阅读说明：本技术 绵羊高渗络合剂精液稀释保存液及其应用方法 (Sheep hypertonic complexing agent semen dilution preserving fluid and application method thereof ) 是由 张永成 周宁聪 李俊龙 何辉杰 刘洁 乌日嘎 屈建军 仇春娟 马云慧 乌德乐呼 于 2021-09-05 设计创作，主要内容包括：本发明公开了一种配制简便的绵羊高渗络合剂精液稀释保存液及其应用方法,该保存液是由2.4g二水柠檬酸三钠、1.0g葡萄糖、0.8g脲、0.05g KCl和0.02g EDTA-Na-(2)与双重蒸馏水定容至100mL配制而成。本发明中的保存液能够供给精子能量来源的养分,提供缓冲剂,增加精液量,便于大量输精,是提高绵羊情期受胎率的新型有效制剂。采用本发明中的稀释保存精液较用常规稀释液,在同等条件下,精子活力提高25％,顶体完整率提高15％。母羊情期受胎率提高10～15％,情期受胎率可达80％以上。本发明中的绵羊精液稀释保存液能够提高种公羊的利用率,扩大良种肉羊覆盖面,提高养殖效益,增加养羊收益。(The invention discloses a sperm dilution preservation solution of sheep hypertonic complexing agent with simple preparation and an application method thereof, and the preservation solution is prepared from 2.4g trisodium citrate dihydrate, 1.0g glucose, 0.8g urea, 0.05g KCl and 0.02g EDTA-Na 2 And then the solution is prepared by diluting the solution with double distilled water to a constant volume of 100 mL. The preservation solution can supply nutrients for sperm energy sources, provide a buffering agent, increase the amount of sperm, facilitate mass insemination, and is a novel effective preparation for improving the estrus conception rate of sheep. Compared with the conventional diluent, the sperm diluted and preserved in the invention has the advantages that the sperm motility is improved by 25 percent and the acrosome integrity is improved by 15 percent under the same condition. The estrus conception rate of the ewes is improved by 10-15%, and the estrus conception rate can reach more than 80%. The sheep semen dilution and preservation solution can improve the utilization rate of stud rams, enlarge the coverage of fine breed mutton sheep, improve the breeding benefit and increase the sheep breeding benefit.)

1. The sheep hypertonic complexing agent semen dilution preserving fluid is characterized in that: the sheep hypertonic complexing agent semen dilution preservative solution is prepared from 2.2g to 2.6g of trisodium citrate dihydrate, 0.8g to 1.2g of glucose, 0.6g to 1.0g of urea, 0.03g to 0.07g of KCl and 0.01g to 0.03g of EDTA-Na₂And then the solution is prepared by diluting the solution with double distilled water to a constant volume of 100 mL.

2. The sheep hypertonic complexing agent semen dilution preservative fluid as claimed in claim 1, which is characterized in that: the sheep hypertonic complexing agent semen dilution preservative solution is prepared from 2.2g to 2.6g of trisodium citrate dihydrate, 0.8g to 1.2g of glucose, 0.6g to 1.0g of urea, 0.03g to 0.07g of KCl and 0.01g to 0.03g of EDTA-Na₂And then the solution is prepared by diluting the solution with double distilled water to a constant volume of 100 mL.

3. The sheep hypertonic complexing agent semen dilution preservative fluid as claimed in claim 2, which is characterized in that: the sheep hypertonic complexing agent semen dilution preservation solution is prepared from 2.4g of lemons dihydrateTrisodium acid, 1.0g glucose, 0.8g urea, 0.05g KCl and 0.02g EDTA-Na₂And then the solution is prepared by diluting the solution with double distilled water to a constant volume of 100 mL.

4. The method for applying the sheep hypertonic complexing agent semen dilution preservation solution according to the claim 1, 2 or 3, is characterized in that: the application method specifically comprises the following steps:

(1) preparing a vessel:

washing glass and ceramic utensils with deionized water, and sterilizing at high temperature and high pressure; the rubber appliance is sterilized with 75% alcohol by mass concentration, and then evaporated in an incubator equipped with an ultraviolet lamp at 30 deg.C for more than 8 h.

