CN107668026A - Application of the rutin as livestock semen Cryoprotectant - Google Patents
Application of the rutin as livestock semen Cryoprotectant Download PDFInfo
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- CN107668026A CN107668026A CN201711067623.0A CN201711067623A CN107668026A CN 107668026 A CN107668026 A CN 107668026A CN 201711067623 A CN201711067623 A CN 201711067623A CN 107668026 A CN107668026 A CN 107668026A
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- rutin
- semen
- sperm
- livestock
- cryoprotectant
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Abstract
The invention discloses application of the rutin as livestock semen Cryoprotectant, the present invention also provides a kind of livestock semen freezen protective liquid, including cryoprotective extender, egg yolk, rutin and glycerine, the present invention is used for pig, sheep and ox semen cryopreservation using rutin as antifreeze first, suitable for semen deposition after the long-term preservation after pig, sheep and the artificial insemination of ox Straw Frozen Semen and seminal fluid superfreeze and long-distance transport, pig after freeze-thaw, sheep and ox sperm motility rate, acrosomal integrity and plasm membrane integrity are remarkably improved.
Description
Technical field
The invention belongs to the Ultra-cryofreezing preservation technical field of mammal seminal fluid, and in particular to rutin is as freeze proof
Application of the agent in domestic animal semen cryopreservation.
Background technology
Development of the sperm freezing technology for domestic animal has great importance, and not only solves the length of buck seminal fluid
Phase preserves problem, is easy to preserve genetic resources, and application is convenient, is not limited by time and space, is carrying out inter-provincial, state
The cooperation of animal husbandry between border, played an important role establishing excellent species gene pool in the whole world etc..Sperm freezing technology
Extensive use, be advantageous to improve the utilization rate of outstanding sire, reduce the number of animals raised of male animal, reduce financial cost, improve poultry
Animal husbandry benefit, especially played an important role in terms of artificial insemination field.Herding of the Semen freezing technique to China and the world
The supply of the satisfaction of industry sustainable development and livestock products has important practical significance.
Sperm is more sensitive to temperature change, and during frozen cooling, sperm is subject to freezing injury, mainly
Including physical injury, chemical lesion and oxidative damage.Compared with fresh semen, the sperm after freezing-defrosting, its
Motility rate, acrosomal integrity, mitochondria activity, DNA percentage of head rices and film percentage of head rice are remarkably decreased.And the quality of sperm quality, it is and pregnant
Rate, parturition rate and the nest litter size of being pregnent are closely related.So far, frozen semen artificial insemination technology has been applied to cowboying life
Production, apply in terms of cow reproduction and improvement and popularize the most.But after freeze-thaw, still there are 40%-50% sperms to lose work
Property, seriously reduce the utilization rate of breeding bull.And Boar spermatozoa and sheep sperm stimulate more sensitive, essence after defrosting for freezing
Sub- anabiosis rate is low, and abnormal spermium is more, and conception rate is less than normal conception rate, therefore Boar spermatozoa and sheep sperm freezing are also located in the world
In experimental stage.
During freezing, due to the reduction of temperature, Extracellular solution can be initially formed the ice crystal of a part, work as cell
After outer ice crystal produces, cause cell appearance again into hypertonic solution environment, so as to cause the abjection of ICW.With continuous
Cooling, also have the formation of ice crystal into the cell., can be to the cell of sperm after extracellular and intracellular ice crystal largely produces
Film, cell interior structure and organelle cause significant mechanical to damage, and destroy cell function, the integrality of cell membrane, and acrosome
Destruction, additionally it is possible to cause the complete of sperm DNA structure to sexually revise, cause DNA damage, so as to cause the death of sperm, reduce
Survival rate.It is inevitable that ice crystal, which is formed, in refrigerating process, and the degree of damage has close associate with the size of ice crystal.
