CN101999342B - Novel preparation method of yolk diluent - Google Patents
Novel preparation method of yolk diluent Download PDFInfo
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- CN101999342B CN101999342B CN2010102992906A CN201010299290A CN101999342B CN 101999342 B CN101999342 B CN 101999342B CN 2010102992906 A CN2010102992906 A CN 2010102992906A CN 201010299290 A CN201010299290 A CN 201010299290A CN 101999342 B CN101999342 B CN 101999342B
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Abstract
The invention discloses a novel preparation method of yolk diluent, belonging to the field of animal reproductive biology. The novel preparation method comprises the following steps of: preparing yolk: preparing 4.6 times of Tris-A mother solution and 6 times of Tris-B mother solution and carrying out the aseptic filtration; diluting into 0.5 time of Tris-A mother solution and Tris-B mother solution with sterile water; respectively mixing with the yolk, stirring, standing, centrifuging and obtaining supernatant; respectively adjusting the concentration of the supernatant with 4.6 times of Tris-A mother solution and 6 times of Tris-B mother solution to obtain 1 time of Tris-A mother solution and Tris-B mother solution; and respectively adding antibiotic and sterile glycerin, adjusting the pH value and storing the mixture at 4DEG C for later use. The method omits the filtering step and is simple; fewer granules exist in the yolk diluent; and the method ensures the motility rate of cryopreservation sperms, improves the quality of froze sperm products and reduces the production cost.
Description
Technical field:
The invention belongs to animal reproduction biological technical field, be applicable to that ox freezes the preparation of dilution in smart the production, a kind of compound method of yolk diluent mainly is provided.
Background technology:
Dilution is the main and necessary reagent during cow frozen semen is produced, and yolk diluent is widely used in the production of obstinacy control frozen semen because of sperm there being the better protection effect.The quality of a lot of granule foreign composition influence dilutions and the detection of product are arranged in the yolk diluent, mainly remove these impurity particles at present through the centrifugal filtration means that add.Because of containing a large amount of organic principles in the yolk, filter with the sterile filters of 0.22 μ m, not only take time and effort, and also with high costs, developed should technology for this reason.
Summary of the invention:
The present invention provides a kind of yolk diluent compound method that ox is frozen smart production that is used for; This know-why is to utilize salt ionic concentration that the gathering of particulate component in the yolk is played an important role; Low salt ionic concentration helps agglomeration of particles, thereby can so just can save the step of filtration through the most of particle of centrifugal removal; Reduce cost, use manpower and material resources sparingly.
The objective of the invention is to realize through following technical scheme: a kind of compound method of yolk diluent is characterized in that: it comprises the steps:
1) preparation of yolk: choose new fresh hen egg, with eggshell with 75% alcohol disinfecting, shell breaking, egg white under aseptic condition; Blot with the egg white of filter paper of sterilizing, extract yolk with asepsis injector with the vitellinae membrana surface;
2) preparation of 4.6 times of Tris-A and 6 times of Tris-B mother liquors:
Prepare 4.6 times of Tris-A mother liquors according to above-mentioned prescription with distilled water, 6 times of Tris-B mother liquors, adjust pH is 6.80 ± 0.02 respectively, filters with 0.22 μ m sterile filters;
3) preparation of 0.5 times of Tris-A solution and 0.5 times of Tris-B solution: using sterile water that 4.6 times of Tris-A mother liquors and 6 times of Tris-B mother liquors are diluted respectively is 0.5 times of Tris-A solution and 0.5 times of Tris-B solution, and the solution for preparing is preserved under 4 ℃ of environment, and is subsequent use;
4) preparation of Tris-A, Tris-B dilution
1. the preparation of Tris-A dilution
0.5 times of Tris-A solution and the yolk volume ratio by 7: 2 is added in the sterile chamber; Stirred 2 hours with 400-500 rev/min with magnetic stirring apparatus down in 4 ℃ of environment, leave standstill 20h again, then with 4 ℃ of supernatants, the centrifugal 60min of 3850g; Get supernatant; And press 4.6 times of Tris-A mother liquors of volume ratio adding of 1: 9.4, and shake up, add antibiotic: gentamicin final concentration 500 μ g/ml; Lincomycin final concentration 300 μ g/ml; Miramycin final concentration 600 μ g/ml; Tylosin final concentration 100 μ g/ml, adjust pH is 6.80 ± 0.02, in 4 ℃ of refrigerators, preserves subsequent use;
2. the preparation of Tris-B dilution
0.5 times of Tris-B solution and the yolk volume ratio by 7: 2.3 is added in the sterile chamber, in 4 ℃ of environment down with magnetic stirring apparatus with 400-500 rev/min of stirring 2 hours, leave standstill 20h again; With 4 ℃ of supernatants, the centrifugal 60min of 3850g, get supernatant then, and add 6 times of Tris-B mother liquors by 7: 93 volume ratio; Add aseptic glycerine again; The final ratio of glycerine is 12%v/v, shakes up, and adds antibiotic: gentamicin final concentration 26.6 μ g/ml; Lincomycin final concentration 15.9 μ g/ml; Miramycin final concentration 31.9 μ g/ml; Tylosin final concentration 5.3 μ g/ml, adjust pH is 6.80 ± 0.02, in 4 ℃ of refrigerators, preserves subsequent use.
