CN106818709A - Cattle freezing seminal fluid dilution preparation method - Google Patents
Cattle freezing seminal fluid dilution preparation method Download PDFInfo
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- CN106818709A CN106818709A CN201710065529.5A CN201710065529A CN106818709A CN 106818709 A CN106818709 A CN 106818709A CN 201710065529 A CN201710065529 A CN 201710065529A CN 106818709 A CN106818709 A CN 106818709A
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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
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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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The invention discloses a kind of cattle freezing seminal fluid dilution preparation method, weigh 5~8g of fructose, the g of sodium citrate 19.8~26.6, add the mL of distilled water 800, stirring, then solution filter paper is filtered in conical flask or saline bottle, puts and sterilize in 75~80 DEG C of water-baths 30 min, be cooled to after normal temperature and add the fresh yolk of 180~220 mL, and the IU units of penicillin, streptomysin each 40~500,000 are added, it is referred to as the first liquid by the solution of this proportional arrangement；Take the mL of the first liquid 430 and add 65~75 mL glycerine, 3~5 DEG C of refrigerator, referred to as the second liquid are put into after stirring；Then by semen dilution, freezing, packaging.Outstanding breeding oxen fresh semen dilution effect and sperm motility rate are improved, the permanently effective preservation of frozen semen is reached.
Description
Technical field
The invention belongs to technical field of animal husbandry, it is related to a kind of cattle freezing seminal fluid dilution preparation method.
Background technology
Cow frozen semen has been widely used with technology of artificial insemination in China's cattle-raising production, therefore cow frozen semen
Production is constantly subjected to the concern in industry with quality control.
It was verified that frozen semen production is the core business of various regions Breeding bull station, the production procedure system should be set up
On the basis of standardized work program, including instrument cleaning and the sterilization of production cow frozen semen, diluent preparing, semen collection,
Liquefacient duration, freezing of semen, jelly essence are thawed, freeze smart microscopy and inspection rule, jelly spermatophore dress, freeze the behaviour such as essence storage and jelly essence transport
Make link.
Major part Breeding bull station replaces autogamy semen diluent with import dilution powder at present, and import dilution powder is to a certain degree
On achieve better effects, but take a long view and which increase the production cost of cow frozen semen, reduce economic benefit, while
Also the defect of cattle freezing seminal fluid dilution formula is reflected.
Crowd sees various regions Breeding bull station freezing seminal fluid dilutions, whether autogamy or import, wherein including dilution
Agent, nutritional agents, protective agent and other additives etc..At present, it is glucose-glycerine-yolk liquid that cattle freezing seminal fluid dilution is most,
But diluent, nutritional agents, protective agent have optional difference chemical substance and dosage combination again, most of Breeding bull station reflects import
Dilution is better than autogamy diluted effect.
Ox straw frozen semen quality height with artificial vagina installation and debugging, breeding oxen prepare with instruction, semen collection Personnel Skill Levels,
Normalization, semen quality evaluation and the extension rate that semen collection is operated calculate, dilute formula of liquid and prepare, the rule of semen dilution process
The multinomial factors such as plasticity, dilution freezing of semen process are relevant.Each link operating personnel major part entered vocational training, specification
Property it is stronger, maximum difference is different semen diluent, and sperm motility is relatively low after causing dilution.
First, current ox straw frozen semen dilution nutritional agents selection glucose, sucrose Shortcomings, from being conducive to sperm generation
Thank from the point of view of absorption, fructose should be selected, because fructose simple structure, be under equal conditions more beneficial for sperm metabolism suction
Receive, meet sperm nutritional need；Secondly, as the sodium citrate for maintaining sperm internal and external environment osmotic balance, its consumption is held
It is not accurate, it is impossible to maintain the isotonicity of solution, cause sperm to deform；3rd, mycillin as antibacterial material, be originally in order to
Improve sperm contamination resistance, but consumption it is out of control especially on the high side when can kill on the contrary sperm or reduce sperm motility；4th, egg
Freshness and sterilization control be not tight, nutritive value reduction；5th, frost durability agent glycerine consumption is unreasonable, fails to reach accordingly
Cryoprotective effects, reduce uptake of the sperm to nutriment on the contrary during excess；6th, the details of operation of cow frozen semen production
Lack normative.
