CN102726372B

CN102726372B - Frozen semen diluent formula for flocks and herds as well as making method

Info

Publication number: CN102726372B
Application number: CN201210247522.2A
Authority: CN
Inventors: 尹旭升; 牛华星; 张淑二; 田宁宁; 霍道坦
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-07-18
Filing date: 2012-07-18
Publication date: 2014-06-11
Anticipated expiration: 2032-07-18
Also published as: CN102726372A

Abstract

The invention provides a frozen semen diluent formula for flocks and herds, and the formula of each 100mL of diluent comprises the following components: 1.35-1.55g of citric acid, 2.4-2.6g of trihydroxymethyl aminomethane (tris), 1.0-1.2g of glucose, 4.5-6.8ml of glycerol, 18.0-21ml of yolk, 4.5-7.5ml of bovine serum albumin (BSA), 0.45-0.75ml of vitamin B12, 50000-100000IU of benzylpenicillin potassium, 50000-100000IU of streptomycin sulfate and the balance of distilled water. The formula provided by the invention is low in cost, simple and convenient in preparation and using and operation steps, capable of effectively improving the sperm motility, the acrosome integrity rate and the plasmalemma integrity rate, and capable of reducing the sperm aberration rate.

Description

A kind of ox, freezing seminal fluid dilution formula and preparation method for sheep

Technical field

The present invention relates to a kind of cattle and sheep freezing seminal fluid dilution formula and preparation method.

Background technology

Requisite constituent when dilution is the semen freezing such as ox, sheep plays the effect of dilution, buffering, nutrition and protection in refrigerating process to sperm, effectively reduce the impact of low temperature on sperm.Although domestic frozen semen is made and technology of artificial insemination has been applied for many years, sperm viability after thawing of semen (35% ~ 40%) is still lower than foreign level (50% ~ 60%).Seminal fluid is after superfreeze is thawed, the vigor reduction of sperm, dead quantity and abnormal rate increase, its reason is that sperm is caught a cold to hit and caused plasmalemmae of sperms structure to change, and has a strong impact on vigor and the fertility of sperm after integrality and the recovery thereof of plasma membrane.

Summary of the invention

The object of the present invention is to provide one can effectively improve sperm viability after thawing of semen, acrosomal integrity, reduces freezing seminal fluid dilution formula and the preparation method for cattle and sheep of rate of teratosperm.

The present invention, by add a certain amount of stabilizing agent and active substance in seminal fluid, can play the effect of protection sperm effectively.The present invention adopts and adds certain density BSA(bovine serum albumin) and V _b12parenteral solution can play certain protective effect to sperm, effectively improves sperm viability and acrosomal integrity, reduces rate of teratosperm.

The present invention adopts following technical scheme:

A kind of cattle and sheep are filled a prescription with freezing seminal fluid dilution, the formula of every 100mL dilution is: citric acid 1.35 ~ 1.55g, trishydroxymethylaminomethane (tris) 2.4 ~ 2.6g, glucose 1.0 ~ 1.2g, glycerine 4.5 ~ 6.8ml, yolk 18.0 ~ 21ml, bovine serum albumin(BSA) (BSA) 4.5 ~ 7.5ml, Cobastab ₁₂0.45 ~ 0.75ml, benzylpenicillin potassium 5 ~ 100,000 IU, streptomycin sulphate 5 ~ 100,000 IU, surplus is distilled water.

A preparation method for freezing seminal fluid dilution formula for cattle and sheep, comprises the steps:

(1) preparation of basal liquid: get citric acid, trishydroxymethylaminomethane (tris), glucose, add in distilled water, be mixed with basal liquid A;

(2) preparation of yolk liquid: choose Fresh Egg, by eggshell sterilization, shell breaking, egg white under aseptic condition; The egg white on vitellinae membrana surface is blotted with sterilizing filter paper, extract yolk with asepsis injector;

(3) after basal liquid A is filtered, carry out pasteurization sterilizing 30 minutes at 65 ℃, temperature adds respectively glycerine, the yolk through sterilizing after 30 ℃, add bovine serum albumin(BSA) (BSA) and cobalamin, stir, pH value is adjusted between 6.5 ~ 7.5, be mixed with B liquid;

(4) before using, in B liquid, add respectively benzylpenicillin potassium and streptomycin sulphate, can use.

While using the present invention to fill a prescription, can:

1, according to determining required dilution addition after former smart sperm volume, vigor and density conversion.The present invention's formula can directly add in the former seminal fluid of ox, to reach the order ground of dilution.

2,, can directly this product be added several times in ox seminal fluid and be diluted during lower than 100ml for dilution addition; If when dilution addition exceedes 100ml, can add at twice, add for the first time 3～5 times of former seminal fluid volume, after the seminal fluid after residue dilution and preliminarily diluted is together put into balancing tank balance, first 30 minutes of packing, adds residue dilution in seminal fluid, then puts into balancing tank until packing.

