CN101595866B - Culture-medium of pig semen and method for processing pig semen - Google Patents
Culture-medium of pig semen and method for processing pig semen Download PDFInfo
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Abstract
The invention discloses a culture-medium of pig semen. Per 100ml of the culture-medium contains the following components: 5-20 g of lactose, 0.005-0.05 g of N-acetyl-D-glucosamine, 0.05-0.5 g of DHA, 1.0-5.0 ml of glycerine, 0.2-1.5 ml of OEP, 10-40 ml of yolk and 50,000-200,000 IU of penicillin and/or streptomycin. The invention also discloses a method for processing the pig semen. Proved by detection, the activity of the pig semen processed by the method reaches 0.3-0.6, and the acrosome integrity exceeds 50 percent, thereby completely conforming to the requirement of artificial fertilization at present.
Description
Technical field
The present invention relates to biotechnology, relate in particular to the processing method of pig semen and pig seminal fluid.
Background technology
Semen freezing technique all has extremely important using value for the preservation of the genetic potential of giving full play to good boar and local pig breed genetic resources.The Semen freezing technique research of pig starts from 1956, and the reported first such as Polge are used the pig frozen semen and obtained farrowing.But in decades, compare with domestic animals such as ox, sheep, the frozen semen of pig is used and is gone back limited, and partly cause is because the pig thawn motility is low, conception rate is low, the brighter defective ejaculation of litter size; Partly cause is because the pig sperm volume is large, operation inconvenience in traditional straw (0.25/0.5ml) practical application; And the particle method is freezing, easily pollutes and is difficult for mark.
External herding researcher is exploring the pig high-capacity packaging always, as Semen freezing techniques such as 5ml plastic sack, bassoons (5ml), makes it to accomplish a defeated sow of straw seminal fluid as the domestic animals such as ox, sheep.But when using the bassoon method, also there is no providing of the dilution additive, sperm concentration of desirable pig seminal fluid, freezing and defreezing method etc. at present.
Therefore, this area is in the urgent need to from parameter testing sperm qualities such as sperm viability, motility rate, curved tail rate, acrosomal integrities, with the sperm quality after thawing, artificial insemination's result as evaluation index, filter out best boar semen method and program, thereby efficient, practical boar semen production technology is provided.
Summary of the invention
The present invention aims to provide the cryopreserving liquid of a boar seminal fluid.
Another object of the present invention is to provide a kind of purposes of cryopreserving liquid of pig seminal fluid described above.
A further object of the present invention is to provide the processing method of a boar seminal fluid.
In a first aspect of the present invention, a boar semen is provided, contain in the every 100ml of described cryopreserving liquid:
(a) 5-20g lactose;
(b) 0.005-0.05g 2-acetylamino-2-deoxy-D-glucose;
(c) 0.05-0.5g DHA (Docosahexaenoic acid, DHA);
(d) 1.0-5.0ml glycerine;
(e) 0.2-1.5ml amino-sodium-dodecyl sulphate fat (Orvus Es Paste, OEP)
(f) 10-40ml yolk; With
(g) penicillin of 5-20 million international units and/or streptomycin.
In another preference, contain in the every 100ml of described cryopreserving liquid:
(a) 8-15g lactose;
(b) 0.008-0.04g 2-acetylamino-2-deoxy-D-glucose;
(c)0.08-0.4gDHA；
(d) 1.5-4.0ml glycerine;
(e)0.3-1.0ml OEP；
(f) 15-30ml yolk; With
(g) penicillin of 8-15 million international units and/or streptomycin.
In a second aspect of the present invention, a kind of purposes of cryopreserving liquid as above is provided, described cryopreserving liquid can be used for preserving the pig seminal fluid.
A third aspect of the present invention provides the processing method of a boar seminal fluid, and described method comprises step:
(1) pig seminal fluid and cryopreserving liquid as above are mixed, obtain containing the mixture of pig seminal fluid;
(2) mixture that step (1) is obtained adds the 5ml bassoon; With
(3) bassoon that the pig seminal fluid will be housed drops in liquid nitrogen frozen, obtains the pig seminal fluid of freezing preservation.
In another preference, in the mixture that step (1) obtains, the density of pig seminal fluid is 0.5-4 * 10
9Individual/ml; More preferably, density is 0.8-3.0 * 10
9Individual/ml.
In another preference, described step (1) comprises the following steps:
(1 ') is with pig seminal fluid and lactose, 2-acetylamino-2-deoxy-D-glucose, DHA, yolk and penicillin and/or streptomycin mixing, obtain containing the original mixture of pig seminal fluid, contain in every 100ml original mixture: the 1-8g lactose, 0.008-0.05g 2-acetylamino-2-deoxy-D-glucose, 0.05-0.6gDHA, 3-20ml yolk, the penicillin of 1.5-10 million international units and/or streptomycin; With
(1 ") obtains the mixture that contains the pig seminal fluid described in above-mentioned step (1) with resulting original mixture and lactose, glycerine, OEP, yolk and penicillin and/or the streptomycin mixing that contains the pig seminal fluid of step (1 ').
