CN103141471A - Formula and preparation method of Kunming wolf dog semen refrigerating fluid - Google Patents
Formula and preparation method of Kunming wolf dog semen refrigerating fluid Download PDFInfo
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- CN103141471A CN103141471A CN2013100398781A CN201310039878A CN103141471A CN 103141471 A CN103141471 A CN 103141471A CN 2013100398781 A CN2013100398781 A CN 2013100398781A CN 201310039878 A CN201310039878 A CN 201310039878A CN 103141471 A CN103141471 A CN 103141471A
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Abstract
The invention discloses a refrigerating fluid formula which can store Kunming wolf dog semen in the long time, the refrigerating fluid comprises, 2.4-2.46g of trimethylol aminomethane, 1.26-1.27g of sodium citrate, 1.50g of bi-ethoxyl-aminoethane sulfoacid, 4 ml of glycerol, 2.0-2.126g of glucose, 20ml of pigeon yolk, 100 thousand units of penicillin, 50 thousand units of streptomycin, and sterile double distilled water is added until the volume of the refrigerating fluid reaches 100ml. Aiming at further improving the method of semen refrigerating and improving refrigerated match conception rates, by the test of influence of sperm activity after thaw by diluent with different formulas and addition of different vitamins of different dosages, an excellent diluent is provided, and the purpose that the sperm survival rate of frozen semen is improved is achieved.
Description
Technical field
The invention belongs to semen freezing liquid technical field, relate in particular to a kind of Kunming hangquan semen freezing formula of liquid and compound method.
Background technology
Dog is the domestic animal of being tamed by the mankind the earliest, and what quantity was maximum at present is also canid, is the domestic animal that appearance difference is maximum in the world, distribution is the widest and kind is maximum.Artificial insemination (AI) is most important to the development of dog industry, and the popularization of technology of artificial insemination depends on the long preservation of seminal fluid.Compare with other animals (as ox, pig), the dog artificial insemination speed of development is slow, and its main cause is that Semen freezing technique does not pass a test, and seminal fluid can not long preservation.
Summary of the invention
For the problems referred to above, the purpose of the embodiment of the present invention is to provide a kind of Kunming hangquan semen freezing formula of liquid and compound method.
The embodiment of the present invention is achieved in that a kind of Kunming hangquan semen freezing formula of liquid, and its constituent is:
Trishydroxymethylaminomethane 2.4～2.46g, sodium citrate 1.26～1.27g, double hydroxyethyl one aminoethane sulphonic acid 1.50g, glycerine 4ml, glucose 2.0～2.126g, pigeon yolk 20ml, add the sterilization distilled water to 100ml, penicillin 100,000 units, streptomycin 50,000 units.
Further, pH is between 6.5～6.8.
Further, the described Kunming of every 100ml hangquan semen freezing formula of liquid can add VB
1212ml, compound VB4ml, V
C0.50g.
Further, the component of compound VB is: every 2ml contains B
120mg, B
22mg, B
62mg, vitamin PP 30mg.
Further, the addition of compound VB is 4mL/100mL, V
CAddition be 0.50g.
For further improving the dog sperm freezing method, improve to freeze and join conception rate, the present invention by on the dilution of different formulations, add the different vitamins of various dose to the test of the rear sperm viability impact of thawing, a kind of good dilution is provided, reach the purpose that improves the frozen semen sperm survival rate, for the quality that further improves frozen semen in the middle of producing provides certain reference.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.
The embodiment of the present invention provides a kind of Kunming hangquan semen freezing formula of liquid and preparation technique, and its constituent is: trishydroxymethylaminomethane 2.4～2.46g, sodium citrate 1.26～1.27g, double hydroxyethyl one aminoethane sulphonic acid 1.50g, glycerine 4ml, glucose 2.0～2.126g, yolk (pigeon) 20ml, add the sterilization distilled water to 100ml, penicillin 100,000 units, streptomycin 50,000 units.PH is between 6.5～6.8.Every 100ml adds VB
1212ml, compound V
B(every 2ml contains B120mg, B22mg, B62mg, vitamin PP 30mg) 4ml, V
C0.50g more help to protect sperm.
The present invention is further illustrated below in conjunction with concrete result of the test.
