CN103548812A - 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 low temperature are preserved dilution formula.
Background technology
Along with the nutritive value of goat milk is approved and accepts by increasing people, milch goat cultivation quantity is also in cumulative year after year, in China, concentrate raising region to be mainly Shaanxi, Shandong, Henan at present, the provinces such as Hebei and Yunnan, 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 has formed gradually the artificial insemination station of herd sire under the penetration and promotion of technology of artificial insemination, it is to accelerating milch goat improved seeds reproductive speed, improve good species male animal availability, reduce production costs, improve raiser's economic benefit and brought into play important function, but some outstanding problems have also been brought under existing situation development, as the preservation after semen collection, conventionally after bright wonderful collection, need to deposit interval semen deposition after 8～12 hours, more some sire center need to be used fresh semen collection by being transported to after a while breeding center, but fresh semen is exposed in air and varies with temperature with holding time prolongation and can greatly reduce spermatozoon activity after gathering, thereby cause artificial insemination conception rate low, reduce the availability of herd sire, therefore for adapt to development needs, under existing situation in the urgent need to developing a kind of liquid storage dilution, for improving male animal station operating efficiency, good species male animal availability and service life, accelerate population reproductive speed, reducing epidemic disease infects, improve raiser's economic benefit and accelerate milch goat industry development technical basis is provided.Simultaneously Semen liquid storage is simple to operate with it, easy to carry with technology of artificial insemination, improves male animal availability, obtains high conception rate and the feature such as economical and practical and more caters to that to take the artificial insemination station of herd sire be the milch goat current situation of leading.
Semen liquid storage dilution, be divided into normal temperature (15～20 ℃) and preserve dilution and low temperature (0～4 ℃) preservation dilution, its function determines the preservation effect to seminal fluid jointly by extension rate, the storage temperature of constituent, osmotic pressure, pH value and the seminal fluid of dilution, and its main component can be divided into buffer, nutritional agents, anti-freezant and additive.The dilution formula of development mainly contains following several both at home and abroad at present: sodium citrate-carbohydrate, Tris-citric acid-carbohydrate and EDTA-sodium citrate-carbohydrate, because composition is different to the protection effect of sperm separately, the formula that researcher develops is also not quite similar.
But there is following problem in existing dilution formula in actual applications:
1) although existing " goat sperm liquid-state preservation method " can (GB) preserve the longer time in the situation that of motility rate 0.3, for milch goat, this motility rate level cannot meet the needs of actual production, and conception rate also cannot guarantee.
2) there is report " a kind of dilution for preserving semen under normal temperature " can make seminal fluid motility rate remain on 0.5-0.6 left and right, but it is limited to maintain the holding time, generally be no more than 24 hours, for expanding the radiation scope at herd sire station and breeding stock availability, can not meet development need.
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 low temperature to preserve dilution.
For achieving the above object, the present invention has adopted following technical scheme.
A dilution for preserving semen under normal temperature, the composition that this normal temperature is preserved dilution comprise trihydroxy methyl-aminomethane of distilled water, 280～400mM, the fructose of the citric acid of 90～130mM, 20～50mM, the vitamin c of the trehalose of 200～300mM, 4～6g/L, the reduced glutathione of the bovine serum albumin(BSA) of 1～12g/L, 4～6mM, the tylosin of the adenosine triphosphate of 1～1.5mM, 0.05～0.055g/L, the lincomycin of the gentamicin of 0.27～0.28g/L, 0.16～0.18g/L and the miramycin of 0.3～0.35g/L.
The composition that described normal temperature is preserved dilution comprise trihydroxy methyl-aminomethane of distilled water, 300mM, the fructose of the citric acid of 94.7mM, 30mM, the vitamin c of the trehalose of 225mM, 5g/L, the reduced glutathione of the bovine serum albumin(BSA) of 3g/L, 5mM, the tylosin of the adenosine triphosphate of 1.2mM, 0.052g/L, the lincomycin of the gentamicin of 0.275g/L, 0.17g/L and the miramycin of 0.33g/L.
Described normal temperature is preserved after dilution mixes with seminal fluid and is preserved under 15～20 ℃ (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 low temperature is preserved dilution, the composition that this low temperature is preserved dilution comprises distilled water, 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, the miramycin of 0.3～0.35g/L and final volume are than the aseptic yolk that is 15～25%.