The harm of ice crystal in refrigerating process how is reduced, freezing ice crystal should be made to be in the state of crystallite as far as possible, cold beat could be reduced
The damage hit.
The content of the invention
In view of the deficienciess of the prior art, the present invention provide a kind of livestock semen Cryoprotectant and its pig,
Application in sheep and ox semen cryopreservation, sperm motility rate is low after solving the problems, such as prior art domestic animal freeze-thaw.
To solve above-mentioned deficiency, the technical solution adopted by the present invention is as follows：
The invention provides rutin as the application in livestock semen Cryoprotectant.
Present invention also offers livestock semen Cryoprotectant, and it includes cryoprotective extender, egg yolk, glycerine and rutin.
The formula of the cryoprotective extender of the present invention is glucose 1.1g, citric acid 1.48g, Tris2.42g, Benzylpenicillin sodium salt
0.06g, streptomycin sulphate 0.1g, distilled water 100ml；Wherein the addition of glycerine is livestock semen Cryoprotectant volume
6%-12%；The volume ratio of cryoprotective extender and egg yolk is 4:1.
In a kind of embodiment, rutin element addition is in 100ml domestic animal semen cryopreservation agent of the present invention
0.010mmol-0.100mmol。
In another embodiment, rutin addition is 0.030mmol- in 100ml domestic animal semen cryopreservation agent of the present invention
0.080mmol。
In another embodiment, rutin addition is 0.050mmol- in 100ml domestic animal semen cryopreservation agent of the present invention
0.100mmol。
The beneficial effects of the invention are as follows：
Rutin is used for pig, sheep and ox semen cryopreservation by the present invention first, and effect is reliable, easy to spread, is applied to
Semen deposition after long-term preservation and long-distance transport after pig, sheep and the artificial insemination of ox Straw Frozen Semen and seminal fluid superfreeze, can
Pig after freeze-thaw, sheep and ox sperm motility rate, acrosomal integrity and plasm membrane integrity are significantly improved, keeps the fertilization energy of sperm
Power.
Brief description of the drawings
After Fig. 1 is addition rutin antifreeze, the comparison diagram of perforatorium integrality and membrane integrity, Fig. 1 (a) is essence
Sub- acrosomal integnity schematic diagram,The sperm damaged for acrosome indicated for the complete sperm of acrosome, △ indicated, Fig. 1 (b) are
Plasmalemmae of sperms integrality schematic diagram, ↑ it is the complete sperm of plasma membrane, △ is damaged sperm.
After Fig. 2 is addition resveratrol antifreeze, the comparison diagram of perforatorium integrality and membrane integrity, Fig. 2 (a) is essence
Sub- acrosomal integnity schematic diagram,The sperm damaged for acrosome indicated for the complete sperm of acrosome, △ indicated, Fig. 2 (b)
For plasmalemmae of sperms integrality schematic diagram, ↑ it is the complete sperm of plasma membrane, △ is damaged sperm.
After Fig. 3 is addition phytic acid antifreeze, the comparison diagram of perforatorium integrality and membrane integrity, Fig. 3 (a) is sperm top
Body integrality schematic diagram,The sperm damaged for acrosome indicated for the complete sperm of acrosome, △ indicated, Fig. 3 (b) is sperm
Membrane integrity schematic diagram, ↑ it is the complete sperm of plasma membrane, △ is damaged sperm.
Embodiment
Rutin (Rutin), also known as rutin sophorin, citrin, Lu Ding, belong to flavonoids, in the plant of nature extensively
In the presence of so that, for predominant amount, through studying, there is also such as hawthorn, ginkgo, matrimony vine contain in some Chinese medicines in root, stem, leaf
There is a certain amount of rutin.Rutin is a kind of powder of yellow crystals, molecular formula C27H30O16, relative molecular mass is
610.51, it is soluble in the organic solvents such as ethanol, dimethyl sulfoxide (DMSO), ether.Rutin has anti-inflammation, radioresistance, regulation hair
The permeability of thin blood vessel, clinically for hypertension, cardiovascular and cerebrovascular etc., there is good therapeutic effect.Contain in the structure of rutin
Multiple hydroxyls, it is presumed that hydroxyl can replace hydrone, crystallization process of the reduction water in refrigerating process, reduce ice crystal pair
The infringement of sperm, the survival rate of sperm is improved, frozen cell may be played a part of protecting well, also can be to metal
Ion plays good complexing.
