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LY/T 3112-2019: Technical regulation of fox artificial insemination
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Technical regulation of fox artificial insemination
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LY/T 3112-2019
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Basic data | Standard ID | LY/T 3112-2019 (LY/T3112-2019) | | Description (Translated English) | Technical regulation of fox artificial insemination | | Sector / Industry | Forestry Industry Standard (Recommended) | | Classification of Chinese Standard | B44 | | Classification of International Standard | 65.020.30 | | Word Count Estimation | 18,138 | | Date of Issue | 2019-10-23 | | Date of Implementation | 2020-04-01 | | Issuing agency(ies) | State Forestry and Grassland Administration | LY/T 3112-2019: Technical regulation of fox artificial insemination---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
(Fox artificial insemination technical regulations)
ICS 65.020.30
B 44
LY
People's Republic of China Forestry Industry Standard
Fox artificial insemination technical regulations
Technical regulation of fox artificial insemination
2019-10-23 released
2020-04-01 implementation
Published by the National Forestry and Grassland Administration
Contents
Foreword ... II
1 Scope ... 1
2 Terms and definitions ... 1
3 Preparation before insemination ... 2
4 Personnel requirements ... 3
5 fox requirements ... 3
6 Insemination operation ... 4
7 Management after insemination ... 7
Appendix A (informative appendix) List of commonly used equipment and drugs for artificial insemination ... 8
Appendix B (informational appendix) Semen quality assessment methods ... 9
Appendix C (informative) Estrus evaluation ... 11
Appendix D (Informative) Commonly Used Record Form for Artificial Insemination ... 12
Foreword
This standard was drafted in accordance with the rules given in GB/T 1.1-2009.
This standard was proposed and managed by the National Technical Committee for Wildlife Conservation, Management, Operation and Utilization (SAC/TC369).
This standard was drafted. Harbin Institute of Specialty Products, Heilongjiang Academy of Agricultural Reclamation Sciences, Heilongjiang Pratt & Whitney Specialty Products Co., Ltd.
The main drafters of this standard. Han Huansheng, Zhao Liping, Liu Zhiping, Wei Ximing, Yin Yajie, Shi Wenqing, Xu Xin, An Rui, Zhai Xuechao,
Yang Yang, Yang Jiao, Zhao Xiaojing, Chai Menglong, Zhang Haiyan
Fox artificial insemination technical regulations
1 Scope
This standard specifies the technical requirements for artificial insemination of blue foxes and silver black foxes. Other foxes can be used in reference.
This standard applies to breeding of artificially-bred foxes in breeding farms and artificial insemination stations.
2 terms and definitions
The following terms and definitions apply to this document.
2.1
Fresh Semen Diluent
Formulated with diluent, nutrient and additives as main ingredients, suitable for fresh sperm that can be fertilized when stored at 33 ℃ ～ 37 ℃ for 3h
Solution.
2.2
Sperm motility
The percentage of sperm in the sperm that moves in a straight line in the semen, commonly known as the sperm motility rate.
2.3
Sperm concentration
The total number of sperm contained in a unit volume of semen is also called the sperm concentration.
2.4
Sperm deformity rate
Percentage of spermatozoa with morphological variation of head, body, and tail in a unit volume of semen.
2.5
Number of effective sperms
The total number of spermatozoa in a normal sperm motion in the semen sample tested, that is, the number of spermatozoa with fertility.
2.6
External observation methods of estrus identification
A method for judging whether estrus and estrus are in accordance with the characteristic behavior, mental state, and vulvar changes of the female fox, referred to as external view
Chafa.
2.7
Artificial insemination
An animal breeding technique that uses auxiliary equipment to artificially deliver semen into the uterus of an estrus female fox to conceive it.
3 Preparation before insemination
3.1 Planning
Before artificial insemination begins, a plan should be prepared, pre-selected supplies (see Appendix A), and design record sheets (see D.1 to D.3 in Appendix D).
