CN102726372A - Frozen semen diluent formula for flocks and herds as well as making method - Google Patents

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, sheep are with freezing seminal fluid dilution prescription and preparation method

Technical field

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

Background technology

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

Summary of the invention

The object of the present invention is to provide and a kind ofly can effectively improve sperm viability after the thawing of semen, acrosomal integrity, the cattle and sheep that reduce rate of teratosperm are with freezing seminal fluid dilution prescription and preparation method.

The present invention can play the effect of protection sperm effectively through in seminal fluid, adding certain amount of stabilizer and active substance.The present invention adopts and adds certain density BSA (bovine serum albumin) and V _B12Parenteral solution can play the certain protection effect to sperm, improves sperm viability and acrosomal integrity effectively, 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 prescription 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 a distilled water.

A kind of cattle and sheep comprise the steps: with the preparation method of freezing seminal fluid dilution prescription

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

(2) preparation of yolk liquid: choose new fresh hen egg, with eggshell sterilization, shell breaking, egg white under aseptic condition; Blot with the egg white of filter paper of sterilizing, extract yolk with asepsis injector with the vitellinae membrana surface;

(3) with after the basal liquid A filtration, under 65 ℃, carry out pasteurization sterilization 30 minutes, temperature is lower than glycerine, the yolk that adds respectively after 30 ℃ through sterilization; Add bovine serum albumin(BSA) (BSA) and cobalamin; Stir, pH value is adjusted between 6.5 ~ 7.5, promptly be mixed with B liquid;

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

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

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

When 2, being lower than 100ml, can directly these article being added in the ox seminal fluid several times and dilute for the dilution addition; If when the dilution addition surpasses 100ml, can add at twice, add 3～5 times of former seminal fluid volume for the first time; After the seminal fluid that will remain behind dilution and the preliminarily diluted together put into the balancing tank balance; Preceding 30 minutes of packing will remain dilution and add in the seminal fluid, put into balancing tank again until packing.

The present invention has following advantage and effect:

1, compare with GB dilution prescription, 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, the inseminatio externalis test shows: sperm is worn the ovum 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 the 68.5ml distilled water, is mixed with basal liquid A.

(2) preparation of yolk liquid: choose new fresh hen egg, with eggshell with 75% alcohol disinfecting, shell breaking, egg white under aseptic condition; Blot with the egg white of filter paper of sterilizing, extract yolk with asepsis injector with the vitellinae membrana surface.

(3) after being filtered, basal liquid A carries out pasteurization sterilization (65 ℃ 30 minutes); Temperature is lower than the glycerine 5.0ml that adds respectively after 30 ℃ through sterilization; Yolk 20ml adds 6ml bovine serum albumin (BSA) and 0.5ml cobalamin, and is even with magnetic stirrer again; PH value is adjusted between 6.5 ~ 7.5, promptly is mixed with B liquid.

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

(5) this prescription 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 the 68.4ml distilled water, is mixed with basal liquid A.

(2) preparation of yolk liquid: choose new fresh hen egg, with eggshell with 75% alcohol disinfecting, shell breaking, egg white under aseptic condition; Blot with the egg white of filter paper of sterilizing, extract yolk with asepsis injector with the vitellinae membrana surface.

(3) after being filtered, basal liquid A carries out pasteurization sterilization (65 ℃ 30 minutes); Temperature is lower than the glycerine 6.5ml that adds respectively after 30 ℃ through sterilization; Yolk 18ml adds 6.5ml bovine serum albumin (BSA) and 0.6ml cobalamin, and is even with magnetic stirrer again; PH value is adjusted between 6.5 ~ 7.5, promptly is mixed with B liquid.

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

Embodiment 3:

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

(2) preparation of yolk liquid: choose new fresh hen egg, with eggshell with 75% alcohol disinfecting, shell breaking, egg white under aseptic condition; Blot with the egg white of filter paper of sterilizing, extract yolk with asepsis injector with the vitellinae membrana surface.

(3) after being filtered, basal liquid A carries out pasteurization sterilization (65 ℃ 30 minutes); Temperature is lower than the glycerine 6.0ml that adds respectively after 30 ℃ through sterilization; Yolk 21ml adds 7.5ml bovine serum albumin (BSA) and 0.7ml cobalamin, and is even with magnetic stirrer again; PH value is adjusted between 6.5 ~ 7.5, promptly is mixed with B liquid.

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

It should be noted that among the present invention:

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

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

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

4, penicillin and streptomycin are optimum with human drugs.

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

Claims (2)

1. cattle and sheep are used the freezing seminal fluid dilution prescription, it is characterized in that, the prescription 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 a distilled water.

2. cattle and sheep as claimed in claim 1 is characterized in that with the preparation method of freezing seminal fluid dilution prescription:

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

(2) preparation of yolk liquid: choose new fresh hen egg, with eggshell sterilization, shell breaking, egg white under aseptic condition; Blot with the egg white of filter paper of sterilizing, extract yolk with asepsis injector with the vitellinae membrana surface;

(3) with after the basal liquid A filtration, under 65 ℃, carry out pasteurization sterilization 30 minutes, temperature is lower than glycerine, the yolk that adds respectively after 30 ℃ through sterilization; Add bovine serum albumin(BSA) (BSA) and cobalamin; Stir, pH value is adjusted between 6.5 ~ 7.5, promptly be mixed with B liquid;

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