CN114767321A - Artificial semen collection and insemination method for Qinling mountain pandas - Google Patents

Abstract

The invention belongs to the technical field of propagation of Qinling mountain pandas, and particularly relates to an artificial semen collection and insemination method of Qinling mountain pandas. The method comprises the following steps: step 1: anesthetizing and fixing male Qinling pandas, inspecting reproductive organs, and collecting sperms by adopting a rectal electrical stimulation method; step 2: checking the quality of the semen of the male pandas collected in the step 1; and step 3: freezing the semen particles of the male pandas in Qinling mountain checked in the step 2, or freezing and storing the semen particles in a thin tube or freezing and storing a test tube; and 4, step 4: and (4) carrying out artificial insemination on the female Qinling mountain pandas by using the semen of the male Qinling mountain pandas stored in the step (3). The artificial insemination is carried out by artificially collecting and freezing the sperm of the giant pandas, unfreezing the frozen sperm at the estrus of the female giant pandas for artificial insemination, and the short insemination time is held, so that the insemination success rate is improved.

Description

Artificial semen collection and insemination method for Qinling mountain pandas

Technical Field

The invention belongs to the technical field of Qinling mountain panda breeding, and particularly relates to an artificial semen collection and insemination method for Qinling mountain pandas.

Background

Qinling mountain is the distribution area with the highest latitude of Chinese pandas, and the population density is the first nationwide. The Qinling mountain pandas are important species groups of the pandas and have unique distribution patterns and species evolution history. The newborn bred by the Qinling mountain panda grows quite immature at birth, the weight is only 0.1 percent of the weight of the mother, the weight of the newborn baby is very light, the average birth weight is about 145 grams, the weight is about one thousandth of that of the adult panda, the lightest weight is 51 grams, the heaviest weight is 225 grams, and the growth cycle is very slow. Most importantly, the self reproductive capacity of the giant pandas in the Qinling mountain is very weak, the natural estrus is only once a year, and the active mating time is 1-3 days, so that the reproductive rate of the giant pandas is further reduced.

Therefore, in order to improve the reproduction rate of pandas, artificial insemination and insemination of pandas are required, so that the reproduction rate of the pandas in the Qinling mountains can be effectively improved, and better species of the pandas in the Qinling mountains can be bred.

Disclosure of Invention

Aiming at the technical problems, the invention provides an artificial insemination method for giant pandas in Qinling mountains, which comprises the steps of artificially collecting the semen of the giant pandas, freezing the semen, unfreezing the frozen semen during the estrus of the female giant pandas to carry out artificial insemination, grasping the short insemination time and improving the insemination success rate.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

an artificial semen collection and insemination method for Qinling mountain pandas comprises the following steps:

step 1: anesthetizing and fixing male Qinling pandas, inspecting reproductive organs, and collecting sperms by adopting a rectal electrical stimulation method;

and 2, step: checking the quality of the semen of the male pandas collected in the step 1;

and step 3: freezing or freezing and storing the male Qinling panda semen particles checked in the step 2 by a thin tube;

and 4, step 4: and (4) carrying out artificial insemination on the female Qinling mountain pandas by using the semen of the male Qinling mountain pandas stored in the step (3).

Preferably, the specific operation of step 1 is:

step 1.1: fasting male Qinling pandas in estrus for 12 hours to promote food in stomach to be emptied;

step 1.2: 4mg/kg-8mg/kg of ketamine is used, and 0.3mg/kg-0.5mg/kg of isoflurane or 0.3mg/kg-0.5mg/kg of pentone is used as an auxiliary for intramuscular injection anesthesia on the neck or the hip of a panda;

step 1.3: checking the reproductive organs of the anesthetized male Qinling pandas in the step 1.2;

step 1.4: cleaning dirt in the foreskin of male Qinling mountain panda with clean gauze, cleaning the inside and outside of the foreskin with 0.1% benzalkonium bromide at 37 ℃, and finally washing with sterilized normal saline at 37 ℃ until no disinfectant residue exists;

step 1.5: connecting an electrical stimulation semen collector, slowly increasing voltage from low to high according to hindlimb contraction degree of male Qinling mountain panda, and continuously stimulating rectum of male Qinling mountain panda for 20-30 times, each time for 2s, until semen flows out;

step 1.6: if the semen is not stimulated or the semen amount is too small in the 1 st time, repeating the step 1.5 for 1 time after 5 min;

step 1.7: holding the semen collecting cup at 38 deg.C, and collecting semen quickly after semen flows out until semen does not flow out.