In a word, each material consumption collocation lacks reasonability in dilution formula of liquid, while, it is necessary to further explore more scientific
Ox straw frozen semen production technology.
The content of the invention
It is an object of the invention to provide a kind of cattle freezing seminal fluid dilution preparation method, solve present in prior art
Problem, improves outstanding breeding oxen fresh semen dilution effect and sperm motility rate, reaches the permanently effective preservation of frozen semen.
The technical solution adopted in the present invention is that a kind of cattle freezing seminal fluid dilution preparation method is entered according to following steps
OK：
Step 1：Semen diluent is configured；
In proportion, 5~8g of fructose, the g of sodium citrate 19.8~26.6 are accurately weighed, the beaker of 1000 mL is moved into without loss
It is interior, the mL of distilled water 800 is added, with the glass bar or magnetic stirrer sterilized, make fructose and sodium citrate fully molten
Solution, then filters in conical flask or saline bottle solution filter paper, ties or stopper bottleneck, in putting 75~80 DEG C of water-baths
Sterilize 30 min, adds the fresh yolk of 180~220mL after being cooled to normal temperature, and add penicillin, streptomysin each 40
~50 ten thousand IU units, the first liquid is referred to as by the solution of this proportional arrangement；
Take the mL of the first liquid 430 and add 65~75 mL glycerine, and be put into 3 DEG C~5 DEG C after being stirred with magnetic stirring apparatus
Refrigerator or biochemical cultivation case in it is stand-by, be referred to as the second liquid by the solution of this proportional arrangement；
Step 2：Semen dilution；
It is slowly added into former qualified seminal fluid with 35 DEG C of the first liquid, is gently shaken up, 35 DEG C of appropriate water are contained with beaker, dilution
Pipe send cooling in 0~5 DEG C of low-temperature cabinet after being put into, at the same time the second liquid is also placed in；It is on the rocks in cup after balance 2h to promote
Its fast cooling, the second liquid is added when being cooled to 10 DEG C, adds more than releveling 30min after the second liquid, could be in low-temperature cabinet
In carry out tubule packing, the tubule after packing can be freezed；
Step 3：Freezing of semen；
To balance, direction restocking of the straw semen by tampon sealing end near operator after encapsulation is piled up, pile up in balancing stand
On, ultrasonic sealing end also should so place away from operator when tubule is put into frigorimeter；A tubule is put on each balancing stand
Label, for the ease of identification, the breeding oxen label of same color；If having not of the same race on same balancing stand
The straw semen of bull, must separately pile up, and separate some distances, in order to avoid obscure；
In straw frozen semen production process, using the full-automatic refrigeration instrument by computer controls, set most in computer first
Good cryogenic temperature curve, frigorimeter should be close proximity to frigorimeter is cooled to 4 DEG C by unlatching liquid nitrogen tank valve, is closed with low-temperature cabinet
The balancing stand for being booked tubule to be frozen, is put down rapidly gently people's frigorimeter by fan power supply after fan stops completely, covers lid, is pressed
Program set in advance is automatically performed refrigerating process；
Step 4：Freeze essence to thaw and sampling observation；
In advance water bath water temperature heating to 38~40 DEG C, with tweezers grip one freezing after straw frozen semen rapidly immersion 38~
Rock in 40 DEG C of water and gently, it is to be dissolved after take out immediately, dry the globule with blotting paper or gauze, then cut off with tubule dedicated scissors
Ultrasonic sealing end, one droplet seminal fluid of drop can carry out microscopy on by the slide of preheating, evaluate sperm motility rate；
Step 5：Freeze spermatophore dress；
In environment below -140 DEG C, packed with capillary count racking machine, its tampon end of the tubule after packaging is in plastics
The bottom of pipe, must not be inverted, and the internal diameter of plastic packing tube answers uniformity, must not use the small plastic tube in big, bottom suitable for reading, packaging
Good straw frozen semen people storehouse storage.
Further, in the step 2, tubule packing is carried out using tubule encapsulation all-in-one machine.
Further, also including step 6, essence storage is frozen；Freeze essence to be stored in the liquid nitrogen of liquid nitrogen container, timing weekly adds once
Liquid nitrogen, when finding that bloom occurs in liquid nitrogen container shell, will should preserve in seminal fluid transfer people other liquid nitrogen containers immediately；Packaged jelly
When being changed between smart liquid nitrogen container, in the liquid nitrogen container outer residence time no more than 3s；Take and to cover liquid nitrogen container plug after depositing jelly essence, picking and placeing
During lid plug, vertically to handle with care, must not overexert, prevent liquid nitrogen container plug from fractureing or damaging；During mobile liquid nitrogen container, should grip
Liquid nitrogen container handle is moved again after lifting tank body, must not be towed on the ground.