We prove test of many times, and during domestic animal freezing of semen, addition rutin antifreeze can play certain guarantor
Shield acts on.At present, relevant rutin is used for the even any mammality freezing of semen of the domestic animals such as pig, sheep and ox as cryoprotector
The research of preservation is both at home and abroad there is not yet relevant report.
Below in an example, involved normal temperature dilution (Betsville Thawing Solution, BTS)
It is formulated and is：Glucose 3.7g, citrate dihydrate trisodium 0.6g, EDTA0.125g, sodium acid carbonate 0.125g, potassium chloride 0.075g,
Benzylpenicillin sodium salt 0.06g, streptomycin sulphate 0.1g, distilled water mix, and are settled to 100ml, adjust PH to 7.2, and osmotic pressure is
310mosm/L。
A kind of livestock semen Cryoprotectant of the present invention, including cryoprotective extender, egg yolk and rutin.
The volume ratio that the amount of glycerine is added in above-mentioned livestock semen Cryoprotectant is 6%-12%, that is, obtains this hair
Another bright livestock semen Cryoprotectant, for example, adding glycerine in above-mentioned 94ml livestock semen Cryoprotectants
Measure as 6ml, that is, another the livestock semen Cryoprotectant for obtaining the present invention is used to preserve pig semen.
The formula of the cryoprotective extender is glucose 1.1g, citric acid 1.48g, Tris2.42g, Benzylpenicillin sodium salt
0.06g, streptomycin sulphate 0.1g, distilled water 100ml.
Embodiment 1：
The preparation method of Cryopreservation of Boar Semen agent of the present invention, comprises the following steps：
Step 1, prepare cryoprotective extender：It is accurate to measure glucose 1.1g, citric acid 1.48g, Tris2.42g, mould
Plain sodium 0.06g, streptomycin sulphate 0.1g, is dissolved in 100ml distilled waters, adjusts pH to 6.21, osmotic pressure 286mosm/L, prepares
Into cryoprotective extender；
Step 2, cryoprotective extender 80ml is accurately measured, add Fresh Egg Huang 20ml, rutin 0.01mmol-
0.10mmol, mix a kind of livestock semen Cryoprotectant (I liquid) for obtaining the present invention.
Take 94ml above-mentioned livestock semen Cryoprotectant to add 6ml glycerine, filtration sterilization, be cooled to room temperature, you can with
Another livestock semen Cryoprotectant (II liquid) for obtaining the present invention is used to preserve pig semen, is put into standby in 4 DEG C of refrigerators stay
Do tests below test.
Inventor has carried out following experiment to the Cryopreservation of Boar Semen dilution using effect of the embodiment：
(1) semen collection
With hand grip semen collection, jelly is filtered to remove through 4 layers of antiseptic gauze, with being carried out at 34-37 DEG C of light microscope often
Advise quality inspection.It is milky to select free from extraneous odour, color and luster, sperm morphology is normal, motility rate more than 0.75, density be " close "
Seminal fluid, 30 DEG C are incubated for testing.
