CN104542573A - Method for manufacturing straw frozen semen of donkey and application of method - Google Patents
Method for manufacturing straw frozen semen of donkey and application of method Download PDFInfo
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- CN104542573A CN104542573A CN201310496150.1A CN201310496150A CN104542573A CN 104542573 A CN104542573 A CN 104542573A CN 201310496150 A CN201310496150 A CN 201310496150A CN 104542573 A CN104542573 A CN 104542573A
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Abstract
The invention discloses a method for manufacturing the straw frozen semen of a donkey and the application of the method. The donkey plays an important role in improvement of horses. The straw frozen semen of the donkey, the frozen semen for short, is prepared by performing special treatment such as semen quality detection, raw semen centrifugal concentration and addition of special freezing dilution protection liquid on the collected fresh semen of the donkey, sealing the semen in a miniature plastic straw of 0.25 ml, freezing the semen according to a specific program and a specific freezing speed by utilizing liquid nitrogen as a cold source, and storing the semen under an ultra-low-temperature environment in a frozen form, so that the aim of storing the semen of the donkey for a long time is achieved. According to the manufacturing and the using of the straw frozen semen of the donkey, the improvement coverage of good breeding donkeys is expanded favorably, the utilization rate is improved, the straw frozen semen of the donkey can be frozen and stored without the limitation of the limited life of a donkey living body, and the service life can be prolonged to the greatest extent.
Description
Technical field:
The Straw Frozen Semen that the present invention relates to the donkey of donkey makes.
Technical background:
Adopt Pseudopyloric metaplasia collection to grow up and plant a jack ass seminal fluid, after flavor evaluation, by 5 kinds of different freezen protective liquid dilutions, balance, freezen protective makes Straw Frozen Semen, to screen suitable donkey testicular fluid freezen protective liquid and cryoprotector.Result shows: lactose-glucose-glycerine-yolk liquid plants freezen protective liquid (P < 0.05) for best; Glycerine is best cryoprotector, and its suitable concentration is 5%, and sperm freezes rear motility rate average out to 0.36, abnormal rate≤27.4%, acrosomal integrity 82.2%, and after thawing, under normal temperature, Sperm survival time is 8.2h.
Summary of the invention:
Donkey has important function in horses improvement; donkey Straw Frozen Semen (hereinafter referred to as freezing essence) refers to the fresh semen of the donkey collected; through semen quality detection, former seminal fluid centrifugal concentrating with after adding the specially treateds such as special chill back protection liquid; seminal fluid is encapsulated in miniature (0.25ml) plastic straw; utilize liquid nitrogen for low-temperature receiver; undertaken freezing by specific program and chilling rate; under making seminal fluid be stored in ultra-low temperature surroundings with the form freezed, reach long-term object of preserving donkey testicular fluid.The making of donkey straw frozen semen and use, be conducive to expanding the improvement coverage rate of good species jack ass and improve its availability, and donkey straw frozen semen freezen protective, can expanded service life to greatest extent by the restriction of donkey live body limited life.
Embodiment:
1. the collection of donkey testicular fluid: donkey Pseudopyloric metaplasia semen collection; select before semen collection to oestrus obviously; the jenny ass of disposition docility or mare as Tai Ma and Baoding in Shelf for keeing; guarantee person poultry safety; again jack ass is led semen collection frame; jack ass mounting is allowed to oestrus dummy teaser for several times; during mounting, semen collection person's Control protection jack ass is cloudy; stem prevents jack ass and dummy teaser from this friendship occurring; when jack ass heat is abundant; penis fully erects and penis is imported ready artificial vagina semen collection, can ejaculate when jack ass penis inserts artificial vagina and twitches for several times.
2. straw frozen semen making apparatus: phase contrast microscope, spectrophotometer, generic centrifuge, MRS-3 automatic packer, program-controlled freezing instrument, wide-mouth liquid nitrogen biological container, liquid nitrogen, mini-straw, thumb pipe, refrigerator, low-temperature operation cabinet, freezing semen frame, slide, graduated cylinder, measuring cup, centrifuge tube, thermostat water bath, pipettor and dilution etc.