3.2 Insemination operation room
3.2.1 The operation room should be located near the wind direction of the fox farm, with an area of 15m2 ~ 25m2, which requires safety, ventilation, heat insulation and sanitation.
Lighting, electrical outlets and consoles.
3.2.2 The operation room shall be divided into a dressing room, a sperm collection and insemination room, and a semen detection and processing room, with a partition in the middle.
3.2.3 The wall and floor of the operating room shall be made of ceramic or hard waterproof materials which are easy to disinfect.
3.2.4 The operating room should be equipped with ultraviolet disinfection lamps and temperature control equipment, and the temperature should be controlled at 18 ℃ ～ 25 ℃.
3.3 supplies preparation
3.3.1 Prepare medicines and equipment before insemination (see Appendix A).
3.3.2 The dressing room shall be equipped with clothes hangers, shoe racks, sinks or sinks.
3.3.3 The sperm collection and insemination room should be equipped with Baoding stand, water basin, towels, washing and disinfecting drugs, vaginal dilation tube, insemination needle, 1mL ～ 10mL
Injectors, scissors, waste collection bins, etc.
3.3.4 The semen detection and processing room shall be equipped with an operating table, a collection cup, a thermometer, a microscope, a glass slide, a cover glass, a dropper, and a thermostatic water bath
Pots and washing and disinfecting supplies.
3.3.5 Estrus identification should be equipped with alcohol cotton balls, cotton swabs, microscopes, glass slides, etc., and resistance testers should be equipped.
3.3.6 The environment and equipment cleaning and disinfection shall be equipped with test tube brushes, continuous syringes, distilled water, washing and disinfecting drugs, drying boxes, disinfection cabinets and
Autoclaves, etc.
3.4 Cleaning and disinfection
3.4.1 Environment
For the first disinfection before the start of artificial insemination, it is advisable to use fire alkali or quicklime for disinfection outside the operating room and 1% to 4% sodium hydroxide or 0.1% to indoor
0.2% Xin Jie Er disinfection solution, in the future indoors should be disinfected with ultraviolet light every day, should not use odor disinfection drugs.
3.4.2 Equipment
3.4.2.1 The artificial insemination equipment should be selectively cleaned and disinfected according to the material.
3.4.2.2 Wash before disinfection. Various equipment and utensils should be cleaned and disinfected before and after use. Wash with water and detergent before cleaning.
Dirt is then rinsed with water. Utensils in contact with semen should be rinsed with distilled water at least twice before drying.
3.4.2.3 After the metal and glass utensils are soaked and brushed with disinfectant, they are dried in a 120 ° C drying oven for more than 2 hours.
3.4.2.4 Plastic products (such as plastic vaginal dilatation tubes) can be washed in accordance with the above procedure, then immersed in 75% alcohol for 1 to 2 hours, and then distilled
Wash with water, and then dry in a drying box at 60 ℃ ～ 80 ℃ according to the material.
3.4.2.5 Towels and work clothes used for wiping the fox's genital area can be sterilized by high-pressure steam sterilizer after washing.
3.4.3 personnel
Operators should strictly disinfect before and after artificial insemination. The sperm collector should cut his nails, wash his hands with soapy water, and rinse with water.
Wash and dry, wear disposable gloves, and work clothes.
4 Staff requirements
4.1 The staff consists of estrus appraisers, Baoding staff, insemination assistants and insemination staff, and no zoonotic disease is required.
4.2 One or two estrus appraisers. The number of personnel should be determined according to the number of female foxes and their working capabilities.
It is necessary to train the breeder to make a preliminary determination of the estrus status of the female fox under his control.
4.3 One Baoding staff member, who is a full-time Baoding fox for sperm collection and insemination in the operating room.
4.4 One insemination assistant assists the insemination worker, and is also responsible for the scrubbing and disinfection of the equipment, indoor hygiene, and records of semen collection and insemination.
