CN110622957B - A Stable and Efficient Pig Frozen Semen Reagent - Google Patents
A Stable and Efficient Pig Frozen Semen Reagent Download PDFInfo
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- CN110622957B CN110622957B CN201910961726.4A CN201910961726A CN110622957B CN 110622957 B CN110622957 B CN 110622957B CN 201910961726 A CN201910961726 A CN 201910961726A CN 110622957 B CN110622957 B CN 110622957B
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- 210000000582 semen Anatomy 0.000 title claims abstract description 75
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- 230000000694 effects Effects 0.000 claims abstract description 18
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 39
- 241000282898 Sus scrofa Species 0.000 claims description 29
- 230000008014 freezing Effects 0.000 claims description 27
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 26
- 238000007710 freezing Methods 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 23
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- 239000008103 glucose Substances 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 239000007983 Tris buffer Substances 0.000 claims description 12
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 12
- 235000018417 cysteine Nutrition 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 10
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 9
- 239000001509 sodium citrate Substances 0.000 claims description 9
- 238000002156 mixing Methods 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
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 claims description 8
- 210000002969 egg yolk Anatomy 0.000 claims description 7
- 102000002322 Egg Proteins Human genes 0.000 claims description 6
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- 235000013345 egg yolk Nutrition 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 238000005138 cryopreservation Methods 0.000 claims description 5
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- 238000012360 testing method Methods 0.000 claims description 4
- 229960000278 theophylline Drugs 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 3
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- RDZTWEVXRGYCFV-UHFFFAOYSA-M sodium 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonate Chemical compound [Na+].OCCN1CCN(CCS([O-])(=O)=O)CC1 RDZTWEVXRGYCFV-UHFFFAOYSA-M 0.000 claims 2
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- VFXZKNGPBLVKPC-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;sodium Chemical compound [Na].OCCN1CCN(CCS(O)(=O)=O)CC1 VFXZKNGPBLVKPC-UHFFFAOYSA-N 0.000 description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
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- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
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- 230000019100 sperm motility Effects 0.000 description 3
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- 239000010902 straw Substances 0.000 description 2
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- 238000005303 weighing Methods 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
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- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
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- 230000002503 metabolic 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
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/126—Physiologically active agents, e.g. antioxidants 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
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
Landscapes
- 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)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a stable and efficient frozen pig semen reagent, and particularly provides a frozen pig semen storage kit for improving storage quality and effect, wherein the frozen pig semen storage kit comprises a pre-diluent, a frozen diluent I, a frozen diluent I I and a unfreezing diluent. The invention can preserve the semen for a long time, effectively improve the utilization rate of boars, reduce the cost of protecting the boars, prevent the drift of excellent genes and the occurrence of infectious diseases, facilitate the deep development of the combined breeding work of the pigs, obtain larger genetic progress, and have important research value and wide application prospect in the aspects of protecting local excellent varieties, accelerating the improvement of the varieties and the like.
Description
Technical Field
The invention belongs to the technical field of improvement of artificial insemination of livestock, and relates to a boar semen storage kit capable of effectively improving the quality and effect of boar semen cryopreservation.
Background
Since Polge et al, British biologist, used glycerol as a cryoprotectant to cryopreserve sperm, the use of frozen sperm for in vitro fertilization and artificial insemination has been widely used in the treatment of infertility in humans and in the breeding of livestock animals. According to modern reproductive biological theory, one male animal sperm can obtain thousands of offspring individuals, so that the high-efficiency preservation of animal sperm can obviously reduce the high burden of maintaining living animal individuals and populations, shorten the breeding period and improve the production efficiency. The pig raising industry is an important part of animal husbandry, and the technology of preserving semen in liquid state at normal temperature for pigs is widely popularized and applied to the production practice of pig raising. Semen freezing preservation is a great revolution of artificial insemination, not only solves the problem of semen long-term preservation, is beneficial to the local pig seed resource protection, but also ensures that the semen is not limited by time and regions, is convenient to carry out inter-provincial and international cooperation and improves the utilization rate of excellent pig; and the service life of the genetic material of the breeding pigs is greatly prolonged, so that the excellent breeding pigs have important meanings in the aspects of short-term descendant determination, semen supply reservation and recovery, pedigree renewal, introduction, production cost reduction and the like. With the intensive development of national and regional joint breeding of breeding pigs, the research on the frozen semen technology of the pigs is more and more urgent. However, in the current domestic and foreign pig raising production, one of the main problems is that the production technology level of the frozen pig semen is low, the conception rate is generally low, and the improvement of pig breeds and the improvement of meat quality are severely restricted.