Preferably, the semen quality inspection of male Qinling mountain pandas in step 2 comprises: the method comprises the following steps of semen odor examination, semen density examination, semen pH value examination, semen sperm motility examination, semen sperm acrosome integrity examination, semen sperm morphology examination and semen sperm plasma membrane integrity examination.

Preferably, the semen sperm acrosome integrity check of step 2 is: aspirate 1. mu.L of seminal fluid + 9. mu.L of LPOPE stain, stain for 90s, smear, and view and rate with 100 Xoil-scope after smear is dry.

Preferably, the particle freezing operation of step 3 is:

step 3.1.1: diluting the semen of the male Qinling mountain panda checked to be qualified in the step 2 by using a frozen diluent, wherein the ratio of the semen to the diluent is 1: 3;

step 3.1.2: balancing the panda semen diluted in the step 3.1.1 in a refrigerator at 4 ℃ for 4 hours.

Step 3.1.3: soaking the frozen semen plate in a metal tank filled with liquid nitrogen for 3 times, lifting the frozen semen plate to a position 5cm away from the liquid nitrogen surface, measuring the temperature of the frozen semen plate by using a thermometer, and quickly dropping the balanced semen on the frozen semen plate by using a micropipette at 40-80 mu L/grain when the temperature is reduced to-80 ℃.

Step 3.1.4: and 3.1.3, after finishing the step for 2min, quickly immersing the frozen semen plate into liquid nitrogen at the temperature of-196 ℃, putting the frozen semen into a glass vial by using balanced tweezers, and putting the glass vial into a liquid nitrogen tank for long-term storage.

Preferably, the capillary freezing operation of step 3 is:

step 3.2.1: diluting the semen of the male panda from Qinling mountain checked and qualified in the step 2 by TEST or SFS in a ratio of 1: 3;

step 3.2.2: and (4) balancing the panda semen diluted in the step 3.1 in a refrigerator at 4 ℃ for 4 hours.

Step 3.2.3: and 3.2.2, sucking the balanced panda semen into a thin plastic tube, wherein each tube is 0.25mL, one end of each tube is plugged with cotton, the other end of each tube is sealed after the semen is injected, and the tubes are sealed by melting the plastic by using a pair of tongs which are heated on an alcohol lamp.

Step 3.2.4: placing the thin plastic tube sealed in the step 3.2.3 on the upper layer of a balanced test tube rack, wherein the temperature is-20 ℃, and the distance from the liquid nitrogen is 7.5cm, and the time is 1 min; then moving the thin plastic tube to the lower layer of the test tube rack, wherein the thin plastic tube is 2.5cm away from liquid nitrogen at the moment, and the temperature is minus 80 ℃ for 1 min; and finally, putting the thin plastic tube into liquid nitrogen for 2min, then putting the thin plastic tube into a paper tube, and putting the paper tube into a liquid nitrogen tank for long-term storage.

Preferably, the test tube freezing operation of step 3 is:

step 3.3.1: adding 200 mu L of collected panda semen into 400 mu L of diluent;

step 3.3.2: putting the diluted panda semen into a 1.5mL plastic test tube, and balancing for 4h in a refrigerator at 4 ℃;

step 3.3.3: and (4) placing the plastic test tube after the balance in the step 3.3.2 on a flat plate 7.5cm high from liquid nitrogen for freezing for 10min, and then placing the flat plate in the liquid nitrogen for long-term storage.