(2) liquefacient duration and freezing
By fresh semen add isothermal normal temperature dilution (30 DEG C, 1：1 dilution), after multilayer gauze wrapped, at 17 DEG C
0.5h-1h is balanced in insulating box.The seminal fluid that Balance Treatment is crossed is centrifuged into (1200rpm, 10min) at 17 DEG C, supernatant is abandoned, adds
The I liquid of the two volumes of isothermal, 1h-1.5h is balanced with being placed in after multilayer gauze wrapped in 4 DEG C of refrigerators.Afterwards by Balance Treatment
The seminal fluid crossed adds 4 DEG C of isometric II liquid, with being placed in releveling 1.5-2.5h in 4 DEG C of refrigerators after gauze wrapped.With special note
Emitter sucks seminal fluid in 0.25ml tubules, quick tube sealing, is placed in Slow-rate freezing instrument slow from 4 DEG C with 1 DEG C/min speed
- 5 DEG C are down to, 2.5-3.5cm above liquid nitrogen is moved to rapidly afterwards and fumigates 10-15min, tubule is put into liquid nitrogen and preserved.
(3) defrosting of straw frozen semen and sperm quality evaluation
1st, sperm motility rate
The 45s that thaws is put into 37 DEG C of water-bath after straw frozen semen is taken out rapidly, is collected in small test tube, is then added
Isometric, 37 DEG C of preheated normal temperature diluteds, are incubated 10min, take 10 μ L seminal fluid in the glass that on slide, closes the lid
Piece, sperm motility rate (linear motion sperm percentage) is evaluated under 400 × inverted microscope.200 essences are at least checked every time
Son, it is repeated 5 times.
2nd, Sperm acrasomal integvity
After being dyed using FITC-PNA dye liquors, fluorescence microscope perforatorium form.
After freezing fine tube defrosting, seminal fluid is added on 2ml 3%PVP liquid levels, 1500rpm centrifugation 6min, abandons supernatant；With
PBS is washed 2 times, 1500rpm centrifugation 6min, abandons supernatant；With (37 DEG C) resuspension sperms of PBS, adjustment density to 1-2 × 106spe/
ml；30 μ l sperm suspensions are drawn, smear, is air-dried, 10min is fixed with methanol；Add 30 μ l FITC-PNA dye liquors, 37
DEG C dark moist environment is incubated 30min；PBS is rinsed, and is air-dried, and drips a small amount of mountant, cover plate, with colourless nail sheet for oil seal,
As early as possible in 400 × fluorescence microscopy Microscopic observation.200 sperms are at least checked every time, are repeated 5 times.
3rd, plasmalemmae of sperms percentage of head rice
Hypotonic swelling detection (HOST) is carried out using fructose-sodium citrate hypotonic medium.The seminal fluid hypotonic medium of defrosting is dilute
Release, adjustment sperm concentration is 1 × l06Sperm/ml, 37 DEG C of incubation 30min, takes 20 μ l seminal fluid to be dripped in suspension in blood count
On plate, 400 × micro- Microscopic observation, curved tail sperm percentage is calculated, at least check 200 sperms every time, be repeated 5 times.
(4) sperm quality evaluation result
It is as follows using the Cryopreservation of Boar Semen antifreeze and freezing of semen-defreezing method, evaluation result of the present invention：
When in pig with 0.010mmol-0.100mmol rutins are added in I liquid of freezing, sperm motility rate reaches after freeze-thaw
65%, acrosomal integrity is up to 70%, and plasm membrane integrity is up to 68%.
Test example result is as shown in table 1 below：
Table 1
| Processing | Sperm motility rate | Acrosomal integrity | Plasm membrane integrity |
| Control group | 30% | 45% | 35% |
| Embodiment 1 | 65% | 70% | 68% |
Comparative example 1：
Quality testing of this comparative example for the freezing agent compounding method of pig semen and to Boar spermatozoa is identical with embodiment 1,
Unlike, phytic acid is 0.02-0.10mmol as antifreeze addition.
Result of the test shows that Boar spermatozoa motility rate is 43%, acrosomal integrity 50%, plasm membrane integrity 48%.