4.5 One insemination worker, with certain professional theoretical foundation and field work experience.
5 fox requirements
5.1 male foxes
Male foxes that can be harvested and bred should meet the following conditions.
a) Have the characteristics and breeding requirements of this variety;
b) entering the body condition during mating period;
c) over 10 months of age;
d) Pure fur color and clear genealogy;
e) Healthy and non-infectious diseases, mainly refers to no deformity of testis, no adhesion of foreskin, no inflammation of reproductive system, no Brucella and Gard
Infectious diseases
f) Reproductive performance requirements. The semen quality should be tested every two weeks during estrus. The quality of fresh essence should meet the requirements of 6.3 of this standard.
5.2 Female Foxes
Breeding female foxes should meet the following conditions.
a) Have the characteristics and breeding requirements of this variety;
b) entering into the mating period with moderate body condition;
c) over 10 months old and under 6 years old;
d) Pure fur color and clear genealogy;
e) Healthy and non-infectious diseases, mainly refers to the absence of malformations and inflammation in the reproductive system, and no infectious diseases such as Brucella and Gardnerella;
f) Reproductive performance requirements. strong motherhood, no vacancy last year, and more litters.
6 Insemination operation
6.1 Estrus evaluation
6.1.1 The estrus identification can be based on the observation of the vulva of the female fox, the estrus identification of the vaginal cells, or the comprehensive determination method supplemented by the resistance tester.
Method (see Appendix C).
6.1.2 In the estrus period, the estrus appraiser performs vulva observation records on the mating female foxes, and regularly observes female foxes with estrus performance, and finally
The time and number of inseminations were determined by vaginal cell estrus identification method or resistance tester, and the number of female foxes that could inseminate each day was reported to the insemination staff.
6.1.3 The best time for insemination is female fox vulva swelling and retraction, wrinkles appear, vaginal cell smears have no more than 80% of keratinocytes, resistance measurement
The resistance value of the meter is changed from high to low.
6.2 Semen collection
6.2.1 Preparation before harvesting
The following preparations should be made before semen collection.
a) Prepare supplies according to the number of female inseminations per day;
b) Put the sterilized insemination needle, sperm collection cup, and vaginal dilation tube into a 33 ℃ ～ 37 ℃ incubator;
c) Wet the sterilized towel with 0.1% ～ 0.2% Xinjieer sterilized warm solution;
d) Preheating the fresh essence diluent in a constant temperature water bath ranging from 33 ° C to 37 ° C for 30 minutes in advance;
e) Determine the number of male foxes.
6.2.2 Baoding
Put the sperm-extracting male fox on the Baoding rack, and the Baoding members fix the head of the fox with neck clamps or clamps, and at the same time hold the tail to make the male fox stand relaxed
Potential.
6.2.3 Cleaning and disinfection
Wipe the abdomen and scrotum of the collected foxes with a disinfected warm and damp towel. The plush is wet and free of dust and dust.
6.2.4 Extraction operation
6.2.4.1 During sperm harvesting, first massage the testicles and penis of the male fox to give the fox a signal of sperm harvesting.
6.2.4.2 After massaging for a few seconds, the thumb and index finger of the sperm collector are pinched on both sides of the male fox penis, the middle finger is pinched on the ventral surface of the penis, and
Press and slide the penis foreskin longitudinally along the penis to rub the penis with friction.
6.2.4.3 The sliding speed should be faster at the beginning of pressing, preferably 4 times/s to 5 times/s, and the moving range should be 7cm ～ 8cm. After pressing for 5s ～ 7s,
The penis is erect, and the corpus cavernosum in the middle of the penis also swells.
6.2.4.4 Then, pull the penis from the rear legs of the male fox to the back, push the foreskin to the back of the corpus cavernosum and continue to squeeze the corpus cavernosum
And the posterior penis, the pressure of the pressure is slowed down, it is advisable to pressure 12 to 13 times per 10s, and the pressure is slightly harder when pressing the spherical sponge, so repeat
Pressure massage (about 10s) until the male fox ejaculates.
6.2.5 Semen collection
Only semi-milk semen was collected during semen collection.
6.2.6 Semen collection frequency
In each estrus season, each male fox can collect sperm from 15 to 25 times, and the frequency of sperm collecting should not exceed 2 times in 3 days.
