CN101062058A - Tubule high-density type method for freezing pig jism and the products thereof - Google Patents
Tubule high-density type method for freezing pig jism and the products thereof Download PDFInfo
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- CN101062058A CN101062058A CNA200710023495XA CN200710023495A CN101062058A CN 101062058 A CN101062058 A CN 101062058A CN A200710023495X A CNA200710023495X A CN A200710023495XA CN 200710023495 A CN200710023495 A CN 200710023495A CN 101062058 A CN101062058 A CN 101062058A
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Abstract
The invention discloses a high density freezing maintain pig seminal fluid method with fistula and product in animal propagation technical domain, which comprises the following steps: collecting fresh seminal fluid; detecting; pre-treating the seminal fluid; defreezing; proceeding assessment procedure of defreeze seminal fluid; diluting with freezing diluting liquid; storing with high density super low temperature with 0. 25mL fistula; stabilizing the defreezing sperm above 0. 5 and average active at 0. 58; reaching the highest at 0. 68; proceeding pig external fertilization with the sperm; getting 54. 3% cleavage ratio and 48. 5% morula developing ratio; meeting one sperm dosage with sperm quantity of one 0. 25mL fistula. This invention possesses larger using latent force and market developing prospect.
Description
One, technical field
The present invention relates to method of the freezing preservation of a kind of tubule high-density type pig seminal fluid and products thereof, belong to the animal reproduction technical field.
Two, technical background
The technical difficulty that pig seminal fluid cryotherapy is preserved is bigger, is for a long time to restrict it to enter the principal element in production practicability stage always.The breakthrough of this technology, not only can solve the problem of pig seminal fluid long preservation, and help to apply the technology of artificial insemination of pig, help the protection of China and even world's pig germ plasm resource, make the application of seminal fluid not be subjected to the restriction of time, region, be convenient to germplasm and exchange and transport, thereby can improve the utilization rate of good species boar greatly; In addition, the boar frozen semen can begin a large amount of supplies again after progeny testing or disease detection, for stable genetic progress and strict bio-safety provide safeguard.Therefore, this technology has the huge application potential and the prospect of marketing.
Accelerating poultry improved variety engineering is an important step of the modern animal husbandry of development, and wherein the production of breeding poultry is the core content of improved variety engineering with supplying with.Technology of artificial insemination is the most fruitful routine techniques during animal breeding is produced, and has been widely used in milch cow and has produced.In other animal cultivation production practices, also obtain promotion and application.Pig is the important source of China's consumption of meat, accounts for 70%～80% of overall consumption meat; The pig kind resource of China is also very abundant.Utilizing frozen semen to carry out artificial insemination is a reproduction technique with extensive potentiality.In Swine Production, can effectively improve the utilization rate of breeding boar, save the expense of raising boar; Help the kind layout, give full play to the effect of good lean meat species boar, accelerate the commodity bacon hogs and produce paces, can accelerate breed improvement rapidly from boar hereditary quality aspect.
At present, at home and abroad in the Swine Production, one of subject matter of existence is that pig frozen semen technical merit is lower, conception rate is low, is seriously restricting improvement of pig kind and germ plasm resource and is preserving.On cattle, can reach the effect of bright smart semen deposition or this friendship with freezing the pedestrian worker's insemination that progresses greatly, and worldwide be widely used.Because the pig sperm is responsive especially to cold shock, compare with the conventional liquid seminal fluid of preserving, thawing, back sperm anabiosis rate is low, abnormal spermium is many, conception rate is low; The vigor and the fertility of freezing after essence is thawed of different breeding boar also have tangible individual variation; Freezing preservation dosage is big.Caused Study on Cryopreservation of Boar Semen to relatively lag behind, still lacked and be similar to the such maturation method of cattle freezing of semen.Up to now; in the artificial insemination of pig; use the liquid seminal fluid of preserving of bright essence and room temperature and estimate still to account for 99% of whole world swine artificial insemination ratio; though smart commercial application that just had in 1975 frozen in pig granule or bassoon; but because of conception rate generally lower; make that freezing essence uses the ratio that accounts for less than 1%, and this ratio there is no big variation in many years.