Comparative example 2：
Quality testing of this comparative example for the freezing agent compounding method of pig semen and to Boar spermatozoa is identical with embodiment 1,
Unlike, resveratrol is added to 0.1-0.3mmol as antifreeze.Result of the test shows that Boar spermatozoa motility rate is 47%, top
Body percentage of head rice is 50%, plasm membrane integrity 56%.
Comparative example 1 and comparative example 2 are that inventor adds the experiment that phytic acid or resveratrol are done in antifreeze,
And the addition of phytic acid or resveratrol is the most suitable addition that test of many times obtains, but test effect is paid no attention to
Think.When rutin is as antifreeze, its motility rate, acrosomal integrity and plasm membrane integrity are significantly better than using phytic acid or white
Veratryl alcohol, it may be possible to due to the rutin as strong antifreeze, play a role apparently higher than phytic acid and resveratrol.
After Fig. 1-Fig. 3 is three kinds of different antifreeze of addition, the comparison diagram of perforatorium integrality and membrane integrity.
According to figure：
It is most of for hat shape dome-type in the acrosomal integnity of rutin, it is the complete sperm of acrosome, it is complete in plasma membrane
Property detection in, most sperms have obvious curved tail phenomenon, and the curved tail of afterbody for the sperm also having is less obvious but plasma membrane compares
Completely, impaired sperm quantity is fewer.
In the acrosomal integnity detection of resveratrol, there is the uneven quantity of fluorescent color and be significantly more than in rutin
Quantity, the integrality of acrosome is significantly lower than the acrosomal integnity after addition rutin.In membrane integrity detection, the curved tail of afterbody
Unconspicuous quantity increase, more than the unconspicuous phenomenon of curved tail after addition rutin.
In the acrosomal integnity detection of phytic acid, occur that hat shape dome-type green fluorescence, fluorescent color are uneven to be had,
It is below the acrosomal integnity of rutin.In the detection of membrane integrity, curved tail substantially exists with unconspicuous, but is less than
Add the curved mantissa amount after rutin.
Three's contrast can be shown that the acrosomal integnity of rutin will be high than phytic acid and resveratrol, membrane integrity
Also above the two, addition rutin can significantly improve the activity of sperm as antifreeze.
Embodiment 2：
The preparation method of sheep semen cryopreservation agent of the present invention, comprises the following steps：
Step 1, cryoprotective extender is prepared, method is the same as embodiment 1；
Step 2, cryoprotective extender 85ml is accurately measured, add Fresh Egg Huang 15ml and rutin 0.030mmol-
0.080mmol, mix a kind of livestock semen Cryoprotectant (I liquid) for being configured to the present invention.
Take 88ml above-mentioned livestock semen Cryoprotectant to add 12ml glycerols to go out into 100ml mixed liquor, filtering
Bacterium, room temperature is cooled to, that is, another livestock semen Cryoprotectant (II liquid) for obtaining the present invention is used to preserve sheep seminal fluid, puts
Enter standby in 4 DEG C of refrigerators to do tests below.
(1) semen collection
With hand grip semen collection, Ordinary fruit quality inspection is carried out at 34-37 DEG C with microscope.It is breast to select free from extraneous odour, color and luster
White, sperm morphology are normal, motility rate more than 0.7, the seminal fluid that density is " close ", 30 DEG C are incubated for testing.
(2) liquefacient duration and freezing
I isometric liquid of isothermal will be added in fresh semen, after multilayer gauze wrapped, balanced in 4 DEG C of refrigerators
2h.II isometric liquid of isothermal is added in the seminal fluid that Balance Treatment is crossed again, with being placed in after multilayer gauze wrapped in 4 DEG C of refrigerators
Balance 1-1.5h.Seminal fluid is sucked in 0.25ml tubules with special syringe, quick tube sealing, is placed in Slow-rate freezing instrument with 1
DEG C/min speed is slowly dropped to -5 DEG C from 4 DEG C, 2.5-3.5cm above liquid nitrogen is moved to rapidly and fumigates 6-8min, and tubule is put into
Preserved in liquid nitrogen.
