CN107396918A - It is a kind of to be used to preserve dilution for hiding sheep seminal fluid and preparation method thereof for a long time - Google Patents

Abstract

The invention discloses a diluent for long-term storage of Tibetan sheep semen, which consists of the following components: double distilled water: 90-110 ml, carbohydrates: 3-5 grams, buffer: 3-4 grams, trihydroxy Methylaminomethane: 0.1 to 0.3 grams, vitamin B12: 3 to 7 milliliters, permeability protectant: 5 to 10 milliliters, egg yolk: 10 to 20 milliliters, penicillin: 80,000 to 120,000 units and streptomycin: 8 to 12 milliliters million units. Its preparation process includes the following steps: S1 A liquid configuration, S2 B liquid configuration, S3 ethylene glycol preparation, S4 semen dilution, S5 balance and S6 freezing. The effects of acrosome integrity rate and estrus conception rate, and at the same time improve the estrus conception rate of high-efficiency breeding of Tibetan sheep artificial insemination, so that the reproductive performance and economic benefits of Tibetan sheep can be significantly improved, and the effect of Tibetan sheep breed resource conservation .

Description

A diluent for long-term storage of Tibetan sheep semen and its preparation method

technical field

The invention relates to the technical field of Tibetan sheep semen diluent, in particular to a diluent for long-term storage of Tibetan sheep semen and a preparation method thereof.

Background technique

Semen cryopreservation is a key technical link in the artificial insemination technology of Tibetan sheep. It solves the problem of long-term storage of semen, makes semen not limited by time, region, and the lifespan of breeding stock, facilitates provincial and international exchanges, maximizes the use of high-quality rams, and accelerates Breeding and improvement of breeds. At the same time, it is very important to measure the descendants of excellent rams in a short period of time, to preserve and restore the characteristics of the excellent genes of Tibetan sheep breeds or individual rams, and to carry out bloodline renewal, introduction, conservation, and reduce production costs. The research on sheep frozen semen insemination in our country began in 1974, when the conception level was very low. At present, the conception rate of domestic sheep in estrus is about 60%. Because cryogenic freezing damages sheep sperm more severely than bovine and goat sperm, about 40-50% of sheep sperm are viable after freezing-thawing, but only 20-30% of sperm remain biologically intact Sex, that is, the integrity rate of sperm acrosome is less than 30%. An injured sperm can move, but not necessarily penetrate the egg membrane to enter the egg cell and fertilize it. Due to the limitations of factors such as the low acrosome integrity rate of sperm due to the cold shock, the special structure of the sheep reproductive tract, and the cost of introducing live breeding animals and some potential safety problems, Tibetan sheep, as one of the strains of sheep breeds, freeze semen Long-term preservation technology has become an important means to change this situation. The important technical problem to be solved by the present invention is to provide a semen diluent for long-term preservation of Tibetan sheep, which can improve the sperm motility, acrosome integrity rate and conception rate of semen after cryopreservation, and the most important thing is to improve the acrosome integrity rate of sperm. At the same time, it improves the conception rate of Tibetan sheep during the estrous period of high-efficiency breeding and artificial insemination, so that the reproductive performance and economic benefits of Tibetan sheep can be significantly improved, and the breed resources of Tibetan sheep can be preserved. For this reason, we invented a diluent for long-term storage of Tibetan sheep semen.

Contents of the invention

The object of the present invention is to provide a diluent for long-term preservation of Tibetan sheep semen and a preparation method thereof, so as to solve the problems raised in the above-mentioned background technology.

In order to achieve the above object, the present invention provides the following technical solution: a diluent for long-term storage of Tibetan sheep semen, consisting of the following components: double distilled water: 90-110 ml, carbohydrates: 3-5 grams, buffer : 3~4g, trishydroxymethylaminomethane: 0.1~0.3g, vitamin B12: 3~7ml, penetration protectant: 5~10ml, egg yolk: 10~20ml, penicillin: 80,000~120,000 units And streptomycin: 80,000 to 120,000 units.

Preferably, the sugars are composed of sucrose and fructose, with 3-4 grams of sucrose and 0.4-0.6 grams of fructose.

Preferably, the buffering agent is sodium citrate.