Preferably, the specific operations of step 4 are:

step 4.1: cleaning vulva and anus of female Qinling giant panda with 0.1% benzalkonium bromide at 37 deg.C, and washing with sterilized normal saline at 37 deg.C until no disinfectant residue exists;

and 4.2: the ball-type glass sperm inseminating device is used for inseminating the particle frozen semen, and the metal sperm inseminating device is used for inseminating the thin-tube frozen semen;

step 4.3: a measuring cylinder-shaped cavity opener made of transparent glass is inserted into a port smooth part at the vaginal end of a female Qinling mountain panda;

step 4.4: inserting the inseminating syringe into the cavity opener, and slowly injecting semen;

step 4.5: making artificial insemination records, and accurately recording the information of females and males, the information of used semen, the information of panda from semen source, date and insemination times in detail.

Compared with the prior art, the invention has the beneficial effects that:

1. fingers lubricated by paraffin oil extend into the rectum to repeatedly massage the abdominal area of the vas deferens, so that the semen collection amount and the semen collection survival rate can be improved, and the semen is prevented from not being collected due to intestinal electrical stimulation;

2. the stepped temperature cryopreservation is adopted, so that the reduction of sperm activity caused by freezing is avoided, and the success rate of cryopreservation is improved;

3. the invention has complete integral technology, can effectively improve the success rate of sperm collection and insemination of pandas, and further improves the breeding rate of the pandas on the Qinling mountain.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

In the drawings:

FIG. 1 is a flow chart of the method of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.

Example (b):

referring to the attached figure 1, the method for artificial semen collection and insemination of Qinling mountain pandas is a flow chart of the method of the invention, and comprises the following steps:

step 1: anesthetizing and fixing male pandas in Qinling mountain, examining reproductive organs, collecting sperms by adopting a rectal electrical stimulation method, and specifically operating as follows:

step 1.1: adult (5.5-20 years old), healthy male Qinling mountain pandas in estrus season (usually 2-5 months in spring) and normal physique. The male panda in Qinling mountain should be bred in the field for more than 30 days, has a population genetic profile established according to LY/T2896-2017, and has detailed and complete basic data, immunization, insect expelling and inspection and quarantine records. Under the natural state of male Qinling mountain pandas, the sports, mental, respiratory and dietary conditions are regularly observed, and the feces condition is checked.

When male Qinling mountain pandas normally estre, the physiological phenomena of regularly or regularly discharging white or yellow viscous liquid can occur, and transient estrus characteristics such as increased activity, changeable cry, swollen testis and the like can also occur. The estrus of male Qinling pandas can last about 30 days. Then fasting male Qinling pandas in estrus for 12 hours to promote food in stomach to be emptied;

step 1.2: the semen collection place has sufficient space, is quiet and warm, and has skid-proof, clean and tidy ground. Checking whether the electrical stimulation semen collector operates normally, selecting disposable sterile package or cleaning and sterilizing all the tools contacting semen, and placing the semen collecting cup in a thermostat at 38 ℃ for later use. All equipment and appliances are required to be sufficiently and properly stored and regularly placed 1 day before semen collection.

Then 4mg/kg-8mg/kg of ketamine is used, and 0.3mg/kg-0.5mg/kg of isoflurane or 0.3mg/kg-0.5mg/kg of entacapone is used as an auxiliary for carrying out intramuscular injection anesthesia on the neck or the hip of the pandas. After anesthesia, panda heart rate, respiratory rate and body temperature were measured. And after the pandas are in a stable anesthesia state, fixing the limbs of the pandas on a veterinarian fixing operating table, and executing subsequent semen collection operation.

Step 1.3: and (3) inspecting reproductive organs of the anesthetized male pandas in Qinling mountain in the step 1.2, touching testicles on two sides of the anesthetized pandas with hands, inspecting the sizes, observing whether red swelling and nodules exist or not, observing whether trauma exists in scrotum or not, and recording.

Step 1.4: an operator searches pudendum of a male panda to be sperm collected, moderately trims the pudendum when the pubic hair is too long, uses clean gauze to clean dirt in the foreskin of the male Qinling panda, uses 0.1% benzalkonium bromide at 37 ℃ to clean the inside and the outside of the foreskin for multiple times, and finally uses sterilized normal saline at 37 ℃ to wash the foreskin until no disinfectant residue exists;

step 1.5: the method comprises the steps of clearing accumulated feces in the intestines of male Qinling pandas by using a conventional enema method, and repeatedly massaging the ampulla region of the vas deferens for 200 times and 400 times by stretching a finger lubricated by paraffin oil into the rectum, so that the semen collection amount and the semen collection survival rate can be improved, and the semen is prevented from not being collected due to intestinal electrical stimulation.

