CN104621097A - Sheep fresh semen preserve diluent and application thereof - Google Patents
Sheep fresh semen preserve diluent and application thereof Download PDFInfo
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Abstract
The invention relates to a livestock semen diluent and the application thereof, and in particular to a sheep fresh semen preserve diluent and the application thereof, aiming at solving the problems that the sheep semen preservation time is short, the vigor of the preserved semen is reduced, the integrity of plasma membrane is destroyed, the fertility rate is low, and the embryo mortality is high in the existing sheep semen preservation method. The sheep fresh semen preserve diluent is prepared from basic liquid, yolk and astaxanthin, wherein the volume ratio of the basic liquid to the yolk in the sheep fresh semen preserve diluent is equal to 8 to 2, and the concentration of the astaxanthin in the sheep fresh semen preserve diluent is 0.5-4 micromol/L. The application method of the sheep fresh semen preserve diluent in sheep fresh semen preservation comprises the steps of 1. preparing the sheep fresh semen preserve diluent; 2. diluting sheep semen; and 3. preserving the semen. The sheep fresh semen preserve diluent can be obtained.
Description
Technical field
The present invention relates to a kind of livestock semen dilution and application thereof.
Background technology
Artificial insemination is that sheep good variety population efficiently expands numerous powerful measure, and the quality of semen quality determines the success or failure of technology of artificial insemination; Efficient Semen routine technology plays an important role at varieties breeding with in selecting.
At present, because ram freezing semen technology is immature, and special genital system structure, most domestic Yang Chang adopts the method short time of 4 DEG C of liquid storages to store seminal fluid, and effective holding time of seminal fluid also only has 1 day ~ 2 days.Along with the prolongation of holding time, liquid storage can cause that sperm viability reduces, membrane integrity destroys, at the decreased survival of gential tract of dam, and then reduces fertilization rate and increase the lethality of embryo.Wherein oxidative stress is the one of the main reasons causing semen quality to reduce.
As far back as nineteen forty-three, MacLeod just finds that sperm can produce ROS.Because plasmalemmae of sperms is rich in polyunsaturated fatty acid, therefore sperm is to oxidative stress quite sensitive.In addition, sperm, as a kind of cell of unique types, loses most cells matter in the process that maturation is grown, and DNA high enrichment, this greatly reduces its endogenous anti-oxidative ability.Therefore, sperm is resisted oxidative stress and is relied on exogenous antioxidant system.In order to improve the preservation quality of seminal fluid, this needs the Conservation environment improving seminal fluid, protects membrane integrity, motility and fertility to be effective means by adding antioxidant.
Summary of the invention
The time that the object of the invention is the Semen routine method existence preservation sheep seminal fluid that will solve existing sheep is short, the energy vigor preserved reduces, the destroy integrity of plasma membrane, the problem that fertilization rate is low and rate of embryonic death is high, and provide the fresh essence of a kind of sheep to preserve dilution and application thereof.
A kind of sheep fresh essence preservation dilution is prepared by basal liquid, yolk and astaxanthin; The volume ratio that the fresh essence of described sheep preserves basal liquid and yolk in dilution is 8:2; The concentration that the fresh essence of described sheep preserves astaxanthin in dilution is 0.5 μm of ol/L ~ 4 μm ol/L.
The fresh essence of sheep preserves the application method of dilution in the fresh essence of sheep is preserved, and completes according to the following steps:
One, prepare the fresh essence of sheep and preserve dilution: basal liquid, yolk and astaxanthin are mixed, obtain the fresh essence of sheep and preserve dilution;
The volume ratio that the fresh essence of sheep described in step one preserves basal liquid and yolk in dilution is 8:2;
The concentration that the fresh essence of sheep described in step one preserves astaxanthin in dilution is 0.5 μm of ol/L ~ 4 μm ol/L;
Two, sheep seminal fluid is diluted:
Utilize artificial vagina method to collect sheep seminal fluid, in the sheep seminal fluid collected, then add the sheep fresh essence preservation dilution that temperature is 35 DEG C ~ 40 DEG C, obtain the sheep seminal fluid after diluting;
The sheep seminal fluid of the collection described in step 2 and temperature are the volume ratio that the fresh essence of the sheep of 35 DEG C ~ 40 DEG C preserves dilution is 1:5;
Three, preserve seminal fluid: the sheep seminal fluid after the dilution using 8 layers ~ 10 layers gauze step 2 to be obtained wraps up, and is being placed into 70h ~ 72h in 4 DEG C of refrigerators, namely complete the fresh essence of a kind of sheep and preserve the application method of dilution in the fresh essence of sheep is preserved.
