CN103548812B - Normal-temperature and low-temperature preservation diluents of milk goat seminal fluid - Google Patents
Normal-temperature and low-temperature preservation diluents of milk goat seminal fluid Download PDFInfo
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Abstract
The invention discloses normal-temperature and low-temperature preservation diluents of milk goat seminal fluid. Each normal-temperature and low-temperature preservation diluent comprises the following main ingredients: tris(hydroxymethyl)aminomethane, citric acid, fructose, mycose, vitamin C, bovine serum albumin, reduced glutathione, triphosadenine and compound antibiotics, and specifically, the low-temperature preservation diluent contains yolk. The normal-temperature and low-temperature preservation diluents provided by the invention can store the seminal fluid in a liquid state effectively for a short time so as to meet the artificial insemination requirement, solve the problems of short storage time limit after semen collection in a male animal station and long transportation distance between semen collection points and semen transportation points, and improve the storage quality and time effectiveness of fresh semen, thereby improving the excellent breeding animal utilization ratio and acquiring a high conception rate; meanwhile, preparation methods of the diluents are simple, the operation is simple, and the diluents are low in production cost and convenient for popularization and application on a large scale.
Description
Technical field
The present invention relates to biological technical field, particularly milch goat semen at normal temperature and Cord blood dilution formula.
Background technology
To be approved by increasing people along with the nutritive value of goat milk and accept, milch goat cultivation quantity is also in cumulative year after year, concentrate raising region to be mainly Shaanxi, Shandong, Henan, the province such as Hebei and Yunnan in China at present, and present a kind of trend to coastal region in east China development.It should be noted that, along with milch goat concentrates culture zone as Shaanxi, Shandong defines the artificial insemination station of herd sire gradually under the penetration and promotion of technology of artificial insemination, it is to quickening milch goat improved seeds reproductive speed, improve good species male animal availability, reduce production cost, improve raiser's economic benefit and play important function, but under existing situation development, also bring some outstanding problems, as the preservation after semen collection, need to deposit interval semen deposition after 8 ~ 12 hours after usual fresh wonderful collection, more some sire center needs to use after fresh semen collection through being transported to breeding center after a while, but fresh semen is exposed in air to vary with temperature and greatly can reduces spermatozoon activity with holding time prolongation after gathering, thus cause artificial insemination conception rate low, reduce the availability of herd sire, therefore for adapt to development needs, in the urgent need to developing a kind of liquid storage dilution under existing situation, for improving male animal station operating efficiency, good species male animal availability and service life, accelerate population reproductive speed, reduce prevailing patient infections, improve raiser's economic benefit and accelerate milch goat industry development and technical basis is provided.Simultaneously Semen liquid storage is simple to operate with it, easy to carry with technology of artificial insemination, raising male animal availability, obtains high conception rate and the feature such as economical and practical and more caters to milch goat current situation based on the artificial insemination station of herd sire.
Semen liquid storage dilution, normal temperature (15 ~ 20 DEG C) is divided into preserve dilution and low temperature (0 ~ 4 DEG C) preservation dilution, its function determines the preservation effect to seminal fluid jointly by the extension rate of the constituent of dilution, osmotic pressure, pH value and seminal fluid, storage temperature, and its main component can be divided into buffer, nutritional agents, anti-freezing agent and additive.The dilution formula of development both at home and abroad mainly contains following several at present: sodium citrate-carbohydrate, Tris-citric acid-carbohydrate and EDTA-sodium citrate-carbohydrate, and because respective composition is different to the protected effect of sperm, the formula that researcher develops also is not quite similar.
But there is following problem in actual applications in existing dilution formula:
1) although existing " goat sperm liquid-state preservation method " (GB) can preserve the longer time when motility rate 0.3, for milch goat, this motility rate level cannot meet the needs of actual production, and conception rate also cannot ensure.
2) there is report " a kind of dilution for preserving semen under normal temperature " that seminal fluid motility rate can be made to remain on about 0.5-0.6, but it is limited to maintain the holding time, generally be no more than 24 hours, for the radiation scope that expansion herd sire is stood and breeding stock availability, can not development need be met.
Summary of the invention
One object of the present invention is to provide a kind of milch goat dilution for preserving semen under normal temperature;
Another object of the present invention is to provide a kind of milch goat seminal fluid Cord blood dilution.
For achieving the above object, present invention employs following technical scheme.
