CN1775020A - Dog-like sperm super-low temperature freezing preservation antifreeze solution - Google Patents
Dog-like sperm super-low temperature freezing preservation antifreeze solution Download PDFInfo
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Abstract
The present invention relates to a canine sperm ultra-low temperature cold storage anti-freezing solution, belonging to the field of animal propagation technology. It is prepared by using amino-sodium-dodecyl sulfate (OEP) 0.05-2.50 ml, N-acetyl-D-glucose amine 0.01-0.10g, fluctose 0.45-3.10g, citric acid 0.50-2.30g, tris (hydroxy methyl) aminomethane 1.10-3.90g, egg yolk 15.0-30.0 ml, glycerine or dimethyl sulphoxide 2.0-8.0 ml, penicillin or gentamycin antimicrobial 1000 UI/ml and distilled water added to 100 ml. The pH value of said antifreezing solution is 6.25-6.95, and its osmotic pressure is 300-350 mOsmo/L.
Description
Technical field
The present invention relates to a kind of dog-like sperm super-low temperature freezing preservation antifreeze solution, be used for that dog-like sperm super-low temperature freezing is preserved and utilize freezing the pedestrian worker that the progresses greatly breed of inseminating, belong to the animal reproduction technical field.
Background technology
Dog is being brought into play distinctive effect as the livestock animals of raising and train the earliest in the human being's production life, the social demand of good species dog grows with each passing day especially.The kind of China dog not only is known in the world with distinctive use and ornamental value, and the formation of world-renowned kind produced significant effects, as the Ma Shidifu (large fierce dog) in Tibet, Shih Tzu, Beijing dog, Lhasa dog (Ai Pusuo dog) etc., as the variety source of precious dog, become the people of the world's common wealth.China urban and rural residents are along with the improvement of economic condition, the people of pet feeding is more and more, people have recognized the great economic benefit that the pet feeding dog is brought, the artificial feeding pet dog is developed as a kind of emerging animal husbandry, pet dog progresses into the Spirit of Man life except entering human material life.Propagating artificially of dog class extensively carried out in present domestic each department, more than one hundred million of existing pet dog.Yet in the long-term breeding process of dog class, because the condition restriction of raising and artificial propagation, particularly the condition restriction of crossbreeding technology directly causes the dog veriety quality degradation of China's artificial feeding, on international pet dog market, lose competitiveness gradually, provisions dog person has caused enormous economic loss, seriously hinders the development of China's dog class animal husbandry.
Can find that in pet dog market, the world famous and precious kind of dog is just accepted by people gradually with its unique personal value, excellent development prospect, the high-quality pet dog will be with its high-quality favor that is subjected to more people.Along with the raising of artificial rearing technology, breeding level, the raising pets kind is more and more.Compare with the dog class that China raises, external dog veriety is various, quality is higher, and especially novel kind costs an arm and a leg.Its main cause is the application of artificial propagation techniques, and is original in numerous dog veriety by the technology of artificial insemination crossbreeding and improvement, can overcome a natural mating difficult problem between different cultivars, can cultivate novel strain by heterosis, hybrid vigor again, produces good economic benefit.Therefore, how further preserving, utilize, develop the kind of dog resource of China, is a very important research topic, carries out research work in this respect in a deep going way, will produce positive role difficult to the appraisal to society, economy, the life of China.The artificial insemination propagation technique comprise utilize the bright seminal fluid after the dilution and thaw after freeze smart semen deposition dual mode, frozen semen and artificial insemination thereof, will improve availability and the value of the public dog of breeding greatly, the guardian technique that wherein relates to is exactly the development of dilution and anti-icing fluid.Studies show that the quality of freeze thawing sperm how, for example the height of vigor, plasma membrane percentage of head rice and acrosomal integrity directly influences artificial insemination's pregnancy rate.The sperm of dog-like sperm and other canid is similar, its in frozen-thaw process to the resistance of low temperature injury a little less than, freezing recovery back sperm viability descends, plasma membrane percentage of head rice and acrosomal integrity also obviously reduce, test-tube pregnancy rate and litter size all are lower than natural propagation (Farstad, W. etc., 1996, Animal Reproduction Science.42:251～260.).Therefore, how long-term low temperature is preserved the high-quality canine semen and made full use of fine provenance is present this area problem demanding prompt solution.