Advantage of the present invention:
One, the astaxanthin that the present invention uses is a kind of antioxidant efficiently, and it is a Carotenoids, is prevalent in fish, shrimps, seaweeds animal and plant body; Astaxanthin can effective scavenging activated oxygen; Its oxidation resistance be the antioxidants such as glutinous flavine, xanthophyll, canthaxanthin, carotin ten times, be 100 times of vitamin E; The present invention preserves in dilution in the fresh essence of sheep and adds astaxanthin, can improve the preservation effect of seminal fluid, extend the holding time of seminal fluid;
Two, the present invention has the features such as efficiency is high, cost is low, simple to operate and applied widely, provides new approach for improving sheep Semen routine effect;
Three, the present invention preserves in dilution in the fresh essence of sheep and adds astaxanthin, Semen routine after 3 days vigor up to 74%, compared with preserving dilution with the common fresh essence of not adding astaxanthin, Semen routine after 3 days vigor improve 10%;
Four, the present invention extended effective holding time of seminal fluid from 1 ~ 2 day to 3 days, even longer;
Five, the present invention can improve the reproductive efficiency that ram is greater than 30%;
Six, the present invention can significantly improve availability, the minimizing number of animals raised, cost-saving, the raising lambing percentage of excellent male animal, can directly help peasants and herdsmen's increasing both production and income, have broad application prospects;
The present invention can obtain the fresh essence of a kind of sheep and preserve dilution.
Accompanying drawing explanation
Fig. 1 is sperm viability time history plot; In Fig. 1,1 is the sheep sperm vigor time history plot using check experiment method to preserve; In Fig. 1,2 is the sheep sperm vigor time history plot that service test one method is preserved; In Fig. 1,3 is the sheep sperm vigor time history plot that service test two method is preserved; In Fig. 1,4 is the sheep sperm vigor time history plot that service test three method is preserved; In Fig. 1,5 is the sheep sperm vigor time history plot that service test four method is preserved;
Fig. 2 is sheep sperm membrane integrity curve over time; In Fig. 2,1 is the sheep sperm membrane integrity curve over time using check experiment method to preserve, 2 is the sheep sperm membrane integrity curve over time that service test one method is preserved, 3 is the sheep sperm membrane integrity curve over time that service test two method is preserved, 4 is the sheep sperm membrane integrity curve over time that service test three method is preserved, and 5 is the sheep sperm membrane integrity curve over time that service test four method is preserved;
Fig. 3 is sperm MDA curve over time; In Fig. 3,1 is the sheep sperm MDA time history plot using check experiment method to preserve; In Fig. 3,2 is the sheep sperm MDA time history plot that service test one method is preserved; In Fig. 3,3 is the sheep sperm MDA time history plot that service test two method is preserved; In Fig. 3,4 is the sheep sperm MDA time history plot that service test three method is preserved; In Fig. 3,5 is the sheep sperm MDA time history plot that service test four method is preserved;
Fig. 4 is sperm ROS curve over time; In Fig. 4,1 is the sheep sperm ROS time history plot using check experiment method to preserve; In Fig. 4,2 is the sheep sperm ROS time history plot that service test one method is preserved; In Fig. 4,3 is the sheep sperm ROS time history plot that service test two method is preserved; In Fig. 4,4 is the sheep sperm ROS time history plot that service test three method is preserved; In Fig. 4,5 is the sheep sperm ROS time history plot that service test four method is preserved.
Embodiment
Embodiment one: present embodiment is that a kind of sheep fresh essence preservation dilution is prepared by basal liquid, yolk and astaxanthin; The volume ratio that the fresh essence of described sheep preserves basal liquid and yolk in dilution is 8:2; The concentration that the fresh essence of described sheep preserves astaxanthin in dilution is 0.5 μm of ol/L ~ 4 μm ol/L.
