CN102696577B - Bovine semen protective agent - Google Patents
Bovine semen protective agent Download PDFInfo
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- CN102696577B CN102696577B CN 201210162654 CN201210162654A CN102696577B CN 102696577 B CN102696577 B CN 102696577B CN 201210162654 CN201210162654 CN 201210162654 CN 201210162654 A CN201210162654 A CN 201210162654A CN 102696577 B CN102696577 B CN 102696577B
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- Prior art keywords
- mother liquor
- seminal fluid
- semen
- hours
- protectant
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- 210000000582 semen Anatomy 0.000 title claims abstract description 88
- 241000283690 Bos taurus Species 0.000 title abstract description 11
- 239000003223 protective agent Substances 0.000 title abstract 4
- 239000012452 mother liquor Substances 0.000 claims abstract description 45
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 210000002969 egg yolk Anatomy 0.000 claims abstract description 9
- 102000002322 Egg Proteins Human genes 0.000 claims abstract description 7
- 108010000912 Egg Proteins Proteins 0.000 claims abstract description 7
- 235000013345 egg yolk Nutrition 0.000 claims abstract description 7
- 230000014759 maintenance of location Effects 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000003860 storage Methods 0.000 claims description 2
- 238000007710 freezing Methods 0.000 abstract description 9
- 230000008014 freezing Effects 0.000 abstract description 9
- 238000002156 mixing Methods 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 230000002035 prolonged effect Effects 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 230000035899 viability Effects 0.000 description 11
- 244000309464 bull Species 0.000 description 10
- 210000005239 tubule Anatomy 0.000 description 10
- 238000012545 processing Methods 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 239000006101 laboratory sample Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 7
- 239000013068 control sample Substances 0.000 description 7
- 238000012856 packing Methods 0.000 description 7
- 231100000225 lethality Toxicity 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000001816 cooling Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- 238000010792 warming Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a protective agent for prolonging the vitality retention period of fresh bovine semen, which is prepared by mixing a mother liquor 1, a mother liquor 2 and fresh egg yolk liquid according to a certain proportion. Due to the adoption of the protective agent provided by the invention, the vitality of fresh bovine semen can still be maintained to be higher than 65% within 16 hours after semen collection, and the semen mortality of the fresh bovine semen is controlled to be 5% to 13% within 16 hours after semen collection. The results show that by using the bovine semen protective agent provided by the invention, the vitality retention period of the fresh bovine semen can be effectively prolonged, so that the long-distance transport is convenient, and a technical support and guarantee is provided for subsequent semen freezing treatment or sex-controlling separation treatment.
Description
Technical field
The present invention relates to ox propagation technique field, specifically, relate to a kind of protectant that the fresh bovine sperm viability keeps the phase that prolongs.
Background technology
The new ox seminal fluid of gathering was generally at room temperature preserved in 8 hours, and seminal fluid can keep enough vigor in order to carry out follow-up precision processing or the property control separating treatment of freezing.But after preservation surpasses 8 hours at normal temperatures after the semen collection, sperm viability will descend rapidly, can't carry out conventional frozen semen preservation again and handle, and also can't be used for the property control separation of flow cytometer more effectively.Therefore, make in fresh semen under the situation of land used and semen collection ground wide apart, how to realize that the long-distance keep-alive transportation of fresh semen becomes the technical problem that needs to be resolved hurrily.
Summary of the invention
The purpose of this invention is to provide a kind of protectant that the fresh bovine sperm viability keeps the phase that prolongs.
In order to realize the object of the invention, a kind of ox seminal fluid protectant of the present invention, it comprises mother liquor 1, mother liquor 2 and egg yolk liquid, the prescription of described mother liquor 1 and mother liquor 2 sees Table 1 and table 2:
The prescription of table 1 mother liquor 1
The prescription of table 2 mother liquor 2
PH value 7.4 is prepared with water.
Preferably, the prescription of mother liquor 1 and mother liquor 2 sees Table 3 and table 4:
The prescription of table 3 mother liquor 1
PH value 6.8 is prepared with water;
The prescription of table 4 mother liquor 2
PH value 7.4 is prepared with water.
Preferably, the volume ratio of mother liquor 1, mother liquor 2 and egg yolk liquid is 5:4:1 in the described ox seminal fluid protectant.
The present invention also provides the above-mentioned protectant method of ox seminal fluid: according to formulated mother liquor 1 and the mother liquor 2 shown in the table, place 4 ℃ of preservations respectively.