At present, the artificial insemination of the dog class especially superfreeze of seminal fluid is preserved and is utilized and freezes smart breeding and be subjected to very much people's attention, but reach livestock animals levels such as ox, sheep far away, about the further research and inquirement of still needing of basic theory and sport technique segment, the wherein preparation of anti-icing fluid, freezing and thaw routine are difficult problems that needs to be resolved hurrily.Anti-icing fluid prescription commonly used both at home and abroad has:
Prescription one: tri methylol amino methane (2.4%), citric acid (1.3%), fructose (1%) glycerine (6.4%), yolk (20%), penicillin, each 500,000 unit of streptomycin.When freezing essence with this prescription making, the average anabiosis rate of sperm is 46.7%, and the freeze thawing sperm viability is 0.28, and the time-to-live after the sperm recovery is more than 3 hours.(Wang Libo etc., HEILONGJIANG ANIMAL SCIENCE AND VETERINARY MEDICINE, 1991,5:35-36.).
Prescription two: tri methylol amino methane (240mM), citric acid (63mM), carbohydrate (70mM), glycerine (8%v/v), yolk (20%v/v), penicillin (100,000 IU), distilled water constant volume 100mL.Make when freezing essence of this prescription, the average vigor of freeze thawing sperm is 0.5, plasma membrane percentage of head rice and acrosomal integrity be respectively 70%, 45% (Yildiz et al, Thefiogenology, 2000,54:579-585).
Prescription three: tri methylol amino methane (2.4g), citric acid (1.4g), glucose (0.8g), glycerine (4mL), yolk (20mL), penicillin (0.06g), distilled water constant volume 100mL.When freezing essence with this making of filling a prescription, freezing the external 39 ℃ of cultivation 4 hourly average vigor in smart recovery back is 0.10, and motility rate and acrosomal integrity are respectively 24.0%, 49.4%; (
Et al, Theriogenology, 1999,50:163-174).The pregnancy rate that smart artificial insemination's breeding is frozen in utilization is 84%.(Rota?et?al，Theriogenology，1999，51：1045-1058)。
Amino-sodium-laurilsulfate Orvus ES Paste (OEP) is a kind of surfactant, it contains lauryl sodium sulfate (SDS) compound in forming, be pig and the dog class animal semen antifreeze composition of dilution (Pursel et al, J.Anim.Sci.47:198-202 commonly used when freezing; Rota et al, Theriogenology, 1999,51:1045-1058;
Et al; Theriogenology; 1999; 50:163-174), main effect is to prevent the damage of low temperature to sperm, and the sperm membrane structure is had protection, repairing effect; can improve freeze thawing sperm viability, survival rate and plasma membrane percentage of head rice (Tsutsui et al; J.Vet.Med.Sci, 2000,62 (5): 533-538).N-acetyl group-D-gucosamine (N-acetyl-D-glucosamine) is chitinous main component; be present in the ectoskeleton of insect and crustacean; belong to the polysaccharide homopolymer; on the top layer, extracellular, space between cells and connective tissue provide main constituent when forming, and makes the flexible and toughness of animal vegetable tissue.Research report N-acetyl group-D-gucosamine by human body peritoneal cell and fibroblast can effectively swash the synthesizing of grape amine glycan in vivo (Breborowicz etc., 1998, Adv.Perit.Dial.14,31-35.).According to another report; N-acetyl group-D-gucosamine has been successfully applied to (Yi etc. in the boar semen dilution; 2002; AnimalReproduction Science.74; 187-194.), but also do not report at present with the research that is used in canine semen antifreezing agent aspect about OEP and N-acetyl group-D-gucosamine.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of dog-like sperm super-low temperature freezing preservation antifreeze solution is provided, make it effectively to prolong dog class freeze thawing survival time of boar sperm, help keeping the membrane integrity and the acrosomal integnity of dog-like sperm, improve the pregnancy rate that the dog class is frozen smart breeding.
For realizing such purpose; the freezing anti-icing fluid of dog-like sperm of the present invention adopts a kind of surfactant amino-sodium-laurilsulfate (OEP) and cell culture fluid composition N-acetyl group-D-gucosamine; add fructose, tri methylol amino methane (Tris), citric acid, yolk, glycerine or dimethyl sulfoxide (DMSO) and bactericide simultaneously, formulated with distilled water.