3. the preparation of donkey freeze-extender: the freeze-extender being applicable to the use of donkey straw frozen semen with Orthogonal Method screening, the main component of dilution is: glucose, glycine, sucrose, glycerine, yolk and distilled water, weigh reagent accurately necessary during preparation, mixing after reagent weighs, completely dissolving, Filter paper filtering and sterilization are stand-by.
4. the process of donkey testicular fluid: a. filters: the absorbent gauze of the donkey testicular fluid collected through sterilization filters, and filters out impurities and part pair thing.B. volume of semen is measured: volume of semen measured by the donkey testicular fluid graduated cylinder after filtration, record volume of semen.C. the mensuration of donkey sperm concentration: get donkey testicular fluid sample 100 microlitre, adds sodium chloride solution 1900 microlitre of 0.7%, fully mixes, and puts into spectrophotometric colo groove and measures sperm concentration, record sperm concentration.D. check Fresh semen activity: get the former smart sample 5-10 microlitre of donkey and drop on slide, examine under a microscope sperm viability with cover glass compressing tablet, the donkey testicular fluid of sperm viability more than 60% can be used for making straw frozen semen.E. the determination of the rear total amount of donkey testicular fluid dilution, determine the total amount of diluting rear seminal fluid according to the former sperm viability of donkey, volume of semen, tubule volume, sperm concentration and every effective number of sperm of dosage (every dosage effective number of sperm 1500-2000 ten thousand), computing formula is: seminal fluid total amount=0.25*0.35* sperm concentration * volume of semen/0.15 after dilution.
5. the centrifugal concentrating of donkey testicular fluid: after donkey testicular fluid dilution seminal fluid total amount determination after, donkey testicular fluid is sub-packed in scale centrifuge tube, puts into centrifuge, with the centrifugation 10 ~ 15 minutes of 2000 revs/min, sucking-off supernatant as far as possible, the supernatant of First Astronautic Research Institute for Measurement and Test's sucking-off.
6. the dilution of donkey testicular fluid: add freeze-extender to dilution in the seminal fluid sucking supernatant after seminal fluid total amount and mix, after the addition of dilution equals dilution, seminal fluid total amount deducts the quantity of centrifugal segregation supernatant.
7. the packing of seminal fluid, the donkey testicular fluid MRS-3 automatic packer after dilution is divided in mini-straw under the room temperature of laboratory, sealing and lettering.
8. the balance of donkey straw semen, the straw semen after packing is preserved 4 ~ 12 hours under being positioned over the temperature of+4 DEG C.
9. donkey straw semen is freezing, donkey testicular fluid after balance is neatly placed on freezing semen frame at about+4 DEG C temperature, then freezing semen frame and seminal fluid are put into program-controlled freezing instrument freezing, freezing curve is :+4 DEG C to-100 DEG C, chilling rate is 60 DEG C/mn.-100 is DEG C to-140 DEG C, chilling rate is 20 DEG C/mn, then the straw frozen semen after freezing is immersed and carries out ultralow temperature in liquid nitrogen and freeze; Wide-mouth liquid nitrogen biological container is freezing: the donkey testicular fluid after balance is neatly placed on freezing semen frame at about+4 DEG C temperature, then freezing semen frame and seminal fluid are put into the freezing 15-20 minute of liquid nitrogen vapor of-120 ~-140 DEG C, then the straw frozen semen after freezing is immersed and carry out ultralow temperature in liquid nitrogen and freeze.
10. thawing of donkey straw frozen semen checks and preserves: the donkey straw frozen semen after freezing is randomly drawed 1-2 and propped up, the water-bath of putting into 40 ± 2 DEG C is immediately thawed, inspection is thawed vigor, and the vigor of thawing can reach 35 ~ 40%, and the donkey straw frozen semen packaging of the vigor more than 35% that thaws is put in the medium-term and long-term preservation of liquid nitrogen.
