CN111374124A - Formula and preparation method of donkey frozen semen diluent - Google Patents
Formula and preparation method of donkey frozen semen diluent Download PDFInfo
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- CN111374124A CN111374124A CN202010413399.1A CN202010413399A CN111374124A CN 111374124 A CN111374124 A CN 111374124A CN 202010413399 A CN202010413399 A CN 202010413399A CN 111374124 A CN111374124 A CN 111374124A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
A diluent for frozen donkey semen is prepared through weighing glucose (1.2 g), citric acid (1.3 g) and tris2.4g), glycerin (5 ml) and distilled water (100 ml), putting them in a medium triangular flask, heating to 95 deg.C for dissolving them completely, cooling to 35 deg.C, adding yolk (20 ml) and penicillin 1 × 10 (10 ml) to obtain a diluent, and mixing with diluent, diluent and additive5IU, streptomycin 1 × 105IU, cooling to 4 ℃ after uniformly mixing, standing and depositing for 12 hours, and taking supernatant as diluent. The advantages are high activity of frozen semen in thin tube after thawing, long survival time of thawed semen, and high conception rate.
Description
The technical field is as follows:
the invention relates to a donkey tubule frozen semen diluent formula and a preparation method thereof.
Background art:
the frozen semen of donkey in China has a history of about twenty years, but the problems of low activity (40-45%) after thawing and low semen deposition and conception rate generally exist, and the preservation and utilization effects of frozen semen of donkey and variety resources are severely restricted.
The invention content is as follows:
the technical problem to be solved by the invention is to provide a donkey frozen semen diluent formula and a preparation method thereof, and the frozen semen has high vitality after being thawed and high average estrus semen deposition and conception rate.
The frozen semen diluent formula comprises the following components: glucose, citric acid, Tris, glycerol, yolk, double distilled water and streptomycin.
The mass ratio of each component is 1.2g of glucose, 1.3g of citric acid, 2.4g of Tris2, 5ml of glycerol, 20ml of yolk, 100ml of double distilled water and 1 × 10 of penicillin5IU, streptomycin 1 × 105IU。
Wherein the glucose is D-glucose, and has a molecular formula of C6H12H2o, molecular weight 198.17; the molecular formula of the citric acid is C6H8O7Molecular weight 192.14; tris refers to Tris (hydroxymethyl) aminomethane and has the molecular formula of (HOCH)2)3CNH2;The molecular weight is 121.14; the pH value of the double distilled water is 6.7-6.8; the streptomycin is streptomycin sodium and streptomycin sulfate, and the yolk is fresh yolk of healthy chicken.
The preparation method of the frozen semen diluent comprises the following steps:
respectively weighing 1.2g of glucose, 1.3g of citric acid and 4.4 g of Tris2 by using a balance, weighing 5ml of glycerol and 100ml of double distilled water, putting the glycerol and the double distilled water into a moderate triangular flask, heating to 95 ℃, completely dissolving the substances, cooling to 35 ℃, and then adding 20ml of egg yolk and 1 × 10 of penicillin5IU, streptomycin 1 × 105IU, cooling to 4 ℃ after uniformly mixing, standing and depositing for 12 hours, and taking supernatant as diluent.
The using method comprises the following steps:
firstly, semen dilution:
heating the diluent to 35 ℃ in a water bath, mixing the semen for standby with the diluent according to the volume ratio of 1: 1-1: 2 for isothermal dilution.
Secondly, cooling and balancing:
the diluted semen is cooled to 0-5 ℃ at the speed of 0.5 ℃/min, stored for 8-14 h and then filled by a thin tube of 0.5ml at the temperature.
Thirdly, freezing:
and (3) putting the filled semen at the temperature of-90 to-145 ℃ for liquid nitrogen fumigation, and immersing the semen in liquid nitrogen for storage after 5 min.
Fourthly, unfreezing and microscopic examination:
randomly tweezing the prepared tubule frozen semen from liquid nitrogen, quickly immersing the tubule frozen semen into warm water at 38-40 ℃, shaking the tubule frozen semen until the tubule frozen semen becomes transparent, taking the tubule frozen semen out, checking the quality of the semen under a microscope of 200-600 times, and storing the tubule frozen semen in a liquid nitrogen tank after the qualified batch of tubule frozen semen is identified through microscopic examination.
The invention has the advantages that: the average activity of the frozen semen of the tubule prepared by the formula after thawing reaches more than 50 percent (on the premise of 70 percent of the activity of the original semen), and the highest thawing activity reaches 62 percent; at the temperature of 38 ℃, the thawed sperms survive for more than 12 hours averagely, the sperm teratogenesis rate is less than 17 percent (the survival time after thawing is reported to be 6-8 hours at present, and the sperm teratogenesis rate is more than 20 percent), and the conception rate is high.