A kind of yolk diluent freezes smart and conventional application of freezing in smart the production in the obstinacy control.
The invention has the beneficial effects as follows: this know-why is to utilize salt ionic concentration that the gathering of particulate component in the yolk is played an important role, and low salt ionic concentration helps agglomeration of particles, thereby can be through the most of particle of centrifugal removal; So just can save the step of filtration; This yolk diluent compound method is simple, and the yolk diluent particle that makes simultaneously is few, guarantees the motility rate of frozen sperm; Improved and frozen smart product quality, reduced production cost.
Description of drawings
Fig. 1 is that ox number 11103832 (X) use novel yolk diluent and former yolk dilution microscope figure.A: novel yolk diluent b: former yolk dilution.
Fig. 2 is that ox YE025 (X) uses novel yolk diluent and former yolk dilution microscope figure.A: novel yolk diluent b: former yolk dilution.
Fig. 3 is that ox number 11102390 (X) use novel yolk diluent and former yolk dilution microscope figure.A: novel yolk diluent b: former yolk dilution.
Embodiment
Embodiment: a kind ofly be used for the compound method that ox is frozen the smart yolk diluent of producing, it comprises the steps:
1, the preparation of yolk: choose new fresh hen egg, with the alcohol disinfecting of eggshell with 75% (v/v), shell breaking, egg white under aseptic condition blot with the egg white of filter paper with the vitellinae membrana surface of sterilizing, extract yolk with asepsis injector;
2, the preparation of 4.6 times of Tris-A and 6 times of Tris-B mother liquors:
Table 1 100ml 4.6 * Tris-A mother liquor prescription
| Nomenclature of drug | Weight (g) |
| Tris?base | 16.25 |
| Citric acid | 7.92 |
| D-Fructose | 5.82 |
Table 2 100ml 6 * Tris-B mother liquor prescription
| Nomenclature of drug | Weight (g) |
| Tris?base | 21.45 |
| Citric acid | 11.99 |
| D-Fructose | 8.83 |
With 4.6 times of Tris-A mother liquors of distilled water preparation, 6 times of Tris-B mother liquors, adjust pH is 6.80 ± 0.02 respectively, filters with 0.22 μ m sterile filters according to last table prescription;
3, the preparation of 0.5 times of Tris-A solution and 0.5 times of Tris-B solution: using sterile water that 4.6 times of Tris-A mother liquors and 6 times of Tris-B mother liquors are diluted respectively is 0.5 times of Tris-A solution and 0.5 times of Tris-B solution, and the solution for preparing preserves under 4 ℃ of environment, subsequent use;
4.Tris-A, the preparation of Tris-B dilution:
(1) preparation of Tris-A dilution
0.5 times of Tris-A solution and the yolk volume ratio by 7: 2 is added in the sterile chamber; Stirred 2 hours with 400-500 rev/min with magnetic stirring apparatus down in 4 ℃ of environment, after leaving standstill 20h under 4 ℃ of conditions, 4 ℃ of supernatants, the centrifugal 60min of 3850g; Get supernatant; And press 4.6 times of Tris-A mother liquors of volume ratio adding of 1: 9.4, and shake up, add antibiotic: gentamicin final concentration 500 μ g/ml; Lincomycin final concentration 300 μ g/ml; Miramycin final concentration 600 μ g/ml; Tylosin final concentration 100 μ g/ml, adjust pH is 6.80 ± 0.02, in 4 ℃ of refrigerators, preserves subsequent use;
(2) preparation of Tris-B dilution
0.5 times of Tris-B solution and the yolk volume ratio by 7: 2.3 is added in the sterile chamber, in 4 ℃ of environment down with magnetic stirring apparatus with 400-500 rev/min of stirring 2 hours, after leaving standstill 20h under 4 ℃ of conditions; With 4 ℃ of supernatants, the centrifugal 60min of 3850g, get supernatant, and add 6 times of Tris-B mother liquors by 7: 93 volume ratio; Add aseptic glycerine in proportion; The final ratio of glycerine is 12% (v/v), shakes up, and adds antibiotic: gentamicin final concentration 26.6 μ g/ml; Lincomycin final concentration 15.9 μ g/ml; Miramycin final concentration 31.9 μ g/ml; Tylosin final concentration 5.3 μ g/ml, adjust pH is 6.80 ± 0.02, in 4 ℃ of refrigerators, preserves subsequent use.