Semen cryopreservation refers to that semen is placed in liquid nitrogen (-196 ℃) or dry ice (-79 ℃) after special treatment, so that the metabolic activity state of the semen is temporarily stopped, and the purpose of preserving the germplasm resources of the species for a long time and practically playing the genetic resources of the elite male livestock can be achieved. The technology can greatly improve the utilization rate of excellent livestock, reduce the feeding amount, save the cost and directly help the agricultural and herdsmen to increase the yield and income. Meanwhile, the problem of food competition between people and livestock in China at present can be relieved, the emission of greenhouse gas is reduced, and the production pressure of the planting industry caused by returning the farmland to the grass is reduced. The technology ensures that the excellent gene is not limited by time and regions, and is an effective means for widely spreading excellent genetic resources.
Although the pig semen freezing technology has important significance in the aspects of livestock breeding, germplasm resource preservation and the like, the pig semen freezing preservation technology is difficult to popularize and apply at present, and mainly because the livestock sperms are particularly sensitive to temperature change and are extremely easy to damage in the freezing process, the production difficulty coefficient of the pig frozen semen is increased, the recovery rate of the thawed sperms is low, and the conception rate of sows is low and the litter size is small. The technical problem seriously restricts the improvement and breeding process of the domestic pig variety. Most of the existing pig frozen semen technologies have low thawing activity which can reach over 60 percent and is extremely small, and the popularization and the use of the pig frozen semen are limited. In terms of reagents, these techniques only focus on freezing fluids, but have little attention on the pre-dilution and thawing processes, and in fact the conditions encountered by the boar semen at different stages of production are not the same and the optimal dilution required is not exactly the same.
Disclosure of Invention
The invention aims to solve the technical problem of low sperm motility after freezing and unfreezing the boar semen. The invention aims to solve the existing problems and defects and provides a pig semen preservation kit which can effectively prolong the survival efficiency of domestic pig sperms in ultralow temperature cryopreservation and is beneficial to maintaining the integrity of sperm acrosome;
the pig semen storage kit comprises a pre-diluent, a freezing diluent I, a freezing diluent II and a thawing diluent;
wherein the pre-diluent component (1L system) is Tris 25-35 g, citric acid 11-8 g, glucose 8-15 g and cysteine 3-8 mM;
the preparation method of the freezing diluent I comprises the following steps: dissolving 22-30 g of glucose, 2.5-4.5 g of citric acid, 4-8 g of sodium citrate, 2-3.2 g of hepes sodium, 4-7 g of Tris, 21.5-4 g of EDTA-Na21, 0.5-2 g of sodium bicarbonate and 0.01-0.2 g of cysteine in 1L of pure water, and adding 250ml of egg yolk to obtain the yolk-containing solution;
the preparation method of the freezing diluent II comprises the following steps: taking 265-280ml of frozen diluent I, adding 15-30 ml of glycerol and 0.1-0.4% of SDS (sodium dodecyl sulfate) by mass fraction, and uniformly mixing to obtain the product;
the preparation method of the thawing diluent comprises the following steps: dissolving 22-30 g of glucose, 2.5-4.5 g of citric acid, 4-8 g of sodium citrate, 2-3.2 g of hepes sodium, 4-7 g of Tris, 21.5-4 g of EDTA-Na21, 0.5-2 g of sodium bicarbonate, 0.01-0.2 g of cysteine and 0.2-4 g of theophylline in 1L of pure water.