Thaw that back sperm survival rate is low, vigor is not enough, produce the capacitation sample and change and shortened the time-to-live because ubiquitous pig is frozen essence, thereby cause the rate of fertilization after the semen deposition to hang down and problems such as litter size is few, do not reach ideal effect even strengthen semen deposition dosage yet, seriously limit pig and frozen smart production application, press for and set up a kind of technical advanced person, reliable, stable, practical Cryopreservation of Boar Semen method on the program.
Three, summary of the invention
Technical problem:
The objective of the invention is at the reality that still lacks the boar semen practical method, method of the freezing preservation of a kind of tubule high-density type pig seminal fluid and products thereof is provided.With 0.25mL tubule freezing pig jism, adopt low-cost, high efficiency freeze-extender, freezing preservation high density pig sperm is obtained stable, high-quality freezing result.
Technical scheme:
The method of the freezing preservation of tubule high-density type pig seminal fluid comprises:
1) bright smart collection and detection
Collect pig under the employing hand grip room temperature and be rich in sperm section seminal fluid, in the beaker of cleaning, after four layers of filtered through gauze, check every index: vigor＞0.7, abnormal rate＜18%, the seminal fluid of plasma membrane percentage of head rice＞70% is used for freezing preservation;
2) pretreatment of seminal fluid
(1) pre-equilibration of seminal fluid: with room-temperature extender ZO liquid or S liquid, 1: 1 by volume dilution seminal fluid, the room temperature lucifuge left standstill 1 hour;
Table 1. room-temperature extender prescription g/100mL
| ZO liquid | S liquid | |
| Glucose natrium citricum citric acid cysteine EDTA BSA Tris NaHCO2 | 1.15 1.17 0.41 0.01 0.23 0.50 0.65 0.125 | 4 0.37 0.24 |
(2) seminal fluid is centrifugal: after leaving standstill, the seminal fluid branch is installed in the 15mL centrifuge tube, with the centrifugal 3min of 2400 * g, remove supernatant, as the freezing processing of back;
3) seminal fluid is freezing
(1) preparation of chill back I liquid: glucose 1.5g, lactose 3g, glycine 0.8g, yolk 24mL adds tri-distilled water to 100mL, and is now with the current;
(2) preparation of freeze-extender II: get the chill back I liquid for preparing, add glycerol, making the quality of glycerol is 9% than concentration, places general refrigerator cold room balance;
(3) freezing preservation: add 1～4mL chill back I liquid in centrifugal, as to go supernatant processing sperm, mixing makes the sperm concentration that suspends again reach (1.5～6) * 10
9Individual/mL, placed 5 ℃ of water-bath balances 1 hour; Simultaneously the 0.25mL tubule of carrying out sign was also obtained the isothermal tubule in 1 hour in 5 ℃ of balances; After balance finishes, add the dilution of chill back II liquid again in the seminal fluid that dilution I liquid diluted, I liquid: II liquid volume ratio is 2: 1, abundant mixing, again 5 ℃ of balances 1 hour, when the also surplus 15min of equilibration time, scoop out liquid nitrogen from liquid nitrogen container and be put into the foam plastics box, there is support at the 3cm place on the liquid nitrogen surface, adds a cover balance; In seminal fluid suction isothermal tubule, after the tubulature, seal with pva powder, the tubule of sealing continues balance 15min at 5 ℃; At last, tubule evenly is laid in the foam box on the liquid nitrogen surface on the 3cm place support, add a cover balance 10min after tubule directly drop into liquid nitrogen freezing.