In another preference, described step (1 ') is at room temperature carried out; Described step (carry out at 2-8 ℃ by 1 "); More preferably, step (carry out at 3-6 ℃ by 1 ").
In another preference, contain penicillin and/or the streptomycin of 2-16g lactose, 0.004-0.03g 2-acetylamino-2-deoxy-D-glucose, 0.03-0.3gDHA, 0.5-4ml glycerine, 0.125-1.0ml OEP, 5-30ml yolk and 2-10 million international units in the every 100ml of the mixture that contains the pig seminal fluid that obtains in step (1).
In another preference, contain penicillin and/or the streptomycin of 3.5-12g lactose, 0.006-0.02g 2-acetylamino-2-deoxy-D-glucose, 0.06-0.25gDHA, 0.8-3ml glycerine, 0.18-0.7ml OEP, 8-20ml yolk and 3-8.5 million international units in the every 100ml of the mixture that contains the pig seminal fluid that obtains in step (1); The penicillin and/or the streptomycin that contain 5.5-8.5g lactose, 0.008-0.013g 2-acetylamino-2-deoxy-D-glucose, 0.10-0.15gDHA, 1.0-2.0ml glycerine, 0.25-0.5ml OEP, 10-15ml yolk and 5-7.5 million international units in the every 100ml of the mixture that contains the pig seminal fluid that obtains in preferred steps (1).
In another preference, described method also comprises step:
The pig seminal fluid of the freezing preservation that (4) step (3) is obtained and thawing solution mix 10-100 second at 25-70 ℃; Contain 2.0-8.0g glucose, 0.05-0.25gEDTA, 0.3-1.2g trisodium citrate, 0.05-0.25g sodium bicarbonate, 0.03-0.20g potassium chloride in described every 100ml thawing solution; Preferably mix 25-80 second at 37-60 ℃.
Accordingly, the invention provides best boar semen method and program, thereby efficient, practical boar semen production technology is provided.
Description of drawings
Fig. 1 has shown the bassoon refrigerating process.
Fig. 2 has shown the main utensil that bassoon is freezing.
Fig. 3 has shown the different thaw points rear sperm quality change curve that thaws.
Embodiment
The inventor has been surprised to find that the freezing preservation liquid of a boar seminal fluid through extensive and deep research, and it can keep vigor, motility rate, membrane integrity and the acrosomal integnity of sperm in the pig seminal fluid for a long time and effectively.In addition, the inventor finds to use bassoon to carry out freezing preservation to the pig seminal fluid of above-mentioned processing, can overcome the conventional granulates method and easily pollute the shortcoming that is difficult for mark.Further, the inventor's impact of temperature and time on above-mentioned thawn motility etc. of also having found to thaw.
Particularly, the inventor carries out frozen in bassoon after finding and to contain the solution mixing of DHA with the pig seminal fluid.Preferably, with the pig seminal fluid by dilute twice after resulting mixed solution carry out frozenly, contain DHA in described mixed solution.
As used herein, " DHA (Docosahexaenoic acid, DHA contain 6 unsaturated bonds) " are a kind of omega-fatty acids, so-called omega-fatty acid refers in fatty acid molecule is closed in many insatiable hungers, apart from two keys of carboxyl distal-most end on the 3rd carbon atom reciprocal.DHA in the present invention can be obtained by mode well-known to those skilled in the art, such as by chemosynthesis or extract from marine organisms (as ocean fish) etc.Can contain other the material that belongs to omega-fatty acid on a small quantity in DHA used in the present invention, such as but not limited to eicosapentaenoic acid (Eicosapen taenoic acid, EPA, contain 5 unsaturated bonds), the content of described a small amount of material is 0-10wt%, being preferably 0-5wt%, is more preferably 0-2wt%.
As used herein, " bassoon " refers to the placement pig seminal fluid that uses and the tubular container of relevant treatment solution in freezing pig jism, and container material can be well-known to those skilled in the art; Its capacity can be 2-10ml, is preferably 4-6ml.In a preference of the present invention, described bassoon is well known in the art can the acquisition by any commercial channel, produces such as but not limited to German Mi Nitu company.
As used herein, " yolk " refers to egg yolk.
As used herein, " room temperature " and " normal temperature " is used interchangeably, and all refers to 10-30 ℃, preferred 15-25 ℃.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can any combination.All features that this case specification discloses can with any composition forms and use, each feature that discloses in specification can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore except special instruction is arranged, the feature that discloses is only the general example of equalization or similar features.