Further, also including step 7：Freeze essence transport；Freezing smart transportation must not lay across, collide and judder, protect
Card freezes essence and is immersed in liquid nitrogen all the time.
Further, in the step, the amount of added first liquid=[（Dilution total amount+qualified semen volume）/ 2]-qualified
Semen volume；
Dilution total amount=original semen volume ×（Extension rate -1）；
Extension rate=(sperm motility after former seminal fluid sperm concentration × original seminal fluid sperm motility × freezing)/every part insemination dose institute
Containing effective sperm count/0.25；
The amount of added second liquid=（The amount of the first liquid added by added dilution total amount one）.
The beneficial effects of the invention are as follows：First, excellent cattle freezing seminal fluid dilution species, at present, Niu Leng have been filtered out
Freeze semen diluent lecithality-phosphate dilution, fructose-yolk-citrate-glycerine dilution, yolk-Tris
Dilution and full-cream homogenized milk and skimmed milk etc. are several, are studied by lot of experiments, as a result show：Former semen quality it is qualified and
On the premise of working specification, the dilution effect of fructose-yolk-sodium citrate-glycerine dilution is best；Second, optimize dilute
The proportioning of chemical substance used in liquid is released, cattle freezing seminal fluid dilution formula is made a general survey of, basic chemical substance used is similar,
But each material proportioning is different, the present invention investigated the optimal of fructose, sodium citrate, yolk, mycillin, glycerine etc. and match somebody with somebody
Side, improves the dilution effect of qualified seminal fluid；3rd, it is the bull semen diluent preparation of further specification, semen dilution process, dilute
The sport technique segments such as embedding, balance cooling, freezing, packaging, storage and the transport of seminal fluid are released, to formulate ox straw frozen semen producting rule
There is provided theoretical foundation and technical support.
Specific embodiment
In below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely retouched
State, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based on the present invention
In embodiment, the every other implementation that those of ordinary skill in the art are obtained under the premise of creative work is not made
Example, belongs to the scope of protection of the invention.
A kind of cattle freezing seminal fluid dilution preparation method, had claimed below before dilution is prepared：
Prepare distilled water：Distilled water is prepared in strict accordance with the operational manual of full-automatic dual pure water distiller, distilled water is best
Stored with clean glassware.
Balance is placed on stable workbench, keeps cleaning drying, and the edge of a knife should be at Light Condition when not using；Weigh
During medicine, pad can be weighed with pan paper behind school zero in disk.Counterweight requirement keeps cleaning, dries, and prevents from making moist or pollutes.
Chemicals specification used by prepared and diluted liquid is pure for analysis, and within the shelf-life, after medicine is taken, should immediately by
Bottleneck is covered tightly, and is properly preserved；Medicine is dealt carefully with once being taken out from bottle, then can not put back to drug bottle and unified collection.
Egg for prepared and diluted liquid should derive from the chicken house without epidemic disease, and fresh, complete, clean, use 75% alcohol swab
Ball carries out thorough disinfection to eggshell surface, and complete yolk is taken out with egg white separator after waiting alcohol to volatilize to the greatest extent（Also can be in egg
Knock a crackle at waist median line open, egg is divided into two, alternately toppled over using two eggshells, remove egg white, leave yolk）,
Then yolk is extracted through membrane of yolk with the syringe of sterilizing.
Specifically follow the steps below：
Step 1：Semen diluent is configured
In proportion, fructose is accurately weighed（Analysis is pure）5~8g, the g of sodium citrate 19.8~26.6 move into 1000 mL without loss
Beaker in, add the mL of distilled water 800, with the glass bar or magnetic stirrer sterilized, fill fructose and sodium citrate
Divide dissolving, then filter in conical flask or saline bottle solution filter paper, tie or stopper bottleneck, put 75~80 DEG C of water-baths
Sterilized 30 min in pot, be cooled to after normal temperature and add the fresh yolk and penicillin, streptomysin each 40~50 of 180~220mL
Ten thousand IU units, this solution is referred to as I liquid；Take the mL of I liquid 430 and add 65~75 mL glycerine, and be sufficiently stirred for magnetic stirring apparatus
It is put into stand-by in 1 DEG C~4 DEG C of refrigerator or biochemical cultivation case after even, the solution is referred to as II liquid.It is worth noting that, preparing
Dilution standing time must not exceed 24 h.
