CN101220345A - Cattle freezing seminal fluid dilution and method for producing the same - Google Patents

Abstract

The invention discloses a frozen bovine semen diluent, and the consumption amounts of all the raw materials in 100ml of the diluent are: 0.8 to 1.2g of fructose, 1.4 to 1.6g of sodium citrate, 2.3 to 2.6g of TRIS, 7.5 to 9.5g of low-density lipoprotein, 5 to 8ml of glycerol, 0.085 to 0.12 million IU of penicillin and the rest is distilled water. The preparation method is that: the fructose, the sodium citrate and the TRIS are dissolved in the distilled water to be prepared into a base liquid; penicillin G sodium and the low-density lipoprotein are added in the base liquid to prepare a I liquid; the glycerol is added in the I liquid to prepare a II liquid; then the pH value is adjusted to 6 to 7.5, then a filtration and a sterilization are carried out, and the liquid is cooled until reaching the room temperature and then arranged in a refrigerator of 2 - 5 DEG C for standby. The frozen bovine semen diluent of the invention has good and reliable effect, which can provide high-quality and excellent straw frozen semen for bovine artificial insemination and have very broad market application prospect.

Description

A kind of cow frozen semen diluent and preparation method thereof

Technical field

The invention belongs to biological technical field, the superfreeze that relates to animal semen is preserved particularly a kind of cow frozen semen diluent and preparation method thereof.

Background technology

Important role is being brought into play in being combined in the modern cattle-raising production of ox semen freezing preservation and artificial fertilization technology, use frozen semen and carry out the restriction that artificial insemination can not be subjected to time, region, cattle breeds and individual physique, and reduce the raising quantity of bull in a large number, reduce production costs, improve the utilization ratio of good species bull, for cattle-raising is brought huge economic benefit.Simultaneously, the development of ox semen freezing technology and the use of cow frozen semen are introduced at cattle breeds, crossbreeding and improvement, and raising reproductive performance, disease control and ox genetic diversity protection aspect have great importance.

The diluent composition that in the making processes of frozen semen, is absolutely necessary, the nutrition that can replenish sperm reduces the seminal fluid stickiness, reduces the influence of foreign matter to sperm, relaxes the damaging effect of meta-bolites simultaneously; Can also protect sperm in addition, strengthen its opposing low temperature and hit and oxidation resistant ability, reduce the freezing damage that causes.At present, in the making processes of frozen semen, usually select to be easy to suppress sperm motility, reduce energy expenditure, prolong the slightly acidic diluent of sperm life.Usually select the main component as diluent such as glucose, Trisodium Citrate, yolk, glycerine, microbiotic for use.The purpose of wherein adding yolk is that sperm suffers the extent of damage of cold shock in 5 ℃ of temperature-fall periods in order to be reduced in, but not all composition is all useful to sperm in the yolk, and wherein some composition can suppress the sperm breathing, reduce sperm motility rate, as high-density lipoprotein (HDL).The effective constituent that in the yolk sperm is shielded then is phosphatide and low-density lipoprotein, if substitute yolk with low-density lipoprotein in diluent, then can improve sperm quality behind the freeze-thaw.

In China, although the technology of ox semen freezing has in recent years had certain development, the frozen semen widespread use, but the cow frozen semen motility rate that uses is still very low at present, generally about 36% (China's cow frozen semen motility rate standard is 30%) is well below external employed motility rate standard (50%～60%).Because China's cow frozen semen motility rate is lower, the feelings phase breeding rate of cow is low after the artificial insemination, and conception rate is low, thereby has had a strong impact on further applying and the raising of production efficiency of China's cow frozen semen.

Summary of the invention

At the relatively low defective of motility rate behind present China ox sperm freezing, the object of the present invention is to provide a kind of cow frozen semen diluent, motility rate reaches 50%～68% effect after using the freezing ox semen thawing of this diluent, has improved China's cow frozen semen production efficiency greatly.