The present invention further provides the application of above-mentioned protectant in the ox seminal fluid is fresh-keeping; it is with mother liquor 1, mother liquor 2 and egg yolk liquid mixed mixed liquor in proportion in 24 hours before use; add this mixed liquor of 0.25～0.5 times of ox seminal fluid volume then in the ox seminal fluid, mixing is placed on 3～6 ℃ of storages.
Adopt protectant provided by the invention that the vigor of fresh bovine seminal fluid is still remained on more than 65% in 16 hours after semen collection, and the sperm lethality that makes the fresh bovine seminal fluid after semen collection 16 hours inner control at 5-13%.The result shows, uses ox seminal fluid protectant of the present invention, can effectively prolong the fresh bovine sperm viability and keep the phase, is convenient to long-distance transport, freezes precision processing or property control separating treatment provides technical support and guarantee for follow-up.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
If do not specialize, the conventional means that used technological means is well known to those skilled in the art among the embodiment, agents useful for same and raw material are the commercial goods.
The preparation of 1 N of seminal fluid protectant 562 of embodiment
The protectant prescription of table 5
The prescription of table 3 mother liquor 1
PH value 6.8 is prepared with water;
The prescription of table 4 mother liquor 2
PH value 7.4 is prepared with water;
Wherein, mother liquor 1 and mother liquor 2 are prepared and in advance in 4 ℃ of preservations; The fresh-laid egg yellow liquor adds in 24 hours before use.
2 Ns of protectant preparations of seminal fluid of embodiment
The protectant prescription of table 5
The prescription of table 6 mother liquor 1
PH value 6.8 is prepared with water;
The prescription of table 7 mother liquor 2
PH value 7.4 is prepared with water;
Wherein, mother liquor 1 and mother liquor 2 are prepared and in advance in 4 ℃ of preservations; The fresh-laid egg yellow liquor adds in 24 hours before use.
3 Ns of protectant preparations of seminal fluid of embodiment
The protectant prescription of table 5
The prescription of table 8 mother liquor 1
PH value 6.8 is prepared with water;
The prescription of table 9 mother liquor 2
PH value 7.4 is prepared with water;
Wherein, mother liquor 1 and mother liquor 2 are prepared and in advance in 4 ℃ of preservations; The fresh-laid egg yellow liquor adds in 24 hours before use.
The application of 4 Ns of seminal fluid protectants 562 of embodiment
1, material and reagent
Seminal fluid source: He Sitan stock bull Rapture, available from Canadian WESTENG company; Chemical reagent is available from Sigma company.
2, experimental technique
The fresh semen 10ml that picks up from the Rapture stock bull is divided into two parts, is respectively charged in the 15ml centrifuge tube, portion is laboratory sample, and another part is control sample.Add the protectant 562 of 1.25ml embodiment 1 in the 5ml seminal fluid of laboratory sample, screw lid, jiggle to make and mix the processing of lowering the temperature then.Cooling step is: the centrifuge tube that seminal fluid and protectant mixture will be housed places in the 500ml plastic bottle that is full of 25 ° of C warm water, screws lid, and this plastic bottle is placed in 4 ° of C refrigerating chambers.
Under 4 ° of C, place the mother liquor of respectively getting after 10 hours, 16 hours, 24 hours, 30 hours among the 10 μ l samples usefulness embodiment 12 and dilute 50 times, get about 20 μ l dilute samples and place on 37 ℃ of warming plates, at the vigor (percentage of forward motile sperm) of test under microscope sperm.The control sample seminal fluid does not add any protectant, places under the room temperature, after 10 hours, 16 hours, 24 hours, 30 hours, adopts identical sampling and diluting method in the test under microscope sperm viability.The method of estimating sperm viability is: the percentage that accounts for whole sperms according to the sperm of the Activity Type of sperm and forward travel is determined; Observing 5 visuals field averages.The results are shown in Table 10.
The vigor that table 10 adds protectant seminal fluid and contrast seminal fluid compares
As can be seen from Table 10, the seminal fluid of the He Sitan stock bull Rapture that handles through protectant semen collection after 16 hours vigor still can reach 65～72%; And after the undressed fresh semen semen collection 16 hours, vigor has been down to 32～37%.And vigor is 32～37% seminal fluid, has been unworthy carrying out follow-up packing freezing processing, also can't carry out effective property control separating treatment.
The application of 5 Ns of seminal fluid protectants 562 of embodiment
1, material and reagent
Seminal fluid source: He Sitan stock bull Blister, available from Canadian WESTENG company; Chemical reagent is available from Sigma company.