1 test material
1.1 experimental animal: 5 of Kunming hangquans, body weight 20～30kg, 2～8 years old age, health, body condition are good.
1.2 test reagent: trihydroxy methyl-aminomethane, two light ethyl-aminoethane sulphonic acid, glucose, lactose, fructose, citric acid, glycerine, mycillin, yolk, Ji's nurse Sa dye liquor, Yihong-nigrosine be dyeing liquor, Cobastab anyway
12, vitamin C, vitamin B compound etc.
1.3 test apparatus: electric light biomicroscope, cell counter, insulating box, centrifuge, liquid nitrogen container, pH test paper etc.
1.4 test site: Sichuan Agricultural University's animal medicine institute embryo engineering base, cow disease recovery centre.
1.5 experimental animal is processed: test and carry out canine distemper, parvovirus vaccine immunity front 2 weeks, the single cage of adaptability is raised, be accompanied by with dog grain mainly that a small amount of raw meat and ox are assorted waits timing, quantitatively, fed by fixing poultry raiser's routine, freely drink water, after semen collection, every dog benefit is eaten two pieces of raw eggs.
2 test methods
2.1 the collection of seminal fluid
Hold the dog penis with thumb and forefinger, gently draw foreskin, be held in bulbus glandis in palm and give suitable pressure, treat that penis fully erects, collect seminal fluid with the collection sperm funnel.
2.2 the routine inspection of semen quality
2.2.1 the smell of seminal fluid and the inspection of color and luster: free from extraneous odour, milky or light oyster white person are normal semen; Otherwise it is undesired to be.
2.2.2 the pH value of seminal fluid checks: get a droplet seminal fluid and with pH value be 5.0～8.4 precision test paper mensuration.
2.2.3 the inspection of sperm viability: seminal fluid is placed in the microscopically of 37 ℃, amplifies 400～600 times of microscopies, make raw score with 10 grades of point systems.Further with the decoration method detection anyway of Yihong one nigrosine (sperm of living is not painted).
The motion number of sperm of sperm motility rate=linearly/total number of sperm * 100%.
2.2.4 the mensuration of sperm concentration (hemacytometry): hemacytometry, range estimation vigor under 400 power microscopes is used the blood cell counting plate bulk density with seminal fluid after with 20 times of dilutions of physiological saline.
2.2.5 acrosomal integrity: with Ji's nurse Sa decoration method, drip compressed tablets, detect (sperm of living is not painted) under 1000 power microscopes.
Sperm acrasomal integvity=Complete acrosomes type number of sperm/sperm sum * 100%.
1.4.5 the inspection of rate of teratosperm: with Ji's nurse Sa decoration method, every sample is made two smears.At each several 100 sperms of about every two different parts, record the teratosperm number at each position with hemacytometer; The average teratosperm number of two smears of every sample represents the teratosperm number of this seminal fluid sample.
Abnormal rate=(sheet 1 left and right deformity sum+sheet 2 left and right sections of section are lopsided total)/400 * 100%.
2.3 the dilution of seminal fluid
Adopt a step dilution method that seminal fluid is diluted according to factors such as the vigor of former sperm, density.Before dilution, first dilution is preheating to 38 ℃ of left and right.Generally seminal fluid density is remained on 1.0 * 10 in test
8About/mL should be not too high, the detection of the rear sperm viability that is conducive to like this to thaw.Dilution is now with the current, shakes up the centrifugal 5min of 1500r/min after preparing.
On the basis of a large amount of preliminary experimenies, filter out effect dilution I, II, III preferably, its formula sees Table 1.
Table 1 dilution formula
2.4 the cooling of seminal fluid and balance:
The seminal fluid that dilution is good is wrapped 8～16 layers of gauze, transfer to the balance 3.5～4.0h that lowers the temperature in the isoperibol of 4 ℃.
2.5 the dog particle freezes refining the work
Change system into fried smoked box with foamed plastics, in establish two Room: one puts the copper gauze on indoor, and lower Sheng liquid nitrogen, and establish a lid is " smoked chamber "; Another is indoor can directly hold liquid nitrogen, is " fried chamber ".Seminal fluid after balance is directly dripped in fried indoor liquid nitrogen, and standing 5～8s after dripping adds a cover stifling 6min in moving into smoked chamber, collect pack, immerses liquid nitrogen.