The composition that described low temperature is preserved dilution comprise trihydroxy methyl-aminomethane of distilled water, 375mM, the fructose of the citric acid of 124mM, 40mM, the vitamin c of the trehalose of 295mM, 5g/L, the reduced glutathione of the bovine serum albumin(BSA) of 10g/L, 5mM, the tylosin of the adenosine triphosphate of 1.2mM, 0.052g/L, the lincomycin of the gentamicin of 0.275g/L, 0.17g/L, the miramycin of 0.33g/L and final volume than the aseptic yolk that is 20%.
Described low temperature is preserved dilution and is mixed with seminal fluid and be cooled to 0～4 ℃ (low temperature) by 2～4 hours, is then placed in vacuum flask and carries out seminal fluid preservation.
After described low temperature preservation 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.
The seminal fluid preservation problem that milch goat seminal fluid preservation dilution of the present invention be take in herd sire station artificial insemination is starting point, trihydroxy methyl-aminomethane-citric acid-the carbohydrate of take is developed the dilution formula (be included in and at least preserve the dilution formula of 36 hours under normal temperature and at least preserve at low temperatures the dilution formula of 3 days) that can efficiently preserve seminal fluid as basic ingredient, dilution of the present invention has good liquid storage effect to milch goat seminal fluid, can effectively solve bright essence test-tube ageing, be conducive to improve improved seeds availability, obtain high conception rate, the Breeds resources of realizing is truly shared, this dilution preparation method is simple, simple to operate simultaneously, and production cost is low, is convenient to large-scale promotion application.
Embodiment
Below in conjunction with embodiment, the present invention is elaborated.
It is starting point that the artificial insemination station of herd sire is take in the present invention, trihydroxy methyl-aminomethane-citric acid-the carbohydrate of take is developed a kind of low temperature that can effectively preserve the seminal fluid normal temperature of 36 hours preservation dilution formula and effectively preserve 3 days as basic ingredient and is preserved dilution formula, greatly solved herd sire station to far and near different problem of semen collection time limit and semen deposition place, given full play to the availability of herd sire (particularly milch goat), the Breeds resources of realizing is truly 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 adding in proportion in dilution of the present invention and citric acid very desirable providing have been provided and have cushioned and equilibrium osmotic pressure action effect.The energy that the nutrition of providing is consumed to supplement sperm existence and motion is provided for fructose and trehalose, maintain the highly active effect of sperm, feature of the present invention is to add a certain proportion of fructose and trehalose can effectively safeguard that plasmalemmae of sperms integrality, reduction perforatorium are undermined maintenance sperm and have impact capacity, thereby 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 can effectively reduce harmful substance active oxygen radical (ROS) according to the above ratio, improve superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPX) activity, there is the sperm motility of raising performance, mediation signal transmission and prevent that lipid peroxidation is to the damage of sperm and the function such as anti-oxidant, thereby greatly improve sperm membrane Membranous lipid fluidity and improve sperm viability.The selection tylosin of composite antibiotic, gentamicin, lincomycin and miramycin can effectively be controlled the colony counts of bacterium in seminal fluid and to sperm nonhazardous effect, thereby safeguard that sperm is compared with 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, preservation is mixed, sealed to each components in certain proportion with bright smart isothermal after fully mixing, seminal fluid and air are completely cut off and be conducive to the damage that sperm is avoided external environment.Deposit at normal temperatures motility rate after 36 hours and still can reach more than 0.65, meet sire station completely to periphery raiser (field) artificial insemination demand.
The invention also discloses a kind of milch goat seminal fluid low temperature and preserve 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 μ m membrane filtration mixes with distilled water in proportion, the final volume mark of aseptic yolk in dilution is 15～25%.
Before each composition of dilution and function, introduce in detail, the present invention filter out suitable trehalose adding proportion can make sperm under cryogenic conditions in Relative High Permeability state, can effectively safeguard membrane integrity, avoid sperm low temperature injury, make it maintain higher vigor.The present invention selects yolk can protect preferably sperm strike of catching a cold under cryogenic conditions as cryoprotective agent; protection plasmalemmae of sperms integrality and acrosomal integnity; can effectively protect sperm in low temperature environment institute's damaged and improve sperm viability, thereby extending the seminal fluid holding time.