The advantage of present embodiment:
One, the astaxanthin that present embodiment uses is a kind of antioxidant efficiently, and it is a Carotenoids, is prevalent in fish, shrimps, seaweeds animal and plant body; Astaxanthin can effective scavenging activated oxygen; Its oxidation resistance be the antioxidants such as glutinous flavine, xanthophyll, canthaxanthin, carotin ten times, be 100 times of vitamin E; Present embodiment is preserved in dilution in the fresh essence of sheep and is added astaxanthin, can improve the preservation effect of seminal fluid, extend the holding time of seminal fluid;
Two, present embodiment has the features such as efficiency is high, cost is low, simple to operate and applied widely, provides new approach for improving sheep Semen routine effect;
Three, present embodiment is preserved in dilution in the fresh essence of sheep and is added astaxanthin, Semen routine after 3 days vigor up to 74%, compared with preserving dilution with the common fresh essence of not adding astaxanthin, Semen routine after 3 days vigor improve 10%;
Four, present embodiment extended effective holding time of seminal fluid from 1 ~ 2 day to 3 days, even longer;
Five, present embodiment can improve the reproductive efficiency that ram is greater than 30%;
Six, present embodiment can significantly improve availability, the minimizing number of animals raised, cost-saving, the raising lambing percentage of excellent male animal, can directly help peasants and herdsmen's increasing both production and income, have broad application prospects.
Present embodiment can obtain the fresh essence of a kind of sheep and preserve dilution.
Embodiment two: the difference of present embodiment and embodiment one is: described basal liquid is prepared by the following method: 3.2g glucose, 3.2g sodium citrate, penicillin 100,000 IU and streptomycin 100,000 IU are dissolved in 100mL ultra-pure water, obtain basal liquid.Other are identical with embodiment one.
Embodiment three: the difference of present embodiment and embodiment one or two is: the concentration that the fresh essence of described sheep preserves astaxanthin in dilution is 0.5 μm of ol/L ~ 1 μm ol/L.Other are identical with embodiment one or two.
Embodiment four: the difference of present embodiment and embodiment one to three is: the concentration that the fresh essence of described sheep preserves astaxanthin in dilution is 1 μm of ol/L ~ 2 μm ol/L.Other are identical with embodiment one to three.
Embodiment five: the difference of present embodiment and embodiment one to four is: the concentration that the fresh essence of described sheep preserves astaxanthin in dilution is 2 μm of ol/L ~ 4 μm ol/L.Other are identical with embodiment one to four.
Embodiment six: present embodiment is that the application method of a kind of sheep fresh essence preservation dilution in the fresh essence of sheep is preserved completes according to the following steps:
One, prepare the fresh essence of sheep and preserve dilution: basal liquid, yolk and astaxanthin are mixed, obtain the fresh essence of sheep and preserve dilution;
The volume ratio that the fresh essence of sheep described in step one preserves basal liquid and yolk in dilution is 8:2;
The concentration that the fresh essence of sheep described in step one preserves astaxanthin in dilution is 0.5 μm of ol/L ~ 4 μm ol/L;
Two, sheep seminal fluid is diluted:
Utilize artificial vagina method to collect sheep seminal fluid, in the sheep seminal fluid collected, then add the sheep fresh essence preservation dilution that temperature is 35 DEG C ~ 40 DEG C, obtain the sheep seminal fluid after diluting;
The sheep seminal fluid of the collection described in step 2 and temperature are the volume ratio that the fresh essence of the sheep of 35 DEG C ~ 40 DEG C preserves dilution is 1:5;
Three, preserve seminal fluid: the sheep seminal fluid after the dilution using 8 layers ~ 10 layers gauze step 2 to be obtained wraps up, and is being placed into 70h ~ 72h in 4 DEG C of refrigerators, namely complete the fresh essence of a kind of sheep and preserve the application method of dilution in the fresh essence of sheep is preserved.
The advantage of present embodiment:
One, the astaxanthin that present embodiment uses is a kind of antioxidant efficiently, and it is a Carotenoids, is prevalent in fish, shrimps, seaweeds animal and plant body; Astaxanthin can effective scavenging activated oxygen; Its oxidation resistance be the antioxidants such as glutinous flavine, xanthophyll, canthaxanthin, carotin ten times, be 100 times of vitamin E; Present embodiment is preserved in dilution in the fresh essence of sheep and is added astaxanthin, can improve the preservation effect of seminal fluid, extend the holding time of seminal fluid;
Two, present embodiment has the features such as efficiency is high, cost is low, simple to operate and applied widely, provides new approach for improving sheep Semen routine effect;
Three, present embodiment is preserved in dilution in the fresh essence of sheep and is added astaxanthin, Semen routine after 3 days vigor up to 74%, compared with preserving dilution with the common fresh essence of not adding astaxanthin, Semen routine after 3 days vigor improve 10%;
Four, present embodiment extended effective holding time of seminal fluid from 1 ~ 2 day to 3 days, even longer;
Five, present embodiment can improve the reproductive efficiency that ram is greater than 30%;
Six, present embodiment can significantly improve availability, the minimizing number of animals raised, cost-saving, the raising lambing percentage of excellent male animal, can directly help peasants and herdsmen's increasing both production and income, have broad application prospects.