Preferably, the permeability protecting agent is ethylene glycol.

Preferably, it consists of the following components: double distilled water: 100ml, sucrose: 3.7g, fructose: 0.5g, sodium citrate: 3.7g, Tris: 0.2g, vitamin B12: 5ml, ethylene glycol Alcohol: 5 ml, egg yolk: 15 ml, penicillin: 100,000 units and streptomycin: 100,000 units.

A method for preparing a diluent for long-term storage of Tibetan sheep semen, comprising the following steps:

S1: Liquid A configuration: Take a 100ml graduated cylinder, put sucrose, sodium citrate, fructose and trishydroxymethylaminomethane into it, and add 50ml of double distilled water to it, stir with a sterilized glass rod, wait until After completely dissolving, add double distilled water to make the volume to 100ml, mix well, filter it with filter paper, put it in a triangular beaker, tie the mouth of the bottle, put it in a water bath at 75°C and boil it for 30 minutes, cool it after the end, take 85ml in In the Erlenmeyer flask, add egg yolk, penicillin and streptomycin, fully stir in a magnetic stirrer, take it out and let it stand for later use;

S2: Liquid B configuration: Take 45 ml of the spare A liquid in step S1, add 5 ml of ethylene glycol, put it in a magnetic stirrer, stir evenly, take it out and let it stand for use;

S3: Ethylene glycol preparation: Accurately measure 5 ml of ethylene glycol with a 20 ml graduated cylinder, put it in a 100 ml Erlenmeyer flask, tie the mouth of the bottle, put it in a 75°C water bath and shake gently until it completely melts;

S4: Semen dilution: collect a drop of semen on the slide, cover the slide, observe that the activity is above 0.70, pour it into a triangular beaker immediately, and slowly add liquid A at 34°C to the semen along the bottle wall for 1:1 Dilute, then shake the semen and diluent evenly in one direction, put the diluted semen triangular beaker into a container filled with 34°C water, then put the container into a 34°C water bath to heat for 10 minutes, and then according to the semen vitality , semen volume, sperm density and 20 million effective sperm counts per dose of thin tube. The sperm density meter gives the final semen dilution amount. After 10 minutes, take out the triangular beaker containing the semen together with the container, and add the remaining liquid A. The amount of liquid A added is half of the total amount of diluent added minus the amount of semen;

S5: Equilibration: put the container into a low-temperature operating cabinet at 4°C for 2-3 hours to balance. The temperature of the semen will drop to 4°C within 1.5 hours at most. Add some ice cubes to the container, and at the same time put liquid B into the low-temperature operation cabinet;

S6: Freezing: add liquid B half an hour before freezing the diluted liquid after balancing in step S5, the amount of liquid B added is the total amount of diluted liquid added minus the amount of liquid A added, and balance for half an hour after adding liquid B , and then pour into 0.25 ml thin tubes in a low-temperature cabinet, and the thin tubes after aliquoting can be frozen.

Preferably, the freezing adopts a program-controlled freezer to freeze, and the best cooling rate of the freezing curve is: 4°C～-10°C (cooling rate 5°C/min), -10°C～-100°C (cooling rate 55°C/min) ), -100°C ~ -140°C (cooling rate 20°C/min), freezing time 7 minutes.

Preferably, the extraction of the yolk is as follows: take fresh whole eggs free of disease, sterilize the surface of the eggshell with 75% alcohol cotton balls, and after the alcohol has evaporated, open a crack along the midline of the egg waist, and divide the egg into pieces. The two halves, poured alternately using the two shells, remove the whites, leaving the yolks, which are then drawn through the yolk membrane with a sterile syringe.

Compared with the prior art, the beneficial effects of the present invention are: in the present invention, the formula has the effects of improving the sperm viability, the acrosome integrity rate of sperm, and the conception rate of estrus after semen cryopreservation, and at the same time improves the high-efficiency breeding of Tibetan sheep. The conception rate of the estrous period of insemination can significantly improve the reproductive performance and economic benefits of Tibetan sheep, and make the Tibetan sheep breed resource conservation effect.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The invention provides a technical solution:

Example 1

A diluent for long-term preservation of Tibetan sheep semen, consisting of the following components: double distilled water:

90 ml, sucrose 3 g, fructose: 0.4 g, sodium citrate: 3 g, tris: 0.1 g, vitamin B12: 3 ml, ethylene glycol: 5 ml, egg yolk: 10 ml, penicillin: 8 10,000 units and streptomycin: 80,000 units.

