Sex pre-selection is a biotechnology that allows adult females to produce sex-specific offspring through human intervention in the normal reproductive process of animals. Sex control technology is of great significance in livestock production. By controlling the sex ratio of the offspring, the maximum economic benefits of production traits limited by sex (such as lactation) and production traits affected by sex (such as growth rate, meat quality, etc.) can be fully utilized. Secondly, controlling the sex ratio of offspring can increase the intensity of selection and speed up the breeding process. Sex control can be achieved through sperm separation or embryo sexing.
Principle
The principle of controlling gender in animals is based mainly on the differences between male and female germ cells, especially animal spermatozoa. In higher animals, X and Y sperms differ considerably in terms of morphology and structure, weight and density, power distribution and electrical properties of the surface membrane charge, antigenic immunity of the sperms, speed and vigor of movement, and acid and alkali resistance.
Operation method
Pig gender control technology
Principle
The principle of controlling gender in animals is based mainly on the differences between male and female germ cells, especially animal spermatozoa. In higher animals, X and Y sperms differ considerably in terms of morphology and structure, weight and density, power distribution and electrical properties of the surface membrane charge, antigenic immunity of the sperms, speed and vigor of movement, and acid and alkali resistance.
Materials and Instruments
Equipment: Move The basic process of porcine sex control can be divided into the following steps: For more product details, please visit Aladdin Scientific website.
① Syringe
② Sperm image analyzer (ceros sperm analyzer Hamilton Thorne research)
③ Flow cytometer (Mo Flo SX, Dakocytomation Fort Collins, Co, USA)
④ Cryo-centrifuge (Eppendorf, 5804R)
Reagents:
① Material: pig
② Sperm dilution solution: glucose, sodium citrate, EDTA-Na, sodium bicarbonate, BSA, HEPES buffer, gentamicin
③ Food coloring reserve solution (25 mg/ml, FD & C40)
④ Chorionic gonadotropin (hCG)
1 . Semen collection and processing
Collect boar semen by hand-holding method and return it to the laboratory as soon as possible. Moderate dilution (each 100 ml of dilution contains: glucose 2.6 g, sodium citrate 0.8 g, EDTA-Na 20.24 g, sodium bicarbonate 0.12 g, BSA 0.25 g, HEPES buffer 0.9 g, gentamycin 0.03 g, pH 7.0) was performed, and sperm viability was measured using a sperm image analyzer, and sperm density was measured using a blood counting machine. Sperm density was measured using a sperm image analyzer. The semen (spermatozoa) was then treated differently according to the needs of the test.
2 . Sperm isolation
(2) Add about 15 μl of H2O (5 mg/ml reserve solution) per ml of diluted semen, stain for 0.5 hours at 34 ℃ in a water bath protected from light, then add 1 μl of food coloring reserve solution (25 mg/ml, FD & C40) and stain for 3-5 minutes, then filter through a 51 μm pore sieve and store at room temperature (20-23 ℃).
(3) Separate the cells by flow cytometry as soon as possible (within 2-3 hours) after adjusting the instrument. Separation conditions: sheath solution (PBS with 0.1% BSA and 0.1% EDTA) at 40 Psi, 150 mW laser, 76 μm inner diameter nozzle, and separation speed of about 3000/s. The collection tube was pre-filled with about 500 mW of BSA and 0.1% EDTA. The collection tube is pre-filled with approximately 500 μl of receiving solution (TEST solution with 1% seminal plasma and 2% yolk; seminal plasma preparation: whole semen is centrifuged at 3500 g for 15 min, the supernatant is filtered twice through a 0.22 μm pore size sieve, and the filtrate is checked for the absence of spermatozoa and set aside), and after collection of approximately 1.0 x 10 sexed spermatozoa, the sample is centrifuged for 15 min at 972 g at 21 °C. The precipitation is made from a heavy suspension (seminal plasma with 1% seminal plasma added), which is then separated from the sample by a laser. （The precipitate is resuspended in a resuspension solution (semen dilution with 1% seminal plasma) and stored at room temperature (20-23 °C) for 1-3 hours for insemination. The purity of sperm separation was determined by flow cytometer reanalysis, and the viability of separated sperm was measured.
3 . Separation of sperm for insemination and farrowing
(1) Preparation of receptor sows: 4 sows (3 Luchuan sows and 1 Guike 1 sow) that were in estrus for 50-60 hours were selected for surgical insemination one after another. On the day of surgery, no feed will be fed, but clean and hygienic drinking water will be guaranteed. Equivalent Luchuan sows were selected for control by conventional artificial insemination.
(2) Surgical insemination: The surgery was performed on one side of the anaesthetized sow. The uterus, fallopian tube and uterine tube were partially exposed, and a suitable disposable infusion needle and infusion tube with syringe were used to inject separated sperm resuspension (about 4.5 million sperms per fallopian tube and 0.5 ml of liquid) into the fallopian tube along the direction of the abdominal pot on the fallopian tube side, and then returned to the abdominal cavity, and the opposite fallopian tube was operated in the same way, and then stitched up and treated the wounds. After the operation, antibiotics were injected for 3-5 days according to the situation. Carry out daily management and wait for the litter.
(3) Measurement of farrowing indexes: counting the number of sows' farrowing (total number of farrowing, number of live piglets) and the sex ratio; observing the piglets' shape, vigor and health during the period of newborn, lactation and weaning, and identifying the sex of the embryo control.