Present embodiment can obtain the fresh essence of a kind of sheep and preserve dilution.
Embodiment seven: the difference of present embodiment and embodiment six is: the basal liquid described in step one is prepared by the following method: 3.2g glucose, 3.2g sodium citrate, penicillin 100,000 IU and streptomycin 100,000 IU are dissolved in 100mL ultra-pure water, obtain basal liquid.Other are identical with embodiment six.
Embodiment eight: the difference of present embodiment and embodiment six or seven is: the concentration that the fresh essence of the sheep described in step one preserves astaxanthin in dilution is 0.5 μm of ol/L ~ 1 μm ol/L.Other are identical with embodiment six or seven.
Embodiment nine: the difference of present embodiment and embodiment six to eight is: the concentration that the fresh essence of the sheep described in step one preserves astaxanthin in dilution is 1 μm of ol/L ~ 2 μm ol/L.Other are identical with embodiment six to eight.
Embodiment ten: the difference of present embodiment and embodiment six to nine is: the concentration that the fresh essence of the sheep described in step one preserves astaxanthin in dilution is 2 μm of ol/L ~ 4 μm ol/L.Other are identical with embodiment six to nine.
Adopt following verification experimental verification beneficial effect of the present invention:
Check experiment: the fresh essence of a kind of sheep preserves the application method of dilution in the fresh essence of sheep is preserved, and completes according to the following steps:
One, prepare the fresh essence of sheep and preserve dilution: by basal liquid and yolk mixing, obtain the fresh essence of sheep and preserve dilution;
Basal liquid described in step one is prepared by the following method: be dissolved in 100mL ultra-pure water by 3.2g glucose, 3.2g sodium citrate, penicillin 100,000 IU and streptomycin 100,000 IU, obtain basal liquid;
The volume ratio that the fresh essence of sheep described in step one preserves basal liquid and yolk in dilution is 8:2;
Two, sheep seminal fluid is diluted:
Utilize artificial vagina method to collect sheep seminal fluid, in the sheep seminal fluid collected, then add the sheep fresh essence preservation dilution that temperature is 40 DEG C, obtain the sheep seminal fluid after diluting;
The sheep seminal fluid of the collection described in step 2 and temperature are the volume ratio that the fresh essence of the sheep of 40 DEG C preserves dilution is 1:5;
Three, preserve seminal fluid: the sheep seminal fluid after the dilution using 10 layers of gauze step 2 to be obtained wraps up, and is being placed into 72h in 4 DEG C of refrigerators, namely complete the fresh essence of a kind of sheep and preserve the application method of dilution in the fresh essence of sheep is preserved.
Test one: the fresh essence of a kind of sheep preserves the application method of dilution in the fresh essence of sheep is preserved, and completes according to the following steps:
One, prepare the fresh essence of sheep and preserve dilution: basal liquid, yolk and astaxanthin are mixed, obtain the fresh essence of sheep and preserve dilution;
Basal liquid described in step one is prepared by the following method: be dissolved in 100mL ultra-pure water by 3.2g glucose, 3.2g sodium citrate, penicillin 100,000 IU and streptomycin 100,000 IU, obtain basal liquid;
The volume ratio that the fresh essence of sheep described in step one preserves basal liquid and yolk in dilution is 8:2;
The concentration that the fresh essence of sheep described in step one preserves astaxanthin in dilution is 0.5 μm of ol/L;
Two, sheep seminal fluid is diluted:
Utilize artificial vagina method to collect sheep seminal fluid, in the sheep seminal fluid collected, then add the sheep fresh essence preservation dilution that temperature is 40 DEG C, obtain the sheep seminal fluid after diluting;
The sheep seminal fluid of the collection described in step 2 and temperature are the volume ratio that the fresh essence of the sheep of 40 DEG C preserves dilution is 1:5;
Three, preserve seminal fluid: the sheep seminal fluid after the dilution using 10 layers of gauze step 2 to be obtained wraps up, and is being placed into 72h in 4 DEG C of refrigerators, namely complete the fresh essence of a kind of sheep and preserve the application method of dilution in the fresh essence of sheep is preserved.