Its preparation method comprises the following steps:

S1: Liquid A configuration: Take a 100ml graduated cylinder, put sucrose, sodium citrate, fructose and trishydroxymethylaminomethane into it, and add 50ml of double distilled water to it, stir with a sterilized glass rod, wait until After completely dissolving, add double distilled water to make the volume to 100ml, mix well, filter it with filter paper, put it in a triangular beaker, tie the mouth of the bottle, put it in a water bath at 75°C and boil it for 30 minutes, cool it after the end, take 85ml in In the Erlenmeyer flask, add egg yolk, penicillin and streptomycin, fully stir in a magnetic stirrer, take it out and let it stand for later use. The shell indicates that it should be sterilized, and after the alcohol has evaporated, a crack is opened along the midline of the egg waist, the egg is divided into two halves, and the two egg shells are poured alternately, the egg white is removed, and the yolk is left, and then passed through with a sterilized syringe Egg yolk is extracted from egg yolk membrane;

S2: Liquid B configuration: Take 45 ml of the spare A liquid in step S1, add 5 ml of ethylene glycol, put it in a magnetic stirrer, stir evenly, take it out and let it stand for use;

S3: Ethylene glycol preparation: Accurately measure 5 ml of ethylene glycol with a 20 ml graduated cylinder, put it in a 100 ml Erlenmeyer flask, tie the mouth of the bottle, put it in a 75°C water bath and shake gently until it completely melts;

S4: Semen dilution: collect a drop of semen on the slide, cover the slide, observe that the activity is above 0.70, pour it into a triangular beaker immediately, and slowly add liquid A at 34°C to the semen along the bottle wall for 1:1 Dilute, then shake the semen and diluent evenly in one direction, put the diluted semen triangular beaker into a container filled with 34°C water, then put the container into a 34°C water bath to heat for 10 minutes, and then according to the semen vitality , semen volume, sperm density and 20 million effective sperm counts per dose of thin tube. The sperm density meter gives the final semen dilution amount. After 10 minutes, take out the triangular beaker containing the semen together with the container, and add the remaining liquid A. The amount of liquid A added is half of the total amount of diluent added minus the amount of semen;

S5: Equilibration: Put the container into a low-temperature operating cabinet at 4°C for 2 hours to balance. The temperature of the semen will drop to 4°C within 1.5 hours at most. Add some ice cubes inside, and at the same time put liquid B into the low-temperature operation cabinet;

S6: Freezing: add liquid B half an hour before freezing the diluted liquid after balancing in step S5, the amount of liquid B added is the total amount of diluted liquid added minus the amount of liquid A added, and balance for half an hour after adding liquid B , then pour 0.25 milliliters of thin tubes into the low-temperature cabinet, and the thin tubes after subpackaging can be frozen. The freezing adopts a program-controlled freezer to freeze, and the best cooling rate of the freezing curve is: 4°C～-10°C (cooling Speed 5°C/min), -10°C～-100°C (cooling rate 55°C/min), -100°C～-140°C (cooling rate 20°C/min), freezing time 7 minutes.

Example 2

A diluent for long-term preservation of Tibetan sheep semen, consisting of the following components: double distilled water:

110ml, sucrose 4g, fructose: 0.6g, sodium citrate: 4g, tris: 0.3g, vitamin B12: 7ml, ethylene glycol: 15ml, egg yolk: 20ml, penicillin: 12 10,000 units and streptomycin: 120,000 units.