Realize that foregoing invention purpose technical scheme is a kind of cow frozen semen diluent, it is characterized in that the consumption of each raw material is in this 100ml diluent:

Fructose 0.8～1.2g, Trisodium Citrate 1.4～1.6g, TRIS 2.3～2.6g, low-density lipoprotein 7.5～9.5g, glycerine 5～8ml, penicillin 8.5～120,000 IU, surplus are distilled water.

Optimum formula of the present invention: the consumption of each raw material is in this 100ml diluent:

Fructose 1.0g, Trisodium Citrate 1.48g, TRIS2.42g, low-density lipoprotein 7.5～9.5g, glycerine 6.4ml, penicillin 100,000 IU, surplus are distilled water.

Low-density lipoprotein of the present invention is the yolk that is extracted from new fresh hen egg.

A further object of the invention provides the method for the above-mentioned ox sperm freezing dilution liquid of preparation, it is characterized in that, comprises the following steps:

1) it is standby to take by weighing various raw material fructose, Trisodium Citrate, TRIS, Benzylpenicillin sodium, glycerine, low-density lipoprotein, distilled water;

2) fructose, Trisodium Citrate, the TRIS with described amount is configured to basal liquid with distilled water;

3) in step 2) Benzylpenicillin sodium and the low-density lipoprotein that add described amount in the basal liquid that disposed be mixed with I liquid;

4) glycerine that adds described amount in the I liquid that step 3) disposed is made II liquid;

5) adjusting the pH value with acid or alkali is 6～7.5, carries out filtration sterilization then, is cooled to room temperature, and it is standby to put into 2～5 ℃ of refrigerators.

(preparation method of the present invention produce have only a kind of product I I liquid, but but be to use I liquid earlier in the using method of back, II liquid is used in the back, it is inconsistent to write context like this)

Illustrate: the finished product are II liquid, but at first will dispose I liquid, the front explain oneself, should be no problem.

The present invention adds low-density lipoprotein in the basic refrigerating fulid at home first; carry out the freezing preservation of ox seminal fluid; obtained good effect; the survival rate of sperm improves greatly after thawing; significantly improved semen quality; the scope of very low temperature biological study has further been expanded in this invention, for the cryoprotection material provides new approach.No matter this method still animal reproduction production aspect, all has its creativeness and practicality in the very low temperature field of biology.

Embodiment

The product preparation method embodiment, the using method embodiment that give below in conjunction with the contriver further specify beneficial effect of the present invention, but protection scope of the present invention is not limited only to this.

The extraction process of embodiment 1 low-density lipoprotein:

After fresh yolk was collected, (0.17M NaCl w/v) with 2～3 times of yolk dilutions, and constantly stirred 1h, behind the centrifugal 45min of 10,000 * g under 4 ℃ the environment, supernatant liquor is separated with throw out then to ooze salts solution with grade earlier.The interference of the small particle that contains for avoiding in the yolk, recentrifuge institute separated liquid supernatant under the similarity condition.The supernatant liquor collected after the secondary centrifuging is mixed with 40% ammoniumsulphate soln, and after fully stirring 1h under 4 ℃ the environment, the centrifugal 45min of mixture (10,000 * g, 4 ℃) makes the solution layering, with the precipitation vitellin(Vt).After centrifugal, discard throw out, supernatant liquor (floating matter) is used distill water dialysis 12h, to remove the ammonium sulfate in the solution.After ammonium sulfate is removed from solution fully, once more with solution centrifugal 45min (10,000 * g, 4 ℃), the remaining floating LDL that is, its purity generally can reach 97%.