(3) defrosting of straw frozen semen and sperm quality evaluation
1st, sperm motility rate
The 25s that thaws is put into 37 DEG C of water-bath after straw frozen semen is taken out rapidly, is collected in small test tube, then use etc.
Volume, 37 DEG C of preheated normal temperature diluteds, 10min is incubated, takes 10 μ L seminal fluid on slide, covered,
Sperm motility rate (linear motion sperm percentage) is evaluated under 400 × inverted microscope.200 sperms, weight are at least checked every time
It is multiple 5 times.
2nd, Sperm acrasomal integvity
After being dyed using FITC-PNA dye liquors, fluorescence microscope perforatorium form.
After freezing fine tube defrosting, seminal fluid is added on 2ml 3%PVP liquid levels, 1500rpm centrifugation 6min, abandons supernatant；With
PBS is washed 2 times, 1500rpm centrifugation 6min, abandons supernatant；With (37 DEG C) resuspension sperms of PBS, adjustment density to 1-2 × 106sperm/
ml；30 μ l sperm suspensions are drawn, smear, is air-dried, 10min is fixed with methanol；Add 30 μ l FITC-PNA dye liquors, 37
DEG C dark moist environment is incubated 30min；PBS is rinsed, and is air-dried, and drips a small amount of mountant, cover plate, with colourless nail sheet for oil seal,
As early as possible in 400 × fluorescence microscopy Microscopic observation.200 sperms are at least checked every time, are repeated 5 times.
3rd, plasmalemmae of sperms percentage of head rice
Hypotonic swelling detection (HOST) is carried out using fructose-sodium citrate hypotonic medium.By the seminal fluid conventional dilution of defrosting
Liquid dilutes, and adjustment sperm concentration is 1 × l06Sperm/ml, 37 DEG C of incubation 30min, takes 20 μ l seminal fluid to be dripped in suspension in haemocyte
On tally, 400 × micro- Microscopic observation, curved tail sperm percentage is calculated, at least check 200 sperms every time, be repeated 5 times.
(4) sperm quality evaluation result
It is as follows using the sheep semen cryoprotectant and freezing of semen-defreezing method, evaluation result of the present invention：
When in sheep with 0.030mmol-0.080mmol rutin is added in I liquid of freezing, sperm motility rate after freeze-thaw
Up to 60%, acrosomal integrity is up to 65%, and plasm membrane integrity is up to 56%.As a result table 2 as shown in table 2 below
| Processing | Sperm motility rate | Acrosomal integrity | Plasm membrane integrity |
| Control group | 43% | 51% | 40% |
| Embodiment 2 | 60% | 65% | 56% |
Comparative example 1：
Quality testing of this comparative example for the freezing agent compounding method of sheep seminal fluid and to Boar spermatozoa is identical with embodiment 1,
Unlike, phytic acid is 0.02-0.05mmol as antifreeze addition.
Result of the test shows that Boar spermatozoa motility rate is 44%, acrosomal integrity 53%, plasm membrane integrity 46%.
Comparative example 2：
Quality testing of this comparative example for the freezing agent compounding method of sheep seminal fluid and to Boar spermatozoa is identical with embodiment 1,
Unlike, resveratrol is added to 0.4mmol as antifreeze.Result of the test shows that sheep sperm motility rate is 49%, and acrosome is complete
Whole rate is 54%, plasm membrane integrity 51%.
Comparative example 1 and comparative example 2 are that inventor adds the experiment that phytic acid or resveratrol are done in antifreeze,
And the addition of phytic acid or resveratrol is the most suitable addition that test of many times obtains, but test effect is paid no attention to
Think, when rutin is as antifreeze, its motility rate, acrosomal integrity and plasm membrane integrity are significantly better than using phytic acid or white
Veratryl alcohol, it may be possible to due to the rutin as strong antifreeze, play a role apparently higher than phytic acid and resveratrol.
