CN102907417B - A Freezing Diluent Formula for Improving the Quality of Freezing Holstein Bull X Sperm - Google Patents
A Freezing Diluent Formula for Improving the Quality of Freezing Holstein Bull X Sperm Download PDFInfo
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Abstract
本发明公开了一种用于提高荷斯坦公牛X精子冻后品质的冷冻稀释液，该冷冻稀释液每100毫升中含有以下物质：柠檬酸钠：0.9～1.2毫克，蔗糖：3～6毫克，低密度脂蛋白：16～18毫克，甘油：4～7毫升，维生素E：500毫克～700毫克，维生素B12：100毫克～200毫克，壮观霉素：0.35毫克，庆大霉素：7～8万单位（IU），蒸馏水补足100毫升。实验数据表明，该冷冻稀释液提高了X精子冻融后的活力和精子顶体完整率，提高了精子冻后品质。The invention discloses a frozen diluent for improving the frozen quality of Holstein bull X sperm. The frozen diluent contains the following substances per 100 ml: sodium citrate: 0.9-1.2 mg, sucrose: 3-6 mg, Low-density lipoprotein: 16-18 mg, glycerin: 4-7 ml, vitamin E: 500 mg-700 mg, vitamin B12 : 100 mg-200 mg, spectinomycin: 0.35 mg, gentamicin: 7- 80,000 units (IU), make up 100 ml with distilled water. Experimental data showed that the freezing diluent improved the viability of X sperm after freezing and thawing and the integrity rate of sperm acrosome, and improved the quality of sperm after freezing.
Description
Technical field
The present invention relates to the test-tube improving technology of a kind of milk cow, relate in particular to and a kind ofly for improving He Sitan bull X sperm, freeze the freezing seminal fluid dilution formula of rear quality.
Background technology
The freezing of livestock semen successfully produced extremely far-reaching influence for the development of people's work insemination techniques, first for expanding availability and the prolongation of outstanding sire, utilize the time to lay a good foundation, secondly the development for artificial insemination and cryobiology provides wide space.Because frozen semen is more convenient in transportation, no matter from the time or from space, all make the breeding efficiency of sire be not fully exerted, accelerated the paces of domestic animal breed improvement.But, the animal of different sexes can meet the needs of people's different aspect, so people need to go to intervene by various ways the normal reproductive process of animal for self, the jenny that makes to grow up is produced the sex offspring that people expect, thereby the gender differences of animal directly affect its market value.Therefore, Sex Control is significant in livestock breeding is produced.In the production of milk cow, realized the separated of X sperm and y sperm at present, by artificial insemination, successfully realized the sex of He Sitanniu has been controlled.But the vigor after He Sitan obstinacy control thawing of semen now and fertilization rate are compared with bright essence, also have very big-difference, research and develop more effective obstinacy control semen cryoprotectant, raising property control sperm Frozen semen activity etc. has great importance.In the X of sexing semen, y sperm separation process, the aquatic foods essence obtaining after qualified after testing will pass through dyeing, separation, balance, centrifugal, XY sperm separation, dilution, packing, a series of processes such as freezing, these programs can cause certain damage to sperm, cause sperm death and vigor to reduce, have a strong impact on semen quality, finally affect fertilization rate, wherein the dilution formula of frozen semen is very important influence factor.Therefore, according to current trend, utilize laboratory condition to further investigate the sexing semen dilution formula of milk cow, the semen diluent that exploitation makes new advances, so both can improve survival rate and fertilization rate after the freeze thawing of He Sitanniu X sperm, accelerate again the improvement dynamics of China milk cow, for improving rapidly improvement and the milk yield of milk cow, increase the economic benefit of dairy, pull place and Regional Economic Development to there is positive effect.
Below the relevant references that applicant retrieves:
【1】Mustafa S, Eserlf D. The effect of ascorbic acid on the freezability of ram semen diluted with extenders containing different proportions of glycerol 【J】. Turkish Jourmal of Verterinary & animal Science, 2004, 28(5): 893-910。
【2】Singh B, Chand D.Siquality of deep frozen murrah buffalo bull (Bubalus bubalis) semen 【J】. Journal of Pairying Food & Home Sciences,1997,14(4):253-259。
【3】Dawson, E.B., Harris,W.A., Teter,M.C., Powell, L.C., 1992. Effect of ascorbic acid supplementation on the sperm quality of smokers. Fertil. Steril. 58,1034–1039。
【4】Partida S L G， Setchell B P，Maxwell W M. Epididymal compounds and antioxidants in diluents for the frozen storage of ram spermatozoa【J】.Reprod Fertil Dev，1997，9(7): 689-696。
【5】Foote R H，Brockett C C，Kaproth M T. Motility and fertility of bull sperm in whole milk extender containing antioxidants 【J】. Anim Reprod Sci，2002， 71(1-2):13-23。
【6】Jones R， Mann T. Damage to ram spermatozoa by peroxidantion of endogenous phospholipids【J】. J Reprod Fertil，1997，50(2): 261-26。
【7】Mustafa S， Esref D. The effect of ascorbic acid on the freezability of ram semen diluted with extenders containing different proportions of glycerol【J】. Turkish Journal of Veterinary & Animal Science，2004，28(5):893-910。
【8】Aitken R J, Clarkson J S. Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa 【J】. J Reprod Fertil, 1998, 81: 459-469.