2, experimental technique
The fresh semen 10ml that picks up from the Blister stock bull is divided into two parts, is respectively charged in the 15ml centrifuge tube, portion is laboratory sample, and another part is control sample.Add the protectant 562 of 2.5ml embodiment 1 in the 5ml seminal fluid of laboratory sample, screw lid, jiggle to make and mix the processing of lowering the temperature then.Cooling step is: the centrifuge tube that seminal fluid and protectant mixture will be housed places in the 500ml plastic bottle that is full of 25 ° of C warm water, screws lid, and this plastic bottle is placed in 4 ° of C refrigerating chambers.
Under 4 ° of C, place the mother liquor of respectively getting after 10 hours, 16 hours, 24 hours, 30 hours among the 10 μ l samples usefulness embodiment 12 and dilute 50 times, get about 20 μ l dilute samples and place on 37 ℃ of warming plates, at the vigor (percentage of forward motile sperm) of test under microscope sperm.The control sample seminal fluid does not add any protectant, places under the room temperature, after 10 hours, 16 hours, 24 hours, 30 hours, adopts identical sampling and diluting method in the test under microscope sperm viability.The method of estimating sperm viability is: the percentage that accounts for whole sperms according to the sperm of the Activity Type of sperm and forward travel is determined; Observing 5 visuals field averages.The results are shown in Table 11.
The vigor that table 11 adds protectant seminal fluid and contrast seminal fluid compares
As can be seen from Table 11, the seminal fluid of the He Sitan stock bull Blister that handles through protectant semen collection after 16 hours vigor still can reach 66～73%; And after the undressed fresh semen semen collection 16 hours, vigor has been down to 30～38%.And vigor is 30～38% seminal fluid, has been unworthy carrying out follow-up packing freezing processing, also can't carry out effective property control separating treatment.
Sperm viability after embodiment 6 tubule packing are freezing
1, material and reagent
Seminal fluid source: He Sitan stock bull Rapture, available from Canadian WESTENG company; Chemical reagent is available from Sigma company.
2, experimental technique
The fresh semen 10ml that picks up from the Rapture stock bull is divided into two parts, is respectively charged in the 15ml centrifuge tube, portion is laboratory sample, and another part is control sample.Add the protectant 562 of 2.5ml embodiment 1 in the 5ml seminal fluid of laboratory sample, screw lid, jiggle to make and mix the processing of lowering the temperature then.Cooling step is: the centrifuge tube that seminal fluid and protectant mixture will be housed places in the 500ml plastic bottle that is full of 25 ° of C warm water; screw lid; this plastic bottle is placed in 4 ° of C refrigerating chambers, place after 16 hours, carry out conventional sperm dilution and tubule packing.
Sperm dilution and tubule packing step: add the 2.5ml Tris-egg yolk liquid (prescription sees Table 12) of 4 ° of C in the seminal fluid, place 4 ° of C after following 30 minutes, adding above-mentioned 4 ° of C dilutions to final concentration in the seminal fluid is every milliliter 60,000,000 sperm.Divide then to install in the 0.25ml plastic straw, making the sperm concentration in each tubule is every milliliter 15,000,000.Tubule is placed the liquid nitrogen gas 3 minutes of liquid nitrogen container top-35 ° C, put in the liquid nitrogen rapidly then.The seminal fluid of control sample does not add any protectant, places 22 ° of C of room temperature after following 16 hours, according to above-mentioned same procedure dilute, packing and freezing processing.
The prescription of table 12Tris-egg yolk liquid (with the water preparation)
The tubule of waiting to be equipped with seminal fluid stops 12 hours in liquid nitrogen after, the taking-up tubule is put in 39 ° of C hot water and was thawed 1 minute, get 10 μ l samples then and dilute 100 times with the mother liquor 1 among the embodiment 1, getting the about 20 μ l of dilute sample again is placed on 37 ° of C warming plates, at the vigor of test under microscope sperm.The method of estimating sperm viability is: the percentage that accounts for whole sperms according to the sperm of the Activity Type of sperm and forward travel is determined; Observing 5 visuals field averages.The results are shown in Table 13.
The vigor that table 13 adds protectant seminal fluid and contrast seminal fluid compares (semen collection is carried out freezing after 16 hours)
As can be seen from Table 13, the ox fresh semen of handling through protection is carried out tubule in semen collection after 16 hours freezing, and the back vigor that thaws reaches 50～55%, satisfies artificial insemination or in-vitro fertilization (IVF) fully to the requirement of sperm viability.And 16 hours vigor after the contrast seminal fluid semen collection, vigor has been down to 35% even lower before the tubule packing, and the tubule that is unworthy carrying out next step is freezing.