Freezing smart defreezing method is:
Take out tubule from liquid nitrogen, drop into rapidly in 37 ℃ of water-baths, cut off the tubule sealing two ends after 30 seconds, be recycled in the 2mL isothermal ZO liquid, mixing is standby gently;
The preservation liquid of sperm after thawing adds 40% refining and 20% yolk on the basis of ZO liquid, placed room temperature preservation 2 hours.Used refining preparation method is: bright essence is got supernatant again through the centrifugal 10min of 2400 * g through the centrifugal 10min of 800 * g, and the collection supernatant is through the filtration of 0.22 μ m filter or add penicillin, each 100IU/mL of streptomycin, and 4 ℃ of refrigerators are preserved standby.
Beneficial effect:
This achievement is at the reality that still lacks at present the boar semen practical method, provide a kind of optimization, practicability freezing pig jism method.Its major advantage and beneficial effect are as follows:
1. have stable, high-quality freezing result: freeze essence thaw the back motility of sperm be stabilized in more than 0.5, be up to 0.68; Freeze the capable pig in-vitro fertilization that progresses greatly with this, obtain 54.3% spilting of an egg rate and 48.5% morula developmental rate, with the external fertilization result who uses bright essence to obtain quite (53.6% spilting of an egg rate and 47.4% morula developmental rate); The good reproducibility of freezing save routine.
2. production practicality is good: with 0.25mL tubule freezing pig jism, adopt low-cost, high efficiency freeze-extender, freezing preservation high density (1 * 10
9Individual/mL and 4 * 10
9Individual/mL) pig sperm, make the smart amount of freezing of 1 tubule satisfy 1 semen deposition dosage, conveniently freeze smart production application.
3. have the supporting essence back that thaws of freezing and preserve liquid: pig is frozen and is stored in after essence is thawed in the ZO liquid that adds 40% refining and 20% yolk, can place in room temperature still to keep original vigor in 2 hours, and motility of sperm was still more than 0.4 in 4 hours; Be very easy to the semen deposition operation in the production.
4. quality evaluation index science more: except traditional vigor inspection, adopt the integrity evaluation of external fertilization experiment and nuclear to freeze extract Iuality, summarized fertility and development of fertilized ova ability objectively.
5. wide application prospect: the use that can make the pig seminal fluid of applying of this achievement is not subjected to the restriction of time, region, is convenient to exchange and transportation; Can improve the utilization rate of good species boar greatly; The boar frozen semen can begin a large amount of supplies again after progeny testing or disease detection, for stable genetic progress and strict bio-safety provide safeguard.With this achievement is support, can quicken the development of other sperm technology (as property control seminal fluid, transgenic sperm).
Four, description of drawings
Fig. 1: the used 0.25mL tubule of boar semen
Fig. 2: the TG complex staining (1. has acrosome reaction sperm alive; 2. the acrosome reaction Necrospermia is arranged; 3. there is not acrosome reaction sperm alive; 4. there is not the acrosome reaction Necrospermia)
Fig. 3: with freezing smart external fertilization embryo (1.2-somatic embryo of producing; 2.4-～8-somatic embryo; 3.16-somatic embryo; 4. morula)
Five, the specific embodiment
The pig tubule high-density type freezes smart production, the evaluation embodiment is as follows:
1. pig semen collection and inspection:
Conventional method is gathered the pig seminal fluid, checks every index after four layers of filtered through gauze, and the seminal fluid of vigor＞0.7, abnormal rate＜18%, plasma membrane percentage of head rice＞70% is used for freezing preservation.
2. the pretreatment of seminal fluid
1) pre-equilibration of seminal fluid: dilute seminal fluid with room-temperature extender (ZO liquid or S liquid, table 1) 1: 1 (v/v), room temperature (16～28 ℃) lucifuge left standstill 1 hour.
Table 1. room-temperature extender prescription (g/100mL)
| ZO liquid | S liquid | |
| Glucose natrium citricum citric acid cysteine EDTA BSA Tris NaHCO2 | 1.15 1.17 0.41 0.01 0.23 0.50 0.65 0.125 | 4 0.37 0.24 |
2) seminal fluid is centrifugal: after leaving standstill, the seminal fluid branch is installed in the 15mL centrifuge tube, with the centrifugal 3min of 2400 * g, remove supernatant, as the freezing processing of back.