Test two: the fresh essence of a kind of sheep preserves the application method of dilution in the fresh essence of sheep is preserved, and completes according to the following steps:
One, prepare the fresh essence of sheep and preserve dilution: basal liquid, yolk and astaxanthin are mixed, obtain the fresh essence of sheep and preserve dilution;
Basal liquid described in step one is prepared by the following method: be dissolved in 100mL ultra-pure water by 3.2g glucose, 3.2g sodium citrate, penicillin 100,000 IU and streptomycin 100,000 IU, obtain basal liquid;
The volume ratio that the fresh essence of sheep described in step one preserves basal liquid and yolk in dilution is 8:2;
The concentration that the fresh essence of sheep described in step one preserves astaxanthin in dilution is 1 μm of ol/L;
Two, sheep seminal fluid is diluted:
Utilize artificial vagina method to collect sheep seminal fluid, in the sheep seminal fluid collected, then add the sheep fresh essence preservation dilution that temperature is 40 DEG C, obtain the sheep seminal fluid after diluting;
The sheep seminal fluid of the collection described in step 2 and temperature are the volume ratio that the fresh essence of the sheep of 40 DEG C preserves dilution is 1:5;
Three, preserve seminal fluid: the sheep seminal fluid after the dilution using 10 layers of gauze step 2 to be obtained wraps up, and is being placed into 72h in 4 DEG C of refrigerators, namely complete the fresh essence of a kind of sheep and preserve the application method of dilution in the fresh essence of sheep is preserved.
Test three: the fresh essence of a kind of sheep preserves the application method of dilution in the fresh essence of sheep is preserved, and completes according to the following steps:
One, prepare the fresh essence of sheep and preserve dilution: basal liquid, yolk and astaxanthin are mixed, obtain the fresh essence of sheep and preserve dilution;
Basal liquid described in step one is prepared by the following method: be dissolved in 100mL ultra-pure water by 3.2g glucose, 3.2g sodium citrate, penicillin 100,000 IU and streptomycin 100,000 IU, obtain basal liquid;
The volume ratio that the fresh essence of sheep described in step one preserves basal liquid and yolk in dilution is 8:2;
The concentration that the fresh essence of sheep described in step one preserves astaxanthin in dilution is 2 μm of ol/L;
Two, sheep seminal fluid is diluted:
Utilize artificial vagina method to collect sheep seminal fluid, in the sheep seminal fluid collected, then add the sheep fresh essence preservation dilution that temperature is 40 DEG C, obtain the sheep seminal fluid after diluting;
The sheep seminal fluid of the collection described in step 2 and temperature are the volume ratio that the fresh essence of the sheep of 40 DEG C preserves dilution is 1:5;
Three, preserve seminal fluid: the sheep seminal fluid after the dilution using 10 layers of gauze step 2 to be obtained wraps up, and is being placed into 72h in 4 DEG C of refrigerators, namely complete the fresh essence of a kind of sheep and preserve the application method of dilution in the fresh essence of sheep is preserved.
Test four: the fresh essence of a kind of sheep preserves the application method of dilution in the fresh essence of sheep is preserved, and completes according to the following steps:
One, prepare the fresh essence of sheep and preserve dilution: basal liquid, yolk and astaxanthin are mixed, obtain the fresh essence of sheep and preserve dilution;
Basal liquid described in step one is prepared by the following method: be dissolved in 100mL ultra-pure water by 3.2g glucose, 3.2g sodium citrate, penicillin 100,000 IU and streptomycin 100,000 IU, obtain basal liquid;
The volume ratio that the fresh essence of sheep described in step one preserves basal liquid and yolk in dilution is 8:2;
The concentration that the fresh essence of sheep described in step one preserves astaxanthin in dilution is 4 μm of ol/L;
Two, sheep seminal fluid is diluted:
Utilize artificial vagina method to collect sheep seminal fluid, in the sheep seminal fluid collected, then add the sheep fresh essence preservation dilution that temperature is 40 DEG C, obtain the sheep seminal fluid after diluting;
The sheep seminal fluid of the collection described in step 2 and temperature are the volume ratio that the fresh essence of the sheep of 40 DEG C preserves dilution is 1:5;
Three, preserve seminal fluid: the sheep seminal fluid after the dilution using 10 layers of gauze step 2 to be obtained wraps up, and is being placed into 72h in 4 DEG C of refrigerators, namely complete the fresh essence of a kind of sheep and preserve the application method of dilution in the fresh essence of sheep is preserved.