Its preparation method comprises the following steps:

S1: Liquid A configuration: Take a 100ml graduated cylinder, put sucrose, sodium citrate, fructose and trishydroxymethylaminomethane into it, and add 50ml of double distilled water to it, stir with a sterilized glass rod, wait until After completely dissolving, add double distilled water to make the volume to 100ml, mix well, filter it with filter paper, put it in a triangular beaker, tie the mouth of the bottle, put it in a water bath at 75°C and boil it for 30 minutes, cool it after the end, take 85ml in In the Erlenmeyer flask, add egg yolk, penicillin and streptomycin, fully stir in a magnetic stirrer, take it out and let it stand for later use. The shell indicates that it should be sterilized, and after the alcohol has evaporated, a crack is opened along the midline of the egg waist, the egg is divided into two halves, and the two egg shells are poured alternately, the egg white is removed, and the yolk is left, and then passed through with a sterilized syringe Egg yolk is extracted from egg yolk membrane;

S2: Liquid B configuration: Take 45 ml of the spare A liquid in step S1, add 5 ml of ethylene glycol, put it in a magnetic stirrer, stir evenly, take it out and let it stand for use;

S3: Ethylene glycol preparation: Accurately measure 5 ml of ethylene glycol with a 20 ml graduated cylinder, put it in a 100 ml Erlenmeyer flask, tie the mouth of the bottle, put it in a 75°C water bath and shake gently until it completely melts;

S4: Semen dilution: collect a drop of semen on the slide, cover the slide, observe that the activity is above 0.70, pour it into a triangular beaker immediately, and slowly add liquid A at 34°C to the semen along the bottle wall for 1:1 Dilute, then shake the semen and diluent evenly in one direction, put the diluted semen triangular beaker into a container filled with 34°C water, then put the container into a 34°C water bath to heat for 10 minutes, and then according to the semen vitality , semen volume, sperm density and 20 million effective sperm counts per dose of thin tube. The sperm density meter gives the final semen dilution amount. After 10 minutes, take out the triangular beaker containing the semen together with the container, and add the remaining liquid A. The amount of liquid A added is half of the total amount of diluent added minus the amount of semen;

S5: Equilibration: Put the container into a low-temperature operating cabinet at 4°C for 3 hours to balance. The temperature of the semen will drop to 4°C within 1.5 hours at most. Add some ice cubes inside, and at the same time put liquid B into the low-temperature operation cabinet;

S6: Freezing: add liquid B half an hour before freezing the diluted liquid after balancing in step S5, the amount of liquid B added is the total amount of diluted liquid added minus the amount of liquid A added, and balance for half an hour after adding liquid B , then pour 0.25 milliliters of thin tubes into the low-temperature cabinet, and the thin tubes after subpackaging can be frozen. The freezing adopts a program-controlled freezer to freeze, and the best cooling rate of the freezing curve is: 4°C～-10°C (cooling Speed 5°C/min), -10°C～-100°C (cooling rate 55°C/min), -100°C～-140°C (cooling rate 20°C/min), freezing time 7 minutes.

Example 3

A diluent for long-term preservation of Tibetan sheep semen, consisting of the following components: double distilled water:

Double distilled water: 100ml, sucrose 3.7g, fructose: 0.5g, sodium citrate: 3.7g, tris: 0.2g, vitamin B12: 5ml, ethylene glycol: 10ml, egg yolk: 15ml, Penicillin: 100,000 units and streptomycin: 100,000 units.

Its preparation method comprises the following steps:

S1: Liquid A configuration: Take a 100ml graduated cylinder, put sucrose, sodium citrate, fructose and trishydroxymethylaminomethane into it, and add 50ml of double distilled water to it, stir with a sterilized glass rod, wait until After completely dissolving, add double distilled water to make the volume to 100ml, mix well, filter it with filter paper, put it in a triangular beaker, tie the mouth of the bottle, put it in a water bath at 75°C and boil it for 30 minutes, cool it after the end, take 85ml in In the Erlenmeyer flask, add egg yolk, penicillin and streptomycin, fully stir in a magnetic stirrer, take it out and let it stand for later use. The shell indicates that it should be sterilized, and after the alcohol has evaporated, a crack is opened along the midline of the egg waist, the egg is divided into two halves, and the two egg shells are poured alternately, the egg white is removed, and the yolk is left, and then passed through with a sterilized syringe Egg yolk is extracted from egg yolk membrane;

S2: Liquid B configuration: Take 45 ml of the spare A liquid in step S1, add 5 ml of ethylene glycol, put it in a magnetic stirrer, stir evenly, take it out and let it stand for use;