2.6 thawing of seminal fluid
Test tube is preheating to 35 ℃～40 ℃, takes out the particle seminal fluid in the liquid nitrogen container and drop in vitro rapidly, shake gently, be melted at 2/3 o'clock until particle, namely take out from water-bath, continue to shake and make its whole thawings.
3 result of the tests
3.1 the impact of different diluent type on thaw rear Sperm acrasomal integvity, motility rate and abnormal rate
Shown in table 2, three kinds of dilutions of I, II, III are made, and to freeze the essence rear motility rate that thaws all the highest greater than the rear average motility rate of freezing of 30.00%, I liquid, but I and II liquid difference not significantly (P＞0.05), all with III liquid significant difference (P＜0.05); Acrosomal integrity and abnormal rate I liquid are better than II, III liquid, and significant difference (P＜0.05), II liquid and III liquid difference be remarkable (P＞0.05) not.
The impact of table 2 different diluent type on thaw rear Sperm acrasomal integvity, motility rate and abnormal rate
Annotate: subscript letter identical table differential different not significantly (P＞0.05) in same column, subscript letter neighboar lists differential different significantly (P＜0.05), the alternate expression difference of subscript letter be (P＜0.01) (lower with) extremely significantly; Below testing and all selecting prescription I is square foundation.
3.2 add the Cobastab of various dose
12Impact on thaw rear Sperm acrasomal integvity, motility rate and abnormal rate
Table 3 as can be known, along with Cobastab
12The increase of amount, the motility rate of the rear sperm that thaws, Sperm acrasomal integvity change little, and difference is not significantly (P＞0.05) all, and abnormal rate is lower with 12mL group and 16mL group, organizes significant difference (P＜0.05) with other.
Table 3 adds the Cobastab of various dose
12Impact on thaw rear Sperm acrasomal integvity, motility rate and abnormal rate
3.3 add the vitamin B compound of various dose to the impact of thaw rear Sperm acrasomal integvity, motility rate and abnormal rate
(every 2mL contains B120mg to vitamin B compound, B22mg, B62mg, vitamin PP 30mg) interpolation such as table 4 are as can be known, the motility rate of 4mL/100mL group is on average higher than other groups, with control group and 6mL/100mL, 8mL/100mL significant difference (P＜0.05), acrosomal integrity is similar to the result of sperm motility rate, and rate of teratosperm is the highest with the 8mL/100mL group, with other group significant differences (P＜0.05), 4mL/100mL, 2mL/100mL group is minimum in 23.58～24.15 scopes, with other group significant differences (P＜0.05).
Table 4 adds the vitamin B compound of various dose to the impact of thaw rear Sperm acrasomal integvity, motility rate and abnormal rate
3.4 add the vitamin C of various dose to the impact of thaw rear Sperm acrasomal integvity, motility rate and abnormal rate
Different vitamin C (0.25g/2mL) concentration amounts are added the average motility rate 0.50g/100mL group of sperm higher than other groups (seeing Table 5) and 1.00g/100mL significant difference (P＜0.05), with other interpolation groups and control group difference not significantly (P＞0.05).Acrosomal integrity is the highest with 0.50g/100mL, 0.75g/100mL group, is respectively 74.58 ± 1.59,74.39 ± 1.78, and two group differences are remarkable (P＞0.05) not, both all organizes significant difference (P＜0.05) with other.Rate of teratosperm is the highest by 31.81 ± 2.30 with control group, and after adding vitamin C, abnormal rate decreases, and wherein the 0.75g/100mL group is minimum is 25.57 ± 2.49, with control group significant difference (P＜0.05).
Table 5 adds the vitamin C of various dose to the impact of thaw rear Sperm acrasomal integvity, motility rate and abnormal rate
3.5 dog frozen semen artificial insemination
Every bitch is oestrused and carry out the in utero semen deposition of 3 second sons from being accepted mating, uses homemade inseminating syringe, and each insemination dose is 2.5ml, every minor tick 24 hours.The artificial insemination result of the test is as shown in table 6, is 80.00% through bright smart conception rate after the semen deposition of uterus, and the frozen semen fertilization rate is 76.00%.