Sperm viability detects:
Respectively by check experiment, test one, test two, test three and test four at 4 DEG C, uses 10 layers of gauze parcel, the sheep sperm vigor be kept in sheep fresh essence preservation dilution detects; The viability examination of sheep sperm utilizes eosin W or W S/nigrosine colouring method to assess; 1.67g eosin W or W S, 10g nigrosine and 2.9g sodium citrate configuration dyeing liquor is dissolved at 100mL distilled water; Add respectively preserve at 4 DEG C in check experiment, test one, test two, test three and test four preserve dilution mixture 100 μ l containing the fresh essence of the sheep of sheep sperm and drip and be layered on cover glass, under × 400 enlargement ratios, observe 200 sperms.Sperm is partly or completely painted is considered to dead, and non-staining sperm is considered to survival; Experiment in triplicate; As shown in Figure 1, Fig. 1 is sperm viability time history plot; In Fig. 1,1 is the sheep sperm vigor time history plot using check experiment method to preserve; In Fig. 1,2 is the sheep sperm vigor time history plot that service test one method is preserved; In Fig. 1,3 is the sheep sperm vigor time history plot that service test two method is preserved; In Fig. 1,4 is the sheep sperm vigor time history plot that service test three method is preserved; In Fig. 1,4 is the sheep sperm vigor time history plot that service test four method is preserved.
Plasmalemmae of sperms integrity detection:
Respectively by check experiment, test one, test two, test three and test four at 4 DEG C, uses 10 layers of gauze parcel, the sheep sperm membrane integrity be kept in sheep fresh essence preservation dilution detects;
Get the fresh essence of the sheep containing sheep sperm preserved at 4 DEG C in check experiment respectively and preserve dilution mixture 50 μ L, the hypotonic solution re-using 300 μ L dilutes, then hatches 60 minutes at 37 DEG C, obtains seminal fluid mixture A; Again 100 μ L seminal fluid mixture A are dripped and be layered on cover glass, use and observe 200 sperms with under light microscope (ZEISS, Japan) 1000 × multiplication factor; Record sperm morphology (swelling or curling).Experiment in triplicate; 1.351g fructose and 0.735g sodium citrate are dissolved in the distilled water of 100mL by the preparation method of described hypotonic solution, obtain hypotonic solution.
In test one, test two, test three and test four at 4 DEG C, use 10 layers of gauze parcel, be kept at method that the fresh essence sheep sperm membrane integrity preserved in dilution of sheep carries out detecting detects the plasmalemmae of sperms integrality of preserving at 4 DEG C method with check experiment; Fig. 2 is sheep sperm membrane integrity curve over time; In Fig. 2,1 is the sheep sperm membrane integrity curve over time using check experiment method to preserve, 2 is the sheep sperm membrane integrity curve over time that service test one method is preserved, 3 is the sheep sperm membrane integrity curve over time that service test two method is preserved, 4 is the sheep sperm membrane integrity curve over time that service test three method is preserved, and 5 is the sheep sperm membrane integrity curve over time that service test four method is preserved.
Sperm MDA detects:
Respectively by check experiment, test one, test two, test three and test four at 4 DEG C, uses 10 layers of gauze parcel, is kept at the sheep sperm MDA Concentration Testing in sheep fresh essence preservation dilution:
Sperm MDA Concentration Testing is completed, in strict accordance with specification description operation by MDA MDA kit.Experiment in triplicate; Described MDA MDA kit buys from Chinese green skies biotech company.
Fig. 3 is sperm MDA curve over time; In Fig. 3,1 is the sheep sperm MDA time history plot using check experiment method to preserve; In Fig. 3,2 is the sheep sperm MDA time history plot that service test one method is preserved; In Fig. 3,3 is the sheep sperm MDA time history plot that service test two method is preserved; In Fig. 3,4 is the sheep sperm MDA time history plot that service test three method is preserved; In Fig. 3,5 is the sheep sperm MDA time history plot that service test four method is preserved.
Sperm ROS detects:
Respectively by check experiment, test one, test two, test three and test four at 4 DEG C, uses 10 layers of gauze parcel, is kept at the sheep sperm ROS Concentration Testing in sheep fresh essence preservation dilution:
Sperm ROS horizontal detection is completed by ROS kit, in strict accordance with specification description operation.Experiment in triplicate; Described ROS kit buys from Chinese green skies biotech company.