Major advantage of the present invention is:
1, pig semen provided by the invention can make frozen pig seminal fluid maintain vigour;
2, the seminal fluid after utilizing the present invention freezing reaches 0.3-0.6 through the vigor that detects its sperm, and Sperm acrasomal integvity surpasses 50%, meets present test-tube requirement fully.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage and umber by weight.
Unless otherwise defined, all specialties of using in literary composition are identical with the meaning that scientific words and one skilled in the art are familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
Sperm viability detection method, sperm curved tail rate detection method and Sperm acrasomal integvity detection method can be consulted " animal thremmatology ", Jiangsu science tech publishing house, Wang Yuanxing, Lang Jiejin chief editor, 1997,327-331 page.
Above-mentioned detection method is all to carry out after thawing.
The mensuration of sperm viability: adopt dull and stereotyped pressed disc method, get a seminal fluid (10 μ l) on the slide of preheating (38 ℃), covered, microscopy under 400 * phase contrast microscope, estimation vigor (ten grades of point systems).
The inspection of membrane integrity: adopt the hypotonic swelling method, get 50 μ l seminal fluid and add in 1ml hypotonic medium (37 ℃ of preheatings), 37 ℃ of waters bath with thermostatic control 30 minutes.Get (20 a μ l) mixed liquor on blood cell counting plate, observe 5 visuals field under 40 * phase contrast microscope, microscopy is counted 200 sperms, calculates the percentage of curved tail sperm.
The crooked number of sperm of curved tail rate=afterbody/total number of sperm * 100%
The inspection of acrosomal integnity: after Ji's nurse Sha dyeing, phase contrast microscope 1000 * 200 sperms of lower random observation calculate Acrosome abnormity rate.
Ji's nurse Sa stained preparation: 1. film-making: get 10 μ l seminal fluid in one of slide, with the neat slide in an edge be 35 ° of angles with the even mop of seminal fluid sample on slide, natural air drying is made semen smear.2. fixing: draw immediately 10% formalin fixer 1-2ml and drip on the smear of natural air drying, fixing 15min, water rinses gently, washes away fixer, natural air drying.3. dyeing: Ji's nurse Sa is dyed drop on smear, dyeing 1.5h, then water rinses gently, dries standby inspection.
The source of the main matter in the embodiment of the present invention is:
| Material | Available from |
| Lactose | Shanghai permanent letter chemical reagent Co., Ltd |
| Glucose | Chemical Reagent Co., Ltd., Sinopharm Group |
| EDTA | Genview company product is available from Beijing ancient cooking vessel state biotechnology Co., Ltd |
| 2-acetylamino-2-deoxy-D-glucose | Sigma company product is available from Beijing ancient cooking vessel state biotechnology Co., Ltd |
| DHA | LiZhu Medicine Group Co., Ltd |
| Trisodium citrate | Shanghai reagent one factory |
| Sodium bicarbonate | Upper marine rainbow photoinitiator chemical factory |
| Potassium chloride | Shanghai examination one chemical reagent Co., Ltd |
| Glycerine | Sigma company product is available from Beijing ancient cooking vessel state biotechnology Co., Ltd |
| Orvus ES Paste | NOVA CHEMICAL SALES，INC. |
| Yolk | Chicken house under animal and veterinary research institute of Shanghai Academy of Agricultural Sciences |
| Penicillin | Shanghai Xinxianfeng Pharmaceutical Co., Ltd. |
| Streptomycin | Shanghai Xinxianfeng Pharmaceutical Co., Ltd. |
General operation in the embodiment of the present invention:
One, seminal fluid is prepared
Available from the Shanghai White pig on Nanhui District of Shanghai watt bits pig farm, 11 grow up, physical health, be highly sexed, the normal boar of semen collection frequency, semen collection in turn, semen collection is 23 times altogether;
Two, semen collection
The dense part of stage casing seminal fluid is collected in the hand grip semen collection, through four layers of filtered through gauze, removes jelly.Do dilution in 1: 1 with HARMONY dilution (available from Shanghai livestock technology Co., Ltd of the Eastern Jin Dynasty) under isothermy, be stored in vacuum flask, transport the laboratory back in 2 hours.
Three, seminal fluid pretreatment and quality inspection
Under room temperature (15-25 ℃) standing 2 hours, make seminal fluid be slow cooling to room temperature (20-23 ℃).Carry out the Ordinary fruit quality inspection under 37 ℃ with microscope.Select that free from extraneous odour, color and luster milky, sperm morphology are normal, vigor 0.7 or more, density be that the seminal fluid of " close " (every milliliter contains 200,000,000 sperms more than is " close ") supplies to try.
