WO2008007463A1 - Composition de dilution et de stockage de sperme - Google Patents
Composition de dilution et de stockage de sperme Download PDFInfo
- Publication number
- WO2008007463A1 WO2008007463A1 PCT/JP2007/000745 JP2007000745W WO2008007463A1 WO 2008007463 A1 WO2008007463 A1 WO 2008007463A1 JP 2007000745 W JP2007000745 W JP 2007000745W WO 2008007463 A1 WO2008007463 A1 WO 2008007463A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- semen
- composition
- storage according
- semen dilution
- storage
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
-
- 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
Definitions
- the present invention relates to a composition for semen dilution preservation. More specifically, the present invention relates to a composition for semen dilution storage that does not contain natural animal-derived components such as serum and that can maintain semen vitality comparable to or higher than that of conventional dilution solutions.
- pork semen In pork meat, pork semen retains its semen vitality (fertility) only for about 2 days if the raw semen remains as it is, and the fertility rate drops rapidly after the 3rd day.
- the sperm concentration is lower than that of cattle, a large amount of injected semen is required, and it is extremely vulnerable to low temperature sensitization, so it is not easy to preserve. For this reason, there are very few farms that use artificial insemination for pork.
- the freezing method includes the tablet method (pellet method) and the straw method.
- fertilization performance is said to be inferior to that of natural mating if semen with a semen vitality of at least 3 5 ++ after thawing is fertilized at a suitable time to females with good estrus. Only However, cryopreservation of swine semen is more difficult than cattle and its practical application is delayed.
- Patent Document 1 a protective agent that does not impair the sperm's physiological function even after cold shock treatment that rapidly cools sperm to 2 to 3 ° C
- lauryl alcohol Physiological functions of sperm and cells can be substantially preserved by containing long-chain alcohols having a carbon number of 10 to 14 such as 10 to 14 and lipids as well as ushi serum albumin (hereinafter referred to as BSA).
- BSA ushi serum albumin
- Patent Document 2 Cell cold injury prevention liquid
- Patent Document 1 Japanese Patent Application Laid-Open No. 8-26 90 1
- Patent Document 2 Japanese Patent Laid-Open No. 10-2 7 9 4 0 1
- Patent Document 3 Japanese Patent No. 3 3 6 4 6 80
- Patent Document 4 Japanese Patent No. 3 6 3 6 6 0 9
- the present invention provides a composition for diluting and storing a semen that does not contain a natural animal-derived component such as a serum-derived component and that can maintain a semen vitality equivalent to or higher than that of a conventional diluting solution. Offering is an issue. Another object of the present invention is to provide a method for storing semen using this composition for semen dilution storage.
- the present inventors removed animal-derived components such as serum and adjusted other components and concentrations to obtain animal-derived components.
- the present inventors have found a composition for semen dilution storage that does not contain components and can maintain semen vitality comparable to or higher than that of a conventional storage dilution, etc., and has completed the present invention.
- save method was completed by adjusting a semen using the composition for semen dilution preservation
- the composition for semen dilution preservation of the present invention is characterized in that it is difficult to foam when dissolved and mixed with the original semen and is well mixed with semen.
- the semen diluted stock solution using the composition for semen dilution preservation of the present invention is characterized in that the semen concentrated portion hardly precipitates and the dispersibility is good. For this reason, the labor of mixing the stored semen once or twice a day, which was required for conventional storage solutions, can be reduced to about once every three days.
- the present invention relates to a method for storing semen using the following composition for semen dilution storage of (1) to (15).
- a composition for diluting and storing a semen containing a protective agent, a saccharide, and a pH adjusting agent, and containing no natural animal-derived components.
- composition for semen dilution storage according to (1) above further comprising an antioxidant.
- composition for semen dilution storage according to the above (1) further comprising an amino acid.
- composition for semen dilution storage according to any one of the above (1) to (3), wherein the natural animal-derived component is a serum-derived component.
- Sugars are glucose, trehalose, sucrose, and lac!
- the pH modifier contains citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDTA (ethylenediaminetetraaceticacid), and ash! Hydrogen Na.
- composition for semen dilution storage according to any one of (2) to (8) above, wherein the antioxidant is ascorbic acid, dibutylhydroxytoluene, or vitamin E.
- the amino acid is at least one selected from the group consisting of L-cystine, glutathione (reduced form), cystine, L (+)-glutamic acid Na_hydrate and glycine.
- the composition for semen dilution storage according to any one of (9).
- composition for semen dilution storage according to (9) or (10) above, containing 0.1 to 4.2% by weight of an antioxidant or an amino acid.