A kind of milch goat dilution for preserving semen under normal temperature, the composition that this normal temperature preserves dilution comprises the miramycin of distilled water, the trihydroxy methyl-aminomethane of 280 ~ 400mM, the citric acid of 90 ~ 130mM, the fructose of 20 ~ 50mM, the trehalose of 200 ~ 300mM, the vitamin c of 4 ~ 6g/L, the bovine serum albumin(BSA) of 1 ~ 12g/L, the reduced glutathione of 4 ~ 6mM, the adenosine triphosphate of 1 ~ 1.5mM, the tylosin of 0.05 ~ 0.055g/L, the gentamicin of 0.27 ~ 0.28g/L, the lincomycin of 0.16 ~ 0.18g/L and 0.3 ~ 0.35g/L.
The composition that described normal temperature preserves dilution comprises the miramycin of distilled water, the trihydroxy methyl-aminomethane of 300mM, the citric acid of 94.7mM, the fructose of 30mM, the trehalose of 225mM, the vitamin c of 5g/L, the bovine serum albumin(BSA) of 3g/L, the reduced glutathione of 5mM, the adenosine triphosphate of 1.2mM, the tylosin of 0.052g/L, the gentamicin of 0.275g/L, the lincomycin of 0.17g/L and 0.33g/L.
Described normal temperature is preserved after dilution mixes with seminal fluid and is preserved under 15 ~ 20 DEG C (normal temperature).
After described normal temperature preservation dilution mixes with seminal fluid, the density of sperm is diluted to 4 ~ 6 × 10
8individual/mL, the holding time is at least 36 hours.
A kind of milch goat seminal fluid Cord blood dilution, the composition of this Cord blood dilution comprises distilled water, trihydroxy methyl-the aminomethane of 280 ~ 400mM, the citric acid of 90 ~ 130mM, the fructose of 20 ~ 50mM, the trehalose of 200 ~ 300mM, the vitamin c of 4 ~ 6g/L, the bovine serum albumin(BSA) of 1 ~ 12g/L, the reduced glutathione of 4 ~ 6mM, the adenosine triphosphate of 1 ~ 1.5mM, the tylosin of 0.05 ~ 0.055g/L, the gentamicin of 0.27 ~ 0.28g/L, the lincomycin of 0.16 ~ 0.18g/L, the miramycin of 0.3 ~ 0.35g/L and final volume are than the aseptic yolk being 15 ~ 25%.
The composition of described Cord blood dilution comprises distilled water, the trihydroxy methyl-aminomethane of 375mM, the citric acid of 124mM, the fructose of 40mM, the trehalose of 295mM, the vitamin c of 5g/L, the bovine serum albumin(BSA) of 10g/L, the reduced glutathione of 5mM, the adenosine triphosphate of 1.2mM, the tylosin of 0.052g/L, the gentamicin of 0.275g/L, the lincomycin of 0.17g/L, the miramycin of 0.33g/L and final volume than the aseptic yolk being 20%.
Be cooled to 0 ~ 4 DEG C (low temperature) through 2 ~ 4 hours after described Cord blood dilution mixes with seminal fluid, be then placed in vacuum flask and carry out Semen routine.
After described Cord blood dilution mixes with seminal fluid, the density of sperm is diluted to 4 ~ 6 × 10
8individual/mL, the time of preservation is at least 72 hours.
Milch goat Semen routine dilution of the present invention with herd sire station artificial insemination in Semen routine problem for starting point, based on trihydroxy methyl-aminomethane-citric acid-carbohydrate, composition develops the dilution formula (at least preserve the dilution formula of 36 hours under being included in normal temperature and at least preserve the dilution formula of 3 days at low temperatures) efficiently preserving seminal fluid, dilution of the present invention has good liquid storage effect to milch goat seminal fluid, can effectively solve fresh essence test-tube ageing, be conducive to improving improved seeds availability, obtain high conception rate, the Breeds resources realized truly is shared, this dilution preparation method is simple simultaneously, and simple to operate, production cost is low, is convenient to large-scale promotion application.
Embodiment
Below in conjunction with embodiment, the present invention is elaborated.
The present invention with the artificial insemination station of herd sire for starting point, based on trihydroxy methyl-aminomethane-citric acid-carbohydrate, composition develops a kind of Cord blood dilution formula effectively can preserved the seminal fluid normal temperature of 36 hours and preserve dilution formula and effectively preserve 3 days, greatly solve herd sire station to semen collection time limit and semen deposition place far and near different problem, given full play to the availability of herd sire (particularly milch goat), the Breeds resources realized truly is shared.
