabbit semen is obtained by removing gel components contained in the collected rabbit semen.
Preferably, the survival rate of the rabbit semen is more than 0.65.
The invention has the following technical characteristics:
1) the cryopreservation diluent adopts hyaluronic acid as a non-permeable cryoprotectant, can avoid toxic action on sperms, and plays a role in protecting active cells in the freezing process.
2) The invention adds antioxidant superoxide dismutase and inositol into the freezing preservation diluent, which is favorable for avoiding the sperm from being damaged by free radicals.
3) The invention greatly improves the survival rate, the plasma membrane integrity rate and the acrosome integrity rate of frozen sperms by combining hyaluronic acid, superoxide dismutase and inositol, and is beneficial to improving the breeding efficiency and the breeding speed of rabbits.
Detailed Description
The following specific examples are further illustrative of the methods and techniques provided by the present invention and should not be construed as limiting the invention thereto.
First, preparation of diluent for cryopreservation
Accurately weighing 4.54g of glucose, 0.38g of sodium citrate, 2g of hyaluronic acid, 4000IU of superoxide dismutase, 0.5g of inositol and 5mL of fresh egg yolk, adding 100mL of purified water, and uniformly stirring to prepare the rabbit semen cryopreservation diluent.
Second, semen collection and viability evaluation
The male rabbit semen was collected by the pseudo-vaginal method. Under the microscope, the survival rate of the sperms detected by a visual method reaches more than 0.65, and the sperms are used for the cryopreservation test. Generally, 0.2-2 mL of semen can be collected each time, and the semen contains gel components and needs to be removed before a semen freezing experiment.
Semen freezing program
Heating the frozen preservation solution to 37 ℃ in water bath at room temperature, slowly pouring the semen subjected to viability evaluation according to the proportion of the diluent to the semen of 1:1, and slightly shaking while adding to fully mix the semen and the diluent. Subpackaging the diluted semen by using a 0.25mL thin tube, and balancing at 5 ℃ for 30-60 min. Pouring liquid nitrogen into a prepared foam box, putting a graduated iron frame 2-5 cm away from the liquid nitrogen surface, putting the balanced thin tube on the iron frame, covering a cover to fumigate for 15min, and then quickly putting into the liquid nitrogen for preservation.
Fourthly, unfreezing frozen semen
When thawing, quickly taking out the thin tube in liquid nitrogen, putting into water with the temperature of 37 ℃, slightly shaking for 30s, taking out, and immediately sucking dry and packaging with dry gauze. The two ends of the thin tube are cut off, the thin tube is dripped on a glass slide which is preheated to 37 ℃ for microscopic examination, and the sperm motility rate is recorded.
Fifth, relevant index detection
Sperm motility: taking a rabbit semen freezing tubule, unfreezing the rabbit semen in water bath at 37 ℃ for 30s, taking 10 mu L of semen on a glass slide, covering the glass slide, and subjectively evaluating the sperm motility rate under a 400 multiplied inverted microscope.
Sperm plasma membrane integrity rate: performing hypotonic expansion detection by using fructose-sodium citrate hypotonic solution. Diluting the thawed semen with hypotonic solution, and adjusting sperm density to 1 × 106mL, incubation at 37 ℃ for 30min, taking 20 μ L of sperm suspension and dropping on a blood cell counting plate, observing under a 400X microscope, calculating the percentage of curvy-tail sperm, and calculating at least 200 sperm each time.
Sperm acrosome integrity rate: after staining the sperm with FITC-PNA staining solution, the sperm acrosome morphology was observed with a fluorescence microscope. Spreading the thawed semen on the surface of PVP liquid, centrifuging for 6min at 800 r, and removing the supernatant; resuspending the pelleted sperm in PBS and adjusting the sperm density to 1X 106and/mL, taking 30 mu L of smear from the raw materials, naturally drying in the air, fixing with methanol for 10min, then adding 30 mu L of FITC-PNA dye solution, incubating for 30min at 37 ℃ in a dark humid environment, washing with PBS solution, naturally drying, adding a small amount of brightener, sealing with colorless nail polish, and immediately observing under a fluorescence microscope.
Sixth, test results
The addition of hyaluronic acid, superoxide dismutase and inositol to the dilution significantly improved the sperm motility, plasma membrane integrity and acrosome integrity after freezing, and the specific results are shown in table 1.
The control diluent was prepared as follows: 4.54g of glucose, 0.38g of sodium citrate and 5mL of fresh egg yolk are added into 100mL of purified water and stirred uniformly.
Table 1: effect of adding hyaluronic acid, superoxide dismutase and inositol in diluent on rabbit semen freezing
Note: different numbers on the same column indicate significant difference (P < 0.05).
As can be seen from the table, compared with the control group, the frozen and preserved diluent of the embodiment has obviously improved sperm motility, plasma membrane integrity and acrosome integrity after the rabbit semen is balanced and frozen, and is beneficial to improving the breeding efficiency and the breeding speed of rabbits.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (5)
1. A rabbit semen cryopreservation diluent, which is characterized in that the components in each 100ml of purified water of the cryopreservation diluent consist of the following substances: 4.54g of glucose, 0.38g of sodium citrate, 2g of hyaluronic acid, 4000IU of superoxide dismutase, 0.5g of inositol and 5mL of fresh egg yolk; the rabbit semen cryopreservation diluent is prepared by adding glucose, sodium citrate, hyaluronic acid, superoxide dismutase and inositol into purified water, fully stirring, and uniformly mixing.
2. The method of using a rabbit semen cryopreservation diluent as claimed in claim 1, comprising the steps of:
(1) heating the frozen and preserved diluent to 37 ℃ in a water bath, slowly pouring the frozen and preserved diluent into the rabbit semen, and slightly shaking while adding to fully mix the frozen and preserved diluent with the rabbit semen;
(2) subpackaging the diluted rabbit semen obtained in the step (1) by using a thin tube, and balancing at 5 ℃ for 30-60 min;
(3) and (3) freezing and storing the rabbit semen balanced in the step (2) by liquid nitrogen.
3. The use according to claim 2, wherein the volume ratio of the cryopreservation diluent to rabbit semen is 1: 1.
4. Use according to claim 3, wherein said rabbit semen is obtained by removing the gel components contained in the collected rabbit semen.
5. The use according to claim 4, wherein the rate of viability of said rabbit semen is above 0.65.
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| CN108719272B true CN108719272B (en) | 2021-01-26 |
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|---|---|---|---|---|
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| CN103004750A (en) * | 2012-11-27 | 2013-04-03 | 云南省畜牧兽医科学院 | Livestock semen freezing diluent as well as preparation method and application thereof |
| CN103651333A (en) * | 2013-12-23 | 2014-03-26 | 伊犁德瑞骏发生物科技有限公司 | Frozen semen diluent of equus animals and preparation method thereof |
-
2018
- 2018-04-11 CN CN201810320469.1A patent/CN108719272B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1025835A2 (en) * | 1999-02-01 | 2000-08-09 | Coty B.V. | Cosmetic composition for protecting the scalp from free radicals |
| CN103004750A (en) * | 2012-11-27 | 2013-04-03 | 云南省畜牧兽医科学院 | Livestock semen freezing diluent as well as preparation method and application thereof |
| CN103651333A (en) * | 2013-12-23 | 2014-03-26 | 伊犁德瑞骏发生物科技有限公司 | Frozen semen diluent of equus animals and preparation method thereof |
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| Title |
|---|
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