CN103704202A - Pony semen cryopreservation method - Google Patents
Pony semen cryopreservation method Download PDFInfo
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- CN103704202A CN103704202A CN201310674230.1A CN201310674230A CN103704202A CN 103704202 A CN103704202 A CN 103704202A CN 201310674230 A CN201310674230 A CN 201310674230A CN 103704202 A CN103704202 A CN 103704202A
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Abstract
The invention discloses a pony semen cryopreservation method. The method comprises the following steps: mixing a pony semen with an especially-developed cryopreservation diluent, carrying out programmed cooling of the above obtained mixture, and preserving the cooled mixture in liquid nitrogen. The cryopreservation diluent can effectively prolong the service life of the pony semen; and the programmed cooling effectively controls the cooling speed, avoids the damages of sperms, and improves the refrigeration effect and the quality of unfrozen sperms.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method that animal semen is preserved, relate in particular to a kind of method of pony semen cryopreservation.
Background technology
Semen cryopreservation is the metabolic activity that utilizes ultralow temperature Inhibit sperm cell, makes it, in almost static state, to preserve for a long time, after thawing, can be returned to normal physiological state again.Semen freezing can fully expand the availability of outstanding sire in conjunction with technology of artificial insemination after thawing, and accelerates breed improvement process and fast-propagation breeding livestock and poultry.The development to Modern Animal Husbandry of improving of seminal fluid cryopreservation method plays extremely important facilitation.
The freezing Techniques of preserving area research of animal semen with a long history, developed the seminal fluid cryopreservation method of many animals perhaps, but the store method of different plant species there are differences.About the semen cryopreservation of horse, there is the bibliographical information of tens of kinds of horse sperm freezing dilution liquid formulas abroad, domestic Xinjiang Agricultural Univ, Xinjiang herding research institute of the herding academy of sciences, Tarim University etc. have carried out the research of the freezing preservation aspect of horse seminal fluid.Freezing preservation liquid and the freezing method of existing horse seminal fluid are still not mature enough; mainly by liquid nitrogen fumigate carry out freezing; cooling rate can not be controlled well; violent cooling may cause Sperm lesion even dead; frozen sperm is of poor quality; anabiosis rate is low, and refrigerating effect is unstable, is difficult to large-scale promotion application.
Pony refers to grow up height the horse below 106 centimetres, is referred to as pony(corresponding with the horse that specially refers to large horse in English).Because it is small and exquisite, intelligent by natural endowments, disposition is docile and very popular.Can be used for viewing and admiring, amusement, experiment and forced labour, be also child and the elderly well " friend ".Because quantity is rare, more seem particularly precious, can be described as the treasured of Malaysia and China.From literature search, also do not relate to the research of pony semen cryopreservation aspect at present.
Summary of the invention
The object of the invention is to for above-mentioned technical problem, a kind of method that can effectively improve the quality of pony semen cryopreservation and the pony semen cryopreservation of effect is provided.
For achieving the above object, the technical solution adopted in the present invention is:
A method for pony semen cryopreservation, is characterized in that, comprises the following steps:
(1) by the yolk of egg after high speed centrifugation, get upper strata yolk standby;
(2) prepare freezing preservation dilution, in the freezing preservation dilution of every 100mL, comprise glucose 3.4～3.8g, lactose 7.0～7.4g, sodium citrate 0.2～0.6g, upper strata yolk 4.8～5.2mL, dimethylformamide 4.8～5.2mL, vitamin E 0.04～0.08g, penicillin 8～12Wan unit, streptomycin 8～12Wan unit; During preparation first by other components dissolved except the yolk of upper strata in appropriate sterile distilled water, then add upper strata yolk, stir 25～35min, then with the filter filtration of aperture 0.3～0.5 μ m, obtain freezing preservation dilution.