The preparation method of embodiment 2 cow frozen semen diluents

Accurately take by weighing 1.0g fructose, 1.48g Trisodium Citrate, 2.42g TRIS with electronic analytical balance, it be dissolved in the 50ml distilled water, with magnetic stirrer evenly after, be configured to the 100ml basal liquid.The Benzylpenicillin sodium and the 8g low-density lipoprotein that add 100,000 IU in basal liquid are mixed with I liquid; The glycerine that adds 6.4ml at I liquid is mixed with II liquid.Adjusting pH with accurate pH meter again is 6.37, and the filter membrane with 0.22 μ m filters then, and it is standby to put into 2～5 ℃ of refrigerators.I, II liquid need matching while using.

The preparation method of embodiment 3 cow frozen semen diluents

Accurately take by weighing 0.5g fructose, 2g Trisodium Citrate, 1.5g TRIS with electronic analytical balance, it be dissolved in the 50ml distilled water, with magnetic stirrer evenly after, be configured to the 100ml basal liquid.The Benzylpenicillin sodium and the 5g low-density lipoprotein that add 50,000 IU in basal liquid are mixed with I liquid; The glycerine that adds 8ml at I liquid is mixed with II liquid.Adjusting pH with accurate pH meter again is 6.4, and the filter membrane with 0.22 μ m filters then, and it is standby to put into 2～5 ℃ of refrigerators.I, II liquid need matching while using.

The preparation method of embodiment 4 cow frozen semen diluents

Accurately take by weighing 1.5g fructose, 1g Trisodium Citrate, 3g TRIS with electronic analytical balance, it be dissolved in the 50ml distilled water, with magnetic stirrer evenly after, be configured to the 100ml basal liquid.The Benzylpenicillin sodium and the 10g low-density lipoprotein that add 120,000 IU in basal liquid are mixed with I liquid; The glycerine that adds 5ml at I liquid is mixed with II liquid.Adjusting pH with accurate pH meter again is 6.2～6.5, and the filter membrane with 0.22 μ m filters then, and it is standby to put into 2～5 ℃ of refrigerators.I, II liquid need matching while using.

The test example is preserved with the holstein cow semen freezing and is described.

(1) preparation of freeze-extender

Accurately take by weighing 1.0g fructose, 1.48g Trisodium Citrate, 2.42g TRIS with electronic analytical balance, it be dissolved in the 50ml distilled water, with magnetic stirrer evenly after, be configured to the 100ml basal liquid.The Benzylpenicillin sodium of interpolation 100,000 IU and the low-density lipoprotein of 8g are mixed with I liquid in basal liquid; The glycerine that adds 6.4ml at I liquid is mixed with II liquid.Adjusting pH with accurate pH meter again is 6.37, carries out filtration sterilization then, is cooled to room temperature, and it is standby to put into 2～5 ℃ of refrigerators.I, II liquid need matching while using.

(2) collection of seminal fluid

Utilize the artificial vagina acquisition method to gather bull semen, after the semen collection, immediately at 37.5 ℃ of following microscopies, observe motility of sperm, and carry out colorimetric at the 550nm place, calculate sperm concentration with spectrophotometer.The seminal fluid of being gathered is oyster white or milk yellow, free from extraneous odour, and sperm morphology is normal, and density is normal, and vigor is made the tubule frozen semen in can be used to more than 0.7.

(3) semen freezing

1. will seal the back balance 1.5～2h in 5 ℃ of refrigerators or thermostat container that finishes with the quick tubulature of seminal fluid (0.25ml tubule) of the II liquid dilution that contains glycerine.

2. after balance finished, the dress tubule was transferred to refrigeration chamber and carries out freezing.Freezing with the program frigorimeter earlier, its program is: the speed according to 3 ℃/min cooling is cooled to-6 ℃ from 5 ℃

3. after straw semen is cooled to-6 ℃ through the program frigorimeter, tubule is taken out from frigorimeter rapidly, be placed on the fast cooling from 2～3cm place, liquid nitrogen liquid top, its rate of temperature fall is-140 ℃/min, drops into liquid nitrogen freezing then rapidly and preserves.

(4) straw frozen semen thaws and semen assessment

After the tubule of superfreeze taken out rapidly from liquid nitrogen, dropped in 36～40 ℃ of waters 30 seconds, then seminal fluid is mixed with PBS liquid, under 37 ℃ temperature, evaluate the semen quality index.