【9】Marti a, L. Mara b, J.I. Marti c, T. Seasonal variations in antioxidant enzyme activity in ram seminal plasma E 【J】. Theriogenology 67 (2007) 1446–1454。
【10】 Medearos CMO, Forell F, Oliveira ATD, et al. Current status of sperm crypereseration: Why isn’t it better 【J】. Theriogenology, 2002，57：327～344。
【11】Ukeda H，Maeda S, Ishii T，Sawamura M. Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3’-{1-【(Phenylamino)-carbonyl】 -34- tetrazolium}-bis(4-methoxy-6-nitro)benzenesulphonic acid hydrate reduction by xanthinexanthine oxidase. Anal Biochem 1997；251： 206-209。
【12】Talevi R，Pelosi S，Sansone G，et al. Effects of different pre-freezing rates on buffalo sperm Motility and ultrastructure preservation 【J】.Buffalo. 1999（43）：537-539。
【13】 Nabil Mansour, Mary A. McNiven, Gavin F. Richardson. The effect of dietary supplementation with blueberry, a-tocopherol or astaxanthin on oxidative stability of Arctic char (Salvelinus alpinus) semen. Theriogenology 66 (2006) 373–382。
【14】Mariola S owinska, Halina Karol, Andrzej Ciereszko. Comet assay of fresh and cryopreserved bull spermatozoa. Cryobiology 56 (2008) 100–102。
【15】M. Moussa V. Martinet, A. Trimeche, D. Tainturier and M. Anton Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen–thawed bull semen Theriogenology 57 (2002) 1695–1706。
【16】Talevi R，Pelosi S，Sansone G，et al. Effects of different pre-freezing rates on buffalo sperm Motility and ultrastructure preservation 【J】.Buffalo. 1999（43）: 537-539。
【17】Partida SLG，Setchell BP，Maxwell WM. Epididymal compounds and antioxidants in diluents for the frozen storage of ram spermatozoa 【J】. Reprod Fertil Dev, 1997, 9(7): 689-696。
【18】B. A. Ball, V. Medina, C.G. Gravance, I. Baumber. Effect of antionxidants on presernation of motity viability and acrosomal integrity of equine spermatozoa during storage at 5 °C . Theriogenology，2001, 56: 577-569。
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[20] Ha Fu, Zhao Youzhang. in sheep freezing seminal fluid dilution, add vitamin B compound improve freeze essence thaw after the research [J] of quality. Gansu Agriculture University's journal, 2003,3 (1): 17-19.
[21] Luo Hailing, Jia Zhihai, Zhu Shien, etc. the impact of Vitamin E Supplementation in Diluent onFrozen-thawed Semen Quality of Sheep [J]. Chinese herding magazine, 2004,40 (11): 7-10.
[22] Li Xiaodong, Bian Fengyu, fourth can be etc. the experimental study of ox extender of freezing semen formula. Chinese milk cow, 1998(3): 15～17.
[23] Wang Xiaoke, Yang Jian. affect factor [J] Inner Mongol herding science of domestic animal frozen semen quality, 2002(6): 20～21.
[24] Wang Lihong, Wang Shouxun, Zhang Wei, the comparison of bull semen detection method, Chinese milk cow, 2002(3): 45～48.
[25] Zhang Zhongcheng. livestock thremmatology [M]. Beijing: Chinese agriculture publishing house, 2004.
[26] cow frozen semen quality of production administrative manual (trying). national livestock husbandry veterinarian general station, cow frozen semen quality supervision and test test center of the Ministry of Agriculture. 2000,21～23.
[27] Qiu Dianrui. affect cow frozen semen product qualitative factor [J]. Chinese herding magazine, 2002,35 (6): 49～50.
[28] Liu Yutang. the analysis of Influential Factors of semen cryopreservation [J]. Heilungkiang animal produces magazine, 2003,11 (2): 10～14.
Summary of the invention
For X sperm in refrigerating process because its processing mode is compared with common semen freezing pre-treatment mode, program very complicated, processing time is relatively long, in processing procedure, sperm lethality is higher, cause sperm viability low, the low defect of pregnancy rate after breeding, the object of the invention is to, and provides a kind of for improving the novel semen diluent formula of quality and fertilization rate after the freeze thawing of He Sitanniu X sperm freezing seminal fluid.This dilution can obviously improve separated later X sperm and freeze rear motility rate and acrosomal integrity and other indexs of correlation, thereby improves quality and fertilization rate after the freeze thawing of X sperm.