Step 2：Semen dilution
It is slowly added into former qualified seminal fluid with 35 DEG C of the first liquid, is gently shaken up, 35 DEG C of appropriate water are contained with beaker, dilution
Pipe send cooling in 0~5 DEG C of low-temperature cabinet after being put into, at the same time the second liquid is also placed in；It is on the rocks in cup after balance 2h to promote
Its fast cooling, the second liquid is added when being cooled to 10 DEG C, adds more than releveling 30min after the second liquid, could be in low-temperature cabinet
In carry out tubule packing, the tubule after packing can be freezed；
The amount of added first liquid=[（Dilution total amount+qualified semen volume）/ 2]-qualified semen volume；
Dilution total amount=original semen volume ×（Extension rate -1）；
Extension rate=(sperm motility after former seminal fluid sperm concentration × original seminal fluid sperm motility × freezing)/every part insemination dose institute
Containing effective sperm count/0.25；
The amount of added second liquid=（The amount of the first liquid added by added dilution total amount one）.
Step 3：Freezing of semen
To balance, direction restocking of the straw semen by tampon sealing end near operator after encapsulation is piled up in balancing stand
On, ultrasonic sealing end also should so place away from operator when tubule is put into frigorimeter.One is put on each balancing stand to be similar to
In the label of tubule, for the ease of identification, the best breeding oxen label of same color.If same balancing stand
On have the straw semen of different breeding oxens, it is necessary to separately pile up, and to separate some distances, in order to avoid obscure.
In straw frozen semen production process, using the full-automatic refrigeration instrument by computer controls, set first in computer
Good optimal cryogenic temperature curve, frigorimeter and low-temperature cabinet should be close proximity to, open liquid nitrogen tank valve and frigorimeter is cooled to 4 DEG C,
Fan power supply is closed, the balancing stand for being booked tubule to be frozen is put down rapidly gently people's frigorimeter after fan stops completely, cover lid
Son, refrigerating process is automatically performed by program set in advance.
Step 4：Freeze essence to thaw and sampling observation
In advance water bath water temperature heating to 38~40 DEG C, with tweezers grip one freezing after straw frozen semen rapidly immersion 38~
Rock in 40 DEG C of water and gently, it is to be dissolved after take out immediately, dry the globule with blotting paper or gauze, then cut off with tubule dedicated scissors
Ultrasonic sealing end, one droplet seminal fluid of drop can carry out microscopy on by the slide of preheating, evaluate sperm motility rate.
Step 5：Freeze spermatophore dress
In environment below -140 DEG C, packed with capillary count racking machine, its tampon end of the tubule after packaging is in plastics
The bottom of pipe, must not be inverted.The internal diameter of plastic packing tube answers uniformity, must not use the small plastic tube in big, bottom suitable for reading.Packaging
Good straw frozen semen people storehouse storage.
Step 6：Freeze essence storage
Freeze essence to be stored in the liquid nitrogen of liquid nitrogen container, taken care of by special messenger, timing plus a liquid nitrogen, answer running check liquid nitrogen container weekly
Situation, when finding that bloom occurs in liquid nitrogen container shell, will should preserve in seminal fluid transfer people other liquid nitrogen containers immediately.Packaged jelly
When being changed between smart liquid nitrogen container, in the liquid nitrogen container outer residence time no more than 3s.Take and to cover liquid nitrogen container plug after depositing jelly essence, picking and placeing
During lid plug, vertically to handle with care, must not overexert, prevent liquid nitrogen container plug from fractureing or damaging.During mobile liquid nitrogen container, should grip
Liquid nitrogen container handle is moved again after lifting tank body, must not be towed on the ground.
Step 7：Freeze essence transport
Freezing in smart transportation will have special messenger to guard, and notice that hold-up vessel must not lay across, collide and judder, it is ensured that freeze essence and begin
It is immersed in liquid nitrogen eventually.