Four, the dilution of seminal fluid and balance
Under room temperature standing 2 hours, rocked gently sperm storing bottle every 0.5 hour.With large centrifuge tube (250ml) packing seminal fluid, centrifugal under room temperature, 1730 rev/mins, 10 minutes, abandon supernatant.Adopt double dilution method to dilute, first at room temperature use dilution I liquid (seeing Table 1) to do the dilution of certain volume ratio, then will dilute seminal fluid and dilution II liquid is put into 4 ℃ of refrigerators together, be slow cooling to 4 ℃ (approximately 2 hours).Make certain volume than dilution with dilution II liquid (seeing Table 1), obtain containing the mixture of pig seminal fluid.Check sperm viability, the vigor of getting directly carries out freezing at the seminal fluid more than 0.6.
The composition of table 1 dilution I liquid, dilution II liquid and thawing solution
| Composition | Dilution I liquid | Dilution II liquid | BTS (thawing solution) |
| Lactose (g) | √ | √ | |
| Glucose (g) | √ | ||
| EDTA(g) | √ | ||
| 2-acetylamino-2-deoxy-D-glucose (g) | √ | ||
| DHA(ml) | √ | ||
| Trisodium citrate (g) | √ | ||
| Sodium bicarbonate (g) | √ | ||
| Potassium chloride (g) | √ | ||
| Glycerine (ml) | √ | ||
| Orvus ES Paste(ml) | √ | ||
| Yolk (ml) | √ | √ | |
| Penicillin, streptomycin (IU) | √ | √ | |
Five, make frozen semen
The bassoon method: the 5ml bassoon is produced by German Mi Nitu company.First with an end closure of 5ml bassoon, carry out mark with small ball on tube wall, then in 4 ℃ of precoolings 2 hours.Draw with the 20ml syringe seminal fluid that 5ml handles well, do not seal an end from bassoon and inject, stop injecting during one section blank pipe of surplus approximately 3cm, also with small ball with this end closure.Dry the globule of large tube outside, the inclination bassoon makes the air bubble section be positioned at the central authorities of bassoon.Bassoon is placed on the pipe support of abundant precooling in the wide-mouth refrigerated container, apart from the above 3-5cm of liquid nitrogen surface place (take 3cm as best), stifling 20 minutes, then drops in the liquid nitrogen container pail and preserve.See Fig. 1 and Fig. 2.
Particle method (contrast): the seminal fluid that balance is good, freezing with the aluminium lid.Inject 2/3 left and right liquid nitrogen in the wide-mouth refrigerated container (macrofoam polystyrene box), aluminium lunch box is flown on liquid nitrogen surface, then be placed on lunch box the aluminium lid is counter.Balance to be cooled approximately 5 minutes after the abundant precooling of freezer surface, is drawn the good seminal fluid of dilution with the 20ml syringe, drips continuously on the aluminium lid and freezes, and every drops amount is about 0.1ml, drips the time of freezing generally in 40-50 second, after dripping 30 left and right, treats that particle fully solidifies.Under the particle shovel, good gauze bag input liquid nitrogen is preserved to put into mark.
Six, the operation of thawing
Bassoon method: with tweezers, bassoon is taken out from liquid nitrogen container rapidly, place certain hour in the water-bath in uniform temperature.Approximately during 1/2-2/3, take out bassoon until seminal fluid dissolving, cut off two ends, seminal fluid is mixed with 37 ℃ of thawing solutions of preheating.
Particle method (contrast): wet freezing process, contain a certain amount of thawing solution with test tube, then 39 ℃ of water-baths, are frozen essence with long forceps with particle and are taken out from liquid nitrogen container rapidly, stop after 10 seconds in air and drop in thawing solution, shake fast test tube and make it to melt.Do freezing process, clean aluminium box in 39 ℃ of water-baths, is frozen essence with long forceps with particle and takes out from liquid nitrogen container rapidly, put into the aluminium box, after particle melts, mix with the equality of temperature thawing solution.
Seven, artificial insemination
Get the good seminal fluid that thaws, pour in the semen deposition bottle, more slowly pour synthermal room-temperature extender into, be diluted to 80ml, freeze essence thaw after each semen deposition dosage be 40ml, the number of sperm of living reaches more than 3,000,000,000.Accept with sow the estrus time that mounting begins to calculate the acceptor sow, semen deposition twice, semen deposition is first carried out in 12-14 hour after oestrusing, and is once defeated again after 12 hours.
Embodiment 1-4
The above-mentioned general operation step 1 to four of process obtains the mixed solution that contains the pig seminal fluid as the working concentration of each composition of table 2; Then carry out the bassoon method according to step 5 freezing.
Table 2
Effect example 1-4
The frozen semen that embodiment 1-4 is obtained thaws through the bassoon method of above-mentioned general operation step 6 after (45 ℃ 70 seconds), carries out the detection of sperm viability, curved tail rate and acrosomal integrity, result such as table 3.