Then, an electric stimulation semen collector is connected according to the use instruction, a rectum probe (the diameter is 2.6mm) with 3 stainless steel electrodes (the length is 6.5cm, the width is 0.3cm, and the electrodes are arranged at 45 degrees, 90 degrees and 135 degrees) and a 60Hz sine wave stimulator is used for continuously stimulating the rectum of the male panda for 20-30 times (2 s each time) under the condition of low voltage (2V-8V) according to the hind limb contraction degree of the male panda, and the voltage is slowly increased from low to high until the semen flows out.

Step 1.6: if no or too little semen is stimulated at the 1 st time, repeat the 1.5 steps 1 time after 5min interval.

Step 1.7: an operator wears the double-layer glove and holds the semen collecting cup preheated to 38 ℃, and after semen flows out, the operator quickly collects the semen until the semen does not flow out. The temperature of the semen collection cup is controlled between 38 ℃ and 40 ℃, and if the ambient temperature is too low, the temperature can be properly increased, and the maximum temperature cannot exceed 42 ℃. And closing the semen collection cup after collecting the semen, placing the cup into a 37 ℃ water bath box for short time storage to be tested, and making a semen collection record.

After semen collection, keeping the semen collection place quiet and warm, and continuously observing until the male pandas naturally revive. If the patient does not wake up within 60min, the relevant veterinarian is informed to check in time.

And 2, step: the quality inspection of the male Qinling mountain panda semen collected in the step 1, the same inspection of taking out the frozen semen for artificial insemination is also carried out, and the method mainly comprises the following steps: the method comprises the following steps of semen odor examination, semen density examination, semen pH value examination, semen sperm motility examination, semen sperm acrosome integrity examination, semen sperm morphology examination and semen sperm plasma membrane integrity examination.

Firstly, preparing to use the apparatus, placing all instruments in contact with the semen in a constant temperature box at 32-37 ℃, placing the diluent in a constant temperature water bath box at 32-35 ℃ for standby, and paying attention to the ambient temperature in the checking process. The metering equipment (thermometer, balance, pipettor, etc.) is accurate, checked periodically and calibrated. And observing whether the semen collecting cup is complete and has no damage, whether the color and luster of the semen are normal, and whether purulent secretion, blood and other foreign matters and dirt are mixed. The inspection of fresh sperm should be controlled within 80min, and the inspection of frozen sperm should be controlled within 120 min.

2.1 semen collection measurement

2.1.1 semen weight determination

Weighing the semen collecting cup in advance, placing the semen collecting cup containing semen on an electronic balance, weighing (to the accuracy of 0.1g), and subtracting the two weighing to obtain the semen collecting weight.

2.1.12 semen volume determination

The volume of the semen is directly measured by a micropipette.

2.3 semen odor examination

The normal panda semen has slight fishy smell, and the abnormal semen with abnormal smell is abnormal semen.

2.4 semen Density examination

Semen density was directly observed under an optical microscope using a blood cell counting plate.

2.5 semen pH examination

Taking a small amount of semen, and detecting by using pH test paper or a pH meter, wherein the normal semen is alkalescent.

2.6 sperm motility test

A small amount of semen is placed on a glass slide which is kept at 37 ℃, and all the sperm which can move forward are viable sperm and are expressed in percentage.

2.7 sperm motility test

Dropping 1 drop of semen on a glass slide with the temperature of 37 ℃, dropping the same volume of Pap staining solution beside the glass slide, mixing the staining solution with the glass slide, flatly coating the staining solution, observing and counting the staining solution under a microscope after the staining solution is dried, wherein white sperms are live sperms, pink sperms are dead sperms, and the sperm motility rate is expressed by percentage.