The freezing anti-icing fluid of dog-like sperm of the present invention's development specifically comprises following composition:
0.05～2.50 milliliter in amino-sodium-laurilsulfate (OEP)
N-acetyl group-D-gucosamine 0.01～0.10 gram
Fructose 0.45～3.10 gram
Citric acid 0.50～2.30 gram
Tri methylol amino methane 1.10～3.90 grams
15.0～30.0 milliliters in yolk
2.0～8.0 milliliters of glycerine or dimethyl sulfoxide (DMSO)s
Bactericide 1000UI/ milliliter
100 milliliters of distilled water constant volumes.
Anti-icing fluid pH value is 6.25～6.95, and osmotic pressure is 300-350mOsmo/L.
Bactericide of the present invention can be selected penicillin or gentamicin, also can have the antibiotic of bactericidal action for other.
Preferred anti-icing fluid (1) contains following composition:
0.50 milliliter in amino-sodium-laurilsulfate (OEP)
N-acetyl group-D-gucosamine 0.06 gram
Fructose 1.20 grams
Citric acid 1.65 grams
Tri methylol amino methane 3.15 grams
20.0 milliliters in yolk
3.0 milliliters of glycerine
Bactericide 1000UI/ milliliter
100 milliliters of distilled water constant volumes.
Anti-icing fluid pH value is 6.65, and osmotic pressure is 320mOsmo/L.
Preferred anti-icing fluid (2) contains following composition:
0.75 milliliter in amino-sodium-laurilsulfate (OEP)
N-acetyl group-D-gucosamine 0.05 gram
Fructose 1.65 grams
Citric acid 2.25 grams
Tri methylol amino methane 1.85 grams
20.0 milliliters in yolk
4.0 milliliters of dimethyl sulfoxide (DMSO)s
Bactericide 1000UI/ milliliter
100 milliliters of distilled water constant volumes.
Anti-icing fluid pH value is 6.60, and osmotic pressure is 325mOsmo/L.
Sperm freezing anti-icing fluid of the present invention can add mentioned component respectively by described content by the conventional method preparation of this area, and adding distil water is made into 100 ml solns and gets final product again.The dilution pH value that is mixed with is 6.25～6.95, and osmotic pressure is 300～350mOsmo/L, can be the external survival of dog-like sperm the optimum substrate condition is provided.
Fructose composition in the sperm freezing anti-icing fluid of the present invention is the energy source of sperm in external metabolism, and tri methylol amino methane, citric acid can be kept the osmotic pressure balance of environment in anti-icing fluid environment and the sperm.In addition, in anti-icing fluid of the present invention, also add penicillin or gentamicin (1000UI/ milliliter) bactericide, improve the normal temperature and the low temperature preservation effect of this anti-icing fluid.
The present invention uses by amino-sodium-laurilsulfate (OEP); the anti-icing fluid that the N-acetyl group-multiple one-tenth such as D-gucosamine are grouped into; dog-like sperm is carried out the straw frozen semen test; and carry out artificial insemination with the freeze thawing sperm; study of the influence of this anti-icing fluid to dog class frozen sperm time-to-live and freeze thawing sperm quality; the result shows: by amino-sodium-laurilsulfate (OEP); the anti-icing fluid that the N-acetyl group-multiple one-tenth such as D-gucosamine are grouped into; can effectively prolong dog class freeze thawing survival time of boar sperm; the integrality of protection freeze thawing sperm membrane structure has shown good sperm protective effect and antifreezing effect.
Compared with prior art, anti-icing fluid of the present invention has following advantage:
1, can effectively prolong dog class freeze thawing survival time of boar sperm, improve the plasma membrane percentage of head rice and the acrosomal integrity of dog class freeze thawing sperm, improve test-tube pregnancy rate;
2, contained N-acetyl group-D-gucosamine composition in this anti-icing fluid can effectively promote the emulsification function of amino-sodium-laurilsulfate to strengthen the protective effect of amino-sodium-laurilsulfate and yolk;
3, preparation is simple, draws materials easily, and cost price is cheap, is easy to promote.