(2) Preparing a preservation solution:

2.4g of trisodium citrate dihydrate, 1.0g of glucose, 0.8g of urea, 0.05g of KCl and EDTA-Na₂0.02g and double distilled water are added to the volume of 100mL to prepare a preserving fluid; filtering, sterilizing, and bottling under sterile condition.

(3) Semen collection:

when semen is collected, the artificial vagina is coated with a lubricant, the loading and unloading of the semen collecting cup are carried out at a position 20cm above the flame of the alcohol lamp, sterile gauze is used for covering before and after semen collection of the penis of the artificial vagina, and the semen collection procedure of sterile operation is strictly fulfilled.

(4) Semen dilution:

semen collected according to the aseptic operation requirement is taken, the sperm motility is more than 0.7, the density is medium, and the sperm aberration rate is lower than 15 percent; diluting with prepared preservative solution and semen at 16 deg.C under isothermal condition, wherein the dilution factor of semen is determined according to semen quality and requirement.

(5) Preservation of diluted semen:

and (3) sucking the diluted semen into a sterilized glass syringe, wearing a suction nozzle of the syringe by using a bent and closed needle head to prevent air from contacting the semen, buffering for 30min at room temperature, wrapping by using 3-4 layers of towels, and then putting into an environment at 0-8 ℃ for storage.

(6) And (3) recovering diluted semen:

taking out the diluted semen from the environment condition of 0-8 ℃, placing the diluted semen for 30min at room temperature, and then incubating the diluted semen for 30min at the temperature of 38 ℃.

(7) Insemination:

after incubation for 30min at 38 ℃, the sperm activity is checked to be not less than 0.6, the sperm motility is normal, the preserved semen is not revived, the semen is directly transfused into the cervix of the ewe, and the deeper the semen is, the better the semen is.

(8) Receiving a tire:

and (4) observing three oestrus cycles by behaviors, and if oestrus is not found, carrying out ultrasonic examination on the placenta by using a B ultrasonic instrument.

5. The application method of the sheep hypertonic complexing agent semen dilution preservation solution according to claim 4, characterized by comprising the following steps: in the step (7), the number of effective sperms per insemination of the semen preserved by the sheep hypertonic complexing agent is 4000-8000 ten thousand.

Technical Field

The invention belongs to the technical field of sheep semen preserving fluid, and particularly relates to sheep hypertonic complexing agent semen diluting preserving fluid and an application method thereof.

Background

In the mutton sheep breeding industry, one problem commonly existing in the prior art is that improved variety rams are relatively lack, so that the breeding benefit is low, and the bottleneck for restricting the high-quality development of the mutton sheep industry is also caused. The preservation of the semen of the domestic animals is one of the main measures for fully utilizing the excellent breeds of male animals, the sperm dormancy is an important condition for prolonging the preservation period of the semen, the semen in the frozen semen is in a frozen dormant state, and the frozen semen of the domestic animals represented by cattle can be preserved for decades at the temperature of-196 ℃, and the technology is widely applied. However, because the semen of sheep has poor freezing resistance, the mating and conception rate of the frozen semen of sheep is low, and therefore, the research of the preservation solution capable of preserving the semen of sheep at normal temperature is inevitable. The general diluting liquid for sheep semen in the prior art is composed of trisodium citrate dihydrate 1.4g, glucose 3.0g and double distilled water to 100ml, and the diluting liquid has short preservation time and low conception rate. Researches show that the sperm of the sire can be enabled to sleep, besides the temperature is reduced, other factors also have the effect of enabling the sperm to sleep, the high osmotic pressure has obvious effect on the sleep of the sheep sperm, and how to utilize the characteristic that the high osmotic pressure influences the sleep of the sheep sperm is researched, so that the preservation solution which can prolong the preservation period of the semen and dilute and expand the amount of the semen at normal temperature is an urgent need of the mutton sheep breeding industry.

Disclosure of Invention

The invention provides a sheep hypertonic complexing agent semen dilution preservation solution and an application method thereof, aiming at overcoming the defects in the prior art.