2.8 semen sperm motility State examination

The sperm motility was observed and evaluated under a microscope and represented by 0 to 5: 0 is inactive; 1 is chatter; 2, turning; 3, the curve moves forwards; 4, linear motion; 5 is a very fast linear motion. The proportion of sperm in each grade is expressed as a percentage.

2.9 semen sperm acrosome integrity check

Pipette 1. mu.L of semen + 9. mu.L of the OPE stain, stain for 90s and smear. After the smear was dry, it was viewed with 100 x oil scope and rated: A. the normal acrosome is dark purple; B. the damaged top body is also dark purple, but the surface of the top is uneven and jagged; C. the head is missing and the top is white; D. the top body is fluffy, the top is white, the surface of the top is uneven, and the damage to the top body is the most serious. The proportion of sperm in each grade is expressed as a percentage.

2.10 sperm morphology examination

mu.L of semen was added to 200. mu.L of 0.3% glyceraldehyde as a solid, and a small amount was put on a slide and observed with an oil mirror.

2.11 sperm plasma Membrane integrity check

A hypotonic swelling experiment is adopted, a small amount of seminal fluid is added into a proper amount of hypotonic solution, if the plasma membrane of the sperms is complete, water enters the sperms to increase the volume of the sperms, swell the acrosomes and bend the tails, and the tail bending rate is calculated and expressed by percentage.

And truly, completely and accurately recording and storing the inspection result, discarding unqualified semen, and reserving qualified semen to execute subsequent operation.

And 3, step 3: and (3) freezing and storing the male Qinling panda semen particles checked in the step (2) or freezing and storing the male Qinling panda semen particles in a thin tube.

The particle freezing operation is specifically as follows:

step 3.1.1: diluting the semen of the male Qinling mountain panda checked to be qualified in the step 2 by using a frozen diluent, wherein the ratio of the semen to the diluent is 1: 3;

step 3.1.2: and (3) balancing the diluted panda semen obtained in the step (3.1.1) in a refrigerator at 4 ℃ for 4 hours.

Step 3.1.3: soaking the frozen semen plate in a metal tank filled with liquid nitrogen for 3 times, lifting the frozen semen plate to a position 5cm away from the liquid nitrogen surface, measuring the temperature of the frozen semen plate by using a thermometer, and quickly dropping the balanced semen on the frozen semen plate by using a micropipette at 40-80 mu L/grain when the temperature is reduced to-80 ℃.

Step 3.1.4: and 3.1.3, after finishing the step for 2min, quickly immersing the frozen semen plate into liquid nitrogen at the temperature of 196 ℃ below zero, filling the frozen semen into glass vials by using balanced tweezers, and putting the glass vials into a liquid nitrogen tank for long-term storage.

The specific operation of the thin tube freezing is as follows:

step 3.2.1: diluting the semen of the male Qinling panda checked to be qualified in the step 2 by TEST or SFS in a ratio of 1: 3;

step 3.2.2: balancing the panda semen diluted in the step 3.1 in a refrigerator at 4 ℃ for 4 hours.

Step 3.2.3: and 3.2.2, sucking the balanced panda semen into a thin plastic tube, wherein each tube is 0.25mL, one end of each tube is plugged with cotton, the other end of each tube is sealed after the semen is injected, and the tubes are sealed by melting the plastic by using a pair of tongs which are heated on an alcohol lamp.

Step 3.2.4: placing the thin plastic tube sealed in the step 3.2.3 on the upper layer of a balanced test tube rack at the temperature of minus 20 ℃ and 7.5cm away from liquid nitrogen for 1 min; then moving the thin plastic tube to the lower layer of the test tube rack, wherein the thin plastic tube is 2.5cm away from liquid nitrogen at the moment, and the temperature is minus 80 ℃ for 1 min; and finally, putting the thin plastic tube into liquid nitrogen for 2min, then putting the thin plastic tube into a paper tube, and putting the paper tube into a liquid nitrogen tank for long-term storage.