The bright essence of table 6 and frozen semen insemination result
4 analyze and discuss
4.1 select different freeze-extenders to play a part very crucial for the quality of dog frozen semen, 3 kinds of dilutions that evidence filters out all can keep the vigor of sperm preferably, motility rate after the thawing of semen that I, II, three groups of dilutions of III are made is all greater than 30.00%, the motility rate of dilution I is for being up to 43.35 ± 0.85,3 all reached can be used for AI freeze the essence minimum standard, indices after the sperm recovery is analyzed, illustrated that these 3 kinds of formulas all can use in the freezing production of dog sperm.Tris in these 3 kinds of dilutions and the mechanism of action of sodium citrate are the pH values of regulating seminal fluid, make the prolonged survival period of sperm.Protective effect to sperm is comparatively obvious.And the sperm viability between I, II group illustrates that without significant difference (P＞0.05) these two kinds of dilutions are basic identical to the protection effect of dog sperm.Wherein glucose, fructose, yolk etc. provide nutrition, the energy that is consumed to replenish sperm.Some molecular energy infiltration sperm membranes participate in sperm metabolism in cell, provide exogenous energy to sperm, slow down the consumption of endogenous material, thereby extend sperm in the time-to-live in the external world.On the basis of forefathers' research, this test has all added glycerine to preserve anti-low temperature strike material as ultralow temperature, and yolk and carbohydrate have also played this effect to a certain extent.Because seminal fluid and dilution are all nutritious materials, the twin control environments of microorganism such as bacterium, their existence may directly have influence on the motility rate of sperm with breeding in seminal fluid preservation process, so the interpolation of antibiotic is essential, generally usually add penicillin and streptomycin, can reach the effect of effective anti-negative bacterium and positive bacteria.
4.2 find obviously to improve the semen quality that freezes after essence is thawed by the interpolation of 3 kinds of vitamins, be beneficial to semen cryopreservation.The active chalcogen that the sperm respiratory metabolism produces, the unsaturated fatty acid on oxidable spermoblast plasma membrane makes plasma membrane that irreversible oxidative damage occur, and lost flowability makes the desmoenzyme disappearance, causes DNA damage.Cobalamin can make oxidized form (S-S-type) glutathione be reduced into reduced form (SH-type) as coenzyme, the glutathione energy reducing activity chalcogen of-SH-type, the oxidative damage of minimizing plasmalemmae of sperms, the integrality of protection plasmalemmae of sperms; As can be seen from the test results, the motility rate mean value of the rear sperm that thaws is all higher than control group, but difference all not significantly (P＞0.05), and along with the increase of cobalamin amount, motility rate changes little.Result of the test shows: the amount of adding vitamin B compound is that 4mL/100mL is more satisfactory, but its motility rate and control group difference not remarkable (P＞0.05), acrosomal integrity is the highest, and rate of teratosperm is minimum, with control group significant difference (P＜0.05).But can improve theoretical foundation that dog freezes rear sperm motility rate and unclear after the interpolation vitamin B compound, the further research that awaits of its complexity and lability; The active oxygen ion that vitamin C produces as antioxidant for clearing cell respiration on the one hand, thus the protection sperm is not subjected to poison due to the freezing free radical that produces; Vitamin C can promote to comprise that in cell, some other antioxidant substances of vitamin E generate on the other hand.This result of the test draws: the vitamin C sperm motility rate of 0.50g/100mL is the highest, but with control group difference not significantly (P＞0.05), acrosomal integrity is the highest, rate of teratosperm is low, with control group significant difference (P＜0.05), illustrate that adding in right amount vitamin C can protect perforatorium, reduces abnormal rate; And during excessive interpolation (1g/100mL), quality can reduce, and may be because vitamin C is acid antioxidant, and excessive interpolation can make that dilution pH value reduces, the osmotic pressure increase, and the acrosome of sperm and plasma membrane are sustained damage.
