CN102578079A - Base fluid for diluting semens of equus animals and preparation method and use method thereof - Google Patents
Base fluid for diluting semens of equus animals and preparation method and use method thereof Download PDFInfo
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Abstract
The invention discloses base fluid for diluting semens of equus animals and a preparation method and a use method thereof. The base fluid contains A fluid and skimmed milk. The A fluid contains glucose, raffinose, D-glucopyranose, trisodium citrate, ppotassium citrate and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The preparation method for the base fluid includes weighing various components of the A fluid into a sterile beaker, adding double-distilled water, dissolving, bringing to volume, filtering, split charging, sterilizing, and sealing to obtain the A fluid, and adding same amount of the skimmed milk into the A fluid to be evenly mixed. Users can also add penicillin and dihydrostreptomycin to the base fluid or add penicillin, dihydrostreptomycin, yolk and glycerol into the base fluid. The base fluid is suitable for semen dilution and normal temperature preservation, low temperature preservation and freezing preservation of the semens of the equus animals. The base fluid for diluting semens of equus animals and the preparation method and the use method of the base fluid have the advantages of being simple to prepare, convenient to use, good in preservation effect of the semens of the equus animals, multi-purpose, time-saving, labor-saving and applicable to mass production and application.
Description
Technical field
The present invention relates to the technical field that animal semen is preserved, particularly a kind of equus sperm dilution basal liquid and compound method and method for using.
Background technology
Sperm dilution is the dilution protection liquid that in seminal fluid, adds suitable sperm in vitro survival and keep sperm viability; Its purpose is to enlarge the capacity of seminal fluid; Prolong survival time of boar sperm; Guarantee the motility rate and the perforatorium percentage of head rice of sperm, strengthen fertility, fully improve the availability of seminal fluid and the efficient of good male animal breeding.Sperm dilution should carry out after collection as early as possible, and not diluted seminal fluid is unfavorable for that sperm survival, particularly seminal fluid are in temperature when low (below 20 ℃), and sperm is hit by low temperature, is prone to cold shock.Having only the sperm through dilution just to be suitable for preservation, transportation and semen deposition etc., is an important technical links in the animal artificial insemination so sperm dilution is handled.
At present, breeding mainly of China's equus carried out with natural mating and artificial auxiliary dual mode, but under the situation of natural mating or hand mating, a general stallion can only be joined 20-30 mare, and the availability of outstanding stallion is seriously limited.Therefore, the development of sperm dilution, preservation and technology of artificial insemination and application just seem extremely important.
In recent years, the research of livestock semen dilution constantly obtains gratifying achievement, especially aspect ruminants such as ox, sheep.The research of equus semen diluent is effect day by day also; But still there are many drawbacks; Show that mainly semen diluent is short to the holding time of sperm viability; Such as normal temperature holding time less than 24 hours (motility rate is lower than 0.3), low temperature holding time less than 96 hours (motility rate is lower than 0.3); Conception rate is low; Semen diluent needs fresh, must be existing with join at present, so complex operation is complicated.Therefore it is long to develop the holding time, and preservation effect is good, and cost is low, and the effective semen diluent of being impregnated has important economic benefit to artificial insemination.
Summary of the invention
In order to solve that traditional semen diluent is short to holding time of sperm viability, conception rate is low and existing, the invention provides a kind of equus sperm dilution basal liquid and compound method and method for using with problem such as join at present.
In order to realize the foregoing invention purpose, the invention provides a kind of equus sperm dilution basal liquid, said basal liquid comprises A liquid and skim milk, said A liquid comprises glucose, raffinose, D-lactose monohydrate, trisodium citrate, potassium citrate and HEPES.
Wherein, the mixed volume ratio of said A liquid and said skim milk is preferably 1:1.
In the said A liquid of every 500ml, each components contents is: glucose 22 ~ 26g, raffinose 1.5 ~ 1.7g, D-lactose monohydrate 1.5 ~ 1.7g, trisodium citrate Na
3C
6H
5O
72H
2O 0.2 ~ 0.25g, potassium citrate 0.35 ~ 0.45g, HEPES 4.6 ~ 4.8g.