(3) gather after pony seminal fluid, filter and remove colloid substance, after centrifugal, remove upper strata seminal plasma;
(4) in the ratio of the volume ratio 3:1 of seminal fluid, freezing preservation dilution, freezing preservation dilution is slowly poured in pretreated seminal fluid, jiggle and make seminal fluid fully contact, mix with dilution;
(5) the pony seminal fluid after dilution is divided to be filled in tubule and sealed, service routine cooling instrument carries out sequencing cooling, cooling process is: default 20 ℃ of start-up routines, slow cooling to 5 ℃ stops 5min, fast cooling is to-10 ℃ of stop 2min, fast cooling, is down to after-43 ℃ when temperature subsequently, takes out fast tubule and proceeds in liquid nitrogen and preserve.
Preferably, in step (1), the rotating speed when yolk of egg is centrifugal is 10000r/m, and centrifugation time is 30min.
Preferably, in step (2), in the freezing preservation dilution of every 100mL, comprise glucose 36g, lactose 7.2g, sodium citrate 0.4g, upper strata yolk 5.0mL, dimethylformamide 5.0mL, vitamin E 0.06g, penicillin 10Wan unit, streptomycin 10Wan unit; While preparing during preparation first by other components dissolved except the yolk of upper strata in appropriate sterile distilled water, then add upper strata yolk, stir 30min, then with the filter filtration of aperture 0.4 μ m, obtain freezing preservation dilution.
Preferably, in step (3), gather after pony seminal fluid, with the bilayer 200 order nylon net filters of sterilizing, remove colloid substance, rotating speed when centrifugal is 1200r/min, and centrifugation time is 7min.
Preferably, in step (4), freezing preservation dilution need first be preheating to 37 ℃.
Preferably, in step (5), the pony seminal fluid after dilution is first at 25 ℃ of standing 1h, then divides to be filled in tubule and seal.
By above technical scheme, beneficial effect of the present invention is as follows:
The freezing preservation dilution that the present invention uses can effectively extend the service life of pony sperm; By sequencing, lower the temperature and effectively control cooling rate, avoid sperm to sustain damage, the quality of sperm after improving refrigerating effect and thawing.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Do not deviating from the present invention spirit and essential in the situation that, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
Embodiment
1. prepare freezing preservation dilution
(1) with 75% alcohol wipe sterilization egg shell, after alcohol volatilization is dry, crack eggshell, after removing egg white, complete yolk is poured on aseptic filter paper, with clean filter paper, remaining egg white is blotted, use does not puncture vitellinae membrana with the disposable syringe of syringe needle, draws yolk.Yolk is packed in 10ml centrifuge tube, and 10000r/m high speed centrifugation 30 minutes, draws that yolk is standby at the middle and upper levels;
(2) prepare freezing preservation dilution, in every 100mL pony semen cryopreservation dilution, the content of each component is: glucose 3.6g, lactose 7.2g, sodium citrate 0.4g, upper strata yolk 5.0mL, dimethylformamide 5.0mL, vitamin E 0.06g, penicillin 10Wan unit, streptomycin 10Wan unit; Compound method: above-mentioned component except the yolk of upper strata is first dissolved in appropriate sterile distilled water, then adds upper strata yolk, then use magnetic stirrer dilution 30min, then filter with the filter of aperture 0.4 μ m, make freezing basic dilution.
2. the freezing preservation of pony seminal fluid
(1) 30min left and right before semen collection, is placed in 37 ℃ of water-bath preheatings by semen cryopreservation dilution.
(2) adopt artificial vagina method to collect pony seminal fluid, with the bilayer 200 order nylon net filters of sterilizing, remove colloid substance, be then distributed in centrifuge tube, the centrifugal 7min of 1200r/min, removes upper strata seminal plasma.Vigor is that rectilinear motion sperm ratio reaches more than 70%, can be freezing; Wherein, described pony is Debao pony.
(3) in the ratio of the volume ratio 3:1 of seminal fluid, dilution, freezing preservation dilution is slowly poured in pretreated seminal fluid along chamber wall, jiggle and make seminal fluid fully contact, mix with dilution.
(4) the seminal fluid mixed solution after dilution is placed in to room temperature (25 ℃) after standing 1 hour, is sub-packed in 0.5mL tubule and seals.