Fig. 4 is sperm ROS curve over time; In Fig. 4,1 is the sheep sperm ROS time history plot using check experiment method to preserve; In Fig. 4,2 is the sheep sperm ROS time history plot that service test one method is preserved; In Fig. 4,3 is the sheep sperm ROS time history plot that service test two method is preserved; In Fig. 4,4 is the sheep sperm ROS time history plot that service test three method is preserved; In Fig. 4,5 is the sheep sperm ROS time history plot that service test four method is preserved.
Sperm motility parameters detects:
The kinematic parameter of sperm measures with computer aided pass design (CASA, Minitube, Germany);
Respectively by check experiment, test one, test two, test three and test four at 4 DEG C, uses 10 layers of gauze parcel, the sheep sperm being kept at 72h in sheep fresh essence preservation dilution is diluted to 2.0 × 10
7/ ml, then balance 5 minutes 37 DEG C of water-baths; The semen sample got after the dilution of 5 μ l drops on slide warm in advance, each sample random observation 5 visuals field.Record total motile (TM, %) and rectilinear motion sperm (PM, %) ratio, as shown in table 1.
Table 1
| Concentration | TM(％) | PM(％) |
| Check experiment | 61.43±3.29 | 54.57±2.18 |
| Test one | 59.09±4.19 | 55.99±2.87 |
| Test two | 62.82±3.73 | 53.64±3.10 |
| Test three | 70.99±3.81 | 63.43±3.25 |
| Test four | 69.39±2.09 | 64.91±1.49 |
From Fig. 1, Fig. 2, Fig. 3, Fig. 4 and table 1, check experiment is preserved in dilution in the fresh essence of sheep and is not added astaxanthin, and test one, test two, test three and test four add astaxanthin, and the astaxanthin concentration that the fresh essence of sheep is preserved in dilution in test one, test two, test three and test four is respectively 0.5 μm of ol/L, 1 μm of ol/L, 2 μm of ol/L and 4 μm ol/L; Test three and test four are preserved in dilution in the fresh essence of sheep and are added astaxanthin, significantly improve the ratio of the plasm membrane integrity of the sperm of sheep seminal fluid, the motility rate of sperm and motile, effectively reduce MDA concentration and the ROS level of seminal fluid;
The sheep seminal fluid of test three and test four is 4 DEG C of preservations after 3 days, and sperm motility rate is up to 74%, and check experiment on average improves 9 percentage points; Plasmalemmae of sperms percentage of head rice reaches 75%, and check experiment on average improves 8 percentage points, just presents obvious reduction after MDA concentration and ROS level preserve 24 hours at 4 DEG C.Therefore, astaxanthin may be that MDA and ROS concentration owing to reducing sperm causes to the protective effect of sperm.Add astaxanthin at sheep fresh essence preservation dilution in the present invention and can act on sperm rapidly, have extremely strong oxidation resistance, sperm can be protected to avoid oxidativestress damage, and then improve Semen liquid storage effect.
Claims (10)
1. the fresh essence of sheep preserves a dilution, it is characterized in that what a kind of sheep fresh essence preservation dilution was prepared by basal liquid, yolk and astaxanthin; The volume ratio that the fresh essence of described sheep preserves basal liquid and yolk in dilution is 8:2; The concentration that the fresh essence of described sheep preserves astaxanthin in dilution is 0.5 μm of ol/L ~ 4 μm ol/L.
2. the fresh essence of a kind of sheep according to claim 1 preserves dilution, it is characterized in that described basal liquid is prepared by the following method: 3.2g glucose, 3.2g sodium citrate, penicillin 100,000 IU and streptomycin 100,000 IU are dissolved in 100mL ultra-pure water, obtain basal liquid.
3. the fresh essence of a kind of sheep according to claim 1 preserves dilution, it is characterized in that the concentration that the fresh essence of described sheep preserves astaxanthin in dilution is 0.5 μm of ol/L ~ 1 μm ol/L.
4. the fresh essence of a kind of sheep according to claim 1 preserves dilution, it is characterized in that the concentration that the fresh essence of described sheep preserves astaxanthin in dilution is 1 μm of ol/L ~ 2 μm ol/L.
5. the fresh essence of a kind of sheep according to claim 1 preserves dilution, it is characterized in that the concentration that the fresh essence of described sheep preserves astaxanthin in dilution is 2 μm of ol/L ~ 4 μm ol/L.