The test tube freezing operation of the step 3 is as follows:

step 3.3.1: adding 200 mu L of collected panda semen into 400 mu L of diluent;

step 3.3.2: putting the diluted panda semen into a 1.5mL plastic test tube, and balancing for 4h in a refrigerator at 4 ℃;

step 3.3.3: and (4) placing the plastic test tube after the balance in the step 3.3.2 on a flat plate 7.5cm high from liquid nitrogen for freezing for 10min, and then placing the flat plate in the liquid nitrogen for long-term storage.

The label for freezing the panda semen needs to record the source, basic information, genetic background, date, content, sperm quality, freezing method and the like of the semen in detail and keep files.

The liquid nitrogen tank filled with the giant panda frozen semen is required to be placed in a flat, light-proof and safe place. The liquid nitrogen tank is responsible for checking the residual amount of liquid nitrogen in the liquid nitrogen tank and supplementing the liquid nitrogen in time. And making a record. The frozen semen should be transported in a liquid nitrogen tank with sufficient liquid nitrogen, and strong vibration is avoided during transportation.

And 4, step 4: and (3) carrying out artificial insemination on the female Qinling pandas by using the male Qinling panda semen stored in the step (3), and specifically comprising the following steps:

preparation:

healthy, unprogged female pandas aged sexually mature (wild 7.5 years, housed 4.5 years) and in the estrus season (typically 2-5 months in spring). The female giant pandas bred in the captive field are more than 30 days, have population genetic files established according to LY/T2896-.

For the clinical examination of female Qinling mountain pandas, the conditions of sports, spirits, respiration and diet are regularly observed, and the condition of excrement is examined. When pandas are normal, the physiological phenomenon that white or yellow viscous liquid is regularly or regularly discharged occurs.

The estrus identification of female Qinling mountain pandas is generally carried out in 2-5 months in spring every year, and transient estrus characteristics of the female pandas, such as cry change, olfactory mark, vulva change, activity increase and the like, can be observed. The estrus climax of female pandas is 2-6 days, and the optimal hybridization time is 12 hours.

The preparation method comprises the steps of preparing the female Qinling mountain pandas before the artificial insemination and preparing the artificial insemination site, wherein the preparation requirements and the preparation process are the same as those of the male Qinling mountain pandas.

All insemination devices that come into contact with semen and the reproductive tract of female pandas should be carefully cleaned and sterilized.

Comparing the collected panda semen strain information with the to-be-matched female panda population genetic file established according to LY/T2896-.

The same operation as that of the male Qinling mountain panda, the anesthesia of the female Qinling mountain panda is maintained, and then the female Qinling mountain panda keeps the supine position and slightly moves forward, backward and high.

Checking the reproductive organs of female Qinling mountain pandas, checking whether the vulva of the female panda to be matched is normal or not and whether a large amount of abnormal secretion exists or not.

And (3) unfreezing the semen of the frozen male Qinling mountain pandas, wherein the frozen semen of the male Qinling mountain pandas can be unfrozen at any time. And (3) if the frozen semen is used for insemination, putting the frozen semen into a water bath at 37 ℃ for shaking and unfreezing for 45-60s, then diluting the semen into the unfrozen liquid, taking a small amount of semen to check the quality, and the checking operation is the same as the step 2.

Step 4.1: an operator searches the pudendum of a female panda, cleans the pudendum and the anus of the female Qinling panda by using 0.1% New benzalkonium bromide at 37 ℃, and then washes the pudendum and the anus by using sterilized normal saline at 37 ℃ until no disinfectant remains;

step 4.2: the ball-type glass semen deposition device is used for depositing semen of particle frozen semen, before depositing semen, a rubber head is arranged at the rear end of the semen deposition device, and the rubber head is pinched by hands to absorb the semen.

The metal sperm feeder is used for the sperm feeding of the tubule frozen sperm, the tubule frozen sperm is cut off, the mouth is cut off, and the section is uniform. The seminal fluid with the sealed tubule cut off is quickly filled into a vas deferens. The method comprises the following steps: the front end is cut off, the push rod of the sperm feeder is pushed backwards, the thin tube is arranged in the tube, the sperm feeder tube enters the plastic outer sleeve, the tube opening tightly pushes the fixing ring in the outer sleeve, the sperm feeder tube is pushed forwards to the head, the rear part of the outer sleeve is screwed with the thread at the rear part of the sperm feeder, and the two parts are tightly combined.

