CN103704203B - Preparation method and using method for GL and TCM199 serving as bovine semen diluent and bovine semen refrigerating fluid - Google Patents
Preparation method and using method for GL and TCM199 serving as bovine semen diluent and bovine semen refrigerating fluid Download PDFInfo
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- CN103704203B CN103704203B CN201410001099.7A CN201410001099A CN103704203B CN 103704203 B CN103704203 B CN 103704203B CN 201410001099 A CN201410001099 A CN 201410001099A CN 103704203 B CN103704203 B CN 103704203B
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Abstract
The invention discloses a preparation method and a using method for GL and TCM199 serving as bovine semen diluent and bovine semen refrigerating fluid. The preparation method comprises preparation of the bovine semen diluent and preparation of the bovine semen refrigerating fluid. The using method comprises the steps of preparing the bovine semen diluent and preparing the bovine semen refrigerating fluid; collecting semen; performing preliminary dilution; performing secondary dilution; and performing refrigeration storage. According to the preparation method and the using method, the defreezing activity of frozen bovine semen is improved, the bovine artificial insemination conception rate is increased, the viscosity of the bovine semen diluent is reduced, and the granular size of the bovine semen diluent is lowered; the operation is simplified; the preparation method and the using method can be used under various large-scale production conditions; the standardization and practicability level of refrigeration of the bovine semen in various scales and the defreezing activity of bovine capillary refrigerated semen are improved; the aberration rate of the defrozen semen is lowered, and the bovine artificial insemination conception rate is increased.
Description
Technical field
The invention belongs to cattle technology of artificial insemination field, more particularly, to a kind of gl and tcm199 as bull semen diluent and
The compound method of freezing liquid and using method.
Background technology
The modes of reproduction of cattle includes this friendship and artificial insemination, artificial insemination divide again fresh join and jelly is joined, entered using straw frozen semen
Row is long-range and concentrates enforcement artificial insemination to be topmost cows modes of reproduction.Therefore, the quality of straw frozen semen is to ensure that manually
The key link of insemination conception rate.The quality of straw frozen semen is subject to for smart bull health status, raise competence, semen quality, tubule
Freeze the impact of smart making link and preservation condition, can be carried out manually controlling in these links in addition to straw frozen semen making, and
Straw frozen semen making link is affected larger by freezing liquid, freezing method.The cryoprotective agent of conventional freezing liquid is glycerol and ovum
Huang, has that viscosity is excessive, yolk lipochondrion is big and uneven in the application, impact sperm motility speed and conception rate.
In other cells and Embryo Cryopreservation Technology research process, find that buffer agent trishydroxymethylaminomethane (tris) delays
Substantially, Sodium Pyruvate can meet the portion of energy of cell and embryo cutting, it is developed and has played notable work for punching effect
With.We utilize the biochemical characteristic of both materials, are introduced in the dilution and cryoprotection system of cattle seminal fluid, and by tight
Lattice test measure it was demonstrated that they dilution and protection to cattle seminal fluid have obvious effect really.
The shortcoming of prior art:
1st, diluent granule is big, viscosity is high.Based on yolk, yolk is not handled by and direct the diluent of prior art
Use, because yolk protein is big, cause the bulk granule in dilution seminal fluid and frozen semen to increase, sperm is often around bulk granule
Assemble and move, reduce the movement velocity of sperm, decrease Free Sperms, decrease the quantity to ovum motile.
2nd, operating technology is complicated.The operating technology of prior art is excessively complicated, is adapted only to factorial praluction, is not suitable for little model
Enclose use.
Content of the invention
The purpose of the embodiment of the present invention is to provide a kind of gl and tcm199 joining as bull semen diluent and freezing liquid
Method processed and using method are it is intended to solve that existing bull semen diluent granule is big, viscosity is high；The complicated problem of operating technology.