Application example 1: be numbered 11103832 stock bull X sperms with former dilution and novel dilution production; According to freezing smart flow process frozen semen (People's Republic of China's agricultural industry criteria; NY/T1234-2006); Behind the semen freezing 24h, detect seminal fluid each item quality index, comprising the back vigor that thaws, back 4h vigor, time-to-live and bacterial population thaw; The result shows that two kinds of yolk diluents are consistent to the back vigor that freezes, 4h vigor, the cryoprotection effect of time-to-live of same stock bull seminal fluid, and bacterial population does not have marked difference; And novel yolk diluent amounts of particles obviously is less than former yolk dilution.
The different yolk diluents of table 3 are to freezing the influence of extract Iuality
| Yolk diluent | Freeze the back vigor | 4 hours vigor | Time-to-live | Bacterial population |
| New dilution | 0.60 | 0.30 | T＝5.5 | 0 |
| Former dilution | 0.61 | 0.20 | T＝5.5 | 11 |
Application examples 2: be numbered YE025 stock bull X sperm with former dilution and novel dilution production; According to freezing smart flow process frozen semen; Behind the semen freezing 24h, detect seminal fluid each item quality index, comprising the back vigor that thaws, back 4h vigor, time-to-live and bacterial population thaw.The result shows that two kinds of yolk diluents are consistent to the back vigor that freezes, 4h vigor, the cryoprotection effect of time-to-live of same stock bull seminal fluid, and bacterial population does not have marked difference.And novel yolk diluent amounts of particles obviously is less than former yolk dilution.
The different yolk diluents of table 4 are to freezing the influence of extract Iuality
| Yolk diluent | Freeze the back vigor | 4 hours vigor | Time-to-live | Bacterial population |
| New dilution | 0.58 | 0.28 | T＝5 | 1 |
| Former dilution | 0.55 | 0.26 | T＝5.5 | 15 |
Application examples 3: be numbered 11102390 stock bull X sperms with former dilution and novel dilution production; According to freezing smart flow process frozen semen; Behind the semen freezing 24h, detect seminal fluid each item quality index, comprising the back vigor that thaws, back 4h vigor, time-to-live and bacterial population thaw.The result shows that two kinds of yolk diluents are consistent to the back vigor that freezes, 4h vigor, the cryoprotection effect of time-to-live of same stock bull seminal fluid, and bacterial population does not have marked difference.And novel yolk diluent amounts of particles obviously is less than former yolk dilution.
The different yolk diluents of table 5 are to freezing the influence of extract Iuality
| Yolk diluent | Freeze the back vigor | 4 hours vigor | Time-to-live | Bacterial population |
| New dilution | 0.54 | 0.20 | T＝5 | 3 |
| Former dilution | 0.55 | 0.27 | T＝5 | 0 |
Claims (1)
1. the compound method of a yolk diluent, it is characterized in that: it comprises the steps:
1) preparation of yolk: choose new fresh hen egg, with eggshell with 75% alcohol disinfecting, shell breaking, egg white under aseptic condition; Blot with the egg white of filter paper of sterilizing, extract yolk with asepsis injector with the vitellinae membrana surface;
2) preparation of 4.6 times of Tris-A and 6 times of Tris-B mother liquors:
Prepare 4.6 times of Tris-A mother liquors according to above-mentioned prescription with distilled water, 6 times of Tris-B mother liquors, adjust pH is 6.80 ± 0.02 respectively, filters with 0.22 μ m sterile filters;
3) preparation of 0.5 times of Tris-A solution and 0.5 times of Tris-B solution: using sterile water that 4.6 times of Tris-A mother liquors and 6 times of Tris-B mother liquors are diluted respectively is 0.5 times of Tris-A solution and 0.5 times of Tris-B solution, and the solution for preparing is preserved under 4 ℃ of environment, and is subsequent use;
4) preparation of Tris-A, Tris-B dilution
1. the preparation of Tris-A dilution
0.5 times of Tris-A solution and the yolk volume ratio by 7: 2 is added in the sterile chamber; Stirred 2 hours with 400-500 rev/min with magnetic stirring apparatus down in 4 ℃ of environment, leave standstill 20h again, then with 4 ℃ of supernatants, the centrifugal 60min of 3850g; Get supernatant; And press 4.6 times of Tris-A mother liquors of volume ratio adding of 1: 9.4, and shake up, add antibiotic: gentamicin final concentration 500 μ g/ml; Lincomycin final concentration 300 μ g/ml; Miramycin final concentration 600 μ g/ml; Tylosin final concentration 100 μ g/ml, adjust pH is 6.80 ± 0.02, in 4 ℃ of refrigerators, preserves subsequent use;
2. the preparation of Tris-B dilution
0.5 times of Tris-B solution and the yolk volume ratio by 7: 2.3 is added in the sterile chamber, in 4 ℃ of environment down with magnetic stirring apparatus with 400-500 rev/min of stirring 2 hours, leave standstill 20h again; With 4 ℃ of supernatants, the centrifugal 60min of 3850g, get supernatant then, and add 6 times of Tris-B mother liquors by 7: 93 volume ratio; Add aseptic glycerine again; The final ratio of glycerine is 12%v/v, shakes up, and adds antibiotic: gentamicin final concentration 26.6 μ g/ml; Lincomycin final concentration 15.9 μ g/ml; Miramycin final concentration 31.9 μ g/ml; Tylosin final concentration 5.3 μ g/ml, adjust pH is 6.80 ± 0.02, in 4 ℃ of refrigerators, preserves subsequent use.
