CN105055045A - Frozen semen artificial insemination method for increasing conception rate of donkeys - Google Patents

Abstract

The invention discloses a frozen semen artificial insemination method for increasing the conception rate of donkeys. The method includes the steps of fresh semen freezing treatment, frozen semen unfreezing treatment and ovulation induction timing insemination. In the process of implementation, first, collected semen of the male donkeys is diluted and placed into a full-automatic freezing instrument to automatically complete freezing, wherein the motility rate is larger than or equal to 70%; before artificial insemination is performed on the female donkeys to be pregnant, the semen is rapidly placed into a water bath of 38 DEG C to be instantly unfrozen, and the step of ovulation induction timing insemination is completed within 1 hour. By the adoption of the method, the technical measure of ovulation induction timing insemination is adopted, so that the conception rate at estrus reaches 70%.

Description

Increase the freezing smart artificial insemination method of donkey conception rate

Technical field

The present invention relates to domestic animal artificial insemination method.

Technical background

Donkey belongs to equus, in order to increase the amount of breeding, generally takes artificial insemination method.Due to the particularity of the ovarian structure of equus, after the performance that occurs oestrusing, duration of estrus, changes greatly, generally can continue 3 ~ 14 days, the ovulation period of donkey also can because of the difference of ovarian follicular growth position on ovary, ovulation period also there will be larger difference, many oestrusing 7th ~ 10 days of ovulation period, not ovulate not etc. in 12 ~ 24 hours before stopping oestrusing, this just causes donkey Inseminated time and is difficult to determine.Conventional art is by the rectal palpation to ovary, to feel that follicle softness is arrived in follicular development flexible, semen deposition is implemented time after tool fluctuation, i.e. the 3rd phase of examination per rectum and the 4th phase, but rectal palpation technology needs technical staff's experience accumulation for a long time, the judgement of different people to the follicular development stage also there are differences, even if accurately judge the stage of follicular development, also be difficult to determine ovulation period accurately, the follicle of 3 phases can maintain 1 ~ 3 day, the follicle of 4 phases also can continue 1-3 days, also be difficult to determine ovulation period, this just causes the ovulation of feelings phase and is difficult to control, the processing procedure of adding the freezing essence of donkey is improper, the motility rate of freezing essence is low, once after artificial defeated freezing essence conception rate less than 25%, need to donkey repeatedly artificial insemination could improve conception rate, waste time and energy.

Summary of the invention

In order to improve the test-tube conception rate of donkey, the object of the invention is to propose a kind of freezing smart artificial insemination method increasing donkey conception rate.

The object of the invention is to reach like this:

Increase a freezing smart artificial insemination method for donkey conception rate, it is characterized in that, it comprises thaw processing procedure and the induced ovulation timing semen deposition process of the freezing processing process of fresh essence, freezing essence, specifically:

The freezing processing process of fresh essence is as follows:

1, artificial vagina is adopted to gather the seminal fluid of jack ass, seminal fluid filters through 4 layers of antiseptic gauze of sterilizing, removing jelly, detect by an unaided eye the color of former seminal fluid, normal semen color and luster is milky white, canescence or faint yellow, get normal semen and record former semen volume, the phase contrast microscope being furnished with 38 DEG C of thermostats evaluates motility rate, getting the former row dilution that progresses greatly of motility rate >=70%; Dilution is diluted in the ratio isothermal of former essence and diluent 1:1; Diluent is by following composition proportion: Tris33-34g, citric acid 8-9g, glucose 15-16.g, glycerol 70-71mL, penicillin 50,000 unit, streptomycin 50,000 unit:

2, the fresh hard-cover after dilution enters in centrifuge tube, places 10 minutes, centrifuge leaves the heart 15 by per minute 1850 per minute, remove the supernatant in centrifuge tube, diluted by atherosclerotic seminal fluid freeze-extender in the ratio of 1:1 at 22 DEG C of constant temperature; First add 1/4 of freeze-extender amount of dilution during dilution, the seminal fluid through preliminarily diluted is placed 10 minutes at 22 DEG C, then, adds remaining diluent and dilutes completely; That dilutes completely carries out subpackage with 0.5mL tubule, then ultrasonic sealing; Straw semen after coating-dividing sealing is put into opaque plastic casing, then plastic casing put into 4 DEG C refrigerator balance 3 ~ 4 hours; Open simultaneously liquid nitrogen container valve full-automatic refrigeration instrument be cooled to 4 DEG C for subsequent use; Straw semen after balance is put into rapidly full-automatic refrigeration instrument, completes refrigerating process according to best freezing curve Automatic Program; Put into liquid nitrogen container after freezing refrigerating process to preserve; Described chill back liquor manufacture method is as follows:

Get following composition: trihydroxy trimethyl-methane 33-34g, citric acid 8-9g, glucose 15-16g, glycerol 70-71mL, penicillin 50,000 unit, streptomycin 50,000 unit are mixed with diluent, appropriate diluent is loaded graduated cylinder, the yolk of 20% parts by volume is added again in graduated cylinder, after sealed membrane sealing, to turn upside down mixing, be placed in 4 DEG C of refrigerator overnight layerings; Afterwards, remove upper foam, get diluent middle level and pour in beaker and precipitate, discard lower sediment thing; Through 4000 revs/min centrifugal 3 times, each 30 minutes, discard top layer impurity and bottom sediment thing, pour in bottle, add qs glycerin, stir with magnetic stirring apparatus after centrifugal, be placed in 4 DEG C of refrigerators and deposit for subsequent use, the resting period was no more than 24 hours.

The processing procedure of thawing of freezing essence is: before treating pregnant jenny ass artificial insemination, semen thin tube is taken out in liquid nitrogen container, be placed in rapidly that 38 DEG C of water-baths are instantaneous thaws, tubule is dried with absorbent paper after thawing, cut off the sealing of tubule without tampon one end, be arranged on spermatic gun, be then incubated, and complete semen deposition process within an hour;

Induced ovulation timing semen deposition process is: occur oestrusing in 7th ~ 10 days after showing donkey, manually rectum Ultrasonic-B probe is delivered to ovary position through donkey rectum, by the scanning plane of adjustment transrectal probe at ovary position, detect the size dimension of follicular development on ovary, when finding that follicular diameter reaches 3.4-3.5cm, lutropin or the chorionic gona dotropin of 300 units is injected to donkey, donkey time of concentration is ovulated, reduce the probability of locking in follicular development, and before ovulation 10 little up to ovulation latter 6 hours in, twice artificial insemination is implemented with spermatic gun, each semen deposition dosage 5mL, guarantee that donkey sperm and donkey ovum have met fertilization process within the time with fertility, donkey is impregnated.

The present invention adopts the technical measures of induced ovulation timing semen deposition, and good effect is as follows:

1, B ultrasonic technology for detection ovary is utilized, clearly can see the size of follicle, compared with traditional rectal palpation technology, be convenient to technical staff grasp and Technique Popularizing, do not need long experience accumulation and long-time training, avoid testing staff to the erroneous judgement in follicular development stage simultaneously, reduce the dependence to traditional rectal palpation technology.

2, after follicle reaches specific size, HORMONE TREATMENT is utilized, the probability of locking in follicular development can be reduced, the time of the ovulation of follicle generation is simultaneously relatively concentrated, can realize ovulation latter 6 hours in or ovulate first 10 hours timing carry out semen deposition, decrease the problem that traditional rectal palpation technology is difficult to determine ovulation period.

3, ensure that the activity of frozen semen; By twice semen deposition at the appointed time, ensure that donkey sperm and donkey ovum have all met fertilization within the time with fertility, while due to semen deposition number of times few, decrease the rejection in jenny ass uterus, conception rate can be improved to a certain extent, particularly frozen semen conception rate.

Here is the methods experiment Contrast on effect of the present invention and traditional rectal palpation:

Processing mode	Process quantity (head)	Non-return rate %
			Tradition rectal palpation mode	30	24.2％
The inventive method	50	70％

It can be seen from the table, the present invention is adopted to adopt the technical measures non-return rate of induced ovulation timing semen deposition to reach 70%.

Detailed description of the invention

The fresh wonderful collection that those skilled in the art describe according to summary of the invention, sterilization dilution process process and induced ovulation timing semen deposition process can be implemented, and are not described in detail at this.