Canine semen anti-icing fluid of the present invention is applicable to long preservation and long-distance transport back semen deposition after dog class straw frozen semen artificial insemination breeding and the canine semen superfreeze, and it can obviously improve the survival index of dog-like sperm, keeps its insemination vigor.
Embodiment
Below by specific embodiment technical scheme of the present invention is further described.The effect of following examples should be understood that it is to annotation of the present invention but not to the restriction of any way of the present invention.
Embodiment 1 (anti-icing fluid 1)
Take by weighing 0.06g N-acetyl group-D-gucosamine earlier, be dissolved in the 10ml distilled water, it is dissolved fully.Measure 20mL yolk, 3mL glycerine, 0.50mL amino-sodium-laurilsulfate (OEP) then respectively and the three is evenly mixed.Taking by weighing 1.20g fructose then is dissolved in the 50ml distilled water, add 3.15g tri methylol amino methane, 1.65g citric acid after treating to dissolve fully respectively, add one by one by said sequence, it is a kind of to treat to add after the reagent that adds earlier dissolves fully the back during interpolation again, and mix three kinds of preparations mutually the dissolving back, adds 100,000 unit penicillin or gentamicins then, last distilled water constant volume 100ml, the pH value is 6.65 behind the constant volume, and osmotic pressure is 320mOsmo/L, puts into 5 ℃ of refrigerators and preserves.All preparation process are all carried out under aseptic condition.The effect of anti-low temperature injury was splendid when this prescription was preserved the canine semen superfreeze.Mix mutually with canine semen in 1: 3 ratio during use, the temperature that will keep anti-icing fluid simultaneously with treat that the frozen semen temperature is identical.
Use the anti-icing fluid of pressing the preparation of embodiment 1 method, when using, measure frozen sperm recovery back freeze thawing survival time of boar sperm, sperm viability (adopting phase contrast microscope to detect sperm viability), plasmalemmae of sperms percentage of head rice and acrosomal integrity (CTC-Hoechst 33258 living body fluorescent dyestuff combined staining methods detect the perforatorium percentage of head rice) with the canine semen superfreeze.The result shows: the freeze thawing survival time of boar sperm is 58 hours, preserves after 4 hours that the freeze thawing sperm viability is 0.22, plasmalemmae of sperms percentage of head rice average out to 31% under 22 ℃ of conditions, and acrosomal integrity is 53%; Utilizing this to freeze essence (back 0.5 hour of freezing recovery) artificial insemination, to breed the bitch pregnancy rate be 85.8%.
Embodiment 2 (anti-icing fluid 2)
Take by weighing 0.05g N-acetyl group-D-gucosamine earlier, be dissolved in the 10ml distilled water, it is dissolved fully.Measure 20mL yolk, 4mL dimethyl sulfoxide (DMSO) (DMSO), 0.75mL amino-sodium-laurilsulfate (OEP) then respectively and the three is evenly mixed.Taking by weighing 1.65g fructose then is dissolved in the 50ml distilled water, add 1.85g tri methylol amino methane, 2.25g citric acid after treating to dissolve fully respectively, add one by one by said sequence, it is a kind of to treat to add after the reagent that adds earlier dissolves fully the back during interpolation again, and mix three kinds of preparations mutually the dissolving back, adds 100,000 unit penicillin or gentamicins then, last distilled water constant volume 100ml, the pH value is 6.60 behind the constant volume, and osmotic pressure is 325mOsmo/L, puts into 5 ℃ of refrigerators and preserves.All preparation process are all carried out under aseptic condition.The effect of anti-low temperature injury was splendid when this prescription was preserved the canine semen superfreeze.Mix mutually with canine semen in 1: 3 ratio during use, the temperature that will keep anti-icing fluid simultaneously with treat that the frozen semen temperature is identical.
Use the anti-icing fluid of pressing the preparation of embodiment 3 methods, when using, measure frozen sperm recovery back freeze thawing survival time of boar sperm, sperm viability (adopting phase contrast microscope to detect sperm viability), plasmalemmae of sperms percentage of head rice and acrosomal integrity (CTC-Hoechst 33258 living body fluorescent dyestuff combined staining methods detect the perforatorium percentage of head rice) with the canine semen superfreeze.The result shows: the freeze thawing survival time of boar sperm is 55 hours, preserves after 4 hours that the freeze thawing sperm viability is 0.20, plasmalemmae of sperms percentage of head rice average out to 32% under 22 ℃ of conditions, and acrosomal integrity is 51%; Utilizing this to freeze essence (back 0.5 hour of freezing recovery) artificial insemination, to breed the bitch pregnancy rate be 83.4%.