Table 3
| Embodiment | Vigor | Motility rate (%) | Curved tail rate (%) | Acrosomal integrity (%) |
| |
0.38±0.07 a | 51.43±7.03 | 49.28±6.55 | 49.30±6.31 a |
| |
0.41±0.03 a | 50.07±3.27 | 46.64±6.96 | 50.78±9.85 a |
| |
0.41±0.03 b | 52.74±6.09 | 48.60±8.66 | 61.96±5.76 b |
| |
0.48±0.08 b | 47.68±8.18 | 52.08±8.68 | 59.47±7.53 b |
Annotate: the different expression of same column shoulder mark lowercase significant difference (P＜0.05), the different expression of capitalization difference is (P＜0.01) extremely significantly, and test repeats 10 times.Below each the table with.
Result shows, adds 0.15% in dilution I liquid, 0.3%DHA is on rear sperm viability and the acrosomal integnity impact significantly (P＞0.05) of thawing.
Embodiment 5-9
The above-mentioned general operation step 1 to four of process obtains the mixed solution that contains the pig seminal fluid as the working concentration of each composition of table 4; Then carry out the bassoon method according to step 5 freezing.
Table 4
Effect example 5-9
The frozen semen that embodiment 5-9 is obtained thaws through the bassoon method of above-mentioned general operation step 6 after (45 ℃ 70 seconds), carries out the detection of sperm viability, curved tail rate and acrosomal integrity, result such as table 5.
Table 5
| Embodiment | Vigor | Motility rate (%) | Curved tail rate (%) | Acrosomal integrity (%) |
| |
0.36±0.08 ab | 43.44±8.13 ab | 53.83±7.03 | 54.69±4.53 ab |
| |
0.42±0.04 a | 50.56±2.69 a | 52.73±7.38 | 62.72±7.64 a |
| |
0.41±0.06 a | 50.94±5.49 a | 53.61±7.40 | 61.69±7.81 a |
| |
0.36±0.03 ab | 44.21±1.54 ab | 44.75±3.92 | 48.25±6.53 b |
| |
0.32±0.03 b | 38.74±6.35 b | 46.23±4.23 | 47.36±6.92 b |
Result shows, in the dilution seminal fluid, effectively to protect plasmalemmae of sperms, sperm to freeze palintrope tail rate be 4%, 5% dilution seminal fluid higher than glycerol concentration to the glycerol concentration of 1%-3%, but difference not significantly (P＞0.05).2%, 3% glycerol concentration has protective effect preferably to perforatorium, and acrosomal integnity is significantly higher than 4%, 5% glycerol concentration (P＜0.05).
Embodiment 10-14
The above-mentioned general operation step 1 to four of process obtains the mixed solution that contains the pig seminal fluid as the working concentration of each composition of table 6; Then carry out the bassoon method according to step 5 freezing.
Table 6
Effect example 10-14
The frozen semen that embodiment 10-14 is obtained thaws through the bassoon method of above-mentioned general operation step 6 after (45 ℃ 70 seconds), carries out the detection of sperm viability, curved tail rate and acrosomal integrity, result such as table 7.
Table 7
| Embodiment | Vigor | Motility rate (%) | Curved tail rate (%) | Acrosomal integrity (%) |
| |
0.42±0.04 | 51.59±8.87 | 50.85±2.97 | 57.88±9.92 b |
| |
0.43±0.04 | 48.68±6.94 | 53.30±7.47 | 61.58±6.63 ab |
| |
0.40±0.05 | 54.21±3.11 | 49.97±3.19 | 70.83±7.80 a |
| |
0.45±0.05 | 53.76±5.80 | 48.24±7.47 | 73.59±4.87 a |
| |
0.40±0.06 | 48.22±6.18 | 51.64±5.15 | 61.41±3.37 ab |
Result shows; in dilution, the 2-acetylamino-2-deoxy-D-glucose of interpolation various dose affects not significantly (P＞0.05) to vigor, motility rate and the curved tail rate of sperm; final concentration is that 0.05% and 0.1% group of 2-acetylamino-2-deoxy-D-glucose has protective effect to perforatorium, and acrosomal integrity is significantly higher than not interpolation group (P＜0.05).
Embodiment 15-19
The above-mentioned general operation step 1 to four of process obtains the mixed solution that contains the pig seminal fluid as the working concentration of each composition of table 8; Then carry out the bassoon method according to step 5 freezing.
Table 8
Effect example 15-19
The frozen semen that embodiment 15-19 is obtained thaws through the bassoon method of above-mentioned general operation step 6 after (45 ℃ 70 seconds), carries out the detection of sperm viability, curved tail rate and acrosomal integrity, result such as table 9.