The invention discloses a kind of milch goat dilution for preserving semen under normal temperature formula, described dilution is by trihydroxy methyl-aminomethane 280 ~ 400mM, citric acid 90 ~ 130mM, fructose 20 ~ 50mM, trehalose 200 ~ 300mM, vitamin c 4 ~ 6g/L, bovine serum albumin(BSA) 1 ~ 12g/L, reduced glutathione 4 ~ 6mM, adenosine triphosphate 1 ~ 1.5mM and composite antibiotic (tylosin of 0.05 ~ 0.055g/L, the gentamicin of 0.27 ~ 0.28g/L, the lincomycin of 0.16 ~ 0.18g/L and the miramycin of 0.3 ~ 0.35g/L) mix with distilled water.
Trihydroxy methyl-aminomethane and citric acid have avirulence, pH modulability and good buffering stability, and the trihydroxy methyl-aminomethane added in proportion in dilution of the present invention and citric acid reach very desirable providing and cushion and equilibrium osmotic pressure action effect.Fructose and trehalose have provides nutrition with supplementary sperm existence and the energy consumed that moves, maintain the highly active effect of sperm, feature of the present invention is to add a certain proportion of fructose and trehalose effectively can be safeguarded plasmalemmae of sperms integrality, reduce perforatorium and undermined and keep sperm to have impact capacity, thus improves sperm viability.Vitamin c, reduced glutathione, bovine serum albumin(BSA) and adenosine triphosphate have extremely strong anti-oxidant, anti-ageing and prevent the characteristics such as Apoptosis, the present invention adds the peroxidatic reaction of lipid that effectively can reduce harmful substance active oxygen radical (ROS) according to the above ratio, improve superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPX) is active, have and improve sperm motility performance, mediation signal transmission and prevent lipid peroxidation to the damage of sperm and the function such as anti-oxidant, greatly improve sperm membrane Membranous lipid fluidity thus improve sperm viability.The selection tylosin of composite antibiotic, gentamicin, lincomycin and miramycin effectively can control the colony counts of bacterium in seminal fluid and to sperm nonhazardous effect, thus safeguard sperm comparatively high-servival rate.
Further illustrate, another feature of above-mentioned milch goat dilution for preserving semen under normal temperature formula is simple to operate and economical and practical, mix with fresh smart isothermal after each components in certain proportion is fully mixed, seal and preserve, seminal fluid and air exclusion are conducive to the damage of sperm from external environment.After depositing 36 hours at normal temperatures, motility rate still can reach more than 0.65, meets sire station completely to periphery raiser (field) artificial insemination demand.
The invention also discloses a kind of milch goat seminal fluid Cord blood dilution formula, described dilution is by trihydroxy methyl-aminomethane 280 ~ 400mM, citric acid 90 ~ 130mM, fructose 20 ~ 50mM, trehalose 200 ~ 300mM, vitamin c 4 ~ 6g/L, bovine serum albumin(BSA) 1 ~ 12g/L, reduced glutathione 4 ~ 6mM, adenosine triphosphate 1 ~ 1.5mM, composite antibiotic (the tylosin of 0.05 ~ 0.055g/L, the gentamicin of 0.27 ~ 0.28g/L, the lincomycin of 0.16 ~ 0.18g/L and the miramycin of 0.3 ~ 0.35g/L), aseptic yolk after 0.45 μm of membrane filtration mixes with distilled water in proportion, the final volume mark of aseptic yolk in dilution is 15 ~ 25%.
Introduce in detail before each composition of dilution and function, the present invention filters out suitable trehalose adding proportion can make sperm be in Relative High Permeability state under cryogenic, effectively can safeguard membrane integrity, from sperm low temperature injury, makes it maintain higher vigor.The present invention select yolk as cryoprotective agent can protect sperm under cryogenic preferably strike of catching a cold; protection plasmalemmae of sperms integrality and acrosomal integnity; available protecting sperm can improve sperm viability in low temperature environment institute's damaged, thus extend the Semen routine time.