In the said A liquid of every 500ml, each components contents is: glucose 22 ~ 26g, raffinose 1.5 ~ 1.7g, D-lactose monohydrate 1.5 ~ 1.7g, trisodium citrate Na
3C
6H
5O
75H
2O 0.25 ~ 0.3g, potassium citrate 0.35 ~ 0.45g, HEPES 4.6 ~ 4.8g.
Said glucose is DEXTROSE ANHYDROUS, and its molecular formula is C
6H
12O
6The molecular formula of said raffinose is C
18H
32O
65H
2O; The molecular formula of said D-lactose monohydrate is C
12H
22O
11H
2O; The molecular formula of said potassium citrate is K
3C
6H
5O
7H
2O; The molecular formula of said HEPES is C
8H
18N
2O
4S.
The present invention also provides the compound method of above-mentioned any one equus sperm dilution basal liquid, and said compound method comprises:
(1) take by weighing glucose, raffinose, D-lactose monohydrate, trisodium citrate, potassium citrate and the HEPES of requirement, put into aseptic beaker, add the distilled water dissolving after, constant volume, filtration, packing, sterilization, sealing promptly get A liquid;
The A liquid mark that (2) will pass through strict sterilization, sealing is prepared the time, places 0 ~ 5 ℃ of preservation, ready access upon use;
(3) the A liquid of getting requirement adds after an amount of skim milk mixes, and promptly gets basal liquid.
The present invention also provides a kind of method for using of equus sperm dilution basal liquid, it is characterized in that, places 37 ℃ of thermostat water baths after 1 hour said basal liquid, and the equus fresh semen is diluted, and is used for bright smart artificial insemination.
The present invention provides a kind of method for using of equus sperm dilution basal liquid again, and when seminal fluid needed normal temperature or low temperature to preserve, the method for using of said basal liquid was:
(1) in said basal liquid, adds dihydrostreptomycin by the amount interpolation penicillin of 1000 units/ml and the amount of 1000ug/ml respectively, obtain dilution after mixing;
(2) place 37 ℃ of thermostat water baths after 1 hour the dilution that obtains in the said step (1), the equus fresh semen is diluted, the semen at normal temperature after will diluting is then preserved or low temperature is preserved; The volume ratio of said dilution and seminal fluid is preferably 1:1 ~ 1:10.
The present invention also provides a kind of method for using of equus sperm dilution basal liquid, and when seminal fluid needed freezing preservation, the method for using of said basal liquid was:
(1) place 37 ℃ of thermostat water baths after 1 hour said basal liquid, the equus fresh semen carried out first time dilution, after the dilution through the centrifugal basal liquid that removes;
(2) in said basal liquid, add the amount interpolation glycerine of yolk and 2.5 ~ 3.5% by the amount interpolation penicillin of 1000 units/ml, the amount interpolation dihydrostreptomycin of 1000ug/ml, 2.5 ~ 3.5% amount respectively, obtain dilution after mixing;
(3) dilution that obtains in the said step (2) was placed 22 ℃ of thermostat water baths 1 hour, then the seminal fluid after dilution for the first time in the said step (1) is carried out the dilution second time, the semen freezing after the dilution is preserved; The volume ratio of said dilution is preferably 1:1 ~ 1:10.