The embodiment of the present invention is achieved in that a kind of gl and tcm199 as the preparation of bull semen diluent and freezing liquid
Method, this gl and tcm199 includes as the compound method of bull semen diluent and freezing liquid: the preparation of bull semen diluent and
The preparation of cattle freezing of semen liquid；
The compound method of bull semen diluent comprises the following steps:
Weigh 2 grams of tcm199,40 grams of trishydroxymethylaminomethane, 20 grams of citric acid, Sodium Pyruvate 0.1 with electronic balance
Gram, 5 grams of Fructose, 1 gram of 800,000 units of Penicillin sodium；
Tcm199, trishydroxymethylaminomethane, citric acid, Sodium Pyruvate, Fructose and 800,000 units of Penicillin sodium are loaded
In 1000 milliliters of volumetric flasks, add 500 milliliters of shake dissolvings of distilled water, arrived with distilled water constant volume again after the dissolving of whole solid contents
1000 milliliters；
The good bull semen diluent of constant volume is carried out filtering subpackage, the necessary autoclaving in advance of subpackage vial, dispensed loading amount
For 100 milliliters～200 milliliters；
The compound method of cattle freezing of semen liquid comprises the following steps:
Freezing liquid is divided into the first liquid and the second liquid, and the first liquid directly dilutes fresh essence, and the second liquid is used for seminal fluid and balances；First liquid
Compound method: accurately extract 25 milliliters of bull semen diluent, be injected in 100ml beaker, then extract 5 milliliters of refined vitelline and
0.5 milliliter of glycerol, adds in the beaker filling 25ml bull semen diluent in the lump, with Glass rod agitation 2 minutes counterclockwise, standing
Standby；
Second liquid making method: accurately extract 25 milliliters of bull semen diluent, be injected in 100ml beaker, then extract
5 milliliters of refined vitelline and 3 milliliters of glycerol, add in the beaker filling 25ml bull semen diluent in the lump, counterclockwise with Glass rod
Agitation 2 minutes, stand for standby use.
The another object of the embodiment of the present invention is to provide a kind of gl and tcm199 as bull semen diluent and freezing liquid
Using method, this gl and tcm199 as bull semen diluent and freezing liquid using method comprise the following steps:
The first step, first with electronic balance weigh 2 grams of tcm199,40 grams of trishydroxymethylaminomethane, 20 grams of citric acid,
0.1 gram of Sodium Pyruvate, 5 grams of Fructose, 1 gram of 800,000 units of Penicillin sodium；By trishydroxymethylaminomethane, citric acid, Sodium Pyruvate,
Fructose and 800,000 units of Penicillin sodium load in 1000 milliliters of volumetric flasks, add 500 milliliters of shake dissolvings of distilled water, treat all solid
Distilled water constant volume to 1000 milliliters is used again after the dissolving of shape thing；The good bull semen diluent of constant volume is carried out filtering subpackage, subpackage glass
The necessary autoclaving in advance of glass bottle, dispensed loading amount is 100 milliliters～200 milliliters, determines that preparation cattle is smart according to usage amount and usage frequency
Liquid diluent；
Second step, is divided into the first liquid and the second liquid according to freezing liquid, and the first liquid directly dilutes fresh essence, and the second liquid is used for seminal fluid
Balance；First liquid making method: 25 milliliters of bull semen diluent, 5 milliliters of refined vitelline, 0.5 milliliter of glycerol；Second liquid preparation side
Method: 25 milliliters of bull semen diluent, 5 milliliters of refined vitelline, 3 milliliters of glycerol.
3rd step, semen collection, the fresh semen of the healthy bull of collection；
4th step, primary dilution, by the first liquid having been preheated with 35 DEG C according to 2.5 times of dilutions of volume of semen, couveuse is protected
Deposit or accumulating 1 hour～2 hours；
5th step, secondary dilution, the second liquid is preheating to 35 DEG C, adds same dose of with the first liquid during primary dilution the
To in the primary dilution seminal fluid of isothermal, straw semen is made in tubulature immediately after mixing to two liquid, and straw semen balances in 4 DEG C of cold closetes
Carry out freezen protective after 1.5 hours；
6th step, freezen protective: the straw semen having balanced is freezed with sequencing frigorimeter, freezing is taken out after terminating
Examine and move in liquid nitrogen container and preserved and use.