Table 9
| Embodiment | Vigor | Curved tail rate (%) | Acrosomal integrity (%) |
| |
0.03±0.06 c | 8.31±1.81 c | 59.92±2.94 a |
| Embodiment 16 | 0.40±0.10 a | 28.25±5.67 a | 55.47±5.28 a |
| Embodiment 17 | 0.41±0.05 a | 19.81±4.62 b | 47.14±7.69 ab |
| Embodiment 18 | 0.28±0.05 b | 11.83±2.17 c | 19.97±11.85 b |
| |
0.30±0.10a b | 13.74±8.03 bc | 32.70±12.49 b |
Result shows, the yolk final concentration that adds in frozen solution is respectively 5%, 10%, 20%, 25% and 30%.As shown in Table 3,10% and 20% yolk can effectively be protected sperm viability, plasma membrane and acrosome, and being significantly higher than yolk concentration is 25% and 30% group (P＜0.05).
Embodiment 20-22
The above-mentioned general operation step 1 to four of process obtains the mixed solution that contains the pig seminal fluid as the working concentration of each composition of table 10; Then carry out the bassoon method according to step 5 freezing.
Table 10
Effect example 20-22
The frozen semen that embodiment 20-22 is obtained thaws through the bassoon method of above-mentioned general operation step 6 after (45 ℃ 70 seconds), carries out the detection of sperm viability, curved tail rate and acrosomal integrity, result such as table 11.
Table 11
| Embodiment | Vigor | Motility rate (%) | Curved tail rate (%) | Acrosomal integrity (%) |
| |
0.44±0.05 | 55.35±4.95 | 53.61±4.11 | 62.84±9.04 |
| Embodiment 21 | 0.41±0.05 | 52.14±7.51 | 57.91±3.74 | 67.92±9.84 |
| Embodiment 22 | 0.41±0.05 | 54.18±8.59 | 49.75±7.04 | 60.02±8.01 |
Result shows, sperm concentration is 1.0 * 10
9Individual/during ml, thaw rear sperm viability and motility rate are the highest, but compare difference not significantly (P＞0.05) with other two groups.Sperm concentration is 1.5 * 10
9Individual/during ml, the curved tail rate of the rear sperm that thaws and acrosomal integrity are the highest, compare difference also not significantly (P＞0.05) with other two groups, and under three density, freezing pig sperm difference on effect is not remarkable, and it is feasible carrying out freezing with the high density sperm in this scope.
Embodiment 23
The above-mentioned general operation step 1 to four of process obtains the mixed solution that contains the pig seminal fluid as the working concentration of each composition of table 12; Then carry out the bassoon method according to step 5 freezing.
Table 12
Frozen semen obtained above is thawed through the bassoon method of above-mentioned general operation step 6, but thawing condition is as shown in table 13, then carries out the detection of sperm viability, curved tail rate and acrosomal integrity, result such as table 13 and Fig. 3.
Table 13
| Thaw point (℃) and thawing time (s) | Melt rear temperature (℃) | Vigor | Motility rate (%) | Curved tail rate (%) | Acrosomal integrity (%) |
| 37℃ 30s | 8.6 | 0.23±0.05 De | 33.43± 7.55 Cd | 31.50± 8.34 Dd | 56.44± 8.65 ABab |
| 37℃ 60s | 23.4 | 0.34± 0.07 BCDcd | 43.76± 6.24 ABCbc | 40.64± 5.36 ABCDbcd | 58.90± 7.76 Aab |
| 37℃ 90s | 29.2 | 0.43± 0.07 ABabc | 52.54± 8.14 Aab | 48.03± 3.90 ABCab | 56.35± 3.60 ABab |
| 45℃ 50s | 22.3 | 0.29± 0.04 CDde | 39.10± 7.72 BCcd | 42.69± 8.60 ABCDbc | 58.63± 5.43 Aab |
| 45℃ 60s | 26.8 | 0.39± 0.03 ABCbc | 47.89± 2.43 ABabc | 51.36± 7.07 ABab | 62.51± 6.61 Aa |
| 45℃ 70s | 31.6 | 0.45± 0.04 ABab | 55.98± 4.69 Aa | 51.49± 2.79 ABab | 63.70± 5.70 Aa |
| 52℃ 25s | 8.9 | 0.39± 0.06 ABCbc | 48.65± 4.92 ABab | 51.27± 4.90 ABab | 65.40± 4.86 Aa |
| 52℃ 35s | 21.6 | 0.46± 0.04 ABab | 55.54± 3.68 Aa | 54.35± 8.14 Aa | 65.40± 4.86 Aa |
| 52℃ 45s | 32.4 | 0.45± | 46.82± | 51.49± | 55.40± |
| 0.09 ABab | 7.10 ABabc | 2.79 ABab | 9.51 ABab | ||
| 60℃ 20s | 11.0 | 0.43± 0.07 ABabc | 45.34± 4.29 ABCbc | 34.01± 7.73 CDcd | 56.61± 7.56 ABab |
| 60℃ 30s | 21.4 | 0.48±0.05 Aab | 47.58± 6.13 ABabc | 43.76± 6.24 ABCDabc | 48.76± 6.18 ABCbcd |
| 60℃ 40s | 27.1 | 0.49±0.05Aa | 53.21± 5.07 Aab | 53.86± 5.88 Aa | 53.71± 8.50 ABCab |
| 68℃ 20s | 14.2 | 0.49±0.07Aa | 52.07± 7.55 Aab | 35.77± 5.58 CDcd | 40.47± 5.45 BCd |
| 68℃ 30s | 24.3 | 0.46± 0.03 ABab | 47.89± 2.43 ABabc | 37.22± 7.83 BCDcd | 41.23± 9.25 BCcd |
| 68℃ 40s | 30.8 | 0.47±0.07 Aab | 51.21± 4.06 ABab | 33.86± 6.51 CDcd | 37.45± 9.16 Cd |
Result shows, 45 ℃, 70s, 52 ℃, 35s are that 5ml bassoon defrost result makes up preferably.