(5) programmed cooling instrument is worked in advance and be chilled in advance the initial temperature that program setting is set, the tubule that seminal fluid is housed is inserted to the freezing tank of cooling instrument, carry out sequencing cooling freezing, cooling process is: default 20 ℃ of start-up routines, slow cooling to 5 ℃ stops 5min, and fast cooling is to-10 ℃ of stops 2min, subsequently fast coolings, when temperature, be down to after-43 ℃, take out fast tubule and proceed in liquid nitrogen and preserve.The model of described programmed cooling instrument is CL5500, place of production Australia.
Test example 1 freeze essence for artificial insemination, test
To by the Debao pony of embodiment gained, freeze after essence thaws, adopt rectum hold semen deposition enter the to oestrus uterine body deep of mare.Oestrus 16 of mares of experimental labor insemination, after March, pregnancy check is determined 7 mare pregnancies.
Test example 2 is frozen the development of preserving dilution
(1) by 5 kinds of formulas in table 1, prepare respectively semen cryopreservation basis dilution, freezing preservation Debao pony seminal fluid, and the sperm viability after thawing and sperm motility speed (average space rate VSL, averaged curve speed VCL, average path speed VAP) are evaluated, determine more excellent basic dilution formula.
Table 1 pony semen cryopreservation basis dilution formula
Note: above 5 kinds of units of yolk, glycerine and DMSO at the middle and upper levels of filling a prescription are mL, and the unit of other compositions is g, and the basic dilution volume of each formula is 100mL, dissolves with sterile distilled water, all adds penicillin, each 10Wan unit of streptomycin.
The controlled-rate freezing of the different chill back formula of liquid of table 2 to Debao pony seminal fluid
Note: represent significant difference (P < 0.05) with thering are different subscript letter persons in a line, identical table differential different not remarkable (P > 0.05).
According to table 2 data, consider vigor and the sperm motility speed parameter of seminal fluid after thawing, the basic dilution of the formula freezing preservation of II, for the best results of freezing preservation Debao pony, can be used as the basic components of follow-up study.
(2) in the basic dilution of the formula freezing preservation of II, add glycerine or the impact of dimethylformamide (DMF) on Debao pony semen cryopreservation effect of variable concentrations.
Table 3 adds the impact of variable concentrations glycerine on pony semen cryopreservation effect
Note: represent significant difference (P < 0.05) with thering are different subscript letter persons in a line, identical table differential different not remarkable (P > 0.05).
Table 4 adds the impact of variable concentrations DMF on pony semen cryopreservation effect
Note: represent significant difference (P < 0.05) with thering are different subscript letter persons in a line, identical table differential different not remarkable (P > 0.05).
As shown in Table 3, in the basic dilution of freezing preservation, add 6% Freezing Glycerine and preserve Debao pony seminal fluid, freeze essence thaw after vigor, average movement velocity, abnormal rate, the acrosomal integrity of sperm be all better than the dilution that adds other concentration glycerine.
According to table 4 data, comprehensively analyze Debao pony freeze essence thaw after every evaluation index, the controlled-rate freezing of finding to add in basic dilution 5%DMF is best.
(3) in the basic dilution of above-mentioned improved freezing preservation, the impact of the vitamin E (Vit E) of interpolation variable concentrations on Debao pony semen cryopreservation effect.
Table 5 adds the impact of various concentrations of vitamin E on Debao pony semen cryopreservation effect
Note: represent significant difference (P < 0.05) with thering are different subscript letter persons in a line, identical table differential different not remarkable (P > 0.05).
According to table 5 data, comprehensively analyze Debao pony freeze essence thaw after every evaluation index, find to add in basic dilution 600 μ g/mL vitamin Es can significantly improve thaw after the acrosomal integrity of sperm become live time with external.
Contrast by above-mentioned three tests is optimized, determine the formula of Debao pony semen cryopreservation dilution: in every 100mL dilution, comprise glucose 3.6g, lactose 7.2g, sodium citrate 0.4g, upper strata yolk 5.0mL, dimethylformamide 5.0mL, vitamin E 0.06g, penicillin 10Wan unit, streptomycin 10Wan unit.