- composition for semen dilution storage containing the following components (a) to (d):
- pH regulators include citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDTA and bicarbonate Na
- At least one selected from the group consisting of L-cystine, glutathione (reduced), cystine, L (+)-glutamic acid Na hydrate, ascorbic acid, glycine and arginine Includes more than one.
- composition for semen dilution storage according to (1 2) above which contains ascorbic acid as an antioxidant.
- composition for semen dilution preservation save in any one of said (1)-(13)
- composition for semen dilution preservation established according to the present invention does not contain natural animal-derived components such as serum-derived components, there is no fear of adverse effects on the stored semen and can be obtained at low cost.
- composition for semen dilution storage of the present invention is useful because it can maintain semen vitality comparable to or higher than that of conventional storage dilutions.
- FIG. 1 shows the dispersibility of a composition for semen dilution storage (Example 1).
- the “semen dilution storage composition” of the present invention is a composition for storing semen, which contains a protective agent, a saccharide, and a pH adjusting agent, while containing a natural animal-derived component. A composition that does not contain. Further, the “semen diluted storage composition” of the present invention refers to a composition containing an antioxidant or an amino acid, but not containing a natural animal-derived component.
- the composition for semen dilution storage of the present invention has an appearance that does not change for at least 6 months when stored at room temperature (temperature when stored in a stability test room kept at around 23 ° C) or at 4 ° C. It is preferable that Furthermore, it is preferable that the composition for semen dilution storage of the present invention has little change in pH and osmotic pressure when the powder is dissolved in 1 L of purified water, and the semen vitality by storage does not change.
- the target for storing semen using the “semen dilution-preserving composition” of the present invention is not particularly limited, but cows, pigs, birds, goats and the like are preferable, and these types are not particularly limited. When used for pigs, any kind of pig is acceptable, but it is preferable to use a breeding pig, and the kind of breeding pig is not particularly limited.
- not containing natural animal-derived components in the present invention means not containing components such as serum-derived components. This is because the effects of natural animal-derived components on stored sperm are unknown and may have adverse effects. Semen dilution maintenance of the present invention It is desirable that the existing composition is composed only of components with a clear origin that does not affect the semen in which the composition is stored. Therefore, it is desirable that the composition for semen dilution preservation of the present invention does not contain any natural animal-derived components.
- the "protective agent" of the present invention protects the head and cap so that various degradation enzymes necessary for fertilization contained in the acrosome in the cap of the sperm head are not deleted. This is to prevent damage to the head.
- the “protective agent” of the present invention may be any component that can sufficiently protect semen even when these natural animal-derived components are not used at all.
- methyl cellulose (MC), sodium carboxymethyl cellulose (CMC-Na), polyvinylpyrrolidone (PVP), xanthan gum, sodium alginate and the like can be mentioned.
- the water-soluble compound of a high molecular organic polymer etc. are mentioned.
- compositions for semen dilution storage are preferably contained in an amount of 0.2 to 10.0% by weight, and preferably 0.2 to 8.1% by weight, in the composition for semen dilution storage. This is because the performance may not be maintained unless it is added within this range. Also, if it exceeds 10.0% by weight, there will be a problem of solubility.
- the "saccharide” of the present invention can be any component that can sufficiently protect semen even when no natural animal-derived component is used. Examples include trehalose, sucrose, lactose, glucose and the like. These 50.4 to 59. It is preferable to contain 9 wt% semen dilution storage composition, 52.5 to 59.5 wt 0 / o, especially from 53.0 to 59.5 wt 0 / o free or It is desirable that
- any component that can sufficiently protect semen can be used even when natural animal-derived components are not used at all.
- it preferably contains citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDT A and hydrogen carbonate Na. It also contains hydroxyethyl piperazine ethane sulfonic acid (H EP ES). Also good.
- H EP ES hydroxyethyl piperazine ethane sulfonic acid
- Kuen tribasic N a dihydrate 1 3.3 to 1 5.4 wt 0/0 tris (hydroxymethyl) Aminometan 1 1.2 to 1 2.8 wt 0 / o, citrate anhydride 5.1 to 6.6 wt.
- citrate tri-Na dihydrate 14.0 to 15.2 wt%
- tris (hydroxymethyl) aminomethane 11.5 to 12.5 wt 0 / o
- citrate anhydride 5.9 to 6. It is preferable to contain 4% by weight, EDTA4.8 to 5.2% by weight, and hydrogen carbonate Na 1.5 to 1.7% by weight.
- any component that can sufficiently protect semen can be used even when natural animal-derived components are not used at all.
- ascorbic acid, dibutylhydroxytoluene, vitamin E and the like are preferable.