Embodiment 3:(anti-icing fluid 3)
Take by weighing 0.05g N-acetyl group-D-gucosamine earlier, be dissolved in the 10.0ml distilled water, it is dissolved fully.Measure 20.0mL yolk, 6.0mL glycerine, 0.70mL amino-sodium-laurilsulfate (OEP) then respectively and the three is evenly mixed.Taking by weighing 2.65g fructose then is dissolved in the 50ml distilled water, add 1.15g tri methylol amino methane, 0.95g citric acid after treating to dissolve fully respectively, add one by one by said sequence, it is a kind of to treat to add after the reagent that adds earlier dissolves fully the back during interpolation again, and mix three kinds of preparations mutually the dissolving back, adds 10.0 ten thousand unit penicillin or gentamicins then, last distilled water constant volume 100ml, the pH value is 6.85 behind the constant volume, and osmotic pressure is 335mOsmo/L, puts into 5 ℃ of refrigerators and preserves.All preparation process are all carried out under aseptic condition.The effect of anti-low temperature injury was splendid when this prescription was preserved the canine semen superfreeze.Mix mutually with canine semen in 1: 3 ratio during use, the temperature that will keep anti-icing fluid simultaneously with treat that the frozen semen temperature is identical.
Use the anti-icing fluid of pressing the preparation of embodiment 2 methods, when using, measure frozen sperm recovery back freeze thawing survival time of boar sperm, sperm viability (adopting phase contrast microscope to detect sperm viability), plasmalemmae of sperms percentage of head rice and acrosomal integrity (CTC-Hoechst 33258 living body fluorescent dyestuff combined staining methods detect the perforatorium percentage of head rice) with the canine semen superfreeze.The result shows: the freeze thawing survival time of boar sperm is that 58 hours, freeze thawing sperm viability are 0.52, plasmalemmae of sperms percentage of head rice average out to 58% under 22 ℃ of conditions, and acrosomal integrity is 63%.
Embodiment 4:(anti-icing fluid 4)
Taking by weighing 2.18g fructose earlier is dissolved in the 50ml distilled water, add 1.14g citric acid, 1.65g tri methylol amino methane after treating to dissolve fully respectively, add one by one by said sequence, it is a kind of to treat to add after the reagent that adds earlier dissolves fully the back during interpolation again, dissolving and mixing is also put into 5 ℃ of refrigerators and is preserved, and takes out and be adjusted to the temperature that will preserve seminal fluid or artificial insemination's temperature during use.Take by weighing 0.05g N-acetyl group-D-gucosamine, be dissolved in the anti-icing fluid of adjusting temperature.Measure 20mL yolk, 4mL glycerine, 1.00mL amino-sodium-laurilsulfate (OEP) then respectively and the three is evenly mixed.Above-mentioned two kinds of preparations are mixed mutually, add 100,000 unit penicillin or gentamicins at last, last distilled water constant volume 100ml, the pH value is 6.75 behind the constant volume, osmotic pressure is 315mOsmo/L.All preparation process are all carried out under aseptic condition.The effect of anti-low temperature injury was splendid when this prescription was preserved the canine semen superfreeze.Mix mutually with canine semen in 1: 3 ratio during use, the temperature that will keep anti-icing fluid simultaneously with treat that the frozen semen temperature is identical.
Use the anti-icing fluid of pressing the preparation of embodiment 4 methods, when using, measure frozen sperm recovery back freeze thawing survival time of boar sperm, sperm viability (adopting phase contrast microscope to detect sperm viability), plasmalemmae of sperms percentage of head rice and acrosomal integrity (CTC-Hoechst 33258 living body fluorescent dyestuff combined staining methods detect the perforatorium percentage of head rice) with the canine semen superfreeze.The result shows: the freeze thawing survival time of boar sperm is 46 hours, preserves after 4 hours that the freeze thawing sperm viability is 0.18, plasmalemmae of sperms percentage of head rice average out to 27% under 39 ℃ of conditions, and acrosomal integrity is 46%; Utilizing this to freeze essence (back 0.5 hour of freezing recovery) artificial insemination, to breed the bitch pregnancy rate be 86.1%.