Claims (6)
1. a method for pony semen cryopreservation, is characterized in that, comprises the following steps:
(1) by the yolk of egg after high speed centrifugation, get upper strata yolk standby;
(2) prepare freezing preservation dilution, in the freezing preservation dilution of every 100mL, comprise glucose 3.4～3.8g, lactose 7.0～7.4g, sodium citrate 0.2～0.6g, upper strata yolk 4.8～5.2mL, dimethylformamide 4.8～5.2mL, vitamin E 0.04～0.08g, penicillin 8～12Wan unit, streptomycin 8～12Wan unit; During preparation first by other components dissolved except the yolk of upper strata in appropriate sterile distilled water, then add upper strata yolk, stir 25～35min, then with the filter filtration of aperture 0.3～0.5 μ m, obtain freezing preservation dilution.
(3) gather after pony seminal fluid, filter and remove colloid substance, after centrifugal, remove upper strata seminal plasma;
(4) in the ratio of the volume ratio 3:1 of seminal fluid, freezing preservation dilution, freezing preservation dilution is slowly poured in pretreated seminal fluid, jiggle and make seminal fluid fully contact, mix with dilution;
(5) the pony seminal fluid after dilution is divided to be filled in tubule and sealed, service routine cooling instrument carries out sequencing cooling, cooling process is: default 20 ℃ of start-up routines, slow cooling to 5 ℃ stops 5min, fast cooling is to-10 ℃ of stop 2min, fast cooling, is down to after-43 ℃ when temperature subsequently, takes out fast tubule and proceeds in liquid nitrogen and preserve.
2. according to the method for pony semen cryopreservation claimed in claim 1, it is characterized in that: in step (1), the rotating speed when yolk of egg is centrifugal is 10000r/m, and centrifugation time is 30min.
3. according to the method for pony semen cryopreservation claimed in claim 1, it is characterized in that: in step (2), in the freezing preservation dilution of every 100mL, comprise glucose 36g, lactose 7.2g, sodium citrate 0.4g, upper strata yolk 5.0mL, dimethylformamide 5.0mL, vitamin E 0.06g, penicillin 10Wan unit, streptomycin 10Wan unit; While preparing during preparation first by other components dissolved except the yolk of upper strata in appropriate sterile distilled water, then add upper strata yolk, stir 30min, then with the filter filtration of aperture 0.4 μ m, obtain freezing preservation dilution.
4. according to the method for pony semen cryopreservation claimed in claim 1, it is characterized in that: in step (3), gather after pony seminal fluid, with the bilayer 200 order nylon net filters of sterilizing, remove colloid substance, rotating speed when centrifugal is 1200r/min, and centrifugation time is 7min.
5. according to the method for pony semen cryopreservation claimed in claim 1, it is characterized in that: in step (4), freezing preservation dilution need first be preheating to 37 ℃.
6. according to the method for pony semen cryopreservation claimed in claim 1, it is characterized in that: in step (5), the pony seminal fluid after dilution is first at 25 ℃ of standing 1h, then divides to be filled in tubule and seal.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104161036A (en) * | 2014-07-15 | 2014-11-26 | 西北农林科技大学 | Antifreeze agent and diluent for livestock semen refrigeration preservation and application thereof |
| CN106259307A (en) * | 2016-08-24 | 2017-01-04 | 新疆畜牧科学院畜牧研究所 | A kind of horse sperm low temperature and freezen protective diluent |
| CN107232179A (en) * | 2016-03-29 | 2017-10-10 | 中国农业大学 | A kind of semen diluent for improving seminal fluid low temperature/freezen protective quality and its application |
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2013
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104161036A (en) * | 2014-07-15 | 2014-11-26 | 西北农林科技大学 | Antifreeze agent and diluent for livestock semen refrigeration preservation and application thereof |
| CN107232179A (en) * | 2016-03-29 | 2017-10-10 | 中国农业大学 | A kind of semen diluent for improving seminal fluid low temperature/freezen protective quality and its application |
| CN106259307A (en) * | 2016-08-24 | 2017-01-04 | 新疆畜牧科学院畜牧研究所 | A kind of horse sperm low temperature and freezen protective diluent |
| CN106259307B (en) * | 2016-08-24 | 2019-03-01 | 新疆畜牧科学院畜牧研究所 | A kind of horse sperm low temperature and freezen protective dilution |
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Application publication date: 20140409 |