Herein, sodium citrate prevents the change of pH during liquefacient duration as buffer substance, while to metabolic acid
Poisoning and enzymatic activity reaction have good cushioning effect.Research shows：19.8~26.6 g sodium citrates are added in dilution
Sperm motility highest after recovery, ring sperm motility is minimum.Otherwise frozen semen recovery sperm motility is extremely low, reduces the cow feelings phase
Conception rate.
Sugar has nutrition, the osmotic pressure of increase dilution in dilution and many recasts such as sperm is protected in low temperature environment
With different carbohydrates and its consumption are different to allogenic animal sperm cryopreservation effect, and sugar of the same race and its consumption are to different animals essence
Sub- controlled-rate freezing is also different, and conventional carbohydrate has glucose, sucrose, fructose etc..Research thinks that fructose is biological special according to it
Property, as the composition of supply Niu Jingzi energy, it is more easy to utilize, effect preferably, selects the consumption to replace the water around sperm membrane
Molecule forms hydrogen bond with phosphate group in film, there is firm and repair to sperm membrane structure.
Conventional antifreeze has EDTA, dimethyl sulfoxide (DMSO), low concentration formaldehyde（0.025%）And glycerine.Wherein glycerine is used as sperm
Best freezeproof protectant during superfreeze, with the characteristic for being dissolved in water, is not susceptible to crystallization, while having water imbibition
It is strong and the characteristics of aqueous solution freezing point can be reduced.When semen diluent is prepared, the control of glycerine consumption 13%~14% for best, it is low
Very low in 13% cryoprotective effects, meeting or exceeding 18% can produce toxic action to sperm.
Yolk has protects sperm from the influence of cold shock, the effect that can prevent sperm internal liquid phase from changing, while ovum
Huang can improve acrosome reaction rate by promoting the combination of sperm and egg cell oolemma, be added herein in semen diluent
The yolk of content, makes cow non-return rate up to more than 75%.
In straw frozen semen production, pathogenic and non-pathogenic disease in seminal fluid are controlled with penicillin and streptomysin, it
With antibacterial range is wide, low cost the features such as.According to antibiotic dosage in this semen dilution formula of liquid, by priority control venereal disease
Cow non-return rate is improved with vibriosis.Seminal fluid is diluted during less than 400,000 IU units and easily receives microbiological contamination, exceeded
During 500000 IU units, antibiotic produces too high lethality, both of which reduction sperm motility and the fertilization of cow feelings phase to sperm in itself
Rate 40%~70%.
In a word, bull semen diluent formula in, by it is scientific and reasonable selection diluent, nutritional agents, protective agent and other
The best of breed of additive and its consumption, while each details of specification semen dilution and straw frozen semen production technology.In rule
On the basis of model operation, straw frozen semen solution Frozen semen activity 10%~15% can be improved, maintain cow non-return rate more than 75%, together
When to accelerate ox improvement paces and improve Breeding bull station production freeze essence economic benefit and regional society benefit will produce product
Pole acts on.
Embodiment 1
In proportion, the g of fructose 5, the g of sodium citrate 26.6 are accurately weighed to move into without loss in the beaker of 1000 mL, distillation is added
The mL of water 800, with the glass bar or magnetic stirrer sterilized, makes fructose and sodium citrate fully dissolve, then by solution
Filtered in conical flask or saline bottle with filter paper, tie or stopper bottleneck, put and sterilize in 75 DEG C of water-baths 30 min, be cooled to
Each 400,000 IU units of fresh yolk and penicillin, streptomysin of 180 mL are added after normal temperature, this solution is referred to as I liquid；Take I liquid
430 mL add 65 mL glycerine, and are put into 1 DEG C of refrigerator or biochemical cultivation case after being stirred with magnetic stirring apparatus and treat
With the solution is referred to as II liquid.It is worth noting that, the dilution standing time for preparing must not exceed 24 h.
It is slowly added into former qualified seminal fluid with 35 DEG C of the first liquid, is gently shaken up, 35 DEG C of appropriate water is contained with beaker,
Dilution tube send cooling in 0 DEG C of low-temperature cabinet after being put into, at the same time the second liquid is also placed in；Balance after 2 h rush on the rocks in cup
Make its fast cooling, the second liquid is added when being cooled to 10 DEG C, add more than the min of releveling 30 after the second liquid, could be in low temperature
Tubule packing is carried out in cabinet, the tubule after packing can be freezed.