- the “amino acid” of the present invention may be any component that can sufficiently protect semen even when no natural animal-derived component is used.
- L-cystine glutathione (reduced form), cystine
- L (+) glutamic acid Na hydrate
- glycine glutathione
- L—cystine hydrochloride — hydrate
- lecithin lecithin
- ergotionine arginine It is preferable that the number is at least one.
- L_cystine 0.1 to 4.2% by weight glutathione (reduced form) 0.1 to 4.2% by weight, cystine 0.1 to 4.2% by weight.
- ascorbic acid is preferably contained in an amount of 0.2 to 0.22% by weight as an antioxidant
- L (+) glutamic acid Na_hydrate is contained in an amount of 0.2 to 0.22% by weight as an amino acid.
- antioxidants or amino acids are not added within this range, performance may not be maintained, and additions exceeding 0.22% by weight affect pH and osmotic pressure during dissolution. There is a case.
- the “method for preserving semen” of the present invention refers to mixing an aqueous solution prepared by dissolving the semen dilution storage composition of the present invention in purified water when necessary and mixing semen at a constant temperature. It means saving.
- a mixture of an aqueous solution of a semen diluted storage composition and semen is referred to as a “semen diluted storage solution”.
- the mixing ratio of the semen and the aqueous solution of the semen dilution storage composition varies depending on the number of sperm contained in the semen, but the sperm dilution storage solution contains 0.4 to 100 million semen. It is preferable to mix.
- the storage temperature of the “semen diluted stock solution” may be any temperature as long as the semen vitality can be maintained, but it is 4 ° C to medium temperature (15 to 18 ° C), especially medium temperature (15 ° C). It is most preferable that the temperature is constantly maintained at a medium temperature (15 ° C.) by a thermostat or the like. In addition, antibiotics can be added and stored as necessary.
- the "semen vitality" of the present invention refers to the determination of the survival rate and motility of sperm contained in the raw semen according to the following vitality criteria. Generally, it is said that fertilization results are not inferior to natural mating if semen with a vigor of semen of 60 + + or higher is fertilized at a suitable time in females with good estrus, but survival is comparable to or higher than that of a conventional storage dilution. It is preferable to have rate and mobility. In the determination of semen, such as sushi, there is a dedicated machine that uses it, and those that do not have a dedicated machine, such as pigs, can be determined visually by placing semen on a semen test plate.
- Survival rate is expressed in% (approximately 5% increments).
- the motility was displayed in five levels: + +10, +10, tens, earth, and one.
- “Development composition 1” and “development composition 2” shown in Table 1 were used as compositions for semen dilution preservation.
- BSA Wired Chemical
- MC Wired Chemical
- CMC—Na Wired Chemical
- citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDTA, hydrogen carbonate Na and HEP ES were used.
- Ascorbic acid was used as an antioxidant and amino acid.
- composition was prepared by mixing the above component 1 using each weight so as to obtain the composition shown in Table 1.
- the resulting developed composition was filled into an aluminum bag (1 pack) as a semen diluted storage composition and stored at room temperature (23 ° C) avoiding high temperature and humidity.
- the semen diluted stock solution was prepared by adding each solution in Table 1 to an aqueous solution of semen from domestic pigs (Landlace) so that the sperm power was ⁇ 0.5 billion ZmL.
- Amikacin was added to each semen-diluted stock solution to 10 OmgZL and stored at medium temperature (15 ° C).
- the semen vitality was examined over time from the beginning to 7 days, and the results obtained are shown in Table 2. The determination of semen vitality was performed according to the above criteria using a semen property inspection board.
- Protective agent At least one selected from the group consisting of high molecular weight polymers such as PVP, xanthan gum and alginic acid was selected.
- pH adjuster At least one selected from the group consisting of citrate tri-Na dihydrate, tris (hydroxymethyl) aminomethane, citrate anhydride, EDT A, and hydrogen carbonate Na was used. .
- L_cystine at least one selected from the group consisting of glutamic acid Na hydrate, ascorbic acid and glycine Was used.
- composition for semen dilution preservation It adjusted by combining the said 1 component and mixing so that it might become the composition for semen dilution preservation
- the obtained composition for diluting and storing semen was filled in an aluminum bag (1 packet) and stored at room temperature (23 ° C) avoiding high temperature and humidity.
- 1 sachet was added to 1 L of purified water and completely dissolved to prepare an aqueous solution of a composition for semen dilution preservation.