Claims (4)
1, a kind of dog-like sperm super-low temperature freezing preservation antifreeze solution; it is characterized in that by amino-sodium-0.05～2.50 milliliter of laurilsulfate, N-acetyl group-D-gucosamine 0.01～0.10 gram, fructose 0.45～3.10 gram, citric acid 0.50～2.30 gram, tri methylol amino methane 1.10～3.90 grams, 15.0～30.0 milliliters in yolk, glycerine or 2.0～8.0 milliliters of dimethyl sulfoxide (DMSO)s, bactericide 1000UI/ milliliter; adding distil water to 100 is milliliter formulated; the pH value of anti-icing fluid is 6.25～6.95, and osmotic pressure is 300～350mOsmo/L.
2, according to the dog-like sperm super-low temperature freezing preservation antifreeze solution of claim 1; it is characterized in that by amino-sodium-0.50 milliliter of laurilsulfate, N-acetyl group-D-gucosamine 0.06 gram, fructose 1.20 grams, citric acid 1.65 grams, tri methylol amino methane 3.15 grams, 20.0 milliliters in yolk, 3.0 milliliters of glycerine, bactericide 1000UI/ milliliter; adding distil water to 100 is milliliter formulated; the pH value of anti-icing fluid is 6.65, and osmotic pressure is 320mOsmo/L.
3, according to the dog-like sperm super-low temperature freezing preservation antifreeze solution of claim 1; it is characterized in that by amino-sodium-0.75 milliliter of laurilsulfate, N-acetyl group-D-gucosamine 0.05 gram, fructose 1.68 grams, citric acid 2.25 grams, tri methylol amino methane 1.85 grams, 20.0 milliliters in yolk, 4.0 milliliters of dimethyl sulfoxide (DMSO)s, bactericide 1000UI/ milliliter; adding distil water to 100 is milliliter formulated; the pH value of anti-icing fluid is 6.60, and osmotic pressure is 325mOsmo/L.
4,, it is characterized in that described bactericide is penicillin or gentamicin according to the dog-like sperm super-low temperature freezing preservation antifreeze solution of claim 1 or 2 or 3.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810163A (en) * | 2010-05-10 | 2010-08-25 | 广西大学 | Method for freezing semen of Macaca fascicularis |
| CN101843238A (en) * | 2010-06-12 | 2010-09-29 | 昆明亚灵生物科技有限公司 | Ultralow temperature cryopreservation method of macaca primate semen |
| CN103141471A (en) * | 2013-02-01 | 2013-06-12 | 四川农业大学 | Formula and preparation method of Kunming wolf dog semen refrigerating fluid |
| CN107494520A (en) * | 2017-09-25 | 2017-12-22 | 江苏农牧科技职业学院 | A kind of dog freezing of semen diluent and freezing method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010008764A1 (en) * | 1989-07-28 | 2001-07-19 | Roy Ax | Fertility-related proteins, and methods of use thereof |
| CN1232634C (en) * | 2004-04-22 | 2005-12-21 | 上海交通大学 | Diluent for sperm of blue fox |
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2005
- 2005-12-15 CN CNB2005101115200A patent/CN100387125C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810163A (en) * | 2010-05-10 | 2010-08-25 | 广西大学 | Method for freezing semen of Macaca fascicularis |
| CN101843238A (en) * | 2010-06-12 | 2010-09-29 | 昆明亚灵生物科技有限公司 | Ultralow temperature cryopreservation method of macaca primate semen |
| CN103141471A (en) * | 2013-02-01 | 2013-06-12 | 四川农业大学 | Formula and preparation method of Kunming wolf dog semen refrigerating fluid |
| CN103141471B (en) * | 2013-02-01 | 2015-09-30 | 四川农业大学 | A kind of Kunming hangquan semen freezing formula of liquid and compound method |
| CN107494520A (en) * | 2017-09-25 | 2017-12-22 | 江苏农牧科技职业学院 | A kind of dog freezing of semen diluent and freezing method |
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