The use of 11. donkey straw frozen semens, namely the donkey straw frozen semen that after freeze-thaw, vigor reaches more than 35% can be used for artificial insemination, a feelings phase semen deposition twice, uses straw frozen semen 2 ~ 3 at every turn, and over-all conception rate can reach 60 ~ 70%.
Claims (11)
1. the collection of donkey testicular fluid; it is characterized in that donkey Pseudopyloric metaplasia semen collection; select before semen collection to oestrus obviously; the jenny ass of disposition docility or mare as Tai Ma and Baoding in Shelf for keeing; guarantee person poultry safety; again jack ass is led semen collection frame; jack ass mounting is allowed to oestrus dummy teaser for several times; during mounting, semen collection person's Control protection jack ass is cloudy; stem prevents jack ass and dummy teaser from this friendship occurring; when jack ass heat is abundant, penis fully erects and penis is imported ready artificial vagina semen collection, can ejaculate when jack ass penis inserts artificial vagina and twitches for several times.
2. straw frozen semen making apparatus, is characterized in that phase contrast microscope, spectrophotometer, generic centrifuge, MRS-3 automatic packer, program-controlled freezing instrument, wide-mouth liquid nitrogen biological container, liquid nitrogen, mini-straw, thumb pipe, refrigerator, low-temperature operation cabinet, freezing semen frame, slide, graduated cylinder, measuring cup, centrifuge tube, thermostat water bath, pipettor and dilution etc.
3. the preparation of donkey freeze-extender, it is characterized in that the freeze-extender being applicable to the use of donkey straw frozen semen with Orthogonal Method screening, the main component of dilution is: glucose, glycine, sucrose, glycerine, yolk and distilled water, weigh reagent during preparation accurately necessary, mixing after reagent weighs, completely dissolving, Filter paper filtering and sterilization are stand-by.
4. the process of donkey testicular fluid, it is characterized in that a filters: the absorbent gauze of the donkey testicular fluid collected through sterilization filters, filter out impurities and part pair thing, b measures volume of semen: volume of semen measured by the donkey testicular fluid graduated cylinder after filtration, record volume of semen, the mensuration of c donkey sperm concentration: get donkey testicular fluid sample 100 microlitre, add sodium chloride solution 1900 microlitre of 0.7%, abundant mixing, put into spectrophotometric colo groove and measure sperm concentration, record sperm concentration, d checks Fresh semen activity: get the former smart sample 5-10 microlitre of donkey and drop on slide, sperm viability is examined under a microscope with cover glass compressing tablet, the donkey testicular fluid of sperm viability more than 60% can be used for making straw frozen semen, the determination of total amount after the dilution of e donkey testicular fluid, according to the former sperm viability of donkey, volume of semen, tubule volume, sperm concentration and the effective number of sperm of every dosage (every dosage effective number of sperm 1500-2000 ten thousand) determine the total amount of diluting rear seminal fluid, computing formula is: seminal fluid total amount=0.25*0.35* sperm concentration * volume of semen/0.15 after dilution.
5. the centrifugal concentrating of donkey testicular fluid, after it is characterized in that the determination of the rear seminal fluid total amount of donkey testicular fluid dilution, is sub-packed in scale centrifuge tube donkey testicular fluid, put into centrifuge, with the centrifugation 10 ~ 15 minutes of 2000 revs/min, sucking-off supernatant as far as possible, the supernatant of First Astronautic Research Institute for Measurement and Test's sucking-off.
6. the dilution of donkey testicular fluid, after it is characterized in that adding freeze-extender to dilution in the seminal fluid sucking supernatant seminal fluid total amount and mix, after the addition of dilution equals dilution, seminal fluid total amount deducts the quantity of centrifugal segregation supernatant.
7. the packing of seminal fluid, is characterized in that the donkey testicular fluid MRS-3 automatic packer after diluting is divided in mini-straw under the room temperature of laboratory, sealing and lettering.