6.3 Semen test
The collected semen should be tested and recorded (see Table D.1 in Appendix B and Appendix D). The quality of fresh sperm should meet.
a) The color of fresh essence is milky white or slightly yellow;
b) no urine, blood and dirt;
c) semen volume ≥0.3mL;
d) sperm motility ≥ 0.7;
e) sperm deformity rate ≤15%;
f) Sperm density ≥400 million/mL.
6.4 Semen dilution
6.4.1 Dilution multiple
Place the sperm collection cup into the thermostatic water bath of 33 ℃ ～ 37 ℃, and determine the dilution according to the sperm motility, sperm density and semen volume.
multiple.
6.4.2 Isothermal Dilution
Use isothermal fresh sperm diluent for stepwise dilution. The method is to slowly add the diluent to the semen along the wall of the sperm collection cup, and gently mix it. It is strictly prohibited.
Quickly flush the diluted solution into the semen and shake vigorously to ensure that there is at least 60 million effective sperm per mL in the diluted semen.
Medium density is better (see Appendix B).
6.5 Semen preservation
6.5.1 Semen should be infused immediately after the semen is diluted. The semen should not be stored for more than 3 hours in a 33 ℃ ～ 37 ℃ water bath.
6.5.2 If the semen needs to be postponed, it should be diluted with 15% -20% egg yolk fresh sperm dilution solution, and then stored in a refrigerator at 4 ℃ ～ 6 ℃. 2
After the day is used up, the temperature of the microscope heating plate should be controlled at 35 ℃ ～ 37 ℃ before use.
6.6 Artificial insemination
6.6.1 Female Fox Baoding
Grab the female fox to be inseminated, fix it on the Baoding rack, and place the female fox upside down or stand in Baoding.
6.6.2 Cleaning and disinfection
Wipe the vulva and its surroundings with a sterile warm and damp towel.
6.6.3 Insemination operation
6.6.3.1 Slowly insert the vaginal dilatation tube into the vagina of the female fox, and its front end reaches the cervix. The left mouth of the tiger is supported on the lower abdomen of the female fox.
Fingers, index and middle fingers touch the front of the vaginal dilatation tube.
6.6.3.2 The cervix is fixed in the left hand, and the insemination needle is gently inserted through the lumen of the vaginal dilatation tube in the right hand, with the front end touching the cervix.
6.6.3.3 Search the cervix with your left and right hands. After the insemination needle passes through the cervix, it will be fixed at a position of 1cm to 2cm in the uterus.
6.6.3.4 The assistant inserts the syringe that sucks semen on the insemination needle, and pushes the syringe to slowly inject the semen into the uterus.
A semen-suctioning syringe is inserted into the insemination needle in advance, and the inseminater directly injects the semen.
6.6.3.5 When sucking semen with a syringe, after sucking to a fixed scale, a little more air should be sucked in to ensure that all semen is infused during insemination
Into the uterus.
6.6.4 Dose for insemination
Each female fox should have an insemination dose of 0.8 mL to 1.0 mL.
6.6.5 Frequency of insemination
It is advisable for each female fox to lose 2 to 3 times during each estrus. It can be inseminated every other day, or it can be stopped for one day and then lost again.
6.6.6 Work after insemination
The following tasks should be performed after the artificial insemination.
a) Clean and disinfect the operation room and destroy the waste products;
b) Clean and disinfect the instruments in the same way as 3.4.2, and store the equipment uniformly;
c) Organize the records and arrange the next insemination plan.
7 Management after insemination
7.1 Insemination fox
7.1.1 Infertile female foxes should avoid fright, to prevent abnormal fetal position, abortion and dystocia.
7.1.2 The feed of inseminated female foxes should not be greatly changed, and must not be fed with moldy, icy feeds.
7.1.3 Make relevant records of infertile female fox disease, physical condition, and abortion (see Table D.2 in Appendix D).