Have the coagulase (EYCE) in report yolk to have the active hydrolyzable lecithin of phosphatidylinositols A2 (PC) generation lysolecithin (LPC) both at home and abroad, 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 after it is added into dilution with 0.45 μ m membrane filtration and with final volume mark 20% left and right, preserves seminal fluid and can greatly avoid the toxic action of the generation of LPC to sperm under 0～4 ℃ of condition.
Another feature of above-mentioned milch goat seminal fluid low temperature preservation dilution formula is simple to operate and the seminal fluid holding time is long, each components in certain proportion is fully mixed, mix with the smart isothermal of aquatic foods after sealing between slow cooling 2h to 0～4 ℃, after utilizing vacuum flask to deposit 72h at this temperature, motility rate still can reach more than 0.55, meets seminal fluid long-distance transport requirement and sire station completely to the more remote raiser of periphery (field) artificial insemination demand.
Embodiment 1
The preparation of milch goat dilution for preserving semen under normal temperature, 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, 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.After fully being mixed, mentioned component and distilled water obtain milch goat dilution for preserving semen under normal temperature.Dilution pH value 6.8～7.0.
Embodiment 2
Milch goat seminal fluid low temperature is preserved 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 to mixture A, select yolk (egg), it is added in said mixture A after with 0.45 μ m membrane filtration, fully mix, obtain milch goat seminal fluid low temperature and preserve dilution.The volume fraction of yolk in dilution is 20%, dilution pH value 6.8～7.0.
The bright smart store method of milch goat
Bright wonderful collection and performance rating: adopt artificial vagina method to gather adult healthy stud ram seminal fluid, apparent motility rate to sperm at 37 ℃, motility rate and density are carried out performance rating, under 100 times of visuals field of microscope, observe the apparent motility rate of bright essence, be divided into 0～5 grade, 0 grade is not fluctuation, and 5 grades is quick whirlpool shape fluctuation.Under 400 times of visuals field, evaluate bright smart motility rate, being greater than 0.8 sample can preserve, and by after 1000 times of aquatic foods essence dilutions, calculates seminal fluid density on blood counting chamber.
Normal temperature store method: according to bright essence (temperature is 37 ℃) volume and the density of adopt, add the described milch goat dilution for preserving semen under normal temperature (embodiment 1) of isothermal, making sperm concentration is 5 * 10
8individual/mL, puts upside down and mixes sealing, can be kept in 15～20 ℃ of environment and be 36h according to concrete room temperature with heat-preservation cotton parcel or by thermos cup water-bath adjusting temperature, during can be as the case may be repeatedly for artificial insemination.
Cryopreservation: according to bright essence (temperature is 37 ℃) volume and the density of adopt, add the milch goat seminal fluid low temperature of isothermal to preserve dilution (embodiment 2), making sperm concentration is 5 * 10
8individual/mL, put upside down and mix sealing, with heat-preservation cotton parcel, be placed in cold compartment of refrigerator 2 hours between slow cooling to 0～4 ℃, maintain on this temperature basis and can continue to be kept in cold compartment of refrigerator or be transferred in the vacuum flask that fills mixture of ice and water to transport for long-distance, the holding time reaches 3 days.During use, seminal fluid is rapidly heated and can be used for artificial insemination after 35～37 ℃, during Reusability as the case may be.
Milch goat seminal fluid is preserved rear performance rating
Sperm motility rate and density measurement: before semen deposition, get 15 μ L samples and under 400 times of visuals field, evaluate bright 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 citrate through containing formaldehyde, calculates every 200 Sperm acrasomal integvities at random under 1000 times or phase contrast microscope.
Plasmalemmae of sperms integrity detection: adopt hypotonic swelling test (HOST) method to detect.With hypotonic medium, adjust sperm concentration to 2 * 10
6individual sperm/mL, hatches 30min for 37 ℃, gets 10 μ L seminal fluid and is engraved in and under 400 times of phase contrast microscopes, calculates sperm swelling situation, statistics plasmalemmae of sperms percentage of head rice.