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| CN2010102992906A CN101999342B (en) | 2010-10-08 | 2010-10-08 | Novel preparation method of yolk diluent |
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| CN2010102992906A CN101999342B (en) | 2010-10-08 | 2010-10-08 | Novel preparation method of yolk diluent |
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| CN101999342B true CN101999342B (en) | 2012-11-07 |
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| CN103518706B (en) * | 2013-10-21 | 2015-04-01 | 西北农林科技大学 | Diluent of frozen semen of milk goat, and preparation method and diluting method thereof as well as preparation method of frozen semen of milk goat in thin pipe |
| CN105285665A (en) * | 2015-10-25 | 2016-02-03 | 钟静涛 | Fishiness removing method of egg powder |
| CN106538511A (en) * | 2016-09-30 | 2017-03-29 | 马伟 | A kind of manufacture method of antioxidative sheep freezing seminal fluid dilution |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1927226A (en) * | 2006-09-11 | 2007-03-14 | 华中农业大学 | Cow sex-control sperm freezing preservation seminal fluid dilution |
| CN101167747A (en) * | 2007-10-16 | 2008-04-30 | 上海金晖家畜遗传开发有限公司 | Bull semen diluent and its configuration method and adding method |
| CN101411722A (en) * | 2008-10-22 | 2009-04-22 | 李喜和 | Freezing mixed sperm for controlling X/Y gender of milk cattle and preparation method thereof |
| CN101554152A (en) * | 2009-05-21 | 2009-10-14 | 西北农林科技大学 | Frozen semen diluent and preparation method thereof |
| CN101810163A (en) * | 2010-05-10 | 2010-08-25 | 广西大学 | Method for freezing semen of Macaca fascicularis |
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- 2010-10-08 CN CN2010102992906A patent/CN101999342B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1927226A (en) * | 2006-09-11 | 2007-03-14 | 华中农业大学 | Cow sex-control sperm freezing preservation seminal fluid dilution |
| CN101167747A (en) * | 2007-10-16 | 2008-04-30 | 上海金晖家畜遗传开发有限公司 | Bull semen diluent and its configuration method and adding method |
| CN101411722A (en) * | 2008-10-22 | 2009-04-22 | 李喜和 | Freezing mixed sperm for controlling X/Y gender of milk cattle and preparation method thereof |
| CN101554152A (en) * | 2009-05-21 | 2009-10-14 | 西北农林科技大学 | Frozen semen diluent and preparation method thereof |
| CN101810163A (en) * | 2010-05-10 | 2010-08-25 | 广西大学 | Method for freezing semen of Macaca fascicularis |
Non-Patent Citations (2)
| Title |
|---|
| 不同种公牛X/Y精子分离效果及对体外受精能力的影响;吴冬生等;《中国奶牛》;20091231(第4期);第11-15页 * |
| 吴冬生等.不同种公牛X/Y精子分离效果及对体外受精能力的影响.《中国奶牛》.2009,(第4期),第11-15页. |
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Address after: 011517 the Inner Mongolia Autonomous Region Helingeer County of Hohhot city Shengle Economic Zone Development Avenue North Building 6 floor of Mengniu Applicant after: Saikexing Reproductive Biotechnology CO., Ltd. Address before: 011517 the Inner Mongolia Autonomous Region Helingeer County of Hohhot city Shengle Economic Zone Development Avenue North Building 6 floor of Mengniu Applicant before: Mengniu Reproductive Biltechnology Co., Ltd. |
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Address after: 011517 the Inner Mongolia Autonomous Region Helingeer County of Hohhot city Shengle economic Park gathering service area management committee building 4 New South Building Room 4422 Patentee after: Inner Mongolia saikexing reproductive biotechnology (Group) Limited by Share Ltd Address before: 011517 the Inner Mongolia Autonomous Region Helingeer County of Hohhot city Shengle Economic Zone Development Avenue North Building 6 floor of Mengniu Patentee before: Saikexing Reproductive Biotechnology CO., Ltd. |