5 conclusions
5.1I, three kinds of dilutions of II, III make to freeze the essence rear motility rate that thaws all the highest greater than the rear average motility rate of freezing of 30.00%, I liquid, but I and II liquid, difference is significantly (P＞0.05) not, III liquid is minimum, significant difference (P＜0.05); Acrosomal integrity and abnormal rate I liquid all are better than II, III liquid, and significant difference (P＜0.05), II liquid and III liquid difference be remarkable (P＞0.05) not.The I dilution is best dilution in this test, and after its thawing of semen, motility rate can reach 43.35 ± 0.85.
5.2 add Cobastab in dilution
12, the motility rate of the rear sperm that thaws, Sperm acrasomal integvity are changed little, but mean value is all higher than control group, and along with Cobastab
12The increase of amount, rate of teratosperm have the reduction of continuation trend.
5.3 interpolation vitamin B compound, vitamin C all have protective effect preferably to Kunming hangquan sperm, but its addition is not The more the better, add vitamin B compound 4mL, vitamin C 0.50g with every 100ml liquid and are added to suitable.
The above is only preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., within all should being included in protection scope of the present invention.
Claims (4)
1. but the freezing liquid formula of long preservation Kunming hangquan seminal fluid, is characterized in that, its constituent is:
Trishydroxymethylaminomethane 2.4～2.46g, sodium citrate 1.26～1.27g, double hydroxyethyl one aminoethane sulphonic acid 1.50g, glycerine 4ml, glucose 2.0～2.126g, pigeon yolk 20ml, add the sterilization distilled water to 100ml, penicillin 100,000 units, streptomycin 50,000 units.
2. hangquan semen freezing formula of liquid in Kunming as claimed in claim 1, is characterized in that, pH is between 6.5～6.8.
3. hangquan semen freezing formula of liquid in Kunming as claimed in claim 1, is characterized in that, every 100ml described Kunming hangquan semen freezing liquid adds VB
1212ml, compound VB4ml, V
C0.50g can better improve preservation effect.
4. hangquan semen freezing formula of liquid in Kunming as claimed in claim 1, is characterized in that, the component of compound VB is: every 2ml contains B
120mg, B
22mg, B
62mg, vitamin PP 30mg.
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Cited By (7)
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| CN104621097A (en) * | 2015-01-29 | 2015-05-20 | 中国科学院东北地理与农业生态研究所 | Sheep fresh semen preserve diluent and application thereof |
| 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 |
| CN108293982A (en) * | 2018-04-10 | 2018-07-20 | 公安部南昌警犬基地 | A kind of preparation method and applications of dog sperm freezen protective dilution |
| CN108651440A (en) * | 2018-04-18 | 2018-10-16 | 中南大学 | A kind of supper-fast freezen protective system of human seminal fluid |
| CN114403131A (en) * | 2022-01-21 | 2022-04-29 | 河北科技师范学院 | Semen cryopreservation liquid and application thereof |
| CN115735904A (en) * | 2022-11-30 | 2023-03-07 | 海关总署北京缉私犬基地 | Sodium dodecyl benzene sulfonate-containing frozen semen diluent for dogs as well as use method and application thereof |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103558200A (en) * | 2013-07-30 | 2014-02-05 | 广西壮族自治区水产科学研究院 | Evaluation method for survival rate and quality of prawn sperms |
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| 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 |
| CN108293982A (en) * | 2018-04-10 | 2018-07-20 | 公安部南昌警犬基地 | A kind of preparation method and applications of dog sperm freezen protective dilution |
| CN108651440A (en) * | 2018-04-18 | 2018-10-16 | 中南大学 | A kind of supper-fast freezen protective system of human seminal fluid |
| CN114403131A (en) * | 2022-01-21 | 2022-04-29 | 河北科技师范学院 | Semen cryopreservation liquid and application thereof |
| CN114403131B (en) * | 2022-01-21 | 2023-02-24 | 河北科技师范学院 | Semen cryopreservation liquid and application thereof |
| CN115735904A (en) * | 2022-11-30 | 2023-03-07 | 海关总署北京缉私犬基地 | Sodium dodecyl benzene sulfonate-containing frozen semen diluent for dogs as well as use method and application thereof |
| CN115735904B (en) * | 2022-11-30 | 2024-02-02 | 海关总署北京缉私犬基地 | Sodium dodecyl benzene sulfonate-containing frozen semen diluent for dogs as well as application method and application thereof |
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