3. seminal fluid is freezing
1) preparation of chill back I liquid: glucose 1.5g, lactose 3g, glycine 0.8g, yolk 24mL adds tri-distilled water to 100mL, needs now with the current.
2) preparation of freeze-extender II: get the chill back I liquid for preparing, add glycerol, making the concentration of glycerol is 9%, places general refrigerator cold room (4～5 ℃) balance.
3) freezing preservation: add 1～4mL (using the 15mL centrifuge tube) chill back I liquid in centrifugal, as to go supernatant processing sperm, mixing makes the sperm concentration that suspends again reach (1.5～6) * 10
9Individual/mL, placed 5 ℃ of water-bath balances 1 hour; Simultaneously carrying out the 0.25mL tubule of sign also in 5 ℃ of balances.After 1 hour balance finishes, add partly measure the dilution of chill back II liquid (I liquid: II liquid is 2: 1, v/v), abundant mixing, 5 ℃ of balances 1 hour, should make sperm concentration after these the 2 times dilutions was (1～4) * 10 again
9Individual/mL.When the also surplus 15min of equilibration time, to scoop out the foam plastics box that liquid nitrogen is put into appropriate depth from liquid nitrogen container, there is support at the 3cm place on the liquid nitrogen surface, adds a cover balance.In seminal fluid suction isothermal tubule, after the tubulature, there is pva powder to seal.The tubule of sealing continues balance 15min at 5 ℃.At last, tubule evenly is laid in the foam box on the liquid nitrogen surface on the 3cm place support, directly drops into liquid nitrogen after adding a cover balance 10min.
4. freeze smart thawing
Take out tubule from liquid nitrogen, drop into rapidly in 37 ℃ of water-baths, cut off the tubule sealing two ends after 30 seconds, be recycled in the 2mL isothermal ZO liquid, mixing is standby gently.
It is obviously smart short than bright that pig is frozen the smart post-thaw survival time, greatly influenced the fertility of sperm, and the too short time-to-live also can increase the semen deposition difficulty, reduce conception rate.Present technique has been improved the preservation liquid of the back sperm that thaws for this reason, on the basis of ZO liquid, add 40% refining (preparation method: bright smart through the centrifugal 10min of 800 * g, get supernatant again through the centrifugal 10min of 2400 * g, the collection supernatant is through the filtration of 0.22 μ m filter or add penicillin, each 100IU/mL of streptomycin, 4 ℃ of refrigerators are preserved standby) and 20% yolk, the back placement room temperature preservation of thawing 2 hours, sperm still keeps original vigor; Motility of sperm remained on more than 0.4 in 4 hours.
5. sperm quality evaluation
(1) evaluation methodology and index
After the bright essence or the essence of freezing are thawed, all carry out the evaluation of indexs such as vigor, acrosomal integnity, membrane integrity, external fertilization spilting of an egg rate and morula developmental rate and nuclear integrity, its quality of overall merit.Concrete operations are as follows:
1) vigor detects
Back sperm sample to be checked is diluted to 1 * 10 thawing
7Individual/mL, get 5 μ l seminal fluid and drop on the warm microscope slide, rapidly covered is placed on counting phase contrast microscope under, counts 200 sperms at least, the total sperm count of calculating motility of sperm=straight-line sperm count/count.
2) acrosomal integnity
Adopt motility rate and acrosomal integnity complex staining, estimate the acrosome status of the sperm of living.Concrete operations are as follows:
(1) preparation of main solution
Platform is expected blue liquid: be made into 1% concentration with the isotonic solution that does not contain bovine serum albumin (BSA), use after 37 ℃ of preheatings, face and use preceding preparation.