S3: Ethylene glycol preparation: Accurately measure 5 ml of ethylene glycol with a 20 ml graduated cylinder, put it in a 100 ml Erlenmeyer flask, tie the mouth of the bottle, put it in a 75°C water bath and shake gently until it completely melts;

S4: Semen dilution: collect a drop of semen on the slide, cover the slide, observe that the activity is above 0.70, pour it into a triangular beaker immediately, and slowly add liquid A at 34°C to the semen along the bottle wall for 1:1 Dilute, then shake the semen and diluent evenly in one direction, put the diluted semen triangular beaker into a container filled with 34°C water, then put the container into a 34°C water bath to heat for 10 minutes, and then according to the semen vitality , semen volume, sperm density and 20 million effective sperm counts per dose of thin tube. The sperm density meter gives the final semen dilution amount. After 10 minutes, take out the triangular beaker containing the semen together with the container, and add the remaining liquid A. The amount of liquid A added is half of the total amount of diluent added minus the amount of semen;

S5: Equilibration: Put the container into a low-temperature operating cabinet at 4°C for 2.5 hours to balance. The temperature of the semen will drop to 4°C within 1.5 hours at most. Add some ice cubes inside, and at the same time put liquid B into the low-temperature operation cabinet;

S6: Freezing: add liquid B half an hour before freezing the diluted liquid after balancing in step S5, the amount of liquid B added is the total amount of diluted liquid added minus the amount of liquid A added, and balance for half an hour after adding liquid B , then pour 0.25 milliliters of thin tubes into the low-temperature cabinet, and the thin tubes after subpackaging can be frozen. The freezing adopts a program-controlled freezer to freeze, and the best cooling rate of the freezing curve is: 4°C～-10°C (cooling Speed 5°C/min), -10°C～-100°C (cooling rate 55°C/min), -100°C～-140°C (cooling rate 20°C/min), freezing time 7 minutes.

According to the present invention, the formula has the effect of improving sperm viability, sperm acrosome integrity rate and estrous conception rate after semen cryopreservation, and simultaneously improves the estrous conception rate of Tibetan sheep with high-efficiency breeding and artificial insemination, so that the reproductive performance and The economic benefits can be significantly improved, and the Tibetan sheep breed resources can be preserved effectively.

Experiment with different formulations

Tibetan sheep thin tube frozen semen diluent formula table

Statistical table of sperm motility rate and acrosome integrity rate of frozen semen in different dilutions

Conclusion: test group and control group former sperm live rate difference is not significant (P＞0.05), after thawing sperm motility, acrosome integrity rate, estrus conception rate three measure indexes, test group is all higher than contrast, and the present invention finally Due to the combined effect of buffer, nutrient (vitamin B ₁₂ ) and osmoprotectant, formula 3 has significantly higher sperm motility, acrosome integrity rate, and conception rate in estrus than formula 1, formula 2 and the control group. Significantly (p<0.01).

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (8)

1. a kind of be used to preserve the dilution for hiding sheep seminal fluid for a long time, it is characterised in that is made up of following deal component：Double distillations Water：90~110 milliliters, carbohydrate：3~5 grams, buffer：3~4 grams, trishydroxymethylaminomethane：0.1~0.3 gram, vitamin B12：3~7 milliliters, permeability protective agent：5~15 milliliters, yolk：10~20 milliliters, penicillin：8~120,000 units and strepto- Element：8~120,000 units.

A kind of 2. dilution for being used to preserve Tibetan sheep seminal fluid for a long time according to claim 1, it is characterised in that：The carbohydrate It is made up of sucrose and fructose, and 3~4 grams of sucrose, 0.4~0.6 gram of fructose.

A kind of 3. dilution for being used to preserve Tibetan sheep seminal fluid for a long time according to claim 1, it is characterised in that：The buffering Agent is sodium citrate.

A kind of 4. dilution for being used to preserve Tibetan sheep seminal fluid for a long time according to claim 1, it is characterised in that：The infiltration Property protective agent is ethylene glycol.