1. measure the various motion correlation parameters of sperm with the colored sperm quality detection system of mighty force (WLJY-9000).Comprise: sperm motility rate, vigor, preceding tropism (STR), rectilinearity (LIN), curve speed (VCL), space rate (VSL), side-sway amplitude (ALH), whip the percentage of frequency (HZ), swing property (WOB), average path speed (VAP), average move angle (MAD) and a, b, c, d level Four sperm etc.;

2. adopt the back integrity of peanut agglutinin dyeing of FITC mark with perforatorium behind the fluorescent microscope detection freeze-thaw.Straw semen after will thawing is adjusted sperm concentration with 37 ℃ PBS solution dilution, draws 30 μ L semen smears with liquid-transfering gun, after the seasoning, with the fixing 10min of methyl alcohol, dripping the FITC-PNA dye liquor on each semen smear respectively then under the room temperature, under 37 ℃, dark wet environment, hatching.With the flushing of PBS liquid, drip optical brightener after the seasoning afterwards, covered is used colourless nail varnish mounting again.With the acrosome situation of fluorescence microscopy sperm, and take pictures with digital camera.

3. utilize hypotonic expansion test (HOST) to check the integrity of plasmalemmae of sperms.Drawing the seminal fluid sample with liquid-transfering gun mixes with hypotonic solution, after hatching 30min under 37 ℃, get 15 μ L seminal fluid samples on blood cell counting plate, 400 * inverted microscope is observed 5 visuals field of different sites down, whether presents the integrity that the end of reel is judged plasma membrane according to sperm.

(6) evaluation result

Use the freeze-thaw method of freeze-extender of the present invention and seminal fluid, its evaluation result is as follows:

1. sperm motility rate reaches more than 50～68% behind the freeze-thaw;

2. the sperm point-to-point speed reaches 30～35 μ m/s behind the freeze-thaw; The sperm rectilinearity reaches more than 55%; Sperm average path speed reaches 35～38 μ m/s; The percentage of a level sperm reaches more than 35%;

3. the acrosomal integrity of sperm reaches more than 72%;

4. the plasma membrane percentage of head rice of sperm reaches more than 55%.

Claims (4)

1. a cow frozen semen diluent is characterized in that, the content of each raw material is in this 100ml diluent:

Fructose 0.8～1.2g, Trisodium Citrate 1.4～1.6g, TRIS2.3～2.6g, low-density lipoprotein 7.5～9.5g, glycerine 5～8ml, penicillin 8.5～120,000 IU, surplus are distilled water.

2. according to the described cow frozen semen diluent of claim 1, the content of each raw material is in this 100ml diluent:

Fructose 1.0g, Trisodium Citrate 1.48g, TRIS2.42g, low-density lipoprotein 8.0g, glycerine 6.4ml, penicillin 100,000 IU, surplus are distilled water.

3. according to the described cow frozen semen diluent of claim 1, it is characterized in that described low-density lipoprotein is the yolk that is extracted from new fresh hen egg.

4. prepare the method for the described ox sperm freezing dilution liquid of claim 1, it is characterized in that, comprise the following steps:

1) it is standby to take by weighing various raw material fructose, Trisodium Citrate, TRIS, Benzylpenicillin sodium, glycerine, low-density lipoprotein, distilled water;

2) fructose, Trisodium Citrate, the TRIS with described weight is configured to basal liquid with distilled water;

3) in step 2) Benzylpenicillin sodium and the low-density lipoprotein that add described amount in the basal liquid that disposed be mixed with I liquid;

4) glycerine that adds described amount in the I liquid that step 3) disposed is made II liquid;

5) adjusting the pH value with acid or alkali is 6～7.5, carries out filtration sterilization then, is cooled to room temperature, and it is standby to put into 2～5 ℃ of refrigerators.