Claims (1)

1. increase a freezing smart artificial insemination method for donkey conception rate, it is characterized in that, it comprises thaw processing procedure and the induced ovulation timing semen deposition process of the freezing processing process of fresh essence, freezing essence, wherein:

The freezing processing process of fresh essence is as follows:

(1) artificial vagina is adopted to gather the seminal fluid of jack ass, seminal fluid filters through 4 layers of antiseptic gauze of sterilizing, removing jelly, detect by an unaided eye the color of former seminal fluid, normal semen color and luster is milky white, canescence or faint yellow, get normal semen and record former semen volume, the phase contrast microscope being furnished with 38 DEG C of thermostats evaluates motility rate, getting the former row dilution that progresses greatly of motility rate >=70%; Dilution is diluted in the ratio isothermal of former essence and diluent 1:1; Diluent is by following composition proportion: Tris33-34g, citric acid 8-9g, glucose 15-16g, glycerol 70-71mL, penicillin 50,000 unit, streptomycin 50,000 unit:

(2) the fresh hard-cover after dilution enters in centrifuge tube, places 10 minutes, centrifuge leaves the heart 15 by per minute 1850 per minute, remove the supernatant in centrifuge tube, diluted by atherosclerotic seminal fluid freeze-extender in the ratio of 1:1 at 22 DEG C of constant temperature; First add 1/4 of freeze-extender amount of dilution during dilution, the seminal fluid through preliminarily diluted is placed 10 minutes at 22 DEG C, then, adds remaining diluent and dilutes completely; That dilutes completely carries out subpackage with 0.5mL tubule, then ultrasonic sealing; Straw semen after coating-dividing sealing is put into opaque plastic casing, then plastic casing put into 4 DEG C refrigerator balance 3 ~ 4 hours; Open simultaneously liquid nitrogen container valve full-automatic refrigeration instrument be cooled to 4 DEG C for subsequent use; Straw semen after balance is put into rapidly full-automatic refrigeration instrument, completes refrigerating process according to best freezing curve Automatic Program; Put into liquid nitrogen container after freezing refrigerating process to preserve; Described chill back liquor manufacture method is as follows:

Get following composition: trihydroxy trimethyl-methane 33-34g, citric acid 8-9g, glucose 15-16g, glycerol 70-71mL, penicillin 50,000 unit, streptomycin 50,000 unit are mixed with diluent, appropriate diluent is loaded graduated cylinder, the yolk of 20% parts by volume is added again in graduated cylinder, after sealed membrane sealing, to turn upside down mixing, be placed in 4 DEG C of refrigerator overnight layerings; Afterwards, remove upper foam, get diluent middle level and pour in beaker and precipitate, discard lower sediment thing; Through 4000 revs/min centrifugal 3 times, each 30 minutes, discard top layer impurity and bottom sediment thing, pour in bottle, add qs glycerin, stir with magnetic stirring apparatus after centrifugal, be placed in 4 DEG C of refrigerators and deposit for subsequent use, the resting period was no more than 24 hours;

The processing procedure of thawing of freezing essence is: before treating pregnant jenny ass artificial insemination, semen thin tube is taken out in liquid nitrogen container, be placed in rapidly that 38 DEG C of water-baths are instantaneous thaws, tubule is dried with absorbent paper after thawing, cut off the sealing of tubule without tampon one end, be arranged on spermatic gun, be then incubated, and complete semen deposition process within an hour;

Induced ovulation timing semen deposition process is: occur oestrusing in 7th ~ 10 days after showing donkey, manually rectum Ultrasonic-B probe is delivered to ovary position through donkey rectum, by the scanning plane of adjustment transrectal probe at ovary position, detect the size dimension of follicular development on ovary, when finding that follicular diameter reaches 3.4-3.5cm, lutropin or the chorionic gona dotropin of 300 units is injected to donkey, donkey time of concentration is ovulated, reduce the probability of locking in follicular development, and before ovulation 10 little up to ovulation latter 6 hours in, twice artificial insemination is implemented with spermatic gun, each semen deposition dosage 5mL, guarantee that donkey sperm and donkey ovum have met fertilization process within the time with fertility, donkey is impregnated.