In order to realize above-mentioned task, the present invention takes following technical solution:
Improve He Sitan bull X sperm and freeze a chill back formula of liquid for rear quality, it is characterized in that, in every 100 milliliters of this freeze-extender of making, contain following material:
Sodium citrate: 0.9～1.2 milligram, sucrose: 3～6 milligrams, low-density lipoprotein: 16～18 milligrams, glycerine: 4～7 milliliters, vitamin E: 500 milligrams～700 milligrams, Cobastab
12: 100 milligrams～200 milligrams, spectinomycin: 0.35 milligram, gentamicin: 7～80,000 units (IU), distilled water is supplied 100 milliliters.
Preferred formula of the present invention, in 100 milliliters of freeze-extenders, each content of material is: sodium citrate: 0.9 milligram, sucrose: 3.3 milligrams, low-density lipoprotein: 16 milligrams, glycerine: 5 milliliters, vitamin E: 500 milligrams, Cobastab
12: 100 milligrams, spectinomycin: 0.35 milligram, gentamicin: 80,000 units (IU)
Above-mentioned compound method of freezing the freeze-extender of rear quality for improving He Sitan bull X sperm, is characterized in that, follows these steps to carry out:
1) accurately take sodium citrate, sucrose, low-density lipoprotein, vitamin E, the Cobastab of formula ratio
12and glycerine, adding distil water to 100 milliliter stirs and stirs for 60 minutes on magnetic stirring apparatus, then puts into 4 ℃ of refrigerator overnight;
2) before sperm dilution, add spectinomycin and gentamicin and stir 10 minutes, with acid or alkali, adjust the acid-base value of dilution, the pH that measures dilution with pH meter is between 6.5～7.5, then with 0.22 micron of filter, dilution is considered to bacterium and process, it is standby that worry bacterium is put into 37.5 ℃ of water-baths after processing.
The present invention is first vitamin E, Cobastab
12joined in freeze-extender, and the compound method of freeze-extender is revised, experimental data shows, of the present inventionly for improving He Sitan bull X sperm, freeze the freeze-extender of rear quality, improved vigor and Sperm acrasomal integvity after the freeze thawing of X sperm, improved sperm and frozen rear quality.
Embodiment
According to technical scheme of the present invention, the present embodiment provides and a kind ofly for improving He Sitan bull X sperm, freezes the freeze-extender of rear quality, contains following material in every 100 milliliters of this freeze-extender of making:
Sodium citrate: 0.9～1.2 milligram, sucrose: 3～6 milligrams, low-density lipoprotein: 16～18 milligrams, glycerine: 4～7 milliliters, vitamin E: 500 milligrams～700 milligrams, Cobastab
12: 100 milligrams～200 milligrams, spectinomycin: 0.35 milligram, gentamicin: 7～80,000 units (IU), distilled water is supplied 100 milliliters.
Its compound method is:
1, take raw material: sodium citrate, sucrose, low-density lipoprotein, glycerine, vitamin E, cobalamin, distilled water.
2, by above sodium citrate, sucrose, low-density lipoprotein, vitamin E, Cobastab
12, glycerine, is configured to basal liquid with distilled water, uses magnetic stirrer 60 minutes, spends the night standby in 4 ℃ of refrigerators.
3, before sperm dilution, add gentamicin, spectinomycin, then magnetic stirrer 10 minutes.
4, with acid or alkali, adjusting dilution pH value is 6.5～7.5, then with 0.22 micron filter, considers bacterium, puts into 37.5 ℃ of water-baths standby after worry bacterium.
During use, leaving in and be equipped with in the freeze-extender having prepared through pretreatment the XY sperm after flow cytometric sorting, balance checked sperm viability after five minutes, and vigor starts to make frozen semen according to common seminal fluid preparation method normally.And carry out relative recording.
In this test, after the sexing semen of measuring above-mentioned freeze-extender and conventional formulation thaws, in A level sperm percentage, B level sperm percentage, acrosomal integrity, average path speed, average move angle, curvilinear velocity, linearity, swing, survey amplitude of oscillation degree, point-to-point speed compares.Amplitude of oscillation degree is surveyed in A level sperm percentage, B level sperm percentage, acrosomal integrity, average path speed, average move angle, curvilinear velocity, linearity, swing, and point-to-point speed adopts sperm automatic analyzer to carry out automatic analysis.Acrosomal integrity and detection method are as follows:
(1) after semen thawing, at the temperature of 35 ℃～37 ℃, carry out motility rate inspection immediately.During evaluation, with glass bar, dip 1 seminal fluid, drip on slide, add cover glass, make to be full of seminal fluid between cover glass and slide, be then placed under 400 times～600 power microscopes and observe.In field of microscope, by seminal fluid upper, middle and lower-ranking liquid level sperm motility information summary is observed, evaluation sperm motility rate.
