CN100383237C

CN100383237C - Low-temp freezing method for storing sperm of gayal

Info

Publication number: CN100383237C
Application number: CNB2004100220188A
Authority: CN
Inventors: 司维; 苏雷; 季维智
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Zoology of CAS
Priority date: 2004-03-12
Filing date: 2004-03-12
Publication date: 2008-04-23
Anticipated expiration: 2024-03-12
Also published as: CN1559440A

Abstract

The present invention relates to a low-temperature freezing method for storing semen of gayals, which belongs to the technical field of biology. In the present invention, a droplet freezing method is utilized to store the semen of the gayals by freezing; freezing liquid is prepared from 15 to 20%v/v of yolk, 3 to 6%v/v of glycerol, 1.0 to 1.5M of lactose and 1.0 to 1.5M of sucrose; antifreeze and the semen are diluted in the proportion of 1: 10, and the diluted semen is put at room temperature, balanced for one hour, transferred to a refrigerator with the temperature of 4 DEG C and balanced for three hours; the quality judging indexes of the frozen semen comprise: the unfreezing recovery rate is not less than 70%; after the semen is put in an incubator with the temperature of 37 DEG C and CO2 of 5% to be cultured for one hour when the semen is unfrozen during the survival time in vitro, the mobility is not less than 30%, and the penetration rate of naked ova of golden hamsters is not less than 38%; when the semen is cultured for one hour in 50 mOsm/kg of hypotonic solution and in a culture phase with the temperature of 38 DEG C and CO2 of 5%, the swelling rate of the semen is not less than 39%. The present invention has the advantages of practicality and reliable effect, scientific and normative provided decision methods and decision indexes for the quality of the frozen semen of the gayals, etc.

Description

Gayal seminal fluid cryogenic freezing store method

Technical field:

The present invention relates to a kind of gayal seminal fluid cryogenic freezing store method, belong to biological technical field.

Background technology:

Freezing being kept at of seminal fluid plays crucial effect in the modern livestock industry.Use frozen semen can not be subjected to time, region and the restriction in breeding stock life-span, reduce male animal and raise quantity, reduce production costs, improve the utilization ratio of good poultry kind, for livestock industry production brings huge economic benefit.Simultaneously, the use of the freezing and frozen semen of seminal fluid all has extremely important meaning in poultry kind of introduction, blood relationship renewal, disease control, endangered breed and genetic diversity protection.

Gayal (Bos frontalis) is the precious animal varieties in a kind of semi-wild, the half domestic world, has characteristics such as crude feed tolerance, strong stress resistance, fine and tender taste, be a kind ofly remain to be developed, kind that the utmost point has promotion prospect.Because no plan slaughters, quantity falls sharply, and is on the brink of extinction, is classified as the special-protection-by-the-State kind by the Ministry of Agriculture in 1997.

Gayal is because discovery is later, about research also only sees cell caryogram, origin and aspects such as evolution and genetic diversity.The inventor searches for comparison with method of the present invention and result in the pertinent literature database, find no any identical record.

Summary of the invention:

The objective of the invention is the fundamental research based on forefathers, the flavor evaluation index of a kind of suitable gayal seminal fluid cryogenic freezing preservation and frozen semen is provided, is that species conservation and utilization rationally provide useful technology.

In order to realize purpose of the present invention, the invention provides following technical scheme:

One, the cryogenic freezing of gayal seminal fluid is preserved

1) collection of seminal fluid and routine inspection: the existing method by milk cow is carried out.

2) deicing fluid configuration: yolk 15%-20% (v/v), glycerine 3%-6% (v/v), lactose 1.0M-1.5M, sucrose 1.0M-1.5M.Take by weighing lactose and sucrose according to quantity, be dissolved in the 60-70 milliliter ultrapure water.Get new fresh hen egg, with 75% ethanol disinfection eggshell, remove egg white, the yolk that is surrounded by complete vitelline membrane is placed on the clean filter paper, blot egg white on the vitelline membrane with filter paper, hold clean syringe and puncture vitelline membrane, extract yolk, yolk is added in the sugar soln by institute's expense, mix thoroughly, filter once with double gauze again, get filtrate, centrifugal 20 minutes of 10000g.Get supernatant liquor and add penicillin 100IU/ml, Vetstrep 100 μ g/ml add glycerine according to quantity.Constant volume is put in the cryogenic refrigerator and is preserved.

3) dilution of seminal fluid: deicing fluid is preheating to 37 ℃, by 1: 10 dilution (deicing fluid: seminal fluid).Seminal fluid after the dilution was placed at room temperature balance 1 hour, transferred in 4 ℃ of refrigerators balance again 3 hours.

4) the freezing and preservation of seminal fluid: freezing employing droplet vitrification.On the dry ice or be pre-chilled on the copper coin about-80 ℃ and carry out.Every about 0.2ml drops in seminal fluid on the dry ice or on the copper coin and drips in the several minutes and just can harden, and the surface becomes light, transfers at once in the liquid nitrogen after 5-7 minute, and the refrigerated seminal fluid is carried out mark, puts into liquid nitrogen and preserves.

5) thawing of frozen semen: get the 1mlPBS thawing solution and put in vitro, be preheated to 37 ℃, get a frozen semen and put into test tube and recover, shake up gently.

Two, the flavor evaluation method of frozen semen and judgement index

1) the sperm complex-velocity rate of thawing: the sperm count of living is checked in the back that thaws, and the complex-velocity rate is not less than 70%.