Furthermore, the invention provides a method for freezing and preserving the boar semen by using the boar semen preservation kit, which comprises the following steps:
1. dissolving one part of pre-diluent in 1L of water, preheating at about 35 ℃ for later use, wherein the prepared pre-diluent comprises (1L system) Tris 25-35 g, citric acid 11-8 g, glucose 8-15 g and cysteine 3-8 mM.
2. Collecting one part of semen, wherein the activity rate of the semen is better than 90% and the activity is more than 80%, weighing, and measuring the density of the semen. Approximately 1: 1 dilution was performed by semen weight. After mixing uniformly, standing at room temperature for about 1h, and then transferring into a 17 ℃ constant temperature box for balancing for 2-3h, wherein the longest time is not more than 24 h.
3. After the temperature is balanced at 17 ℃, the environmental temperature is controlled at 17 ℃, a large-capacity centrifuge is used for centrifuging at 17 ℃ for 10-15min at 900g, supernatant fluid is removed, and the solution is diluted to 20 hundred million/ml by using a solution I precooled at 17 ℃.
4. Placing the diluted semen in a beaker containing appropriate amount of 17 deg.C water, balancing the beaker in a refrigerator at 4 deg.C for 3-4h, adding 4 deg.C pre-cooled solution II 1: 1 for dilution, and mixing.
5. The final diluted semen was filled into 0.5ml straws and sealed at 4 ℃. The filled tubules are stacked on the stacking frame.
6. The stacked tubules were frozen using a program freezer. Freezing at-1.5 deg.C/min at 4-1 deg.C; 1 to 140 ℃ and 30 ℃ below zero/min; 140 ℃ below zero, 5 min.
7. The tube was then immersed in liquid nitrogen.
Wherein the preparation method of the solution I and the solution II comprises the following steps: dissolving 22-30 g of glucose, 2.5-4.5 g of citric acid, 4-8 g of sodium citrate, 2-3.2 g of hepes sodium, 4-7 g of Tris, 21.5-4 g of EDTA-Na21, 0.5-2 g of sodium bicarbonate and 0.01-0.2 g of cysteine in 1L of pure water, and adding 250ml of egg yolk to obtain solution I. And (3) taking 276ml of the solution I, adding 15-24 ml of glycerol and 0.1-0.4% of SDS (sodium dodecyl sulfate) in mass fraction, and uniformly mixing to obtain a solution II.
Further, the invention provides a method for unfreezing the boar semen by using the boar semen storage kit, which comprises the following steps:
taking out a thin tube stored in liquid nitrogen, thawing in 50 deg.C water bath for 15-16s, wiping off water outside the thin tube, cutting two ends of the thin tube to make semen flow into 4ml centrifuge tube, adding 30 deg.C preheated thawing solution, incubating at 30 deg.C for 15-20min, and observing activity.
The preparation method of the thawing solution comprises the following steps: dissolving 22-30 g of glucose, 2.5-4.5 g of citric acid, 4-8 g of sodium citrate, 2-3.2 g of hepes sodium, 4-7 g of Tris, 21.5-4 g of EDTA-Na21, 0.5-2 g of sodium bicarbonate, 0.01-0.2 g of cysteine and 0.2-4 g of theophylline in 1L of pure water.
Advantageous effects
The pre-diluent, the freezing diluent I, the freezing diluent II and the thawing diluent in the boar semen preservation kit have unique technical effects:
the pre-diluent is used for preserving semen for a short time and is used as a centrifugal protective solution, while the prior art uses common normal-temperature preservation diluent. The frozen diluent takes egg yolk and glycerin as main protective agents, and is divided into a liquid I (cooling protective liquid) without glycerin and a liquid II (freezing protective liquid) with glycerin. In the scheme, the SDS surfactant is also added into the liquid II to improve the freezing effect. The thawing diluent is necessary by adopting a high-density freezing scheme, the activity of the thawed sperms can be recovered to the maximum extent by adopting a unique formula, and the prior art uses a common normal-temperature preservation diluent.
The invention has low cost, easy popularization, simple and easy unfreezing method, easy artificial insemination and suitability for large-scale production.