Orth ' s liquid: 6.8% heavy chromium potassium liquid and formalin close with 4: 1 ratio and mix, and potassium dichromate solution will prepared with preceding 1d at the latest, and Orth ' s liquid is being used preceding preparation.
(2) staining procedure
Expect blue liquid for add equivalent in seminal fluid to be checked 1%, 15min is hatched in 37 ℃ of water-baths; Smear is on the slide of preheating, and 37 ℃ air-dry, and is standby under room temperature after the abundant rinsing of water.Orth ' s is liquid-solid to decide 45min, and the several seconds in 80% ethanol is immersed in washing, air-dry back, more at room temperature with Ji's nurse Sa liquid dyeing 90min, and abundant air-dry, the mounting in washing back, microscopy
(3) painted type
Acrosome reaction sperm alive is arranged: the not painted or nattierblue of postnuclear cap portion, the not painted or part aubergine of acrosome
The acrosome reaction Necrospermia is arranged: postnuclear cap portion darkcyan, the not painted or part kermesinus of acrosome portion
No acrosome reaction sperm alive: the not painted or nattierblue of postnuclear cap portion, acrosome portion aubergine
Do not have the top and not react Necrospermia: postnuclear cap portion darkcyan, the dark violet redness of acrosome portion
(4) detect
Under 1000 * oily mirror, check 100～200 sperm heads of each specimen counting.
3) membrane integrity
Curved tail rate with hypotonic swelling experiment (HOST) mensuration sperm sample is the plasma membrane percentage of head rice.Is the dilution of sperm sample 1 * 10
6Individual/mL, to get 500 μ l and add in the 1mL hypotonic medium (0.73g Sodium Citrate, usp, Dihydrate Powder and 1.35g fructose are dissolved in the 100mL tri-distilled water, and osmotic pressure is 150mOsm/kg), 60min is hatched in 37 ℃ of water-baths.Get 5 μ l seminal fluid and drip on the preheating microscope slide, add coverslip and be placed on counting under the phase contrast microscope, to 100 sperms of minority.
4) external fertilization spilting of an egg rate and embryo development rate
The sperm that will thaw is centrifugal through routine, do external capacitation after the washing handles, and external capacitation sperm and maturation in vitro ovum were hatched 6 hours altogether, takes out and supposes that germ cell puts into the NCSU-23 culture fluid, in 38.5 ℃, 5%CO
2Cultivate under gas phase and the saturated humidity condition, inspection record spilting of an egg situation and fetal development situation are calculated spilting of an egg rate and embryo development rate.
The preparation of NCSU-23: difference weighing NaCl 0.6354g, KCl 0.0356g, MgSO
47H
2O 0.0293g, CaCl
20.0187g, K
2HPO
40.0162g, NaHCO
30.2106g, glucose 0.1000g, glutaminase 0.0146g, taurine 0.0876g; hypotaurine 0.0546g, BSA (Fraction V) 0.4000g, phenol red 0.0010g is dissolved in the tri-distilled water; be settled to 100mL (pH7.2～7.4) then, filtration sterilization, 4 ℃ of preservations are standby.
5) nuclear integrity
Adopt the discrete experiment of staining of sperm matter (SCD), have only DNA complete just can produce the characteristic halo, what contain the DNA fragment does not produce halo.Concrete steps are as follows:
(1) preparation of cell pyrolysis liquid
0.4M Tris-HCl，2M NaCl，1％SDS，0.05M EDTA，pH7.5
(2) film-making
The frosted microscope slide: in order to avoid form the coarse groove vestige on the microscope slide face, gel comes off in processing procedure during mill.Also can be cut into the thin plate of suitable size with 96 orifice plates, be used to spread glue.