2) the sperm in vitro survival time: put into 37 ℃, 5%CO behind the semen thawing ₂After cultivating 1 hour in the incubator, degrees of motion is not less than 30%.

3) yellow suslik ova nuda penetration-detection (SPA): Golden Hamster is through super row, ova nuda preparation, sperm through capacitation, penetrate cultivation.Smart ovum is trained after 3-5 hour fixing altogether, observes penetration coefficient.Penetration coefficient should not be lower than 38%.Penetration coefficient be calculated as ova nuda number/ova nuda sum that penetration coefficient (%)=quilt penetrates) * 100%.

4) the hypotonic swelling test of sperm (HOS test): seminal fluid in the hypotonic solution of 50mOsm/kg, 38 ℃ 5%CO ₂Cultivated 1 hour in cultivating mutually, sperm swelling rate is not less than 39%.Sperm swelling rate (%)=(playing sperm count/total sperm count of swelling reaction) * 100%.

Gayal seminal fluid cryopreservation method of the present invention has practical and reliable for effect, advantages such as the assessment method of the gayal frozen semen quality that provides and judgement index science, standard.

Embodiment:

Embodiment one:

Gayal seminal fluid cryogenic freezing store method is finished through the steps such as flavor evaluation of the thawing of the freezing and preservation of the dilution of the collection of seminal fluid, inspection, deicing fluid configuration, seminal fluid, seminal fluid, frozen semen, frozen semen.The same prior art of part steps.Difference is:

1, the prescription of liquid is yolk 15%v/v, glycerine 3%v/v, and lactose 1.0M, sucrose 1.0M, all the other ultrapure waters are 60 milliliters;

2, the dilution of seminal fluid is for to be preheating to 37 ℃ with refrigerating fulid, and by deicing fluid: semen dilution is to dilute at 1: 10, and the seminal fluid after the dilution was placed at room temperature balance 1 hour, transfers in 4 ℃ of refrigerators balance again 3 hours;

3, the freezing employing droplet vitrification of seminal fluid, promptly on the dry ice or be pre-chilled to the seminal fluid that drips 0.2m on-80 ℃ the copper coin, seminal fluid on the dry ice or on the copper coin drips sclerosis and the surface became light after 5 minutes when dropping in, and puts into liquid nitrogen and preserves;

4, the quality judging index of frozen semen is: the complex-velocity rate of thawing is not less than 70%; The sperm in vitro survival time is put into 37 ℃, 5%CO after thawing ₂After cultivating 1 hour in the incubator, degrees of motion is not less than 30%; Golden Hamster ova nuda penetration coefficient is not less than 38%: seminal fluid in the hypotonic solution of 50mOsm/kg, 38 ℃ 5%CO ₂

Cultivated 1 hour in cultivating mutually, sperm swelling rate is not less than 39%.

Embodiment two:

Substantially with embodiment one.Difference is:

1, the prescription that freezes liquid is yolk 17.5%v/v, glycerine 4.5%v/v, and lactose 1.25M, sucrose 1.25M, ultrapure water are 65 milliliters;

2, seminal fluid on the dry ice or on the copper coin drips sclerosis and the surface became light after 6 minutes when dropping in, and puts into liquid nitrogen and preserves.

Embodiment three:

Substantially with embodiment one.Difference is:

1, the prescription that freezes liquid is yolk 20%v/v, glycerine 6%v/v, lactose 1.5M, sucrose 1.5M, 70 milliliters of ultrapure waters;

2, drip after sclerosis and surface become bright 5-7 minute when dropping in seminal fluid on the dry ice or on the copper coin, put into liquid nitrogen and preserve.

Claims

1. gayal seminal fluid cryogenic freezing store method comprises the thawing of freezing and preservation, frozen semen of dilution, the seminal fluid of collection, inspection, deicing fluid configuration, the seminal fluid of seminal fluid, the flavor evaluation step of frozen semen, it is characterized in that:

(1) prescription of deicing fluid is: yolk 15%-20%v/v, and glycerine 3%-6%v/v, lactose 1.0M-1.5M, sucrose 1.0M-1.5M, all the other are ultrapure water;

(2) dilution of seminal fluid is: dilute with seminal fluid after deicing fluid is preheating to 37 ℃, the Dilution ratio of deicing fluid and seminal fluid is 1: 10, and the seminal fluid after the dilution was placed at room temperature balance 1 hour, transfers in 4 ℃ of refrigerators balance again 3 hours;

(3) seminal fluid freezing and saving as: on the dry ice or be pre-chilled to the seminal fluid that drips 0.2ml on-80 ℃ the copper coin, drip after sclerosis and surface become bright 5-7 minute, put into liquid nitrogen and preserve when dropping in seminal fluid on the dry ice or on the copper coin;

(4) the quality judging index of frozen semen is:

A. the sperm complex-velocity rate of thawing: the sperm count of living is checked in the back that thaws, and the complex-velocity rate is not less than 70%;

B. sperm in vitro survival time: put into 37 ℃, 5%CO behind the semen thawing ₂After cultivating 1 hour in the incubator, degrees of motion is not less than 30%;

C. yellow suslik ova nuda penetration-detection: Golden Hamster is through super row, ova nuda preparation, sperm through capacitation, penetrate cultivation, smart ovum is trained after 3-5 hour fixing altogether, observes penetration coefficient, penetration coefficient should not be lower than 38%;

D. the hypotonic swelling test of sperm: seminal fluid in the hypotonic solution of 50mOsm/kg, 38 ℃ 5%CO ₂Cultivated 1 hour in cultivating mutually, sperm swelling rate is not less than 39%.