The beneficial effect that the technical scheme that the embodiment of the invention provides is brought is: basal liquid of the present invention can directly be used for the dilution of equus fresh semen, is applicable to bright smart artificial insemination; In the time need carrying out normal temperature or low temperature preservation to the fresh semen of equus, an amount of penicillin and the dihydrostreptomycin of adding promptly can be used for the dilution of equus seminal fluid in basal liquid, can carry out normal temperature preservation or low temperature preservation to seminal fluid after the dilution; In the time need carrying out freezing preservation to the fresh semen of equus, an amount of penicillin, dihydrostreptomycin, yolk and the glycerine of adding promptly can be used for the dilution of equus seminal fluid in basal liquid, can carry out freezing preservation to seminal fluid after the dilution; And under normal temperature preservation, low temperature preservation or the condition of freezing preservation; The dilution of using equus sperm dilution basal liquid of the present invention to carry out seminal fluid is preserved, and effect obviously is superior to traditional dilution, and the preservation effect of equus seminal fluid is good; Prolonged survival time of boar sperm in the dilution seminal fluid; Guarantee the motility rate and the perforatorium percentage of head rice of sperm, strengthen fertility, fully improved the availability of seminal fluid and the efficient of good male animal breeding; Basal liquid of the present invention preparation is simple, easy to use, can place 0-5 ℃ of preservation, and ready access upon use and can the many usefulness of a liquid is time saving and energy saving, is suitable for large-scale production and application.
Embodiment
Below in conjunction with embodiment the present invention is done and to further describe.
Embodiment 1
Take by weighing glucose 24g, raffinose 1.6g, D-lactose monohydrate 1.6g, trisodium citrate Na
3C
6H
5O
72H
2O 0.24g, potassium citrate 0.4g and HEPES 4.7g put into aseptic beaker, add distilled water dissolving after, constant volume, filtration, packing, sterilization, sealing promptly get A liquid; To pass through the A liquid mark preparation time of strict sterilization, sealing, place 0 ~ 5 ℃ of preservation, ready access upon use; After getting skim milk that an amount of A liquid adds equivalent volumes and mixing, obtain the basal liquid of present embodiment 1.
After the amount interpolation dihydrostreptomycin that in basal liquid, adds penicillin and 1000ug/ml by the amount of 1000 units/ml respectively mixes, obtain the dilution of present embodiment 1.
Comparative trial 1
With traditional dilution glucose-yolk diluent (control group 1) and milk dilution (control group 2), dilute the seminal fluid of horse respectively with the dilution (embodiment group 1) of embodiment 1.Wherein, place 37 ℃ of thermostat water baths after 1 hour the dilution of embodiment 1,1:5 dilutes the fresh semen (motility rate >=0.8) of horse by volume, and the seminal fluid after will diluting then places under the normal temperature (15 ~ 25 ℃) and preserves.In 0 ~ 48 hour, observe the sperm viability of control group 1, control group 2 and embodiment group 1, whenever measured the motility rate of sperm at a distance from 12 hours, the result is as shown in table 1.
Table 1: different diluent normal temperature is preserved the influence of (15 ~ 25 ℃) seminal fluid to the horse sperm motility rate
| Group | The 0h motility rate | The 12h motility rate | The 24h motility rate | 36 h motility rates | The 48h motility rate |
| Control group 1 | 0.85±0.02 | 0.55±0.03 | 0.25±0.04 | 0 | 0 |
| Control group 2 | 0.85±0.03 | 0.40±0.04 | 0.10±0.02 | 0 | 0 |
| Embodiment group 1 | 0.85±0.02 | 0.75±0.02 | 0.65±0.03 | 0.50±0.03 | 0.4±0.04 |
Data by table 1 can be known; Under the condition that normal temperature is preserved, to compare with traditional semen diluent, the sperm motility rate of utilizing equus semen diluent of the present invention to preserve is obviously higher; The holding time significant prolongation, the motility rate of sperm still maintains about 0.4 when preserving 48h.
Embodiment 2
Take by weighing glucose 23g, raffinose 1.7g, D-lactose monohydrate 1.5g, trisodium citrate Na
3C
6H
5O
75H
2O 0.27g, potassium citrate 0.35g and HEPES 4.6g put into aseptic beaker, add distilled water dissolving after, constant volume, filtration, packing, sterilization, sealing promptly get A liquid; To pass through the A liquid mark preparation time of strict sterilization, sealing, place 0 ~ 5 ℃ of preservation, ready access upon use; After getting skim milk that an amount of A liquid adds equivalent volumes and mixing, obtain present embodiment 2 basal liquids.
After the amount interpolation dihydrostreptomycin that in basal liquid, adds penicillin and 1000ug/ml by the amount of 1000 units/ml respectively mixes, obtain the dilution of present embodiment 2.