The freezing and unfreezing method provided by the invention is simple, easy in material obtaining, reliable in effect and easy to popularize, is suitable for artificial fertilization of the pig thin-tube frozen semen, long-term storage after ultralow-temperature freezing of the pig semen and insemination after long-distance transportation, can obviously improve the sperm motility rate, the acrosome integrity rate and the plasma membrane integrity rate after freezing and unfreezing of the pig semen, and keeps the insemination activity of the pig semen.
Detailed Description
The present invention is further described with reference to specific examples to enable those skilled in the art to better understand the present invention and to practice the same, but the examples are not intended to limit the present invention.
Example 1
A method for freezing and preserving boar semen by using a boar semen preservation kit comprises the following steps:
1. dissolving the pre-diluent part in 1L of water, and preheating for later use at about 35 ℃. Wherein: the pre-diluent comprises 25-35 g of Tris, 11-8 g of citric acid, 8-15 g of glucose and 3-8 mM of cysteine.
2. Collecting one part of semen, wherein the activity rate of the semen is better than 90% and the activity is more than 80%, weighing, and measuring the density of the semen. Approximately 1: 1 dilution was performed by semen weight. After mixing uniformly, standing at room temperature for about 1h, and then transferring into a 17 ℃ constant temperature box for balancing for 2-3h, wherein the longest time is not more than 24 h.
3. After the temperature is balanced at 17 ℃, the environmental temperature is controlled at 17 ℃, a large-capacity centrifuge is used for centrifuging at 17 ℃ for 10-15min at 900g, supernatant fluid is removed, and the solution is diluted to 20 hundred million/ml by using a solution I precooled at 17 ℃.
4. Placing the diluted semen in a beaker containing appropriate amount of 17 deg.C water, balancing the beaker in a refrigerator at 4 deg.C for 3-4h, adding 4 deg.C pre-cooled solution II 1: 1 for dilution, and mixing.
5. The final diluted semen was filled into 0.5ml straws and sealed at 4 ℃. The filled tubules are stacked on the stacking frame.
6. The stacked tubules were frozen using a program freezer. Freezing at-1.5 deg.C/min at 4-1 deg.C; 1 to 140 ℃ and 30 ℃ below zero/min; 140 ℃ below zero, 5 min.
7. The tube was then immersed in liquid nitrogen.
Wherein the preparation method of the solution I and the solution II comprises the following steps: 22-30 g of glucose, 2.5-4.5 g of citric acid, 4-8 g of sodium citrate, 2-3.2 g of hepes sodium, 4-7 g of Tris, EDTA-Na21.5-4 g of sodium bicarbonate, 0.5-2 g of sodium bicarbonate and 0.01-0.2 g of cysteine are dissolved in 1L of pure water, and 250ml of egg yolk is added to obtain the solution I. Taking 265-280ml of I liquid, adding 15-30 ml of glycerol and 0.1-0.4% of SDS by mass fraction, and uniformly mixing to obtain 300ml of II liquid.
8. Taking out a thin tube stored in liquid nitrogen, thawing in 50 deg.C water bath for 15-16s, wiping off water outside the thin tube, cutting two ends of the thin tube to make semen flow into 4ml centrifuge tube, adding 30 deg.C preheated thawing solution, incubating at 30 deg.C for 15-20min, and observing activity. The preparation method of the thawing solution comprises the following steps: dissolving 22-30 g of glucose, 2.5-4.5 g of citric acid, 4-8 g of sodium citrate, 2-3.2 g of hepes sodium, 4-7 g of Tris, 21.5-4 g of EDTA-Na21, 0.5-2 g of sodium bicarbonate, 0.01-0.2 g of cysteine and 0.2-4 g of theophylline in 1L of pure water.
Comparative example
1. Motility rate of sperm
Taking out the frozen semen of the thin tube, quickly putting the frozen semen into a water bath at 37 ℃ for thawing for 45s, collecting the frozen semen in a small test tube, incubating for 5min after thawing, taking 10 mu l of semen on a glass slide, covering the glass slide, and evaluating the sperm motility rate (percentage of linearly moving sperm) under a 400X inverted microscope. At least 200 sperm cells were examined each time, 10 replicates.