Have the coagulase (EYCE) in report yolk to have active hydrolyzable lecithin (PC) of phosphatidylinositols A2 both at home and abroad and produce lysolecithin (LPC), LPC has certain toxic action to sperm, can cause damage to membrane integrity.But this toxicity intensity depends on the quality of yolk, the active temperature of addition and LPC.The yolk that the present invention selects, fully mixes it after being added into dilution with 0.45 μm of membrane filtration, preserve the toxic action of generation to sperm that seminal fluid can avoid LPC greatly under 0 ~ 4 DEG C of condition with final volume mark about 20%.
Another feature of above-mentioned milch goat seminal fluid Cord blood dilution formula is simple to operate and the Semen routine time is long, each components in certain proportion is fully mixed, to mix after sealing between slow cooling 2h to 0 ~ 4 DEG C with fresh smart isothermal, after utilizing vacuum flask to deposit 72h at such a temperature, motility rate still can reach more than 0.55, meet completely seminal fluid long-distance transport require and sire station to the more remote raiser of periphery (field) artificial insemination demand.
Embodiment 1
Milch goat dilution for preserving semen under normal temperature is prepared, its composition optimum content is: trihydroxy methyl-aminomethane 300mM, citric acid 94.7mM, fructose 30mM, trehalose 225mM, vitamin c 5g/L, reduced glutathione 5mM, bovine serum albumin(BSA) 3g/L, the tylosin of adenosine triphosphate 1.2mM, 0.052g/L, the gentamicin of 0.275g/L, the lincomycin of 0.17g/L, the miramycin of 0.33g/L.Namely milch goat dilution for preserving semen under normal temperature is obtained after mentioned component and distilled water fully being mixed.Dilution pH value 6.8 ~ 7.0.
Embodiment 2
Milch goat seminal fluid Cord blood diluent preparing, its composition optimum content is: trihydroxy methyl-aminomethane 375mM, citric acid 124mM, fructose 40mM, trehalose 295mM, vitamin c 5g/L, reduced glutathione 5mM, bovine serum albumin(BSA) 10g/L, adenosine triphosphate 1.2mM, the tylosin of 0.052g/L, the gentamicin of 0.275g/L, the lincomycin of 0.17g/L, the miramycin of 0.33g/L, mentioned component and distilled water are fully mixed to obtain mixture A, select yolk (egg), by it to be added in said mixture A after 0.45 μm of membrane filtration, abundant mixing, namely milch goat seminal fluid Cord blood dilution is obtained.The volume fraction of yolk in dilution is 20%, dilution pH value 6.8 ~ 7.0.
The fresh smart store method of milch goat
Fresh wonderful collection and performance rating: adopt Pseudopyloric metaplasia to gather adult healthy stud ram seminal fluid, to the apparent motility rate of sperm at 37 DEG C, motility rate and density carry out performance rating, the apparent motility rate of fresh essence is observed under microscope 100 times of visuals field, be divided into 0 ~ 5 grade, 0 grade is not fluctuation, and 5 grades is the fluctuation of quick swirling.Under 400 times of visuals field, evaluate fresh smart motility rate, the sample being greater than 0.8 can be preserved, and calculates seminal fluid density by after fresh essence dilution 1000 times on blood counting chamber.
Normal temperature store method: according to adopted fresh essence (temperature is 37 DEG C) volume and density, adds the described milch goat dilution for preserving semen under normal temperature (embodiment 1) of isothermal, makes sperm concentration be 5 × 10
8individual/mL, puts upside down mixing sealing, and according to concrete room temperature heat-preservation cotton parcel or can regulate temperature with thermos cup water-bath, be kept in 15 ~ 20 DEG C of environment and be 36h, period can as the case may be repeatedly for artificial insemination.
Cryopreservation: according to adopted fresh essence (temperature is 37 DEG C) volume and density, adds the milch goat seminal fluid Cord blood dilution (embodiment 2) of isothermal, makes sperm concentration be 5 × 10
8individual/mL, put upside down mixing sealing, to be placed in cold compartment of refrigerator 2 hours between slow cooling to 0 ~ 4 DEG C with heat-preservation cotton parcel, maintain in the vacuum flask that this temperature basis can continue be kept in cold compartment of refrigerator or be transferred to and fill mixture of ice and water and transport for long-distance, the holding time reaches 3 days.Be rapidly heated by seminal fluid during use and can be used for artificial insemination after 35 ~ 37 DEG C, period can Reusability as the case may be.
Performance rating after milch goat Semen routine
Sperm motility rate and density measurement: get 15 μ L samples before semen deposition under 400 times of visuals field, evaluate fresh smart motility rate, sperm motility parameters mainly comprises sperm motility rate, sperm rectilinear motion speed and curve speed.