Comparative trial 2
With traditional dilution glucose-yolk diluent (control group 3) and glucose-tartaric acid first sodium-yolk diluent (control group 4), dilute the seminal fluid of horse respectively with the dilution (embodiment group 2) of embodiment 2.Wherein, place 37 ℃ of thermostat water baths after 1 hour the dilution of embodiment 2,1:10 dilutes the fresh semen (motility rate >=0.8) of horse by volume, and the seminal fluid after will diluting then places under the low temperature (0 ~ 5 ℃) and preserves.In 0 ~ 120 hour, observe the sperm viability of control group 4, control group 5 and embodiment group 2, whenever measured the motility rate of sperm at a distance from 24 hours, the result is as shown in table 2.
Table 2 different diluent low temperature is preserved the influence of (0 ~ 5 ℃) seminal fluid to the horse sperm motility rate
| Group | The 0h motility rate | The 24h motility rate | The 48h motility rate | The 72h motility rate | 96 h motility rates | The 120h motility rate |
| Control group 3 | 0.85±0.02 | 0.70±0.04 | 0.60±0.02 | 0.40±0.03 | 0.20±0.02 | 0.05±0.02 |
| Control group 4 | 0.85±0.03 | 0.75±0.02 | 0.60±0.02 | 0.45±0.03 | 0.30±0.03 | 0.15±0.02 |
| Embodiment group 2 | 0.85±0.02 | 0.75±0.03 | 0.70±0.03 | 0.60±0.02 | 0.55±0.02 | 0.45±0.03 |
Data by table 2 can be known; Under the condition that low temperature is preserved, to compare with traditional semen diluent, the sperm motility rate of utilizing equus semen diluent of the present invention to preserve is obviously higher; The holding time significant prolongation, the motility rate of sperm still maintains about 0.45 when preserving 120h.
Embodiment 3
Take by weighing glucose 25g, raffinose 1.5g, D-lactose monohydrate 1.7g, Na
3C
6H
5O
75H
2O 0.3g, potassium citrate 0.45g and HEPES 4.8g put into aseptic beaker, add distilled water dissolving after, constant volume, filtration, packing, sterilization, sealing promptly get A liquid; To pass through the A liquid mark preparation time of strict sterilization, sealing, place 0 ~ 5 ℃ of preservation, ready access upon use; After getting skim milk that an amount of A liquid adds equivalent volumes and mixing, obtain the basal liquid of present embodiment 3.
In the part basal liquid, add the amount interpolation glycerine of yolk and 2.5 ~ 3.5% by the amount interpolation penicillin of 1000 units/ml, the amount interpolation dihydrostreptomycin of 1000ug/ml, 2.5 ~ 3.5% amount respectively, after mixing, obtain the dilution of present embodiment 3.
Comparative trial 3
With traditional dilution lactose-yolk-glycerine dilution (control group 5) and lactose-EDTA-sodium citrate-yolk-glycerine dilution (control group 6), dilute the seminal fluid of donkey respectively with the dilution (embodiment group 3) of embodiment 3.
Wherein, place 37 ℃ of thermostat water baths after 1 hour basal liquid, equus fresh semen (motility rate>=0.75) carried out first time dilution, after the dilution through the centrifugal basal liquid that removes; Place 22 ℃ of thermostat water baths after 1 hour dilution,, make final sperm concentration reach 1 * 10 carrying out the dilution second time through the seminal fluid after the dilution for the first time
8Individual/ml, the semen freezing after will diluting is then preserved.Observe the sperm viability of control group 5, control group 6 and embodiment group 3 freezing front and back, the result is as shown in table 3.
Table 3 different diluent freezing preservation seminal fluid is to the influence of donkey sperm motility rate
| Group | Sperm motility rate before freezing | Freezing back sperm motility rate |
| Control group 5 | 0.82±0.03 | 0.35±0.01 |
| Control group 6 | 0.80±0.02 | 0.40±0.02 |
| Embodiment group 3 | 0.80±0.02 | 0.55±0.02 |
Data by table 3 can be known, under the condition of freezing preservation, compare with traditional semen diluent, and the sperm motility rate of utilizing equus semen diluent of the present invention to preserve is obviously higher.