Embodiment 24
The above-mentioned general operation step 1 to four of process obtains the mixed solution that contains the pig seminal fluid as the working concentration of each composition of table 14; Then carry out the bassoon method according to step 5 freezing.
Table 14
Frozen semen obtained above is thawed after (45 ℃ 70 seconds) through the bassoon method of above-mentioned general operation step 6, seminal fluid reaches 0.3-0.6 through the vigor that detects its sperm, Sperm acrasomal integvity surpasses 50%, meet present test-tube requirement fully, after the artificial insemination of carrying out above-mentioned general operation step 7, effect shows below:
The Shanghai White pig: the seminal fluid of using the said procedure preparation carries out the artificial insemination, altogether 18 sows of semen deposition.13 sow gestation farrowing (conception rate 72.2%) as a result, 7.8 of average every nest farrowing, 2 of minimum products, 12 of maximum products.
Small-sized plum mountain pig: 22 sows of semen deposition.18 sow gestation farrowing (conception rate 81.8%) as a result, 8.6 of average every nest farrowing, 4 of minimum products, 13 of maximum products.
The above is only preferred embodiment of the present invention, be not to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people complete, if defined identical with the claim scope of application, also or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.
Claims (14)
1. a boar semen, is characterized in that, contains in the every 100ml of described cryopreserving liquid:
(a) 5-20g lactose;
(b) 0.005-0.05g 2-acetylamino-2-deoxy-D-glucose;
(c) 0.05-0.5g DHA;
(d) 1.0-5.0ml glycerine;
(e) 0.2-1.5ml amino-sodium-laurilsulfate;
(f) 10-40ml yolk; With
(g) penicillin of 5-20 million international units and/or streptomycin.
2. cryopreserving liquid as claimed in claim 1, is characterized in that, contains in the every 100ml of described cryopreserving liquid:
(a) 8-15g lactose;
(b) 0.008-0.04g 2-acetylamino-2-deoxy-D-glucose;
(c) 0.08-0.4g DHA;
(d) 1.5-4.0ml glycerine;
(e) 0.3-1.0ml amino-sodium-laurilsulfate;
(f) 15-30ml yolk; With
(g) penicillin of 8-15 million international units and/or streptomycin.
3. the purposes of a cryopreserving liquid as claimed in claim 1, is characterized in that, described cryopreserving liquid is used for preserving the pig seminal fluid.
4. the processing method of a boar seminal fluid, is characterized in that, described method comprises step:
(1) pig seminal fluid and cryopreserving liquid as claimed in claim 1 are mixed, obtain containing the mixture of pig seminal fluid;
(2) mixture that step (1) is obtained adds the 5ml bassoon; With
(3) bassoon that the pig seminal fluid will be housed drops in liquid nitrogen frozen, obtains the pig seminal fluid of freezing preservation.
5. method as claimed in claim 4, is characterized in that, in the mixture that step (1) obtains, the density of pig sperm is 0.5-4 * 10
9Individual/ml.
6. method as claimed in claim 5, is characterized in that, in the mixture that step (1) obtains, the density of pig sperm is 0.8-3.0 * 10
9Individual/ml.
7. method as claimed in claim 4, is characterized in that, described step (1) comprises the following steps:
(1 ') is with pig seminal fluid and lactose, 2-acetylamino-2-deoxy-D-glucose, DHA, yolk and penicillin and/or streptomycin mixing, obtain containing the original mixture of pig seminal fluid, contain in every 100ml original mixture: the 1-8g lactose, 0.008-0.05g 2-acetylamino-2-deoxy-D-glucose, 0.05-0.6g DHA, 3-20ml yolk, the penicillin of 1.5-10 million international units and/or streptomycin; With
(1 ") obtains the mixture that contains the pig seminal fluid described in step as claimed in claim 4 (1) with resulting original mixture and lactose, glycerine, amino-sodium-laurilsulfate, yolk and penicillin and/or the streptomycin mixing that contains the pig seminal fluid of step (1 ').