- PH adjuster Cenic acid tri-Na dihydrate 6.9 g, Tris (hydroxymethyl) aminomethane 5.65 g, Cubic anhydride 2.9 g, EDTA 2.35 g, Hydrogen carbonate N a 0. 75 g
- composition for semen dilution storage prepared in Example 2 the composition for semen dilution storage of Formula 6 was filled in an aluminum bag, avoiding high temperature and humidity, at room temperature (23 ° C) or at 4 ° C. Stored for 6 months.
- the aging stability of the semen diluted storage composition during this storage period was examined by standard tests of the appearance of the semen diluted storage composition and the pH and osmotic pressure characteristics of the aqueous solution adjusted at the time of use.
- semen dilution storage composition of Formula 2 0.5 billion Zm of domestic pig (land race) semen in an aqueous solution of the semen dilution storage composition dissolved in purified water as in Examples 1 and 2 Zm
- the semen diluted stock solution was obtained.
- the aqueous solution was prepared by adding 9 O m I.
- This semen diluted aqueous solution was stored at medium temperature (15 ° C) for 7 days, and then semen vitality was determined. Table 4 shows the results of property and standard tests and storage performance tests.
- composition for semen dilution preservation showed almost no change in properties and specifications even after storage for 6 months, and semen vitality did not decline. Accordingly, it was shown that the composition for semen dilution storage of the present invention can be stored for at least 6 months, and can be adjusted and used as necessary.
- Example 2 The preservation performance of the semen vitality of the composition for semen dilution preservation
- a composition using BSA as a protective agent was prepared and used.
- the composition of the composition used for comparison is shown below.
- PH modifier Cenic acid tri-Na dihydrate 6.9 g, Tris (hydroxymethyl) aminomethane 5.65 g, Cubic anhydride 2.9 g, EDTA 2.35 g, Hydrogen carbonate N a 0. 75 g
- Antioxidants, amino acids, etc . Ascorbic acid 0.1 g
- the semen vitality storage performance of each semen diluted storage composition is the storage described in Example 1. The same as the performance test (semen vitality measurement). The vitality after dilution of semen and the vitality after storage for 7 days at medium temperature (15 ° C) were examined.
- the vitality at the time of dilution of the semen contained in the semen diluted stock solution using the formulations 1 to 6 and the comparative composition was 80 + +10 to 90 +++.
- the vitality of these semen after storage for 7 days was approximately 60 + + or more for the semen diluted storage compositions of Formulas 1 to 6 and 70 + + for those with high vitality.
- those using the comparative composition ranged from 4 5 +10 to 5 5 +++. Therefore, it was confirmed that the composition for semen dilution storage of the present invention can maintain semen vitality comparable to or higher than that of a conventional storage dilution without containing animal-derived components.
- composition for semen dilution storage of the present invention is safe and inexpensive, and can maintain semen vitality comparable to or higher than that of a conventional storage dilution, etc. It can be used by fertilizers and pig farmers.
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Abstract
La présente invention concerne une composition de dilution et de stockage de sperme ne contenant pas de constituant dérivé d'animal naturel tel que du sérum et pouvant maintenir une activité de sperme à un niveau égal ou supérieur comparé à un liquide classique de dilution et de stockage de sperme. Grâce à l'élimination d'un constituant dérivé d'animal naturel tel que du sérum et l'ajustement d'autres constituants et de la concentration, on a pu obtenir une composition de dilution et de stockage de sperme pouvant maintenir une activité de sperme à un niveau égal ou supérieur comparé à un liquide classique de dilution et de stockage de sperme.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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JP2008524719A JP5238501B2 (ja) | 2006-07-12 | 2007-07-10 | 精液希釈保存用組成物 |
Applications Claiming Priority (2)