To sum up, under the condition that no matter preserve at normal temperature, the low temperature preservation still is freezing preservation, the dilution of using equus sperm dilution basal liquid of the present invention to carry out seminal fluid is preserved, and effect obviously is superior to traditional dilution.
The above is merely preferred embodiment of the present invention, and is in order to restriction the present invention, not all within spirit of the present invention and principle, any modification of being done, is equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. equus sperm dilution basal liquid is characterized in that, said basal liquid comprises A liquid and skim milk, and said A liquid comprises glucose, raffinose, D-lactose monohydrate, trisodium citrate, potassium citrate and HEPES.
2. equus sperm dilution basal liquid according to claim 1 is characterized in that the mixed volume ratio of said A liquid and said skim milk is 1:1.
3. equus sperm dilution basal liquid according to claim 1 is characterized in that, in the said A liquid of every 500ml, each components contents is: glucose 22 ~ 26g, raffinose 1.5 ~ 1.7g, D-lactose monohydrate 1.5 ~ 1.7g, trisodium citrate Na
3C
6H
5O
72H
2O 0.2 ~ 0.25g, potassium citrate 0.35 ~ 0.45g, HEPES 4.6 ~ 4.8g.
4. equus sperm dilution basal liquid according to claim 1 is characterized in that, in the said A liquid of every 500ml, each components contents is: glucose 22 ~ 26g, raffinose 1.5 ~ 1.7g, D-lactose monohydrate 1.5 ~ 1.7g, trisodium citrate Na
3C
6H
5O
75H
2O 0.25 ~ 0.3g, potassium citrate 0.35 ~ 0.45g, HEPES 4.6 ~ 4.8g.
5. according to any described equus sperm dilution basal liquid of claim 1-4, it is characterized in that said glucose is DEXTROSE ANHYDROUS, its molecular formula is C
6H
12O
6The molecular formula of said raffinose is C
18H
32O
65H
2O; The molecular formula of said D-lactose monohydrate is C
12H
22O
11H
2O; The molecular formula of said potassium citrate is K
3C
6H
5O
7H
2O; The molecular formula of said HEPES is C
8H
18N
2O
4S.
6. compound method like any described equus sperm dilution basal liquid of claim 1-5 is characterized in that said compound method comprises:
(1) take by weighing glucose, raffinose, D-lactose monohydrate, trisodium citrate, potassium citrate and the HEPES of requirement, put into aseptic beaker, add the distilled water dissolving after, constant volume, filtration, packing, sterilization, sealing promptly get A liquid;
The A liquid mark that (2) will pass through strict sterilization, sealing is prepared the time, places 0 ~ 5 ℃ of preservation;
(3) the A liquid of getting requirement adds after an amount of skim milk mixes, and promptly gets basal liquid.
7. method for using like any described equus sperm dilution basal liquid of claim 1-6; It is characterized in that; Place 37 ℃ of thermostat water baths after 1 hour said basal liquid, the equus fresh semen is diluted, be used for bright smart artificial insemination.
8. the method for using like any described equus sperm dilution basal liquid of claim 1-6 is characterized in that, when seminal fluid needed normal temperature or low temperature to preserve, the method for using of said basal liquid was:
(1) in said basal liquid, adds dihydrostreptomycin by the amount interpolation penicillin of 1000 units/ml and the amount of 1000ug/ml respectively, obtain dilution after mixing;
(2) place 37 ℃ of thermostat water baths after 1 hour the dilution that obtains in the said step (1), the equus fresh semen is diluted, the semen at normal temperature after will diluting is then preserved or low temperature is preserved.