8. method as claimed in claim 7, is characterized in that, described step (1 ') is at room temperature carried out; Described step (carry out at 2-8 ℃ by 1 ").
9. method as claimed in claim 8, is characterized in that, described step (1 ') is at room temperature carried out; Described step (carry out at 3-6 ℃ by 1 ").
10. method as claimed in claim 4, it is characterized in that, contain penicillin and/or the streptomycin of 2-16g lactose, 0.004-0.03gN-acetyl-D-aminoglucose, 0.03-0.3g DHA, 0.5-4ml glycerine, 0.125-1.0ml amino-sodium-laurilsulfate, 5-30ml yolk and 2-10 million international units in the every 100ml of the mixture that contains the pig seminal fluid that obtains in step (1).
11. method as claimed in claim 10, it is characterized in that, contain penicillin and/or the streptomycin of 3.5-12g lactose, 0.006-0.02g 2-acetylamino-2-deoxy-D-glucose, 0.06-0.25g DHA, 0.8-3ml glycerine, 0.18-0.7ml amino-sodium-laurilsulfate, 8-20ml yolk and 3-8.5 million international units in the every 100ml of the mixture that contains the pig seminal fluid that obtains in step (1).
12. method as claimed in claim 11, it is characterized in that, contain penicillin and/or the streptomycin of 5.5-8.5g lactose, 0.008-0.013g 2-acetylamino-2-deoxy-D-glucose, 0.10-0.15g DHA, 1.0-2.0ml glycerine, 0.25-0.5ml amino-sodium-laurilsulfate, 10-15ml yolk and 5-7.5 million international units in the every 100ml of the mixture that contains the pig seminal fluid that obtains in step (1).
13. method as claimed in claim 4 is characterized in that, described method also comprises step:
The pig seminal fluid of the freezing preservation that (4) step (3) is obtained and thawing solution mix 10-100 second at 25-70 ℃; Contain 2.0-8.0g glucose, 0.05-0.25gEDTA, 0.3-1.2g trisodium citrate, 0.05-0.25g sodium bicarbonate, 0.03-0.20g potassium chloride in described every 100ml thawing solution.
14. method as claimed in claim 13 is characterized in that, in step (4), the pig seminal fluid of the freezing preservation that step (3) is obtained and thawing solution mix 25-80 second at 37-60 ℃.
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| CN102599147A (en) * | 2012-03-03 | 2012-07-25 | 平顶山瑞祥牧业有限公司 | Diluting powder for artificial fertilization of pigs |
| CN103651332A (en) * | 2013-12-13 | 2014-03-26 | 广东省计划生育科学技术研究所 | Docosahexaenoic acid-glycine-glycerinum compound human seminal fluid cryoprotectant and preparation method thereof |
| WO2015193265A1 (en) | 2014-06-16 | 2015-12-23 | Università degli Studi di Parma | Composition for extenders for the long-term conservation of animal seminal material |
| CN105815307A (en) * | 2016-04-22 | 2016-08-03 | 李志新 | Boar semen storage method |
| CN105794770A (en) * | 2016-04-22 | 2016-07-27 | 李志新 | Special diluent for boar semen |
| CN109258626A (en) * | 2018-10-29 | 2019-01-25 | 北京田园奥瑞生物科技有限公司 | One boar freezes smart thawing solution and preparation method thereof and defreezing method |
| CN111436420A (en) * | 2020-04-14 | 2020-07-24 | 武汉市金驰海科技有限公司 | Long-acting fresh pig semen diluting preservative and using method thereof |
| CN111849872B (en) * | 2020-07-29 | 2022-01-11 | 吉林省农业科学院 | Method for improving in-vitro fertilization effect of thawed pig sperms for non-treatment infertility |
| CN113615682B (en) * | 2021-09-05 | 2022-08-26 | 内蒙古赛诺种羊科技有限公司 | Sheep hypertonic complexing agent semen dilution preserving fluid and application method thereof |
| CN114651815B (en) * | 2022-04-12 | 2023-05-09 | 江苏农牧科技职业学院 | Preservation method of frozen semen of pigs |
| CN115039766B (en) * | 2022-08-13 | 2022-11-18 | 中国农业科学院北京畜牧兽医研究所 | Normal-temperature boar semen diluent |
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| CN101062058A (en) * | 2007-06-05 | 2007-10-31 | 南京农业大学 | Tubule high-density type method for freezing pig jism and the products thereof |
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Non-Patent Citations (2)
| Title |
|---|
| A. Maldjian et al..Changes in sperm quality and lipid composition during cryopreservation of boar semen.《Theriogenology》.2005,第63卷411-421. * |
| 李林林.猪精液5ml细管冷冻保存技术研究.《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》.2006,(第12期),第13-34页. * |
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