The detection of Sperm acrasomal integvity: get 15 μ L sample making smears, encapsulation after liquid-solid fixed, the Ji's nurse Sa dye liquor dyeing of the citrate containing formaldehyde, calculates every 200 Sperm acrasomal integvities at random under 1000 times or phase contrast microscope.
Plasmalemmae of sperms integrity detection: adopt hypo-osmotic swelling test (HOST) method to detect.With hypotonic medium adjustment sperm concentration to 2 × 10
6individual sperm/mL, hatches 30min for 37 DEG C, gets under namely 10 μ L seminal fluid are engraved in 400 times of phase contrast microscopes and calculates sperm swelling situation, statistics plasmalemmae of sperms percentage of head rice.
Specific experiment example
Adopt Pseudopyloric metaplasia to gather 27 adult healthy Xining basin stud ram (1 ~ 4 one full year of life) seminal fluid, to the apparent motility rate of seminal fluid at 37 DEG C, motility rate and density are evaluated.Utilize above-mentioned two kinds of dilutions and method of operating respectively by Semen routine to description time, using traditional trihydroxy methyl-aminomethane conserving liquid as experiment contrast.Performance rating is carried out to every ram seminal fluid.Result shows that the fresh precision of gathered stud ram sample is 1.9 ~ 2.8 × 10
9change within the scope of individual/mL, averag density is 2.5 × 10
9individual/mL, every apparent motility rate of stud ram all reaches peak, and motility rate changes in 0.75 ~ 0.85 scope, and mean value is 0.8.Normal temperature is preserved rear motility rate and is changed in 0.55 ~ 0.7 scope, and mean value is 0.65, and acrosomal integrity mean value is 74%, and plasm membrane integrity mean value is 67%.After Cord blood, motility rate changes in 0.45 ~ 0.6 scope, and mean value is 0.55, and acrosomal integrity mean value is 68%, and plasm membrane integrity mean value is 62%.Data result is in table 1.
The effect of dilution of the present invention mainly can supplement the nutrient consumption of sperm, maintains the osmotic pressure of sperm, reduces foreign matter to the impact of sperm, reduces the harm of sperm metabolism product, can reduce sperm by low temperature strike cause the damage of plasmalemmae of sperms structure.Prove that dilution formula disclosed in this invention has good effect for the preservation of milch goat semen at normal temperature with Cord blood by experiment, (test proves, after the fresh essence of milch goat deposits 36h after dilution adds in normal temperature, motility rate still can reach more than 0.65, acrosomal integrity more than 74%; 72h can be extended under Cord blood condition, motility rate still remains on 0.55, and acrosomal integrity reaches 68%) can effectively keep sperm motility rate, acrosomal integrity, reduce rate of teratosperm, effectively can improve artificial insemination conception rate and inseminatio externalis efficiency, improve artificial insemination production efficiency.This diluent preparing technique is simple simultaneously, and production cost is low and be easy to carry, and is convenient to large-scale promotion application.
Table 1 dilution of the present invention is on the impact of milch goat Semen liquid storage
Claims (8)
1. a milch goat dilution for preserving semen under normal temperature, it is characterized in that: the composition that this normal temperature preserves dilution comprises distilled water, trihydroxy methyl-the aminomethane of 280 ~ 400mM, the citric acid of 90 ~ 130mM, the fructose of 20 ~ 50mM, the trehalose of 200 ~ 300mM, the vitamin c of 4 ~ 6g/L, the bovine serum albumin(BSA) of 1 ~ 12g/L, the reduced glutathione of 4 ~ 6mM, the adenosine triphosphate of 1 ~ 1.5mM, the tylosin of 0.05 ~ 0.055g/L, the gentamicin of 0.27 ~ 0.28g/L, the lincomycin of 0.16 ~ 0.18g/L and the miramycin of 0.3 ~ 0.35g/L.
2. a kind of milch goat dilution for preserving semen under normal temperature according to claim 1, is characterized in that: the composition that described normal temperature preserves dilution comprises the miramycin of distilled water, the trihydroxy methyl-aminomethane of 300mM, the citric acid of 94.7mM, the fructose of 30mM, the trehalose of 225mM, the vitamin c of 5g/L, the bovine serum albumin(BSA) of 3g/L, the reduced glutathione of 5mM, the adenosine triphosphate of 1.2mM, the tylosin of 0.052g/L, the gentamicin of 0.275g/L, the lincomycin of 0.17g/L and 0.33g/L.