5. a kind of dilution for being used to preserve Tibetan sheep seminal fluid for a long time according to claim 1, it is characterised in that by following part Measure component composition：Double distilled water：100 milliliters, 3.7 grams of sucrose, fructose：0.5 gram, sodium citrate：3.7 grams, trihydroxy methyl amino Methane：0.2 gram, vitamin B12：5 milliliters, ethylene glycol：10 milliliters, yolk：15 milliliters, penicillin：100000 units and streptomysin: 100000 units.

6. a kind of preparation method for being used to preserve the dilution for hiding sheep seminal fluid for a long time according to claim 1, its feature exist In：Comprise the following steps：

S1：A liquid configures：100 milliliters of graduated cylinder is taken, sucrose, sodium citrate, fructose and trishydroxymethylaminomethane are put into it In, and 50 milliliters of double distilled water is added thereto, stirred with the glass bar sterilized, add double distilled water again until completely dissolved 100 milliliters are settled to, is filtered after mixing with filter paper in conical beaker, ties bottleneck, 75 DEG C of water-bath interval boiling boilings is put and disappears Poison 30 minutes, cools down after terminating, takes 85 milliliters in conical flask, adds yolk, penicillin and streptomysin, is put into magnetic agitation It is sufficiently stirred in device, takes out stand for standby use；

S2：B liquid configures：Take 45 milliliters of A liquid standby in step S1 to add 5 milliliters of ethylene glycol to be put into magnetic stirring apparatus, uniformly Stirring, take out set aside for use；

S3：Ethylene glycol prepares：5 milliliters of ethylene glycol is accurately measured with 20 milliliters of graduated cylinders, in 100 milliliters of conical flasks, and is tied Bottleneck, put gently rocked to it in 75 DEG C of water-baths melt completely it is standby；

S4：Semen dilution：Collecting semen drop one is dropped in load fragment, and close the lid fragmentation, and observation vigor falls at once more than 0.70 Enter in conical beaker, be slowly added into 34 DEG C of A liquid along bottle wall in seminal fluid and carry out 1：1 dilution, then, shakes toward the direction Even seminal fluid and dilution, diluted seminal fluid conical beaker are put into the container for filling 34 DEG C of water, then container is put into 34 DEG C of water Heated in bath 10 minutes, effective sperm count 2000 is then ensured according to sperm viability, semen volume, sperm concentration and every dose of tubule Ten thousand, sperm concentration instrument provides final semen dilution amount, takes the conical beaker equipped with seminal fluid for one piece together with container after 10 minutes Go out, then add the amount of remaining A liquid, the amount of added A liquid is that added dilution total amount half subtracts semen volume；

S5：Balance：Container is put into 4 DEG C of low-temperature operation cabinet to balance 2~3 hours, the temperature of seminal fluid is at most in 1.5 hours 4 DEG C are dropped to, if cooling is too slow, some ice cubes can be added in container, at the same time when seminal fluid temperature drops to 15 DEG C B liquid is also placed in low-temperature operation cabinet；

S6：Freezing：By the dilution after being balanced in step S5, half an hour addition B liquid before freezing, the amount of added B liquid is added dilute The amount that liquid total amount subtracts added A liquid is released, half an hour is balanced after adding B liquid, 0.25 milliliter of tubule is then poured into low-temperature cabinet, point Tubule after dress can be freezed.

7. a kind of preparation method for being used to preserve the dilution for hiding sheep seminal fluid for a long time according to claim 6, its feature exist In：The freezing is freezed using program-controlled freezing instrument, and the optimal rate of temperature fall of freezing curve is：4 DEG C~-10 DEG C (5 DEG C of rate of temperature fall/ Min), -10 DEG C~-100 DEG C (55 DEG C/min of rate of temperature fall), -100 DEG C~-140 DEG C (20 DEG C/min of rate of temperature fall), during freezing Between 7 minutes.

8. according to a kind of preparation method for being used to preserve the dilution for hiding sheep seminal fluid for a long time as claimed in claim 6, its feature It is：The yolk take for：The fresh complete egg of no epidemic disease is taken, eggshell is shown to carry out disinfection with 75% cotton ball soaked in alcohol and treated A crackle is knocked open along egg waist median line after alcohol volatilization is clean, divide egg one to two halves, alternately toppled over using two eggshells, removed Egg white is removed, leaves yolk, then extracts yolk through membrane of yolk with the syringe of sterilizing.