Embodiment 7 adds the property control separating resulting of protectant seminal fluid and contrast seminal fluid
1, material and reagent
Seminal fluid source: He Sitan stock bull Rapture, available from Canadian WESTENG company; Chemical reagent is available from Sigma company.
2, experimental technique
The fresh semen 10ml that picks up from the Rapture stock bull is divided into two parts, is respectively charged in the 15ml centrifuge tube, portion is laboratory sample, and another part is control sample.Add the protectant 562 of 2.5ml embodiment 1 in the 5ml seminal fluid of laboratory sample, screw lid, jiggle to make and mix the processing of lowering the temperature then.Cooling step is: the centrifuge tube that seminal fluid and protectant mixture will be housed places in the 500ml plastic bottle that is full of 25 ° of C warm water, screws lid, and this plastic bottle is placed in 4 ° of C refrigerating chambers.When placing after 16 hours under 4 ° of C, sample thief carries out conventional fluorescer dyeing, and last machine (MoFlo Cytometer) separates, and records property control separating rate under the best machine location condition and the sperm lethality of machine demonstration.Do not add any protectant in the contrast seminal fluid, place and place under 22 ° of C of room temperature after 16 hours, sample thief carries out conventional fluorescer dyeing, and last machine (MoFlo Cytometer) separates, the sperm lethality that record property control separating rate and machine show.The results are shown in Table 14.
Table 14 adds protectant seminal fluid relatively controls separating resulting (semen collection separated after 16 hours) with the contrast essence
As can be seen from Table 14, through seminal fluid that protectant was handled semen collection after 16 hours the control of carrying out property separate, best result is 4800～5200 sperms of per second (these for separating rate) normally and efficiently from speed; The detected sperm lethality of machine has only 5～13%.And not adding protectant contrast seminal fluid in semen collection after 16 hours, best result only is 1200～2100 sperms of per second from speed; The detected sperm lethality of machine is then up to 50～70%, and this moment, the adjustment location of machine was also very difficult, can't carry out effective property control substantially and separate.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. an ox seminal fluid protectant is characterized in that it comprises mother liquor 1, mother liquor 2 and egg yolk liquid, and the prescription of described mother liquor 1 and mother liquor 2 is:
The prescription of mother liquor 1 is counted with 1L:
The prescription of mother liquor 2 is counted with 1L:
The volume ratio of mother liquor 1, mother liquor 2 and egg yolk liquid is 5:4:1, places 4 ℃ of preservations.
2. the protectant method of the preparation described ox seminal fluid of claim 1 is characterized in that, according to formulated mother liquor 1 and mother liquor 2 shown in the table, places 4 ℃ of preservations respectively.
3. the application of the described protectant of claim 1 in the ox seminal fluid is fresh-keeping.
4. application according to claim 3, it is characterized in that, with mother liquor 1, mother liquor 2 and egg yolk liquid mixed mixed liquor in proportion, add this mixed liquor of 0.25～0.5 times of ox seminal fluid volume then in the ox seminal fluid in 24 hours before use, place 3～6 ℃ of storages.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210162654 CN102696577B (en) | 2011-08-18 | 2012-05-23 | Bovine semen protective agent |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110238343.8 | 2011-08-18 | ||
| CN201110238343 | 2011-08-18 | ||
| CN 201210162654 CN102696577B (en) | 2011-08-18 | 2012-05-23 | Bovine semen protective agent |
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| Publication Number | Publication Date |
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| CN102696577A CN102696577A (en) | 2012-10-03 |
| CN102696577B true CN102696577B (en) | 2013-08-21 |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103654998A (en) * | 2012-09-18 | 2014-03-26 | 北京富通华投资有限公司 | Method for inseminating multiparous cows by using frozen sex-controlled semen |
| CN103843759B (en) * | 2014-03-07 | 2015-05-20 | 四川农业大学 | Chicken seminal fluid diluent applicable to high altitude localities and preparation method and application of diluent |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2813756B1 (en) * | 2000-09-11 | 2003-03-07 | Imv Technologies | DILUENT FOR THE CONSERVATION OF SWINE SPERMATOZOIDES |
| CN101220345A (en) * | 2008-01-23 | 2008-07-16 | 西北农林科技大学 | Cattle freezing seminal fluid dilution and method for producing the same |
| CN101263806A (en) * | 2008-04-11 | 2008-09-17 | 高庆华 | Dilution for preserving semen under normal temperature |
| CN101856016B (en) * | 2010-06-19 | 2013-03-13 | 张尤嘉 | Diluent for preserving porcine semen at normal temperature |
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