Embodiment 2
In proportion, fructose 8g, the g of sodium citrate 26.6 are accurately weighed to move into without loss in the beaker of 1000 mL, distillation is added
The mL of water 800, with the glass bar or magnetic stirrer sterilized, makes fructose and sodium citrate fully dissolve, then by solution
Filtered in conical flask or saline bottle with filter paper, tie or stopper bottleneck, put and sterilize in 80 DEG C of water-baths 30 min, be cooled to
Each 500,000 IU units of fresh yolk and penicillin, streptomysin of 220 mL are added after normal temperature, this solution is referred to as I liquid；Take I liquid
430 mL add 75 mL glycerine, and are put into 4 DEG C of refrigerator or biochemical cultivation case after being stirred with magnetic stirring apparatus and treat
With the solution is referred to as II liquid.It is worth noting that, the dilution standing time for preparing must not exceed 24 h.
It is slowly added into former qualified seminal fluid with 35 DEG C of the first liquid, is gently shaken up, 35 DEG C of appropriate water is contained with beaker,
Dilution tube send cooling in 5 DEG C of low-temperature cabinets after being put into, at the same time the second liquid is also placed in；It is on the rocks in cup after balance 2h to promote
Its fast cooling, the second liquid is added when being cooled to 10 DEG C, adds more than releveling 30min after the second liquid, could be in low-temperature cabinet
In carry out tubule packing, the tubule after packing can be freezed.
Embodiment 3
In proportion, the g of fructose 6, the g of sodium citrate 23 are accurately weighed to move into without loss in the beaker of 1000 mL, distilled water is added
800 mL, with the glass bar or magnetic stirrer sterilized, make fructose and sodium citrate fully dissolve, and then use solution
Filter paper is filtered in conical flask or saline bottle, ties or stopper bottleneck, is put and sterilize in 78 DEG C of water-baths 30 min, is cooled to often
Each 450,000 IU units of fresh yolk and penicillin, streptomysin of 200 mL are added after temperature, this solution is referred to as I liquid；Take I liquid 430
ML adds 70 mL glycerine, and be put into after being stirred with magnetic stirring apparatus it is stand-by in 2 DEG C of refrigerator or biochemical cultivation case,
The solution is referred to as II liquid.It is worth noting that, the dilution standing time for preparing must not exceed 24 h.
It is slowly added into former qualified seminal fluid with 35 DEG C of the first liquid, is gently shaken up, 35 DEG C of appropriate water is contained with beaker,
Dilution tube send cooling in 3 DEG C of low-temperature cabinets after being put into, at the same time the second liquid is also placed in；Balance after 2 h rush on the rocks in cup
Make its fast cooling, the second liquid is added when being cooled to 10 DEG C, add more than the min of releveling 30 after the second liquid, could be in low temperature
Tubule packing is carried out in cabinet, the tubule after packing can be freezed.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the scope of the present invention.It is all
Any modification, equivalent substitution and improvements made within the spirit and principles in the present invention etc., are all contained in protection scope of the present invention
It is interior.
Claims (5)
1. a kind of cattle freezing seminal fluid dilution preparation method, it is characterised in that follow the steps below：
Step 1：Semen diluent is configured；
In proportion, 5~8g of fructose, the g of sodium citrate 19.8~26.6 are weighed, is moved into without loss in the beaker of 1000 mL, plus
Enter the mL of distilled water 800, with the glass bar or magnetic stirrer sterilized, fructose and sodium citrate is fully dissolved, then
Solution filter paper is filtered in conical flask or saline bottle, bottleneck is tied or stopper, is put and sterilize in 75~80 DEG C of water-baths 30
Min, adds the fresh yolk of 180~220 mL, and add penicillin, streptomysin each 40~500,000 after being cooled to normal temperature
IU units, the first liquid is referred to as by the solution of this proportional arrangement；
Take the mL of the first liquid 430 and add 65~75 mL glycerine, and be put into 3~5 DEG C after being stirred with magnetic stirring apparatus
It is stand-by in refrigerator or biochemical cultivation case, it is referred to as the second liquid by the solution of this proportional arrangement；
Step 2：Semen dilution；
It is slowly added into former qualified seminal fluid with 35 DEG C of the first liquid, is gently shaken up, 35 DEG C of appropriate water are contained with beaker, dilution
Pipe send cooling in 0~5 DEG C of low-temperature cabinet after being put into, at the same time the second liquid is also placed in；Balance on the rocks in cup after 2 h promoting