Gl and tcm199 that the present invention provides, will used as the compound method of bull semen diluent and freezing liquid and using method
Tcm199, glycerol, trishydroxymethylaminomethane and Sodium Pyruvate have been incorporated in cattle semen cryopreservation system；Gl and tcm199
Standardized production system as bull semen diluent and freezing liquid is conducive to large-scale production and popularization and application.The present invention improves
The diluent of cattle seminal fluid and frozen solution and its using method, on the premise of not affecting freezing of semen effect, significantly carry
The high defrosting vigor of cow frozen semen, improves cattle artificial insemination conception rate, situation about declining in China's cow quantity
Under, with more real application value, reduce the viscosity of diluent, reducing the granule of diluent, simplify operation it is adaptable to
All kinds of large-scale production conditions use.The method of the present invention is simple, easy to operate, significantly improves the cattle freezing of semen of various scales
Standardization and practical level；Improve the defrosting vigor of cattle straw frozen semen, reduce abnormal rate and the raising of defrosting sperm
The conception rate of cattle artificial insemination.
Brief description
Fig. 1 is the compound method flow chart of bull semen diluent provided in an embodiment of the present invention；
Fig. 2 is the compound method flow chart of cattle freezing of semen liquid provided in an embodiment of the present invention；
Fig. 3 is the using method stream as bull semen diluent and freezing liquid for gl and tcm199 provided in an embodiment of the present invention
Cheng Tu.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not used to
Limit the present invention.
Below in conjunction with the accompanying drawings and specific embodiment is further described to the application principle of the present invention.
As shown in figure 1, the compound method of the bull semen diluent of the embodiment of the present invention comprises the following steps:
S101: with electronic balance accurately weigh 2 grams of tcm199,40 grams of trishydroxymethylaminomethane, 20 grams of citric acid, third
0.1 gram of keto acid sodium, 5 grams of Fructose, 1 gram of 800,000 units of Penicillin sodium；
S102: said components are loaded in 1000 milliliters of volumetric flasks, adds 500 milliliters of shake dissolvings of distilled water, treat all
Distilled water constant volume to 1000 milliliters is used again after solid content dissolving；
S103: the good bull semen diluent of constant volume is carried out filtering subpackage, the necessary autoclaving in advance of subpackage vial, point
Loading amount is 100 milliliters～200 milliliters, determines according to usage amount and usage frequency.
As shown in Fig. 2 the compound method of the cattle freezing of semen liquid of the embodiment of the present invention comprises the following steps:
S201: freezing liquid is divided into the first liquid and the second liquid, and the first liquid directly dilutes fresh essence, and the second liquid is used for seminal fluid and balances；
First liquid making method: 25 milliliters of bull semen diluent, 5 milliliters of refined vitelline, 0.5 milliliter of glycerol；
S202: the second liquid making method: 25 milliliters of bull semen diluent, 5 milliliters of refined vitelline, 3 milliliters of glycerol.
As shown in figure 3, gl and tcm199 of the embodiment of the present invention is as the using method of bull semen diluent and freezing liquid
Comprise the following steps:
S301: prepare gl and tcm199 as bull semen diluent and freezing liquid；
S302: semen collection, the fresh semen of the healthy bull of collection；
S303: primary dilution, by the first liquid having been preheated with 35 DEG C according to 2.5 times of dilutions of volume of semen, couveuse preserves
Or accumulating 1 hour～2 hours；
S304: secondary dilution, the second liquid is preheating to 35 DEG C, adds and the first liquid same dose of second during primary dilution
To in the primary dilution seminal fluid of isothermal, straw semen is made in tubulature immediately after mixing to liquid, and straw semen balances in 4 DEG C of cold closetes
Carry out freezen protective after 1.5 hours.
S305: freezen protective: the straw semen having balanced is freezed with sequencing frigorimeter, freezing is inspected by random samples after terminating
And move into and preserved in liquid nitrogen container and use.