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JP2006-191992 | 2006-07-12 | ||
JP2006191992 | 2006-07-12 |
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WO2008007463A1 true WO2008007463A1 (fr) | 2008-01-17 |
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PCT/JP2007/000745 WO2008007463A1 (fr) | 2006-07-12 | 2007-07-10 | Composition de dilution et de stockage de sperme |
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WO (1) | WO2008007463A1 (fr) |
Cited By (13)
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JP2014128219A (ja) * | 2012-12-28 | 2014-07-10 | Hiroshima Univ | 精子保存液、精子保存方法及び人工授精方法 |
EP3007556A4 (fr) * | 2013-06-13 | 2017-03-08 | Biomatrica, INC. | Stabilisation de cellules |
US9845489B2 (en) | 2010-07-26 | 2017-12-19 | Biomatrica, Inc. | Compositions for stabilizing DNA, RNA and proteins in saliva and other biological samples during shipping and storage at ambient temperatures |
US9999217B2 (en) | 2010-07-26 | 2018-06-19 | Biomatrica, Inc. | Compositions for stabilizing DNA, RNA, and proteins in blood and other biological samples during shipping and storage at ambient temperatures |
US10064404B2 (en) | 2014-06-10 | 2018-09-04 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
FR3072247A1 (fr) * | 2017-10-13 | 2019-04-19 | Biodesiv Efnium | Polymere antimicrobien pour semences animales |
US10568317B2 (en) | 2015-12-08 | 2020-02-25 | Biomatrica, Inc. | Reduction of erythrocyte sedimentation rate |
JP2020150899A (ja) * | 2019-03-22 | 2020-09-24 | 国立研究開発法人産業技術総合研究所 | 受精用精子液の製造方法及び凍結精子ストローの製造方法 |
WO2021132655A1 (fr) | 2019-12-27 | 2021-07-01 | 国立研究開発法人国立成育医療研究センター | Inhibiteur de la fragmentation d'œufs fertilisés |
CN113273566A (zh) * | 2021-05-31 | 2021-08-20 | 西北农林科技大学 | 一种甘氨酸作为公猪精液常温保存液中有效成分的应用 |
CN114794086A (zh) * | 2022-06-08 | 2022-07-29 | 杭州东源生物科技有限公司 | 一种抗凝集的猪精液稀释粉及其应用 |
CN115251042A (zh) * | 2022-09-08 | 2022-11-01 | 安徽科技学院 | 一种家禽精液稀释缓冲液及其制备和家禽精液冷存方法 |
US12121022B2 (en) | 2023-05-26 | 2024-10-22 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
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US9845489B2 (en) | 2010-07-26 | 2017-12-19 | Biomatrica, Inc. | Compositions for stabilizing DNA, RNA and proteins in saliva and other biological samples during shipping and storage at ambient temperatures |
US9999217B2 (en) | 2010-07-26 | 2018-06-19 | Biomatrica, Inc. | Compositions for stabilizing DNA, RNA, and proteins in blood and other biological samples during shipping and storage at ambient temperatures |
JP2014128219A (ja) * | 2012-12-28 | 2014-07-10 | Hiroshima Univ | 精子保存液、精子保存方法及び人工授精方法 |
EP3007556A4 (fr) * | 2013-06-13 | 2017-03-08 | Biomatrica, INC. | Stabilisation de cellules |
US10064404B2 (en) | 2014-06-10 | 2018-09-04 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
US10772319B2 (en) | 2014-06-10 | 2020-09-15 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
US11672247B2 (en) | 2014-06-10 | 2023-06-13 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
US11116205B2 (en) | 2015-12-08 | 2021-09-14 | Biomatrica, Inc. | Reduction of erythrocyte sedimentation rate |
US10568317B2 (en) | 2015-12-08 | 2020-02-25 | Biomatrica, Inc. | Reduction of erythrocyte sedimentation rate |
US12089588B2 (en) | 2015-12-08 | 2024-09-17 | Biomatrica, Inc. | Reduction of erythrocyte sedimentation rate |
FR3072247A1 (fr) * | 2017-10-13 | 2019-04-19 | Biodesiv Efnium | Polymere antimicrobien pour semences animales |
JP2020150899A (ja) * | 2019-03-22 | 2020-09-24 | 国立研究開発法人産業技術総合研究所 | 受精用精子液の製造方法及び凍結精子ストローの製造方法 |
JP7212886B2 (ja) | 2019-03-22 | 2023-01-26 | 国立研究開発法人産業技術総合研究所 | 受精用精子液の製造方法及び凍結精子ストローの製造方法 |
WO2021132655A1 (fr) | 2019-12-27 | 2021-07-01 | 国立研究開発法人国立成育医療研究センター | Inhibiteur de la fragmentation d'œufs fertilisés |
CN113273566A (zh) * | 2021-05-31 | 2021-08-20 | 西北农林科技大学 | 一种甘氨酸作为公猪精液常温保存液中有效成分的应用 |
CN114794086A (zh) * | 2022-06-08 | 2022-07-29 | 杭州东源生物科技有限公司 | 一种抗凝集的猪精液稀释粉及其应用 |
CN115251042A (zh) * | 2022-09-08 | 2022-11-01 | 安徽科技学院 | 一种家禽精液稀释缓冲液及其制备和家禽精液冷存方法 |
CN115251042B (zh) * | 2022-09-08 | 2023-06-02 | 安徽科技学院 | 一种家禽精液稀释缓冲液及其制备和家禽精液冷存方法 |
US12121022B2 (en) | 2023-05-26 | 2024-10-22 | Biomatrica, Inc. | Stabilization of thrombocytes at ambient temperatures |
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