The specific implementation mode is as follows:
the frozen semen diluent formula comprises the following components: glucose, citric acid, Tris, glycerol, yolk, double distilled water and streptomycin;
the mass ratio of each component is 1.2g of glucose, 1.3g of citric acid, 2.4g of Tris2, 5ml of glycerol, 20ml of yolk, 100ml of double distilled water and 1 × 10 of penicillin5IU, streptomycin 1 × 105IU。
Wherein the glucose is D-glucose, and has a molecular formula of C6H12H2o, molecular weight 198.17; the molecular formula of the citric acid is C6H8O7Molecular weight 192.14; tris refers to Tris (hydroxymethyl) aminomethane and has the molecular formula of (HOCH)2)3CNH2;The molecular weight is 121.14; the pH value of the double distilled water is 6.7-6.8; the streptomycin is streptomycin sodium and streptomycin sulfate, and the yolk is fresh yolk of healthy chicken.
The preparation method of the frozen semen diluent comprises the following steps:
respectively weighing 1.2g of glucose, 1.3g of citric acid and 4.4 g of Tris2 by using a balance, weighing 5ml of glycerol and 100ml of double distilled water, putting the glycerol and the double distilled water into a moderate triangular flask, heating to 95 ℃, completely dissolving the substances, cooling to 35 ℃, and then adding 20ml of egg yolk and 1 × 10 of penicillin5IU, streptomycin 1 × 105IU, cooling to 4 ℃ after uniformly mixing, standing and depositing for 12 hours, and taking supernatant as diluent.
The using method comprises the following steps:
firstly, semen dilution:
heating the diluent to 35 ℃ in a water bath, mixing the semen for standby with the diluent according to the volume ratio of 1: 1-1: 2 for isothermal dilution.
Secondly, cooling and balancing:
the diluted semen is cooled to 0-5 ℃ at the speed of 0.5 ℃/min, stored for 8-14 h and then filled by a thin tube of 0.5ml at the temperature.
Thirdly, freezing:
and (3) putting the filled semen at the temperature of-90 to-145 ℃ for liquid nitrogen fumigation, and immersing the semen in liquid nitrogen for storage after 5 min.
Fourthly, unfreezing and microscopic examination:
randomly tweezing the prepared tubule frozen semen from liquid nitrogen, quickly immersing the tubule frozen semen into warm water at 38-40 ℃, shaking the tubule frozen semen until the tubule frozen semen becomes transparent, taking the tubule frozen semen out, checking the quality of the semen under a microscope of 200-600 times, and storing the tubule frozen semen in a liquid nitrogen tank after the qualified batch of tubule frozen semen is identified through microscopic examination.
And (3) comparing the sperm microscopic examination effects:
the tubule frozen semen manufactured by the invention is used as a test group, the donkey semen tubule frozen semen existing in the market is used as a control group, microscopic examination is carried out after thawing, and the data of the test group and the control group are compared as follows:
table 1 frozen donkey semen diluent thawing effect comparison, unit: % min,. degree.C.;
note: the upper mark A of the test group is obviously different from the control group, the upper mark AA of the test group is obviously different from the control group, and the non-upper mark means that the difference is not obvious or the same with the control group.
Experiments show that the difference between the test group and the control group is very obvious no matter the activity after thawing or the survival time after thawing; the sperm aberration rate after unfrozen is obviously different between the test group and the control group.
Estrus identification of donkey:
the delivery and distribution period of the female donkey is determined by adopting a method of combining external observation and rectal examination. Selecting the female donkey for estrus by an external observation method, wherein the female donkey for estrus has gloomy vulvae, frequent urination, trumpet-shaped and clawed mouth, low head and closed ear, and is willing to be actively connected into the male donkey; then detecting the follicular development of the female donkey by rectal examination, pulling the female donkey into a six-column fence for fixation, pulling the donkey tail to one side by an assistant, drawing the five fingers of an examiner together, slowly inserting the female donkey anus to take out the night soil for a plurality of times and then searching for the ovary. The follicle volume is increased to 5-6 cm, the elasticity is weakened, the fluctuation is obvious, the persimmon which is well cooked has the feeling of breaking by touch, and the donkey can be used for the first semen deposition. When the follicular wall becomes thin, follicular fluid gradually loses the sensation of two layers of skin, and a second insemination is performed.
Insemination:
firstly, the female donkey is drawn into the six-column fence for fixation, the donkey tail is pulled to one side by an assistant, 2% of the donkey tail is used for disinfecting the pudendum, the pudendum is cleaned by warm boiled water, and finally, the pudendum is wiped clean by using a piece of sterile gauze. Then an operator holds the semen transporter which sucks the semen in advance with the left hand, the five fingers are conical, the instrument is slowly inserted into the vagina, the cervical orifice is found, the semen transporter is guided into the cervix for 10-15 cm, then the instrument is fixed, the left hand lifts the instrument filled with the semen, all the semen is injected into the cervix under the action of gravity, the right hand grabs a screwdriver with the right hand and shakes the cervix gently while the semen transporter is pulled out, the semen backflow is prevented, and the semen transporting frequency is controlled to be 2-3 times.