The invention is realized by the following technical scheme: the invention discloses a sheep hypertonic complexing agent semen diluting and preserving solution which is prepared from 2.2-2.6 g trisodium citrate dihydrate, 0.8-1.2 g glucose, 0.6-1.0 g urea, 0.03-0.07 g KCl and 0.01-0.03 g EDTA-Na₂And then the solution is prepared by diluting the solution with double distilled water to a constant volume of 100 mL.

As a preferred embodiment of the invention, the semen dilution preservative solution of the sheep hypertonic complexing agent in the invention is prepared from 2.2g to 2.6g of trisodium citrate dihydrate, 0.8g to 1.2g of glucose, 0.6g to 1.0g of urea, 0.03g to 0.07g of KCl and 0.01g to 0.03g of EDTA-Na₂And then the solution is prepared by diluting the solution with double distilled water to a constant volume of 100 mL.

As the best mode of the invention, the semen dilution preservative solution of the sheep hypertonic complexing agent in the invention is prepared from 2.4g of trisodium citrate dihydrate, 1.0g of glucose, 0.8g of urea, 0.05g of KCl and 0.02g of EDTA-Na₂And then the solution is prepared by diluting the solution with double distilled water to a constant volume of 100 mL. The osmotic pressure is 350-370 mosm/L; EDTA-Na₂Is a complexing agent with the chemical formula of C₁₀H₁₄N₂Na₂O₈It has six coordination atoms, and has the functions of heavy metal detoxication, antioxidant synergist, stabilizer and softening agent, and can prevent the precipitation of glass-like crystal.

The invention also discloses an application method of the sheep hypertonic complexing agent semen dilution preservative fluid, which specifically comprises the following steps:

(1) preparing a vessel: washing glass and ceramic utensils with deionized water, and sterilizing at high temperature and high pressure; the rubber appliance is sterilized with 75% alcohol by mass concentration, and then evaporated in an incubator equipped with an ultraviolet lamp at 30 deg.C for more than 8 h.

(2) Preparing a preservation solution: 2.4g of trisodium citrate dihydrate, 1.0g of glucose, 0.8g of urea, 0.05g of KCl and EDTA-Na₂0.02g and double distilled water are added to the volume of 100mL to prepare a preserving fluid; filtering, sterilizing, and bottling under sterile condition.

(3) Semen collection: bacterial contamination can reduce the semen preservation effect, when collecting semen, the artificial vagina is coated with a lubricant, the loading and unloading of the semen collecting cup are carried out at a position 20cm above the flame of the alcohol lamp, sterile gauze is used for covering before and after semen collection of the penis of the artificial vagina, and the semen collection procedure of sterile operation is strictly fulfilled.

(4) Semen dilution: semen collected according to the aseptic operation requirement is taken, the sperm motility is more than 0.7, the density is medium, and the sperm aberration rate is lower than 15 percent; diluting with prepared preservative solution and semen at 16 deg.C under isothermal condition, wherein the dilution factor of semen is determined according to semen quality and requirement.

(5) Preservation of diluted semen: and (3) sucking the diluted semen into a sterilized glass syringe, wearing a suction nozzle of the syringe by using a bent and closed needle head to prevent air from contacting the semen, buffering for 30min at room temperature, wrapping by using 3-4 layers of towels, and then putting into an environment at 0-8 ℃ for storage.

(6) And (3) recovering diluted semen: taking out the diluted semen from the environment condition of 0-8 ℃, standing for 30min at room temperature, and then incubating for 30min at the temperature of 38 ℃; the sperm moving speed and moving shape are improved after incubation.

(7) Insemination: after incubation for 30min at 38 ℃, checking that the sperm activity is not less than 0.6, if the sperm movement morphology is normal, the preserved semen is not revived, and the semen is directly infused into the cervix of the ewe, and is naturally inserted as far as possible; the effective number of sperms per insemination of the sperms preserved by the sheep hypertonic complexing agent is 4000-8000 ten thousand.

(8) Receiving a tire: and (4) observing three oestrus cycles by behaviors, and if oestrus is not found, carrying out ultrasonic examination on the placenta by using a B ultrasonic instrument.