6. the fresh essence of sheep preserves an application for dilution, it is characterized in that the fresh essence of a kind of sheep is preserved the application method of dilution in the fresh essence of sheep is preserved and completed according to the following steps:
One, prepare the fresh essence of sheep and preserve dilution: basal liquid, yolk and astaxanthin are mixed, obtain the fresh essence of sheep and preserve dilution;
The volume ratio that the fresh essence of sheep described in step one preserves basal liquid and yolk in dilution is 8:2;
The concentration that the fresh essence of sheep described in step one preserves astaxanthin in dilution is 0.5 μm of ol/L ~ 4 μm ol/L;
Two, sheep seminal fluid is diluted:
Utilize artificial vagina method to collect sheep seminal fluid, in the sheep seminal fluid collected, then add the sheep fresh essence preservation dilution that temperature is 35 DEG C ~ 40 DEG C, obtain the sheep seminal fluid after diluting;
The sheep seminal fluid of the collection described in step 2 and temperature are the volume ratio that the fresh essence of the sheep of 35 DEG C ~ 40 DEG C preserves dilution is 1:5;
Three, preserve seminal fluid: the sheep seminal fluid after the dilution using 8 layers ~ 10 layers gauze step 2 to be obtained wraps up, and is being placed into 70h ~ 72h in 4 DEG C of refrigerators, namely complete the fresh essence of a kind of sheep and preserve the application method of dilution in the fresh essence of sheep is preserved.
7. the fresh essence of a kind of sheep according to claim 6 preserves the application of dilution, it is characterized in that the basal liquid described in step one is prepared by the following method: be dissolved in 100mL ultra-pure water by 3.2g glucose, 3.2g sodium citrate, penicillin 100,000 IU and streptomycin 100,000 IU, obtain basal liquid.
8. the fresh essence of a kind of sheep according to claim 6 preserves the application of dilution, it is characterized in that the concentration that the fresh essence of sheep described in step one preserves astaxanthin in dilution is 0.5 μm of ol/L ~ 1 μm ol/L.
9. the fresh essence of a kind of sheep according to claim 6 preserves the application of dilution, it is characterized in that the concentration that the fresh essence of sheep described in step one preserves astaxanthin in dilution is 1 μm of ol/L ~ 2 μm ol/L.
10. the fresh essence of a kind of sheep according to claim 6 preserves the application of dilution, it is characterized in that the concentration that the fresh essence of sheep described in step one preserves astaxanthin in dilution is 2 μm of ol/L ~ 4 μm ol/L.
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| CN106172372A (en) * | 2016-07-07 | 2016-12-07 | 中国科学院东北地理与农业生态研究所 | The making of a kind of sheep frozen spermatic fluid preserves integrated apparatus and application process thereof |
| CN107821384A (en) * | 2017-11-15 | 2018-03-23 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | The dilution of sheep seminal fluid high power low temperature preserves liquid and preparation method |
| CN108935442A (en) * | 2018-03-27 | 2018-12-07 | 河北农业大学 | A kind of sheep sperm cryo-conservation technology |
| CN111449058A (en) * | 2020-05-26 | 2020-07-28 | 广西大学 | Method for improving cock semen preservation quality |
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| CN103548812A (en) * | 2013-10-30 | 2014-02-05 | 西北农林科技大学 | Normal-temperature and low-temperature preservation diluents of milk goat seminal fluid |
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| CN102524241A (en) * | 2010-12-16 | 2012-07-04 | 宁波大学 | Additive for freezing storage protective liquid of fish spermatid |
| CN103141471A (en) * | 2013-02-01 | 2013-06-12 | 四川农业大学 | Formula and preparation method of Kunming wolf dog semen refrigerating fluid |
| CN103548812A (en) * | 2013-10-30 | 2014-02-05 | 西北农林科技大学 | Normal-temperature and low-temperature preservation diluents of milk goat seminal fluid |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106172372A (en) * | 2016-07-07 | 2016-12-07 | 中国科学院东北地理与农业生态研究所 | The making of a kind of sheep frozen spermatic fluid preserves integrated apparatus and application process thereof |
| CN107821384A (en) * | 2017-11-15 | 2018-03-23 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | The dilution of sheep seminal fluid high power low temperature preserves liquid and preparation method |
| CN108935442A (en) * | 2018-03-27 | 2018-12-07 | 河北农业大学 | A kind of sheep sperm cryo-conservation technology |
| CN111449058A (en) * | 2020-05-26 | 2020-07-28 | 广西大学 | Method for improving cock semen preservation quality |
| CN111449058B (en) * | 2020-05-26 | 2022-01-11 | 广西大学 | Method for improving cock semen preservation quality |
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