2. Percentage of sperm acrosome integrity
After dyeing by using FITC-PNA dye solution, observing the shape of a sperm acrosome by a fluorescence microscope, unfreezing a tubule, adding the semen on a 2ml 3% PVP liquid surface, centrifuging and washing for 2 times at 800 Xg for 3min, and removing the supernatant; resuspending in PBS (37 ℃) to adjust the density to 1-2X 106 sperms/ml; sucking 30 μ l of sperm suspension, smearing, air drying, and fixing with pure methanol for 10 min; then adding 30 mul FITC-PNA dye solution, and incubating for 30min in a dark and humid environment at 37 ℃; PBS rinse, air dry, drip glycerol: PBS (9: 1), cover slip, seal with colorless nail polish, and view as quickly as possible under a 400 Xfluorescence microscope. At least 200 sperm cells were examined each time, 10 replicates.
3. Sperm plasma membrane integrity rate
Hypotonic swelling assay (HOST) was performed using fructose-sodium citrate hypotonic solution. Diluting the thawed semen with hypotonic solution, and adjusting sperm density to 1 × 106Incubating at 37 deg.C for 30min, dropping 20 μ l semen into suspension, observing under 400 × microscope, calculating percentage of curved tail sperm, checking at least 200 sperm each time, and repeating for 10 times.
(IV) sperm quality assessment results
By applying the semen freezing-thawing method and the comparative test example of the invention, the evaluation results are as follows:
the freezing and thawing activity of the TCG formula (Tris3.04g, citric acid 1.7g, glucose 1.25g, dissolved in 100mL of water, added with 25mL of egg yolk and 3% of glycerin) in the literature is 0.3-0.4, and the freezing activity rate of the invention is 0.5-0.7.
Note: all data are expressed as mean ± sem
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
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|---|---|---|---|---|
| CN114651815B (en) * | 2022-04-12 | 2023-05-09 | 江苏农牧科技职业学院 | A kind of preservation method of pig frozen semen |
| CN115918641A (en) * | 2022-11-29 | 2023-04-07 | 湖北省农业科学院畜牧兽医研究所 | Porcine semen cryoprotectant, preparation method, freezing and unfreezing method and application |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1391151A1 (en) * | 2002-08-19 | 2004-02-25 | Stoeterij Zangersheide N.V. | Thinner for conservation of sperm |
| CN101392236A (en) * | 2008-11-06 | 2009-03-25 | 上海交通大学 | Antifreezer for diluting boar semen |
| KR101396925B1 (en) * | 2011-11-14 | 2014-05-20 | 가천의과학대학교 산학협력단 | Cryopreservation technique using antifreeze protein from Leucosporidium sp. |
| CN103918642B (en) * | 2014-04-17 | 2015-10-28 | 中国农业科学院北京畜牧兽医研究所 | A kind of boar semen method and special refrigerating fulid thereof |
| CN104322484A (en) * | 2014-10-10 | 2015-02-04 | 山东鑫基牧业有限公司 | Freezing and unfreezing method for boar semen |
| CN104322485A (en) * | 2014-10-10 | 2015-02-04 | 山东鑫基牧业有限公司 | Boar frozen semen diluted powder |
| CN104663650A (en) * | 2015-03-02 | 2015-06-03 | 天津市宝坻区人民医院 | Sperm diluent preparing method |
| CN108782545A (en) * | 2018-08-30 | 2018-11-13 | 贵州省种畜禽种质测定中心 | One boar freezes essence pre-dilution liquid |
| CN109258626A (en) * | 2018-10-29 | 2019-01-25 | 北京田园奥瑞生物科技有限公司 | One boar freezes smart thawing solution and preparation method thereof and defreezing method |
| CN110278940A (en) * | 2019-07-11 | 2019-09-27 | 河南省农业科学院畜牧兽医研究所 | A kind of Boar spermatozoa preservative agent and its preparation method and application |
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