7.1.4 Before entering the pre-birth period, do prenatal preparations, increase staff, and make preparations for fox rearing, feeding, delivery, and disease prevention.
Make.
7.2 Birth records
Keep farrowing records (see Table D.2 in Appendix D) and archive them.
7.3 Work summary
Summarize the overall work of artificial insemination (see Table D.3 in Appendix D), summarize, and archive.
AA
Appendix A
(Informative appendix)
Table of commonly used equipment for artificial insemination
A.1 List of commonly used equipment for artificial insemination
No. Product Name Unit Quantity Specification
1 Drying box table 1
2 Disinfection counter 1
3 Refrigerator counter 1
4 Constant temperature water bath pot table 1
5 Steam sterilization pot table 1
6 Alcohol bottles with 75% alcohol
7 New Gil disinfectant bottle amount
8 Distilled water bucket
9 appropriate amount of washing liquid bottle
10 amount of sink
11 Towel blocks
12 test tubes
13 continuous syringes 2 ～ 3
15 sets of fine cups
16 insemination needles
17 vaginal dilatation tubes
18 syringes 1mL, 2/2.5mL, 5mL, 10mL
19 microscope stage 1 400x ～ 600x
20 constant temperature heating plate 1
21 Coverslips/Slide Boxes
22 wipe the right amount on paper
23 cotton swabs
24 appropriate amount of disposable gloves/mask pack
25 Scissors Pack 1
26 Appropriate amount of work clothes
27 Ultrasonic cleaner table 1
28 Distilled water table 1
29 Air Conditioner 1
30 UV lamp ≥2W/m2 appropriate amount
Sperm density meter/cytometer
Number board
Desk/piece 1
BB
Appendix B
(Informative appendix)
Semen quality assessment method
B.1 Sperm motility (or sperm motility)
B.1.1 Sperm viability should be assessed by microscopy;
B.1.2 Drop a drop of semen on an ordinary glass slide, and then cover the entire liquid surface with a cover glass to make a microscopic examination of the tablet;
B.1.3 Perform visual evaluation under a microscope with a constant temperature heating plate (33 ° C to 37 ° C) at 400 times to 600 times;
B.1.4 Use a 10-point scoring standard from 0 to 1.0;
B.1.5 Estimate the percentage of spermatozoa moving forward in the semen sample;
B.1.6 100% for straight forward athletes is 1.0 points, that is, sperm vitality is 1.0, 90% for straight forward athletes is 0.9 points, that is, sperm vitality
0.9, and so on.
B.2 Sperm density
B.2.1 Sperm density should be detected by blood cell counting method;
B.2.2 Pipette 0.2mL or 2mL of 3% NaCl solution into a small test tube with a 1mL thin tube;
B.2.3 According to the requirement of the dilution factor, use a blood pipette to aspirate and discard 10µL or 20µL of 3% NaCl solution (or diluent);
B.2.4 Use a blood pipette to aspirate 10 µL or 20 µL of the semen to be tested and inject it into a small test tube and shake well;
B.2.5 Take a drop of diluted semen on the edge of the cover glass on the calculation board to allow the semen to penetrate into the calculation chamber and fill it up.
With bubbles
B.2.6 Sperm in 80 small squares in the 4 middle corners of the calculation room and 5 middle squares in the center are counted under a 400x to 600x microscope
number;
B.2.7 For the sperm that presses the boundary line of the head, the principle of "counting the number and counting the left and not the right" should be followed;
B.2.8 Calculation formula. Sperm density (sperm count in 1mL of original sperm) = sperm count in 5 large squares × 5 (equal to 2 in the calculation room)
Number of spermatozoa in 5 large squares) × 10 (equal to the number of spermatozoa in 1mm3) × 1000 (equal to the number of spermatozoa in a 1mL sample of the test dilution)
× Dilution multiple of semen tested.
B.3 Sperm density estimation method
-Sperm density is divided into "dense", "medium" and "lean" according to the density and distribution of sperm under a microscope at 400x to 600x.
Three levels.