8. the balance of donkey straw semen, is characterized in that the straw semen after packing is preserved 4 ~ 12 hours under being positioned over the temperature of+4 DEG C.
9. donkey straw semen is freezing, it is characterized in that the donkey testicular fluid after balancing neatly is placed on freezing semen frame at about+4 DEG C temperature, then freezing semen frame and seminal fluid are put into program-controlled freezing instrument freezing, freezing curve is :+4 DEG C to-100 DEG C, chilling rate is 60 DEG C/mn.-100 is DEG C to-140 DEG C, chilling rate is 20 DEG C/mn, then the straw frozen semen after freezing is immersed and carries out ultralow temperature in liquid nitrogen and freeze; Wide-mouth liquid nitrogen biological container is freezing: the donkey testicular fluid after balance is neatly placed on freezing semen frame at about+4 DEG C temperature, then freezing semen frame and seminal fluid are put into the freezing 15-20 minute of liquid nitrogen vapor of-120 ~-140 DEG C, then the straw frozen semen after freezing is immersed and carry out ultralow temperature in liquid nitrogen and freeze.
10. thawing of donkey straw frozen semen checks and preserves, it is characterized in that freezing after donkey straw frozen semen randomly draw 1-2 and prop up, the water-bath of putting into 40 ± 2 DEG C is immediately thawed, inspection is thawed vigor, the vigor of thawing can reach 35 ~ 40%, and the donkey straw frozen semen packaging of the vigor more than 35% that thaws is put in the medium-term and long-term preservation of liquid nitrogen.
The use of 11. donkey straw frozen semens, namely the donkey straw frozen semen that after it is characterized in that freeze-thaw, vigor reaches more than 35% can be used for artificial insemination, a feelings phase semen deposition twice, uses straw frozen semen 2 ~ 3 at every turn, and over-all conception rate can reach about 60 ~ 70%.
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| CN201310496150.1A CN104542573A (en) | 2013-10-22 | 2013-10-22 | Method for manufacturing straw frozen semen of donkey and application of method |
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| CN201310496150.1A CN104542573A (en) | 2013-10-22 | 2013-10-22 | Method for manufacturing straw frozen semen of donkey and application of method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105055045A (en) * | 2015-09-02 | 2015-11-18 | 山东农业大学 | Frozen semen artificial insemination method for increasing conception rate of donkeys |
| CN105519519A (en) * | 2015-08-31 | 2016-04-27 | 新疆畜牧科学院畜牧研究所 | Donkey seminal fluid refrigeration preservation method |
| CN109223245A (en) * | 2018-09-26 | 2019-01-18 | 江苏农牧科技职业学院 | A kind of artificial insemination method of the kind goose sperm suitable for refrigeration |
| CN112155785A (en) * | 2020-10-28 | 2021-01-01 | 内蒙古草原御驴科技牧业有限公司 | Healthy breeding method for improving mating success rate of donkey mothers |
-
2013
- 2013-10-22 CN CN201310496150.1A patent/CN104542573A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105519519A (en) * | 2015-08-31 | 2016-04-27 | 新疆畜牧科学院畜牧研究所 | Donkey seminal fluid refrigeration preservation method |
| CN105519519B (en) * | 2015-08-31 | 2018-10-30 | 新疆畜牧科学院畜牧研究所 | A kind of donkey testicular fluid freezing and storing method |
| CN105055045A (en) * | 2015-09-02 | 2015-11-18 | 山东农业大学 | Frozen semen artificial insemination method for increasing conception rate of donkeys |
| CN109223245A (en) * | 2018-09-26 | 2019-01-18 | 江苏农牧科技职业学院 | A kind of artificial insemination method of the kind goose sperm suitable for refrigeration |
| CN112155785A (en) * | 2020-10-28 | 2021-01-01 | 内蒙古草原御驴科技牧业有限公司 | Healthy breeding method for improving mating success rate of donkey mothers |
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Application publication date: 20150429 |