Embodiment 3：
The preparation method of ox semen cryopreservation agent of the present invention, comprises the following steps：
Step 1, cryoprotective extender is prepared, method is the same as embodiment 1；
Step 2, cryoprotective extender 80ml is accurately measured, add Fresh Egg Huang 20ml and rutin 0.050mmol-
0.100mmol, that is, obtain a kind of livestock semen Cryoprotectant (I liquid) of the present invention.
Glycerol adding 6ml in above-mentioned livestock semen Cryoprotectant, mix, filtration sterilization, be cooled to room temperature, that is, obtain
The present invention another livestock semen Cryoprotectant (II liquid) be used for preserve ox seminal fluid, be put into 4 DEG C of refrigerators it is standby do it is following
Experiment.
(1) semen collection
With hand grip semen collection, Ordinary fruit quality inspection is carried out at 37 DEG C with microscope.It is milky white to select free from extraneous odour, color and luster
Color, sperm morphology are normal, motility rate more than 0.7, the seminal fluid that density is " close ", 37 DEG C are incubated for testing.
(2) liquefacient duration and freezing
Fresh semen is added to the II liquid of isothermal, adjustment sperm concentration is 1.5 × 106Individual/ml, after gauze wrapped,
4h is balanced in 4 DEG C of refrigerators.
The seminal fluid that Balance Treatment is crossed, seminal fluid is sucked in 0.25ml tubules with special syringe, quick tube sealing, is placed in
- 5 DEG C are slowly dropped to from 4 DEG C with 1 DEG C/min speed in Slow-rate freezing instrument, 2-3cm above liquid nitrogen is moved to rapidly and fumigates 5-
10min, tubule is put into liquid nitrogen and preserved.
(3) defrosting of straw frozen semen and sperm quality evaluation
1st, sperm motility rate
The 45s that thaws is put into 37 DEG C of water-bath after straw frozen semen is taken out rapidly, is collected in small test tube, is then added
Isometric normal temperature diluted, 10min is incubated, takes 10 μ L seminal fluid on slide, covered, 400 × be inverted
Sperm motility rate (linear motion sperm percentage) is evaluated under microscope.200 sperms are at least checked every time, are repeated 5 times.
2nd, Sperm acrasomal integvity
After being dyed using FITC-PNA dye liquors, fluorescence microscope perforatorium form.
After freezing fine tube defrosting, seminal fluid is added on 2ml 3%PVP liquid levels, 1500rpm centrifugation 6min, abandons supernatant；With
PBS is washed 2 times, 1500rpm centrifugation 6min, abandons supernatant；With (37 DEG C) resuspension sperms of PBS, adjustment density to 1-2 × 106spe/
ml；30 μ l sperm suspensions are drawn, smear, is air-dried, 10min is fixed with methanol；Add 30 μ l FITC-PNA dye liquors, 37
DEG C dark moist environment is incubated 30min；PBS is rinsed, and is air-dried, and drips a small amount of mountant, cover plate, with colourless nail sheet for oil seal,
As early as possible in 400 × fluorescence microscopy Microscopic observation.200 sperms are at least checked every time, are repeated 5 times.
3rd, plasmalemmae of sperms percentage of head rice
Hypotonic swelling detection (HOST) is carried out using fructose-sodium citrate hypotonic medium.By the seminal fluid conventional dilution of defrosting
Liquid dilutes, and adjustment sperm concentration is 1 × l06Individual/ml, 37 DEG C of incubation 30min, takes 20 μ l seminal fluid to be dripped in suspension in hemocytometer
On number plates, 400 × micro- Microscopic observation, curved tail sperm percentage is calculated, at least check 200 sperms every time, be repeated 5 times.