The invention has the beneficial effects that: the invention develops an application project of the sheep hypertonic complexing agent semen preservation diluent, develops a sheep hypertonic complexing agent semen dilution preservative solution which is simple and convenient to prepare, is also a preservative solution for diluting sheep semen at a low temperature by a large time, and consists of sodium citrate, a complex, inorganic salts, a nitrogen-containing compound and the like. Compared with the conventional diluent, the sperm diluted and preserved in the invention has the advantages that the sperm motility is improved by 25 percent and the acrosome integrity is improved by 15 percent under the same condition. The semen preservation diluent of the sheep hypertonic complexing agent can supply nutrients for sperm energy sources, provide a buffering agent, increase the semen amount, facilitate mass semen deposition and be a novel effective preparation for improving the estrus conception rate of sheep. The sperm preservation diluent of the hypertonic complexing agent for sheep utilizes the characteristic of dormancy effect of hypertonic pressure on animal sperms, the storage life of the sperm is prolonged at the low temperature of 0-8 ℃, compared with the sperm insemination diluted by the conventional diluent, the sperm preservation diluent for the hypertonic complexing agent for sheep is used for the sperm insemination, the estrus conception rate of the ewe is improved by 10-15%, and the estrus conception rate can reach more than 80%. The sheep hypertonic complexing agent semen storage diluent can improve the utilization rate of stud rams, enlarge the coverage of fine breed mutton sheep, improve the breeding benefit and increase the sheep raising income. In conclusion, through the research and development and application of the sheep hypertonic complexing agent semen storage diluent, the storage and use period of sheep semen can be greatly prolonged, the improvement and breeding work of livestock can be conveniently carried out, the utilization rate of stud rams can be fully improved, the conception rate of female livestock can be improved, and the sheep hypertonic complexing agent semen storage diluent has great significance for the vigorous popularization of the artificial insemination work of livestock.

The invention can improve the semen preservation effect: sperm dormancy is an important means for prolonging the semen preservation period, and high osmotic pressure has dormancy effect on animal sperm. The invention utilizes the characteristic of dormancy effect of high osmotic pressure on animal sperms, the semen storage life is prolonged at low temperature of 0-8 ℃, the activity of the diluted semen can still reach 0.6-0.7 after 3 days, and the semen is 1: 3, 1: 5 and 1: 10 times. Meanwhile, the high-permeability medium is used for preserving the semen, so that the integrity rate of the sperm acrosome can be improved, the time of the sperm with the same activity can be prolonged, and compared with the preservation by the same-period isotonic medium, the sperm has high movement speed and good movement form. Compared with the equal-permeability solution dilution, the semen dilution and preservation solution of the hypertonic complexing agent for sheep has the advantages that the estrus conception rate of the ewes is improved by 10-15% and can reach more than 80% when the semen is preserved by the hypertonic complexing agent solution.

The invention can improve the utilization of stud rams: the conventional semen in the prior art is diluted by a low power of 1: 2-4, and one ram can be matched with more than 100-300 ewes in one year by an artificial insemination technology, so that the semen is increased by more than 10-20 times compared with ram backcross. The high-permeability complexing agent solution is adopted to dilute and preserve semen according to a high-power dilution ratio of 1: 20-50, and through an artificial insemination technology, one ram can be bred with more than 2000-6000 ewes in one year, which is about 200 times higher than the present mating.

Detailed Description

The present invention will be described in detail with reference to specific embodiments.

Example (b): the invention discloses a sheep hypertonic complexing agent semen dilution preservative solution which is prepared from 2.4g trisodium citrate dihydrate, 1.0g glucose, 0.8g urea, 0.05g KCl and 0.02g EDTA-Na₂And then the solution is prepared by diluting the solution with double distilled water to a constant volume of 100 mL.