Comparison of semen deposition effects:
the frozen semen prepared by the formula of the control group and the frozen semen of the test group are adopted to carry out the transfusion test, and the cervix is used for carrying out the sperm transfusion by a cervical holding method. 1 dose of 0.5ml tubule frozen semen is input each time, 1 semen is input again after 12 hours, the semen is input for 3 times at most, and the frozen semen mating and conception rates of a control group and a test group are shown in table 2.
Table 2 shows the pregnancy rate of frozen semen hybridization between the control group and the test group; unit: only,%.
Through comparison, the difference between the test group and the control group is very obvious, and the frozen semen prepared by the formula disclosed by the invention is proved to have a remarkable effect.
Claims (4)
1. The formula of the donkey frozen semen diluent is characterized in that: comprises the following components: glucose, citric acid, Tris, glycerol, yolk, double distilled water and streptomycin.
2. Frozen semen dilution formulation for donkey according to claim 1The formula is characterized in that the mass ratio of the components is 1.2g of glucose, 1.3g of citric acid, 2.4g of Tris2, 5ml of glycerol, 20ml of yolk, 100ml of double distilled water and 1 × 10 g of penicillin5IU, streptomycin 1 × 105IU。
3. The donkey frozen semen diluent formulation according to claim 1 or 2, characterized in that: wherein the glucose is D-glucose, and has a molecular formula of C6H12H2o, molecular weight 198.17; the molecular formula of the citric acid is C6H8O7Molecular weight 192.14; tris refers to Tris (hydroxymethyl) aminomethane and has the molecular formula of (HOCH)2)3CNH2;The molecular weight is 121.14; the pH value of the double distilled water is 6.7-6.8; the streptomycin is streptomycin sodium and streptomycin sulfate, and the yolk is fresh yolk of chicken.
4. A method for preparing frozen semen diluent of donkey is characterized in that 1.2g of glucose, 1.3g of citric acid and 1.4 g of Tris2.4g are respectively weighed by a balance, 5ml of glycerol and 100ml of double distilled water are weighed, the mixture is put into a moderate triangular flask, heated to 95 ℃, and then 20ml of yolk and 1 × 10 of penicillin are added after the substances are completely dissolved and cooled to 35 DEG C5IU, streptomycin 1 × 105IU, cooling to 4 ℃ after uniformly mixing, standing and depositing for 12 hours, and taking supernatant as diluent.
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| CN202010413399.1A CN111374124A (en) | 2020-05-15 | 2020-05-15 | Formula and preparation method of donkey frozen semen diluent |
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| CN202010413399.1A CN111374124A (en) | 2020-05-15 | 2020-05-15 | Formula and preparation method of donkey frozen semen diluent |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113729007A (en) * | 2021-09-23 | 2021-12-03 | 内蒙古自治区农牧业科学院 | Bacteriostatic uterine horn sperm-infusion diluent suitable for donkey sperm and preparation method thereof |
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| CN103039434A (en) * | 2012-12-10 | 2013-04-17 | 山东天龙牧业科技有限公司 | Duck egg yolk anti-freezing agent for cryopreservation of donkey semen and preparation method thereof |
| 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 |
| 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 |
| CN107980766A (en) * | 2017-12-19 | 2018-05-04 | 天津农学院 | A kind of method of donkey testicular fluid freezen protective |
| CN108669069A (en) * | 2018-01-31 | 2018-10-19 | 广西壮族自治区畜禽品种改良站 | One boar category animal freezing seminal fluid dilution and its preparation method |
| CN110604129A (en) * | 2019-10-28 | 2019-12-24 | 浙江大学 | Hu sheep semen cryopreservation liquid and application thereof |
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2020
- 2020-05-15 CN CN202010413399.1A patent/CN111374124A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103039434A (en) * | 2012-12-10 | 2013-04-17 | 山东天龙牧业科技有限公司 | Duck egg yolk anti-freezing agent for cryopreservation of donkey semen and preparation method thereof |
| 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 |
| 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 |
| CN107980766A (en) * | 2017-12-19 | 2018-05-04 | 天津农学院 | A kind of method of donkey testicular fluid freezen protective |
| CN108669069A (en) * | 2018-01-31 | 2018-10-19 | 广西壮族自治区畜禽品种改良站 | One boar category animal freezing seminal fluid dilution and its preparation method |
| CN110604129A (en) * | 2019-10-28 | 2019-12-24 | 浙江大学 | Hu sheep semen cryopreservation liquid and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113729007A (en) * | 2021-09-23 | 2021-12-03 | 内蒙古自治区农牧业科学院 | Bacteriostatic uterine horn sperm-infusion diluent suitable for donkey sperm and preparation method thereof |
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