Its fast cooling, the second liquid is added when being cooled to 10 DEG C, adds more than the min of releveling 30 after the second liquid, could be in low-temperature cabinet
In carry out tubule packing, the tubule after packing can be freezed；
Step 3：Freezing of semen；
To balance, direction restocking of the straw semen by tampon sealing end near operator after encapsulation is piled up, pile up in balancing stand
On, ultrasonic sealing end also should so place away from operator when tubule is put into frigorimeter；A tubule is put on each balancing stand
Label, for the ease of identification, the breeding oxen label of same color；If having not of the same race on same balancing stand
The straw semen of bull, must separately pile up, and separate some distances, in order to avoid obscure；
In straw frozen semen production process, using the full-automatic refrigeration instrument by computer controls, set most in computer first
Good cryogenic temperature curve, frigorimeter should be close proximity to frigorimeter is cooled to 4 DEG C by unlatching liquid nitrogen tank valve, is closed with low-temperature cabinet
The balancing stand for being booked tubule to be frozen, is put down rapidly gently people's frigorimeter by fan power supply after fan stops completely, covers lid, is pressed
Program set in advance is automatically performed refrigerating process；
Step 4：Freeze essence to thaw and sampling observation；
In advance water bath water temperature heating to 38~40 DEG C, with tweezers grip one freezing after straw frozen semen rapidly immersion 38~
Rock in 40 DEG C of water and gently, it is to be dissolved after take out immediately, dry the globule with blotting paper or gauze, then cut off with tubule dedicated scissors
Ultrasonic sealing end, one droplet seminal fluid of drop can carry out microscopy on by the slide of preheating, evaluate sperm motility rate；
Step 5：Freeze spermatophore dress；
In environment below -140 DEG C, packed with capillary count racking machine, its tampon end of the tubule after packaging is in plastics
The bottom of pipe, must not be inverted, and the internal diameter of plastic packing tube answers uniformity, must not use the small plastic tube in big, bottom suitable for reading, packaging
Good straw frozen semen people storehouse storage.
2. cattle freezing seminal fluid dilution preparation method according to claim 1, it is characterised in that in the step 2, tubule
Packing is carried out using tubule encapsulation all-in-one machine.
3. cattle freezing seminal fluid dilution preparation method according to claim 1, it is characterised in that also including step 6, freezes essence
Storage；Freeze essence to be stored in the liquid nitrogen of liquid nitrogen container, weekly timing plus a liquid nitrogen, when finding that bloom occurs in liquid nitrogen container shell,
To should be preserved in seminal fluid transfer people other liquid nitrogen containers immediately；When being changed between the packaged smart liquid nitrogen container of jelly, stopped outside liquid nitrogen container
Time is no more than 3s；Take and to cover liquid nitrogen container plug after depositing jelly essence, when lid plug is picked and placeed, vertically to handle with care, must not be firmly
It is too quickly, prevent liquid nitrogen container plug from fractureing or damaging；During mobile liquid nitrogen container, should grip after liquid nitrogen container handle lifts tank body and move again, no
Obtain and tow on the ground.
4. cattle freezing seminal fluid dilution preparation method according to claim 1, it is characterised in that also including step 7：Freeze essence
Transport；Freezing smart transportation must not lay across, collide and judder, it is ensured that freeze essence and be immersed in liquid nitrogen all the time.
5. cattle freezing seminal fluid dilution preparation method according to claim 1, it is characterised in that added in the step 2
The amount of the first liquid=[（Dilution total amount+qualified semen volume）/ 2]-qualified semen volume；
Dilution total amount=original semen volume ×（Extension rate -1）；
Extension rate=(sperm motility after former seminal fluid sperm concentration × original seminal fluid sperm motility × freezing)/every part insemination dose institute
Containing effective sperm count/0.25；
The amount of added second liquid=（The amount of the first liquid added by added dilution total amount one）.
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| CN114208813A (en) * | 2021-12-23 | 2022-03-22 | 北京奶牛中心 | Cow frozen semen diluent, preparation method thereof and semen freezing method |
| CN115500346A (en) * | 2022-10-11 | 2022-12-23 | 甘肃省家畜繁育改良管理站 | Preparation method of bovine semen diluent |
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