The invention also discloses an application method of the sheep hypertonic complexing agent semen dilution preservative fluid, which specifically comprises the following steps: (1) preparing a vessel: the invention relates to a device for preparing semen dilution and preservation solution of sheep hypertonic complexing agent, which comprises the following components: the device comprises an ultra-clean workbench capable of guaranteeing sterile operation, an analytical balance for accurate weighing, a water circulating vacuum pump for pumping when filtering solution, a biological microscope for detecting sperm motility and acrosome integrity, an autoclave for high-temperature and high-pressure sterilization and a sand core funnel capable of filtering bacteria and impurities. Washing glass and ceramic utensils with deionized water, sterilizing at high temperature and high pressure, disinfecting rubber utensils with 75% alcohol, and evaporating at 30 deg.C in an incubator with ultraviolet lamp for more than 8 hr; (2) preparing a preservation solution: 2.4g of trisodium citrate dihydrate, 1.0g of glucose, 0.8g of urea, 0.05g of KCl and EDTA-Na₂0.02g and double distilled water are added to the volume of 100mL to prepare a preserving fluid; filtering, sterilizing, and bottling in an ampoule; (3) semen collection: bacterial contamination can reduce the semen preservation effect, when collecting semen, the artificial vagina is coated with a lubricant, the loading and unloading of the semen collecting cup are carried out at a position 20cm above the flame of the alcohol lamp, sterile gauze is used for covering before and after semen collection of the penis of the artificial vagina, and the semen collection procedure of sterile operation is strictly implemented; (4) semen dilution: semen collected according to the aseptic operation requirement is taken, the sperm motility is more than 0.7, the density is medium, and the sperm aberration rate is lower than 15 percent; prepared preservative fluid anddiluting semen under 16 deg.C isothermal condition, wherein the dilution factor of semen is determined according to semen quality and requirement; (5) preservation of diluted semen: sucking the diluted semen into a sterilized glass syringe, wearing a suction nozzle of the syringe by a bent and closed needle head to prevent air from contacting the semen, buffering for 30min at room temperature, wrapping by using 3-4 layers of towels, and storing at 0-8 ℃; (6) and (3) recovering diluted semen: taking out the diluted semen from the environment condition of 0-8 ℃, standing for 30min at room temperature, and then incubating for 30min at the temperature of 38 ℃; the movement speed and the movement form of the incubated sperms are improved; (7) insemination: after incubation for 30min at 38 ℃, checking that the sperm activity is not less than 0.6, if the sperm movement morphology is normal, the preserved semen is not revived, and the semen is directly infused into the cervix of the ewe, and is naturally inserted as far as possible; the effective number of sperms per insemination of the sperms preserved by the sheep hypertonic complexing agent is 4000-8000 ten thousand; (8) receiving a tire: and (4) observing three oestrus cycles by behaviors, and if oestrus is not found, carrying out ultrasonic examination on the placenta by using a B ultrasonic instrument.

(1) Compared with the preservation solution of a control group in the prior art, the preservation solution of the sheep hypertonic complexing agent for diluting semen has the following preservation effects:

test groups: hypertonic complexing agent diluting preserving fluid

Control group: 1.4g of sodium citrate dihydrate, 3.0g of glucose and double distilled water to 100mL

Dilution ratio of 1: 5

Through inspection, the difference P is less than 0.01, and the difference is very obvious.

(2) Compared with the ewe conception effect of the preservative fluid for the control group in the prior art, the sperm dilution preservative fluid for the sheep hypertonic complexing agent has the following advantages:

test groups: hypertonic complexing agent diluting preserving fluid

Control group: 1.4g of sodium citrate dihydrate, 3.0g of glucose and double distilled water to 100mL

Dilution ratio of 1: 5

The sperm acrosome integrity rate is improved by adopting the hypertonic complexing agent diluent in the invention to preserve the sperm, the time for maintaining the equal activity of the sperm is prolonged, and the sperm is restored and then is preserved with higher movement speed, better movement shape and higher conception rate than the sperm preserved by the same period of the same isoosmotic, which is proved. The conception rate of the semen inseminating ewe preserved by the hypertonic complexing agent is measured, so that a basis can be provided for popularization and application of a semen hypertonic complexing agent preservation technology, and the semen preservation problem and popularization and application possibility of the hypertonic complexing agent used in production can be known.

Finally, it should be noted that the above contents are only used to illustrate the technical solutions of the present invention, and do not limit the protection scope of the present invention. Simple modifications and equivalents may be made by those skilled in the art without departing from the spirit and scope of the present invention.