3. a kind of milch goat dilution for preserving semen under normal temperature according to claim 1, is characterized in that: described normal temperature is preserved after dilution mixes with seminal fluid and preserved at 15 ~ 20 DEG C.
4. a kind of milch goat dilution for preserving semen under normal temperature according to claim 3, is characterized in that: described normal temperature is preserved after dilution mixes with seminal fluid, and the density of sperm is diluted to 4 ~ 6 × 10
8individual/mL, the holding time is at least 36 hours.
5. a milch goat seminal fluid Cord blood dilution, it is characterized in that: the composition of this Cord blood dilution comprises distilled water, trihydroxy methyl-the aminomethane of 280 ~ 400mM, the citric acid of 90 ~ 130mM, the fructose of 20 ~ 50mM, the trehalose of 200 ~ 300mM, the vitamin c of 4 ~ 6g/L, the bovine serum albumin(BSA) of 1 ~ 12g/L, the reduced glutathione of 4 ~ 6mM, the adenosine triphosphate of 1 ~ 1.5mM, the tylosin of 0.05 ~ 0.055g/L, the gentamicin of 0.27 ~ 0.28g/L, the lincomycin of 0.16 ~ 0.18g/L, the miramycin of 0.3 ~ 0.35g/L and final volume are than the aseptic yolk being 15 ~ 25%.
6. a kind of milch goat seminal fluid Cord blood dilution according to claim 5, is characterized in that: the composition of described Cord blood dilution comprises distilled water, the trihydroxy methyl-aminomethane of 375mM, the citric acid of 124mM, the fructose of 40mM, the trehalose of 295mM, the vitamin c of 5g/L, the bovine serum albumin(BSA) of 10g/L, the reduced glutathione of 5mM, the adenosine triphosphate of 1.2mM, the tylosin of 0.052g/L, the gentamicin of 0.275g/L, the lincomycin of 0.17g/L, the miramycin of 0.33g/L and final volume than the aseptic yolk being 20%.
7. a kind of milch goat seminal fluid Cord blood dilution according to claim 5, is characterized in that: be cooled to 0 ~ 4 DEG C through 2 ~ 4 hours after described Cord blood dilution mixes with seminal fluid, is then placed in vacuum flask and carries out Semen routine.
8. a kind of milch goat seminal fluid Cord blood dilution according to claim 7, is characterized in that: after described Cord blood dilution mixes with seminal fluid, the density of sperm is diluted to 4 ~ 6 × 10
8individual/mL, the time of preservation is at least 72 hours.
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| CN104621097A (en) * | 2015-01-29 | 2015-05-20 | 中国科学院东北地理与农业生态研究所 | Sheep fresh semen preserve diluent and application thereof |
| CN104585164A (en) * | 2015-02-06 | 2015-05-06 | 塔里木大学 | Improved diluent for long-term preservation of semen of ruminant |
| CN107232180B (en) * | 2017-05-25 | 2021-07-27 | 中国热带农业科学院热带作物品种资源研究所 | Goat semen diluent for relieving high-temperature stress, preparation method and application method thereof |
| CN107668027A (en) * | 2017-11-15 | 2018-02-09 | 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 | Sheep sperm room temperature preservation dilution and preparation method thereof |
| CN108684654A (en) * | 2018-05-22 | 2018-10-23 | 华南农业大学 | A kind of optimization dilution |
| CN109644983A (en) * | 2018-12-17 | 2019-04-19 | 浙江万里学院 | Boar spermatozoa low temperature dilution based on Shelled Turtle Trionyx Sinensis hibernation serum |
| CN110463688B (en) * | 2019-07-10 | 2021-07-27 | 安徽农业大学 | Diluent of sheep semen and preservation method |
| CN111657270B (en) * | 2020-07-21 | 2022-09-13 | 张良斌 | Normal-temperature preservative for livestock semen and preparation method and use method thereof |
| CN112586494A (en) * | 2020-12-18 | 2021-04-02 | 洛阳市洛瑞牧业有限公司 | Fried smoking method frozen beef sperm manufacturing process |
| CN112514890A (en) * | 2020-12-18 | 2021-03-19 | 洛阳市洛瑞牧业有限公司 | Normal-temperature short-term preservation diluent for fresh sheep essence and preparation method thereof |
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