Specific experiment example
Adopt artificial vagina method to gather 27 adult healthy Xi Nongsa energy milch goat stud ram (1～4 one full year of life) seminal fluid, the apparent motility rate to seminal fluid at 37 ℃, motility rate and density are evaluated.Utilize above-mentioned two kinds of dilutions and method of operating respectively seminal fluid to be saved to description time, the traditional trihydroxy methyl-aminomethane of usining is preserved liquid as experiment contrast.Every ram seminal fluid is carried out to performance rating.Result shows that the bright precision of gathered stud ram sample is 1.9～2.8 * 10
9within the scope of individual/mL, change, 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 plasma membrane percentage of head rice mean value is 67%.Low temperature is preserved rear motility rate and is changed in 0.45～0.6 scope, and mean value is 0.55, and acrosomal integrity mean value is 68%, and plasma membrane percentage of head rice 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 the impact of foreign matter on sperm, reduces the harm of sperm metabolism product, can reduce sperm and be subject to low temperature to hit the damage of the plasmalemmae of sperms structure that causes.By experimental results show that dilution formula disclosed in this invention, for milch goat semen at normal temperature, preserve with low temperature and preserve and there is good effect, (evidence, after the bright essence of milch goat is deposited 36h after dilution adds in normal temperature, motility rate still can reach more than 0.65, and acrosomal integrity is more than 74%; Under low temperature preservation condition, can extend to 72h, motility rate still remains on 0.55, and acrosomal integrity reaches 68%) can effectively keep sperm motility rate, acrosomal integrity, reduce rate of teratosperm, can effectively 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.
The impact of table 1 dilution of the present invention on 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 is preserved dilution comprises distilled water, 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 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 is preserved dilution comprise trihydroxy methyl-aminomethane of distilled water, 300mM, the fructose of the citric acid of 94.7mM, 30mM, the vitamin c of the trehalose of 225mM, 5g/L, the reduced glutathione of the bovine serum albumin(BSA) of 3g/L, 5mM, the tylosin of the adenosine triphosphate of 1.2mM, 0.052g/L, the lincomycin of the gentamicin of 0.275g/L, 0.17g/L and the miramycin of 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 ℃.
4. a kind of milch goat dilution for preserving semen under normal temperature according to claim 3, is characterized in that: after described normal temperature is preserved dilution and mixed with seminal fluid, 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 low temperature is preserved dilution, it is characterized in that: the composition that this low temperature is preserved dilution comprises distilled water, 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, the miramycin of 0.3～0.35g/L and final volume are than the aseptic yolk that is 15～25%.
6. a kind of milch goat seminal fluid low temperature is preserved dilution according to claim 5, it is characterized in that: the composition that described low temperature is preserved dilution comprise trihydroxy methyl-aminomethane of distilled water, 375mM, the fructose of the citric acid of 124mM, 40mM, the vitamin c of the trehalose of 295mM, 5g/L, the reduced glutathione of the bovine serum albumin(BSA) of 10g/L, 5mM, the tylosin of the adenosine triphosphate of 1.2mM, 0.052g/L, the lincomycin of the gentamicin of 0.275g/L, 0.17g/L, the miramycin of 0.33g/L and final volume than the aseptic yolk that is 20%.
7. a kind of milch goat seminal fluid low temperature is preserved dilution according to claim 5, it is characterized in that: described low temperature is preserved dilution and mixed with seminal fluid and be cooled to 0～4 ℃ by 2～4 hours, is then placed in vacuum flask and carries out seminal fluid preservation.
8. a kind of milch goat seminal fluid low temperature is preserved dilution according to claim 7, it is characterized in that: after described low temperature preservation 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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| 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 |
| CN110463688A (en) * | 2019-07-10 | 2019-11-19 | 安徽农业大学 | A kind of dilution and store method of sheep sperm |
| CN110463688B (en) * | 2019-07-10 | 2021-07-27 | 安徽农业大学 | Diluent of sheep semen and preservation method |
| CN111657270A (en) * | 2020-07-21 | 2020-09-15 | 张良斌 | Normal-temperature preservative for livestock semen and preparation method and use method thereof |
| 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 |
| 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 |
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