Step 4.3: finding the pudendum of a female Qinling giant panda, and inserting a measuring cylinder-shaped cavity opener made of transparent glass into the port smooth part at the pudendum end of the female Qinling giant panda;

step 4.4: inserting the inseminating syringe into vagina of female giant panda, wherein the position is not easy to be too deep or too shallow, and the inseminating syringe is slightly retreated by about 0.5cm before the inseminating syringe is injected into the semen, and the inseminating syringe is slowly injected into the semen. Preventing and treating the semen suck-back before the vas deferens is drawn out.

The time and the times of artificial insemination need to be about 2-3d according to the specific situation of female individuals.

Step 4.5: making artificial insemination records, and accurately recording the information of females and males, the information of used semen, the information of panda from semen source, date and insemination times in detail.

After artificial insemination, keeping the place quiet and warm, and continuously observing until the female Qinling mountain panda revives naturally. If the patient does not wake up within 60min, the relevant veterinarian is informed to check in time.

The foregoing shows and describes the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (8)

1. An artificial semen collection and insemination method for Qinling mountain pandas is characterized in that: the method comprises the following steps:

step 1: anesthetizing and fixing male Qinling pandas, inspecting reproductive organs, and collecting sperms by adopting a rectal electrical stimulation method;

step 2: checking the quality of the male Qinling panda semen collected in the step 1;

and step 3: freezing the semen particles of the male pandas in Qinling mountain checked in the step 2, or freezing and storing the semen particles in a thin tube or freezing and storing a test tube;

and 4, step 4: and (4) carrying out artificial insemination on the female Qinling mountain pandas by using the semen of the male Qinling mountain pandas stored in the step (3).

2. The artificial insemination and insemination method for the Qinling mountain pandas according to claim 1, wherein the method comprises the following steps: the specific operation of the step 1 is as follows:

step 1.1: fasting the male Qinling pandas in estrus for 12 hours to promote the food in stomach to be emptied;

step 1.2: 4mg/kg-8mg/kg of ketamine is used, and 0.3mg/kg-0.5mg/kg of isoflurane or 0.3mg/kg-0.5mg/kg of entacapone is used as an auxiliary for carrying out intramuscular injection anesthesia on the neck or the hip of a panda;

step 1.3: inspecting reproductive organs of the anesthetized male Qinling pandas in the step 1.2;

step 1.4: cleaning dirt in the foreskin of male Qinling mountain panda with clean gauze, cleaning the inside and outside of the foreskin with 0.1% benzalkonium bromide at 37 ℃ for multiple times, and finally washing with sterile normal saline at 37 ℃ until no disinfectant residue exists;

step 1.5: connecting an electrical stimulation semen collector, slowly increasing voltage from low to high according to hind limb contraction degree of male Qinling mountain pandas, and continuously stimulating rectum of male Qinling mountain pandas for 20-30 times, each time for 2s, until semen flows out;

step 1.6: if no semen is stimulated or the semen amount is too small in the 1 st time, repeating the step 1.5 for 1 time after 5 min;

step 1.7: holding the semen collecting cup at 38 deg.C, and collecting semen quickly after semen flows out until semen does not flow out.

3. The artificial insemination and insemination method for the Qinling mountain pandas according to claim 2, wherein the method comprises the following steps: the step 2 of semen quality inspection of male Qinling mountain pandas comprises the following steps: the method comprises the following steps of semen odor examination, semen density examination, semen pH value examination, semen sperm motility examination, semen sperm acrosome integrity examination, semen sperm morphology examination and semen sperm plasma membrane integrity examination.

4. The artificial insemination and insemination method for the Qinling mountain pandas according to claim 3, wherein the method comprises the following steps: the semen sperm acrosome integrity check of step 2 is as follows: aspirate 1. mu.L of seminal fluid + 9. mu.L of LPOPE stain, stain for 90s, smear, and view and rate with 100 Xoil-scope after smear is dry.