(4) sperm quality evaluation result
It is as follows using the ox semen cryopreservation antifreeze and freezing of semen-defreezing method, evaluation result of the present invention：
When adding rutin 0.050mmol-0.100mmol, sperm motility rate is up to 68% after freeze-thaw, acrosomal integrity
Up to 75%, plasm membrane integrity is up to 62%.As a result it is as shown in table 3.
Table 3
| Processing | Sperm motility rate | Acrosomal integrity | Plasm membrane integrity |
| Control group | 44% | 58% | 52% |
| Embodiment 3 | 68% | 75% | 62% |
Comparative example 1：
Quality testing of this comparative example for the freezing agent compounding method of ox seminal fluid and to Boar spermatozoa is identical with embodiment 1,
Unlike, phytic acid is 0.03-0.09mmol as antifreeze addition.
Result of the test shows that Boar spermatozoa motility rate is 46%, acrosomal integrity 59%, plasm membrane integrity 53%.
Comparative example 2：
Quality testing of this comparative example for the freezing agent compounding method of ox seminal fluid and to Boar spermatozoa is identical with embodiment 1,
Unlike, resveratrol is added to 0.03-0.08mmol as antifreeze.Result of the test shows that ox sperm motility rate is 48%,
Acrosomal integrity is 61%, plasm membrane integrity 56%.
Comparative example 1 and comparative example 2 are that inventor adds the experiment that phytic acid or resveratrol are done in antifreeze,
And the addition of phytic acid or resveratrol is the most suitable addition that test of many times obtains, but test effect is paid no attention to
Think, when rutin is as antifreeze, its motility rate, acrosomal integrity and plasm membrane integrity are significantly better than using phytic acid or white
Veratryl alcohol, it may be possible to due to the rutin as strong antifreeze, play a role apparently higher than phytic acid and resveratrol.
It should be appreciated that for those of ordinary skills, it can according to the above description be improved or be become
Change, and all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
Claims (6)
1. rutin is as the application in livestock semen Cryoprotectant.
2. livestock semen Cryoprotectant described in claim 1, including cryoprotective extender, egg yolk, glycerine and rutin.
3. livestock semen Cryoprotectant as claimed in claim 2, it is characterised in that the formula of the cryoprotective extender is grape
Sugared 1.1g, citric acid 1.48g, Tris2.42g, Benzylpenicillin sodium salt 0.06g, streptomycin sulphate 0.1g, distilled water 100ml；It is described sweet
The addition of oil is the 6%-12% of livestock semen Cryoprotectant volume；The volume ratio of the cryoprotective extender and egg yolk is
4:1。
4. livestock semen Cryoprotectant as claimed in claim 3, it is characterised in that livestock semen freezen protective described in 100ml
Rutin element addition is 0.010mmol-0.100mmol in agent.
5. livestock semen Cryoprotectant as claimed in claim 3, it is characterised in that livestock semen freezen protective described in 100ml
Rutin addition is 0.030mmol-0.080mmol in agent.
6. livestock semen Cryoprotectant as claimed in claim 3, it is characterised in that livestock semen freezen protective described in 100ml
Rutin addition is 0.050mmol-0.100mmol in agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111657265A (en) * | 2020-06-16 | 2020-09-15 | 西北农林科技大学 | Application of taxifolin and kaempferol in preparation of livestock cryopreservation agent |
| CN113615682A (en) * | 2021-09-05 | 2021-11-09 | 内蒙古赛诺种羊科技有限公司 | Sheep hypertonic complexing agent semen dilution preserving fluid and application method thereof |
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Cited By (2)
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| CN111657265A (en) * | 2020-06-16 | 2020-09-15 | 西北农林科技大学 | Application of taxifolin and kaempferol in preparation of livestock cryopreservation agent |
| CN113615682A (en) * | 2021-09-05 | 2021-11-09 | 内蒙古赛诺种羊科技有限公司 | Sheep hypertonic complexing agent semen dilution preserving fluid and application method thereof |
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