The glue plate: the 1st layer of gel, the microscope slide that above-mentioned mill is good, frosting makes progress, about 40 ℃ of preheatings; No Ca with the 100uL 0.65% normal fusing point agarose (NMA) of 45 ℃ of preheatings
2+, Mg
2+Phosphate buffer (PBS) drops on the microscope slide, covers coverslip rapidly No. 1, places 4 ℃ of following 10min that NMA is solidified again.The 2nd layer of gel transfers to sperm concentration (5～10) * 10
6Individual/mL, mix at 37 ℃ and 1% low melting-point agarose (LMA), making the LMA ultimate density is 0.7%.Get 50 μ l and drip on the 1st layer of gel, cover the 2nd coverslip immediately, put 4 ℃ of refrigerator 4min.
Lysis: the lid thin slice is taken away gently, under the room temperature gel level is immersed 25min in the new 10mL cell pyrolysis liquid of preparing, then use deionized water wash 5min, use 70%, 90%, 100% dehydration of alcohol then, each 2min, dyeing at last.
(3) observe
With 40 * microscopic examination, each microscope slide is counted 500 sperms at least.
(2) boar semen evaluation result (table 2)
Adopt the 0.25mL tubule that the pig sperm is carried out the freezing preservation of high density, the back motility of sperm of thawing is stabilized in more than 0.5, and average vigor 0.58 is up to 0.68.Adopt this technology that the pig seminal fluid is carried out freezing preservation, freeze and be used for pig in-vitro fertilization after essence is thawed, obtain 54.3% spilting of an egg rate and 48.5% morula developmental rate; Be as good as with the external fertilization result (53.6% spilting of an egg rate and 47.4% morula developmental rate) who adopts the pig fresh semen to obtain.1 contained sperm of 0.25mL tubule can satisfy 1 semen deposition dosage in the production.
Table 2. boar semen obtains the parameters index
| Pre-diluent | Average vigor | Acrosomal integrity | The plasma membrane percentage of head rice | Abnormal rate | External fertilization | ||
| Spilting of an egg rate | Developmental rate | ||||||
| Bright essence is frozen and is frozen after essence is thawed after essence thaws | ZO liquid S liquid | 0.83 0.58 0.55 | 80.5％ 47.6％ 42.3％ | 76.3％ 61.2％ 60.7％ | 14％ 26.4％ 27.3％ | 53.6％ 54.3％ 54.2％ | 47.4％ 48.5％ 45.7％ |
Claims (4)
1, the method for tubule high-density type freezing pig jism comprises:
1) bright smart collection and detection
Collect pig under the employing hand grip room temperature and be rich in sperm section seminal fluid, in the beaker of cleaning, after four layers of filtered through gauze, check every index: vigor＞0.7, abnormal rate＜18%, the seminal fluid of plasma membrane percentage of head rice＞70% is used for freezing preservation;
2) pretreatment of seminal fluid
(1) pre-equilibration of seminal fluid: with room-temperature extender ZO liquid or S liquid, 1: 1 by volume dilution seminal fluid, the room temperature lucifuge left standstill 1 hour;
Table 1. room-temperature extender prescription g/100mL
ZO liquid S liquid
Glucose natrium citricum citric acid cysteine EDTA BSA Tris NaHCO2 1.15 1.17 0.41 0.01 0.23 0.50 0.65 0.125 4 0.37 0.24
(2) seminal fluid is centrifugal: after leaving standstill, the seminal fluid branch is installed in the 15mL centrifuge tube, with the centrifugal 3min of 2400 * g, remove supernatant, as the freezing processing of back;
3) seminal fluid is freezing
(1) preparation of chill back I liquid: glucose 1.5g, lactose 3g, glycine 0.8g, yolk 24mL adds tri-distilled water to 100mL, and is now with the current;
(2) preparation of freeze-extender II: get the chill back I liquid for preparing, add glycerol, making the quality of glycerol is 9% than concentration, places general refrigerator cold room balance;
(3) freezing preservation: add 1～4mL chill back I liquid in centrifugal, as to go supernatant processing sperm, mixing makes the sperm concentration that suspends again reach 1.5 * 10
9Individual/mL～6 * 10
9Individual/mL, placed 5 ℃ of water-bath balances 1 hour; Simultaneously the 0.25mL tubule of carrying out sign was also obtained the isothermal tubule in 1 hour in 5 ℃ of balances; After balance finishes, add the dilution of chill back II liquid again in the seminal fluid that dilution I liquid diluted, I liquid: II liquid volume ratio is 2: 1, abundant mixing, again 5 ℃ of balances 1 hour, when the also surplus 15min of equilibration time, scoop out liquid nitrogen from liquid nitrogen container and be put into the foam plastics box, in the foam plastics box on the liquid nitrogen surface 3cm place support is arranged, add a cover balance; In seminal fluid suction isothermal tubule, after the tubulature, seal with pva powder, the tubule of sealing continues balance 15min at 5 ℃; At last, tubule evenly is laid in the foam box on the liquid nitrogen surface on the 3cm place support, add a cover balance 10min after tubule directly drop into liquid nitrogen freezing.