5. The artificial insemination and insemination method for Qinling mountain pandas as claimed in claim 4, wherein the method comprises the following steps: the particle freezing operation of the step 3 is as follows:

step 3.1.1: diluting the semen of the male Qinling panda checked to be qualified in the step 2 by using a freezing diluent, wherein the ratio of the semen to the freezing diluent is 1: 3;

step 3.1.2: balancing the panda semen diluted in the step 3.1.1 in a refrigerator at 4 ℃ for 4 hours;

step 3.1.3: soaking the frozen semen plate in a metal tank filled with liquid nitrogen for 3 times, lifting the frozen semen plate to a position 5cm away from the liquid nitrogen surface, measuring the temperature of the frozen semen plate by using a thermometer, and quickly dripping balanced semen on the frozen semen plate at 40-80 mu L/grain by using a micropipette when the temperature is reduced to-80 ℃;

step 3.1.4: and 3.1.3, after finishing the step for 2min, quickly immersing the frozen semen plate into liquid nitrogen at the temperature of-196 ℃, putting the frozen semen into a glass vial by using balanced tweezers, and putting the glass vial into a liquid nitrogen tank for long-term storage.

6. The artificial insemination and insemination method for Qinling mountain pandas as claimed in claim 4, wherein the method comprises the following steps: the detailed operation of the tubule freezing in the step 3 is as follows:

step 3.2.1: diluting the semen of the male panda from Qinling mountain checked and qualified in the step 2 by TEST or SFS in a ratio of 1: 3;

step 3.2.2: balancing the panda semen diluted in the step 3.1 in a refrigerator at 4 ℃ for 4 hours;

step 3.2.3: 3.2.2 sucking the balanced panda semen into a thin plastic tube, wherein each tube is 0.25mL, one end of each tube is plugged with cotton, the other end of each tube is sealed after the semen is injected, and the tubes are sealed by melting the plastic by using a pair of tongs which are heated on an alcohol lamp;

step 3.2.4: placing the sealed thin plastic tube of step 3.2.3 on the upper layer of a balanced test tube rack, wherein the temperature is-20 ℃, the chaotropic nitrogen surface is 7.5cm, and the time is 1 min; then moving the thin plastic tube to the lower layer of the test tube rack, wherein the chaotropic nitrogen surface is 2.5cm at the temperature of minus 80 ℃, and the time is 1 min; and finally, putting the thin plastic tube into liquid nitrogen for 2min, then putting the thin plastic tube into a paper tube, and putting the paper tube into a liquid nitrogen tank for long-term storage.

7. The artificial insemination and insemination method for Qinling mountain pandas as claimed in claim 4, wherein the method comprises the following steps: the test tube freezing operation of the step 3 is as follows:

step 3.3.1: adding 200 mu L of collected panda semen into 400 mu L of diluent;

step 3.3.2: putting the diluted panda semen into a 1.5mL plastic test tube, and balancing for 4h in a refrigerator at 4 ℃;

step 3.3.3: and (4) placing the plastic test tube after the balance in the step 3.3.2 on a flat plate 7.5cm high from liquid nitrogen for freezing for 10min, and then placing the flat plate in the liquid nitrogen for long-term storage.

8. The artificial insemination and insemination method for Qinling mountain pandas as claimed in claim 7, wherein the method comprises the following steps: the specific operation of the step 4 is as follows:

step 4.1: cleaning vulva and anus of female Qinling giant panda with 0.1% benzalkonium bromide at 37 deg.C, and washing with sterilized normal saline at 37 deg.C until no disinfectant residue exists;

step 4.2: the ball-type glass sperm inseminating device is used for inseminating the particle frozen semen, and the metal sperm inseminating device is used for inseminating the thin-tube frozen semen;

step 4.3: a measuring cylinder-shaped cavity opener made of transparent glass is inserted into a port smooth part at the vaginal end of a female Qinling mountain panda;

step 4.4: inserting the inseminating syringe into the cavity opener, and slowly injecting semen;

step 4.5: making artificial insemination records, and accurately recording the information of the female and the male, the information of the used semen, the information of the panda from which the semen comes, the date and the insemination times in detail.

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