Method particularly includes:
The first step, first with electronic balance weigh 2 grams of tcm199,40 grams of trishydroxymethylaminomethane, 20 grams of citric acid,
0.1 gram of Sodium Pyruvate, 5 grams of Fructose, 1 gram of 800,000 units of Penicillin sodium；By tcm199, trishydroxymethylaminomethane, citric acid, third
Keto acid sodium, Fructose and 800,000 units of Penicillin sodium load in 1000 milliliters of volumetric flasks, add 500 milliliters of shake dissolvings of distilled water,
Distilled water constant volume to 1000 milliliters is used again after the dissolving of whole solid contents；The good bull semen diluent of constant volume is carried out filtering and divides
Dress, the necessary autoclaving in advance of subpackage vial, dispensed loading amount is 100 milliliters～200 milliliters, determines according to usage amount and usage frequency
Prepare bull semen diluent；
Second step, is divided into the first liquid and the second liquid according to freezing liquid, and the first liquid directly dilutes fresh essence, and the second liquid is used for seminal fluid
Balance；Freezing liquid is divided into the first liquid and the second liquid, and the first liquid directly dilutes fresh essence, and the second liquid is used for seminal fluid and balances；First liquid is joined
Method processed: 25 milliliters of bull semen diluent, 5 milliliters of refined vitelline, 0.5 milliliter of glycerol；Second liquid making method: cattle seminal fluid is dilute
Release 25 milliliters of liquid, 5 milliliters of refined vitelline, 3 milliliters of glycerol.
3rd step, semen collection, the fresh semen of the healthy bull of collection；
4th step, primary dilution, by the first liquid having been preheated with 35 DEG C according to 2.5 times of dilutions of volume of semen, couveuse is protected
Deposit or accumulating 1 hour～2 hours；
5th step, secondary dilution, the second liquid is preheating to 35 DEG C, adds same dose of with the first liquid during primary dilution the
To in the primary dilution seminal fluid of isothermal, straw semen is made in tubulature immediately after mixing to two liquid, and straw semen balances in 4 DEG C of cold closetes
Carry out freezen protective after 1.5 hours；
6th step, freezen protective: the straw semen having balanced is freezed with sequencing frigorimeter, freezing is taken out after terminating
Examine and move in liquid nitrogen container and preserved and use.
In conjunction with specific examples below, the using effect of the present invention is made additional remarks:
Popularization and application are carried out according to the cattle straw frozen semen that the present invention produces in Gansu Province Pingliang and Qingyang City.Wherein thin
The sperm anabiosis rate that pipe freezes essence is 62.64%, and defrosting vigor is 54.23%, and acrosomal integrity is 79.84%；Accumulative tubule of promoting freezes
99000 pieces of essence, average non-return rate reaches 78.70%, and average calving rate reaches 95.84%, is all remarkably higher than other types of
The application effect of cattle straw frozen semen.
Invention significantly improves the standardization of cattle freezing of semen of various scales and practical level；Improve cattle tubule
Freeze defrosting vigor, the abnormal rate reducing defrosting sperm and the conception rate that improve cattle artificial insemination of essence.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (1)
1. a kind of gl and tcm199 as bull semen diluent and freezing liquid compound method it is characterised in that this gl and
Tcm199 includes as the compound method of bull semen diluent and freezing liquid: the preparation of bull semen diluent and cattle freezing of semen liquid
Preparation；The compound method of bull semen diluent comprises the following steps: weighs 2 grams of tcm199, trihydroxy methyl ammonia with electronic balance
40 grams of methylmethane, 20 grams of citric acid, 0.1 gram of Sodium Pyruvate, 5 grams of Fructose, 1 gram of 800,000 units of Penicillin sodium；By tcm199, three hydroxyls
Aminomethane, citric acid, Sodium Pyruvate, Fructose and 800,000 units of Penicillin sodium load in 1000 milliliters of volumetric flasks, add
500 milliliters of shake dissolvings of distilled water, use distilled water constant volume to 1000 milliliters after the dissolving of whole solid contents again；By the good cattle of constant volume
Semen diluent carries out filtering subpackage, the necessary autoclaving in advance of subpackage vial, and dispensed loading amount is 100 milliliters～200 milliliters；Cattle
The compound method of freezing of semen liquid comprises the following steps: freezing liquid is divided into the first liquid and the second liquid, and the first liquid directly dilutes fresh essence,