(2) get a seminal fluid after thawing and drop on clean slide, with another slide, be 45 degree, seminal fluid is smeared to opposite side, make smear.After natural air drying, with 1～2 milliliter of formalin phosphate buffer, fix 15 minutes, after washing and drying, with Ji's nurse Sa, dye 90 minutes, then remove the dye liquor in smear, air-dry 500 sperms of 1000 times of biology microscope Microscopic observations that are placed on, record sperm sum and Complete acrosomes number of sperm, finally obtain acrosomal integrity.
Be below the concrete experimental example that inventor provides, the adult Holstein bull that physique is healthy and strong, be highly sexed is selected in test, by stock bull feeding and management requirement, raises, and its semen collection frequency is 3 times weekly.Utilize artificial vagina acquisition method to gather bull semen, after semen collection, microscopy at 35 ℃～37 ℃ immediately.The seminal fluid gathering is milky or milk yellow, free from extraneous odour, and sperm morphology is normal, and motility rate reaches more than 0.7 can be for making frozen semen.
Test is provided with three different formulations according to the technical scheme of not applying for, and compares with conventional formulation.
Formula 1: prepare 100 milliliters of freeze-extenders: 0.9 milligram of sodium citrate, 3.3 milligrams of sucrose, 16 milligrams of low-density lipoproteins, 5 milliliters of glycerine, 500 milligrams of vitamin Es, Cobastab
12100 milligrams, 0.35 milligram of spectinomycin, gentamicin 80,000 units (IU).
Formula 2: prepare 100 milliliters of freeze-extenders: 1.0 milligrams of sodium citrates, 4.3 milligrams of sucrose, 17 milligrams of low-density lipoproteins, 6 milliliters of glycerine, 600 milligrams of vitamin Es, Cobastab
12150 milligrams, 0.35 milligram of spectinomycin, gentamicin 80,000 units (IU).
Formula 3: prepare 100 milliliters of freeze-extenders: 1.1 milligrams of sodium citrates, 5.3 milligrams of sucrose, 18 milligrams of low-density lipoproteins, 700 milligrams of vitamin Es, Cobastab
12200 milligrams; 7 milliliters of glycerine, 0.35 milligram of spectinomycin, gentamicin 80,000 units (IU).
Conventional formulation: prepare 100 milliliters of dilutions: 1.2 milligrams of sodium citrates, 6.3 milligrams of sucrose, 20 milliliters, yolk, 5 milliliters of glycerine, 0.35 milligram of spectinomycin, gentamicin 80,000 units (IU).
Strictly by above formula, take ingredient, adding distil water to 100 milliliter.On magnetic stirring apparatus, stir and stir for 60 minutes, then put into 4 ℃ of refrigerator overnight.
Before sperm dilution, add 0.35 milligram of spectinomycin, gentamicin 80,000 units (IU) also stir 10 minutes.With acid or alkali, adjust the acid-base value of dilution, with pH meter, measure dilution pH between 6.7～7.2.With 0.22 micron of filter, dilution is considered to bacterium and processes, after to put into 37.5 ℃ of water-baths standby.
Then according to sexing semen preparation method, make, after thawing, with sperm automatic analyzer, detect semen quality, specific targets see the following form:
Different formulations freezing seminal fluid dilution on freeze thawing after the impact (mean ± standard deviation) of X sperm quality
Note: the different sign significant difference of institute's marking-up parent phase (P<0.05) after colleague's data, institute's marking-up parent phase is with indicating difference not remarkable (P<0.05).
As can be seen from the above table, optimum formula is formula one.In this formula, A level sperm percentage is significantly higher than conventional formulation 16.12%, simultaneously higher than formula two, fill a prescription three; B level sperm percentage is higher than conventional formulation 16.98%, simultaneously higher than formula two, fill a prescription three.Acrosomal integrity is higher than conventional formulation group 6.24%; Simultaneously higher than formula two, fill a prescription three; Average path speed is significantly higher than conventional formulation 7.15%, simultaneously higher than formula two, fill a prescription three; Average move angle is significantly higher than conventional formulation 5.78%, simultaneously higher than formula two, fill a prescription three; Curvilinear velocity is significantly higher than conventional formulation 7.07% higher than traditional control group, simultaneously higher than formula two, fill a prescription three; Linearity, swing property are surveyed amplitude of oscillation degree, and point-to-point speed is more or less the same with control group.
So show that through above analysis formula one product mass-energy to sperm in sperm freezing course of defrosting plays maximum protection effect.
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