--Dense. The entire field of vision is filled with sperm and almost no gap is visible. It is difficult to see a single sperm activity, with a density of about 1 billion/mL.
--Medium. There is a clear gap between sperm in the field of vision equivalent to the length of a sperm, and the activity of a single sperm can be seen, with a density of.200 million/mL
about.
-Thin. The space between sperm in the visual field is longer than the length of more than 3 sperm, and even the total number of sperm can be checked.
-"0". No sperm was found in the entire field of vision.
B.4 Sperm density regression equation estimation method
B.4.1 Regression equation. Y ﹦ 0.0318732 ﹢ 0.0111555X
B.4.2 Y represents the number of spermatozoa per mL, and X represents the number of spermatozoa in a field of view under a 400x microscope;
B.4.3 The estimation range is limited to sperm density less than 100 million per mL, and it should be tested after the original semen is diluted;
B.4.4 Under a 400x microscope, the density of semen increases by 10 million/mL each time the sperm count in a single field increases by 10.
B.5 Sperm deformity rate
B.5.1 Sperm deformity rate should be examined by sperm staining microscopy;
B.5.2 Take a drop of the semen to be tested and place it on a glass slide. When the density is high, dilute it with normal saline.
sheet;
B.5.3 Stain with 0.5% gentian violet alcohol or blue ink for 3 minutes, dry naturally, and wash with water;
B.5.4 Observe under 400x600x 600x microscope after washing, check the number of spermatozoa in different fields, not less than.200, and calculate the deformity
Sperm percentage, repeated average.
B.5.5 Calculation formula. Sperm malformation rate = number of malformed sperm/total sperm count × 100%.
CC
Appendix C
(Informative appendix)
Estrus identification
C.1 Comparison table of three methods for estrus identification. vulva observation, vaginal cell estrus identification, and electrical resistance
Identification method
Estrus
Insemination start time
Early estrus
Vulvar changesa
The pubic hair is separated, the vulva is exposed, and the vulva is swollen
Swelling and flushing, further development, vulvar
Swelling is obvious, and the clitoris is oval.
The vulva is highly swollen and valgus, the vulva is round,
The upper part of the vulva is slightly wrinkled and dark in color.
And there are white or yellow curd-like secretions.
The vulva gradually retracts, becomes lighter, and swells
And eversion disappeared, and finally turned off-white.
The vulva swells to a minimum and becomes soft
When wrinkled.
Changes in vaginal cells
Onset of white blood cells and a small number of nucleated epithelium
Cells, further leukopenia, there
Nucleic epithelial cells increase and further whiten
Very few cells with nucleated and non-nucleated keratinocytes
Cells each half.
Kernel-free keratinocytes dominate, more than 80%, have
A small number of nuclear keratinocytes.
White blood cells appear and gradually increase, there are
Comparing nuclear polygonal keratinocytes with blood cells
many.
When non-nucleated keratinocytes are above 80%.
Vaginal resistance change b
Use the change of vaginal resistance value of female foxes during estrus to determine the degree of estrus. The change of electrical resistance of female foxes during estrus is generally low to high, and then
In the process of decreasing from high, insemination is generally performed after the peak.
The maximum resistance during estrus is usually between 800Ω ～ 1200Ω for blue fox and between 600Ω ～ 800Ω for silver fox. Artificial insemination time
The best point is that the blue fox is usually within 24 hours to 48 hours after the vaginal resistance reaches the maximum value, and the silver fox is within 24 hours.
When the resistance value drops from the highest point by about 20%.
a. Changes in the vulva can be recorded with a "+" sign. The first uplift of the genital area can be marked with "+". When the increase is checked again, it can be marked with "+﹢". When the uplift is maximum, it can be marked with "+﹢﹢"...
b. When the resistance tester is used to measure estrus, when the estrus starts and the vulva is not swollen to an appropriate level, it is not necessary to start measuring the resistance value. Generally, the resistance value is measured when the vulva becomes red and swollen before the estrus.
DD
Appendix D
(Informative appendix)
Commonly used artificial insemination r...
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