9. the method for using like the described equus sperm dilution of claim 1-6 basal liquid is characterized in that, when seminal fluid needed freezing preservation, the method for using of said basal liquid was:
(1) place 37 ℃ of thermostat water baths after 1 hour said basal liquid, the equus fresh semen carried out first time dilution, after the dilution through the centrifugal basal liquid that removes;
(2) in said basal liquid, add the amount interpolation glycerine of yolk and 2.5 ~ 3.5% by the amount interpolation penicillin of 1000 units/ml, the amount interpolation dihydrostreptomycin of 1000ug/ml, 2.5 ~ 3.5% amount respectively, obtain dilution after mixing;
(3) dilution that obtains in the said step (2) was placed 22 ℃ of thermostat water baths 1 hour, then the seminal fluid after dilution for the first time in the said step (1) is carried out the dilution second time, the semen freezing after the dilution is preserved.
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| CN103651333A (en) * | 2013-12-23 | 2014-03-26 | 伊犁德瑞骏发生物科技有限公司 | Frozen semen diluent of equus animals and preparation method thereof |
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| CN106305705A (en) * | 2016-07-28 | 2017-01-11 | 西安乐民反刍动物研究所 | Preparation method for diluent for milk goat artificial insemination technology |
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| CN103039434A (en) * | 2012-12-10 | 2013-04-17 | 山东天龙牧业科技有限公司 | Duck egg yolk anti-freezing agent for cryopreservation of donkey semen and preparation method thereof |
| WO2014089924A1 (en) * | 2012-12-10 | 2014-06-19 | 山东天龙牧业科技有限公司 | Duck egg yolk anti-freezing agent for cryopreservation of donkey semen and preparation method thereof |
| CN103858858A (en) * | 2012-12-11 | 2014-06-18 | 中国农业大学 | Horse semen dilution and its preparation method |
| CN103120155B (en) * | 2013-02-05 | 2014-07-16 | 新疆维吾尔自治区畜牧科学院畜牧科学研究所 | Cryopreservation solution of horse sperms and freezing method thereof |
| CN103120155A (en) * | 2013-02-05 | 2013-05-29 | 新疆维吾尔自治区畜牧科学院畜牧科学研究所 | Cryopreservation solution of horse sperms and freezing method thereof |
| CN104115819A (en) * | 2013-04-25 | 2014-10-29 | 上海农林职业技术学院 | Canine frozen semen diluting solution formula |
| WO2015027354A2 (en) | 2013-08-29 | 2015-03-05 | Universidad Austral De Chile | Method for the cooling and cryopreservation of horse semen and diluents used in said method |
| CN103704202A (en) * | 2013-12-11 | 2014-04-09 | 广西大学 | Pony semen cryopreservation method |
| CN103651333A (en) * | 2013-12-23 | 2014-03-26 | 伊犁德瑞骏发生物科技有限公司 | Frozen semen diluent of equus animals and preparation method thereof |
| CN103843758A (en) * | 2014-02-19 | 2014-06-11 | 新疆维吾尔自治区畜牧科学院畜牧研究所 | Low temperature preservation diluent for horse sperm |
| CN103843758B (en) * | 2014-02-19 | 2015-09-23 | 新疆维吾尔自治区畜牧科学院畜牧研究所 | A kind of horse sperm Cord blood dilution |
| CN107232179A (en) * | 2016-03-29 | 2017-10-10 | 中国农业大学 | A kind of semen diluent for improving seminal fluid low temperature/freezen protective quality and its application |
| CN106305705A (en) * | 2016-07-28 | 2017-01-11 | 西安乐民反刍动物研究所 | Preparation method for diluent for milk goat artificial insemination technology |
| CN107980765A (en) * | 2017-12-06 | 2018-05-04 | 中国农业大学 | A kind of semen diluent for improving donkey testicular fluid Cord blood quality and preparation method and application |
| CN110583630A (en) * | 2019-10-22 | 2019-12-20 | 山东畜牧兽医职业学院 | Donkey semen diluent and preparation method thereof |
| CN112205353A (en) * | 2020-10-19 | 2021-01-12 | 内蒙古草原御驴科技牧业有限公司 | Scientific breeding and healthy breeding method of donkeys |
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Application publication date: 20120718 |