Tool of the present invention has the following advantages and effect:

1, compared with GB dilution formula, cost is low, and preparation and use operating procedure are comparatively easy.

2, can effectively improve vigor (45% ~ 55%), the acrosomal integrity (>=68%) of sperm, plasma membrane completion rate (>=50%), reduces rate of teratosperm (≤15%, wherein sheep≤18%).

3, inseminatio externalis test shows: Sperm penetration of ova ability increases by 145%.

4, the present invention's scope of application of filling a prescription is wider, in the making of beef cattle, milk cow, goat and sheep seminal fluid, all obtain good result, its sperm viability >=45%, acrosomal integrity >=68%, plasma membrane percentage of head rice (>=50%), rate of teratosperm≤15%(sheep sperm abnormal rate≤18%).

Specific embodiment

Embodiment 1:

(1) preparation of basal liquid: get citric acid 1.353g, trishydroxymethylaminomethane (tris) 2.422g, glucose 1.1g, adds in 68.5ml distilled water, is mixed with basal liquid A.

(2) preparation of yolk liquid: choose Fresh Egg, by 75% alcohol disinfecting for eggshell, shell breaking, egg white under aseptic condition; The egg white on vitellinae membrana surface is blotted with sterilizing filter paper, extract yolk with asepsis injector.

(3) after being filtered, basal liquid A carries out pasteurization sterilizing (65 ℃ 30 minutes), temperature adds respectively the glycerine 5.0ml through sterilizing after 30 ℃, yolk 20ml, add 6ml bovine serum albumin (BSA) and 0.5ml cobalamin, even by magnetic stirrer again, pH value is adjusted between 6.5 ~ 7.5, is mixed with B liquid.

(4) before using, in B liquid, add respectively each 50,000 IU of benzylpenicillin potassium and streptomycin sulphate, can use.

(5) this formula result of use is best.

Embodiment 2:

(1) preparation of basal liquid: get citric acid 1.45g, trishydroxymethylaminomethane (tris) 2.56g, glucose 1.0g, adds in 68.4ml distilled water, is mixed with basal liquid A.

(3) after being filtered, basal liquid A carries out pasteurization sterilizing (65 ℃ 30 minutes), temperature adds respectively the glycerine 6.5ml through sterilizing after 30 ℃, yolk 18ml, add 6.5ml bovine serum albumin (BSA) and 0.6ml cobalamin, even by magnetic stirrer again, pH value is adjusted between 6.5 ~ 7.5, is mixed with B liquid.

(4) before using, in B liquid, add respectively each 7.5 ten thousand IU of benzylpenicillin potassium and streptomycin sulphate, can use.

Embodiment 3:

(1) preparation of basal liquid: get citric acid 1.55g, trishydroxymethylaminomethane (tris) 2.48g, glucose 1.2g, adds in 64.8ml distilled water, is mixed with basal liquid A.

(3) after being filtered, basal liquid A carries out pasteurization sterilizing (65 ℃ 30 minutes), temperature adds respectively the glycerine 6.0ml through sterilizing after 30 ℃, yolk 21ml, add 7.5ml bovine serum albumin (BSA) and 0.7ml cobalamin, even by magnetic stirrer again, pH value is adjusted between 6.5 ~ 7.5, is mixed with B liquid.

(4) before using, in B liquid, add respectively each 100,000 IU of benzylpenicillin potassium and streptomycin sulphate, can use.

It should be noted that in the present invention:

1, chemicals used is generally chemical pure or pure for analyzing, and all carries out dissolution filter before use.

2, the egg using should be chosen when daily output fresh hen egg, and carries out surface sterilization through alcohol, will sneak into scarcely albumen and vitellinae membrana while getting yolk.

3, glycerine must be through autoclaving before using, sterilizing pressure 0.1-0.15MPa, 20 minutes.

4, penicillin and streptomycin are with human drugs optimum.

5, the dilution after preparation should be saved in 4～6 ℃ of environment, should note sealing fresh-keeping not contaminated.

Claims

1. a preparation method for freezing seminal fluid dilution formula for cattle and sheep, is characterized in that, the formula of every 100mL dilution is:

(1) preparation of basal liquid: get citric acid 1.353g, trishydroxymethylaminomethane (Tris) 2.422g, glucose 1.1g, adds in 68.5ml distilled water, is mixed with basal liquid A;

(2) preparation of yolk liquid: choose Fresh Egg, by 75% alcohol disinfecting for eggshell, shell breaking, egg white under aseptic condition; The egg white on vitellinae membrana surface is blotted with sterilizing filter paper, extract yolk with asepsis injector;

(3) will after basal liquid A filtration, carry out pasteurization sterilizing, at 65 ℃, sterilize 30 minutes, temperature adds respectively the glycerine 5.0ml through sterilizing after 30 ℃, yolk 20ml, add 6ml bovine serum albumin (BSA) and 0.5ml cobalamin, even by magnetic stirrer again, pH value is adjusted between 6.5 ~ 7.5, be mixed with B liquid;