Second liquid is used for seminal fluid and balances；First liquid making method: accurately extract 25 milliliters of bull semen diluent, be injected into 100ml and burn
In cup, then extract 5 milliliters of refined vitelline and 0.5 milliliter of glycerol, add in the lump in the beaker filling 25ml bull semen diluent, use
Glass rod agitation 2 minutes counterclockwise, stand for standby use；Second liquid making method: accurately extract 25 milliliters of bull semen diluent, by it
In injection 100ml beaker, then extract 5 milliliters of refined vitelline and 3 milliliters of glycerol, add in the lump and fill 25ml bull semen diluent
In beaker, with Glass rod agitation 2 minutes counterclockwise, stand for standby use；
This gl and tcm199 as bull semen diluent and freezing liquid using method comprise the following steps: the first step, first
Weigh 2 grams of tcm199,40 grams of trishydroxymethylaminomethane, 20 grams of citric acid, 0.1 gram of Sodium Pyruvate, Fructose 5 with electronic balance
Gram, 1 gram of 800,000 units of Penicillin sodium；Tcm199, trishydroxymethylaminomethane, citric acid, Sodium Pyruvate, Fructose and 800,000 are single
Position penicillin sodium loads in 1000 milliliters of volumetric flasks, adds 500 milliliters of shake dissolvings of distilled water, after the dissolving of whole solid contents again
With distilled water constant volume to 1000 milliliters；The good bull semen diluent of constant volume is carried out filtering subpackage, subpackage vial must be in advance
Autoclaving, dispensed loading amount is 100 milliliters～200 milliliters, determines according to usage amount and usage frequency and prepares bull semen diluent；Second
Step, is divided into the first liquid and the second liquid according to freezing liquid, the first liquid directly dilutes fresh essence, and the second liquid is used for seminal fluid and balances；First liquid
Compound method: 25 milliliters of bull semen diluent, 5 milliliters of refined vitelline, 0.5 milliliter of glycerol；Second liquid making method: cattle seminal fluid is dilute
Release 25 milliliters of liquid, 5 milliliters of refined vitelline, 3 milliliters of glycerol；3rd step, semen collection, the fresh semen of the healthy bull of collection；4th step,
Primary dilution, by the first liquid having been preheated with 35 DEG C according to 2.5 times of dilutions of volume of semen, couveuse preserves or accumulating 1 hour～2
Hour；5th step, secondary dilution, the second liquid is preheating to 35 DEG C, adds and the first liquid same dose of second during primary dilution
To in the primary dilution seminal fluid of isothermal, straw semen is made in tubulature immediately after mixing to liquid, and straw semen balances in 4 DEG C of cold closetes
Carry out freezen protective after 1.5 hours；6th step, freezen protective: the straw semen having balanced is carried out cold with sequencing frigorimeter
Freeze, freezing is inspected by random samples and moved into after terminating and preserved in liquid nitrogen container and use.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410001099.7A CN103704203B (en) | 2014-01-03 | 2014-01-03 | Preparation method and using method for GL and TCM199 serving as bovine semen diluent and bovine semen refrigerating fluid |
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|---|---|---|---|
| CN201410001099.7A CN103704203B (en) | 2014-01-03 | 2014-01-03 | Preparation method and using method for GL and TCM199 serving as bovine semen diluent and bovine semen refrigerating fluid |
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| CN103704203A CN103704203A (en) | 2014-04-09 |
| CN103704203B true CN103704203B (en) | 2017-01-18 |
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| CN107183007B (en) * | 2017-05-25 | 2021-05-04 | 中国热带农业科学院热带作物品种资源研究所 | Method for making frozen semen of goat through thin tube |
| CN107333752A (en) * | 2017-08-08 | 2017-11-10 | 重庆信首科技有限公司 | Kind ox seminal fluid Cryopreservation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2008117026A1 (en) * | 2007-03-23 | 2008-10-02 | The Royal Veterinary College | Method for enhancing sperm survival |
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2014
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008117026A1 (en) * | 2007-03-23 | 2008-10-02 | The Royal Veterinary College | Method for enhancing sperm survival |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| T CM 1 9 9 对奶牛冻精活力的研究;邱忠玉 等;《中国畜禽种业》;20080228(第2期);第24-26页 * |
| 丙酮酸盐对牛***冷冻效果的影响;胡传活 等;《河南农业科学》;20060731(第7期);第105-108页 * |
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