According to the method for the described tubule high-density type freezing pig jism of claim 1, it is characterized in that 2, the defreezing method that freezes essence is:
Take out tubule from liquid nitrogen, drop into rapidly in 37 ℃ of water-baths, cut off the tubule sealing two ends after 30 seconds, be recycled in the 2mL isothermal ZO liquid, mixing is standby gently;
The preservation liquid of sperm after thawing adds 40% refining and 20% yolk on the basis of ZO liquid, placed room temperature preservation 2 hours.
3, according to the method for claim 1 or 2 described tubule high-density type freezing pig jisms, it is characterized in that, the preservation liquid of sperm after thawing, the refining preparation method of adding on the basis of ZO liquid is: bright smart in the centrifugal 10min of 800 * g, get supernatant again through the centrifugal 10min of 2400 * g, the collection supernatant is through the filtration of 0.22 μ m filter or add penicillin, each 100IU/mL of streptomycin, and 4 ℃ of refrigerators are preserved standby.
4, the 0.25mL tubule freezing pig jism product that method obtained of the described 0.25mL tubule high-density type of one of claim 1-3 freezing pig jism.
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| CN102687715A (en) * | 2011-03-23 | 2012-09-26 | 北京浩邦猪人工授精服务有限责任公司 | Cold-water instant pig seminal fluid dilution powder formula and manufacturing process and application method |
| CN102613168A (en) * | 2012-03-05 | 2012-08-01 | 深圳市安多福动物药业有限公司 | Disinfectant for animal semen and preparation method thereof |
| CN103141472A (en) * | 2013-03-19 | 2013-06-12 | 广西壮族自治区水产研究所 | Ultra-low-temperature freezing preservation and activation method for Hong Kong oyster sperm |
| CN103141472B (en) * | 2013-03-19 | 2014-11-26 | 广西壮族自治区水产研究所 | Ultra-low-temperature freezing preservation and activation method for Hong Kong oyster sperm |
| CN103299987A (en) * | 2013-05-29 | 2013-09-18 | 西北农林科技大学 | Diluent for normal temperature preservation of boar semens and preparation method thereof |
| CN104180601A (en) * | 2014-09-12 | 2014-12-03 | 佘高飞 | Constant-temperature refrigerator used for storing boar semen |
| CN107736339A (en) * | 2017-10-25 | 2018-02-27 | 南京农业大学 | A kind of novel portable temperature regulating device and use its boar semen method |
| CN110786315A (en) * | 2019-10-10 | 2020-02-14 | 北京田园奥瑞生物科技有限公司 | Batch production process of frozen pig semen |
| CN111480646A (en) * | 2020-04-29 | 2020-08-04 | 湖北省农业科学院畜牧兽医研究所 | High-throughput device and method for quickly freezing boar semen |
| CN111619839A (en) * | 2020-06-18 | 2020-09-04 | 安徽农业大学 | Method for manually canning semen in production of thin tube frozen semen |
| CN111619839B (en) * | 2020-06-18 | 2021-12-03 | 安徽农业大学 | Method for manually canning semen in production of thin tube frozen semen |
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