In vitro fertilization in pigs

Summary

In vitro fertilization (IVF) is a technological process in which fertilization is accomplished in an in vitro environment between a mature oocyte and an energized sperm, either in vivo or in vitro. In biology, an animal obtained after the transfer of an IVF embryo to the mother is called a test-tubeanimal. This technology is not only of theoretical significance for the study of gamete development, maturation, fertilization and early embryonic cell differentiation, but also of great significance for the solution of human infertility and other social problems, and also of wide practical value for the acceleration of animal reproduction in production.

Operation method

In vitro fertilization in pigs

Materials and Instruments

Equipment:
① 36 ℃ constant temperature water bath
② 2 500 ml beakers
③ 1 stirring rod, 2 towels
③ 1 stirring rod, 2 towels ④ 3 sperm vials
⑤ 1.5 ml centrifuge tube, 17 ℃ refrigerator.
⑤ 1.5 ml centrifuge tube and 17℃ refrigerator:
① Material: pig ovaries
② Dilution powder (Androhep. Minitube)
DPBS (0.1% BSA) ④ Maturation solution
DPBS (0.1% BSA) ④ Maturation solution
⑤ Hypotonic solution

Move

The basic process of porcine in vitro fertilization can be divided into the following steps:
(a) Fresh semen dilution
Fresh semen can be kept alive for at least 1 hour at a constant temperature of 37 ℃. Therefore, before going to the farm, prepare thermos flasks with 38 ℃ water, and put the bottle into the thermos flasks as soon as possible after getting it to make sure that semen can be diluted and preserved for as short a period of time as possible.
Preparation in the laboratory:
1. Prepare the diluent by adding 200 ml of sterile deionized water to a 500 ml beaker and placing the same beaker into a water bath.
2. When the water has warmed up, weigh 9.3 g of the diluent powder (note: different diluent powders have slightly different values) and add it to the 200 ml of water, stirring the powder with a stirring rod, and allow the diluent to equilibrate for at least 40 minutes.
3. Prepare two 1.5 ml centrifuge tubes by adding 38 °C pre-warmed 9.3 g of 9.3 g diluent powder to the water and adding it to the water. Prepare 2 tubes of 1.5 ml centrifuge tubes and add 990 μl of DPBS pre-warmed at 38 ℃. 10 μl of fresh semen and 10 μl of diluted semen will be added for density and viability. Two 1.5 ml centrifuge tubes were prepared by adding preheated 990 μl of hypotonic solution to test the integrity of the plasma membrane of fresh and diluted semen by tail-bend rate.
4. Feel the temperature of the room, and adjust the air conditioner to a normal room temperature to avoid the room from being too cold or too hot.
When the semen is delivered to the laboratory, the vials are quickly taken out and gently rolled in a clockwise direction (to avoid damage to the sperm tails, do not turn them up and down, shake them or roll them in both directions), 10 μl of the vials are aspirated and added to 990 μl of the prepared DPBS for the density and viability tests, and 10 μl of the vials are aspirated for the tail curvature assay. The remaining part of the semen (80-100 ml) was slowly poured against the wall of the bottle into a preheated 500 ml empty beaker (this beaker was kept in a water bath throughout the dilution). The diluent from the other beaker is slowly poured into the semen along the wall, while the stirring rod is gently and slowly stirred clockwise through the liquid until 200 ml of diluent has been poured.
After slight mixing, pour the diluent equally into the vial. After exhausting the gas, the vials were tightly capped, wrapped in two layers of towels, and placed at room temperature for 20-30 minutes, and then the vials and towels were placed in a refrigerator at 17 ℃ for storage (up to 7 days).
After 1 hour, one tube of vial was taken out, rotated to mix well, and 1 ml of the diluted semen was placed in a 1.5 ml centrifuge tube and placed in a 38 ℃ incubator for 20 minutes, mixing well, and 2 x 10-μl diluted semen were added to DPBS and hypotonic solution (see Table 4 for formulations). Take two 10 μl diluted semen and add them into DPBS and hypotonic solution (see Table 4-8-5) respectively for density, viability and tail curvature.
During semen storage, it is important to ensure that the semen is mixed every 12 hours to ensure that sperms do not die in large numbers due to excessive extrusion.
(ii) Sperm quality related tests
Sperm density, sperm viability, and sperm plasma membrane integrity are currently routinely measured in the laboratory.
1. Sperm density 8-10 μl of spermatozoa in an appropriate amount of diluted spermatozoa (usually 100-fold dilution, i.e., 10 μl of spermatozoa added to 990 μl of DPBS) are spotted on a coverslip and in the gap of the hematocrit plate. When the number of free spermatozoa in each compartment (25 compartments in total) is >10, the total number of spermatozoa in 5 randomly selected compartments is counted, which means that the density of spermatozoa in this dilution is Nx5x10/ml. If the number of spermatozoa in each compartment is less than 10, then the spermatozoa density in the fluid is obtained by counting all the 25 compartments to obtain a density of Nx10/ml.
2

. Sperm viability Same as above but dead spermatozoa will be counted during the counting

process.

As above, but dead sperm were counted during the counting process.

Viability = 100% x [total number of sperm - total number of dead sperm].

3

. Sperm plasma membrane integrity Only sperm with an intact plasma membrane can bend under hypotonicity. A suitable amount of spermatozoa was added to 990 μl of preheated hypotonic solution and placed at 38 ℃ for 30 minutes. 10 μl of spermatozoa were mixed and counted for total spermatozoa and spermatozoa with curved tails, and the curvature rate was measured.

Bent tail rate = 100% x (number of spermatozoa with bent tails/total number of spermatozoa).

(III) Technical procedure of porcine IVF
The day before IVF:
1. 8:00 p.m. Prepare the insemination solution (total 15 ml) 15 ml of mTBM mother liquor, add 0.01 g of caffeine (2 mmol/L) to dissolve. Add 0.03 g BSA (A-7888).
(1) Preparation of fertilization droplets with mTBM liquid: add 4 50-mountain microdroplets to each of 2 35 mm dishes, cover with liquid paraffin, and place in a _CO2 incubator overnight.
(2) IVF egg wash drops: make 3 500 μl mTBM drops in 35 mm dishes. Cover with liquid paraffin and place in a _CO2 incubator overnight.
(3) IVF Sperm Diluent: 11 ml in a 15 ml centrifuge tube covered with a ventilated filter cap and equilibrated together in a _CO2 box for the next day's sperm dilution.
2. Preparation of DPBS Sperm Wash 5 ml of DPBS in a 15 ml tube and 0.005 g [0.1% (w/v)] BSA (A-8022), filtered, and placed in an incubator at 39 ℃.
3.

Prepare porcine embryo culture medium PZM-3:

① 9.7 ml PZM-3 base solution ＋ 200 μl BEM ＋ 100 μl MEM ＋ 0.03 g BSA, dissolved, filtered (4 ℃, 1 week shelf life).

② Culture drop: 5-6 wells of 24-well plate, 500 μl per well.

③ Wash drops: 3 x 500 μl in 35 mm dish. _CO2 incubator, overnight.

IVF day:
After 42 hours of oocyte culture, mature oocytes are obtained by digestion as before. After washing the mature eggs with IVF egg wash drops, add the eggs to the IVF fertilization droplets (30-35 eggs/drop) and place the dish in a _CO2 incubator.
1. Prepare sperm Remove the vial containing the diluted semen, gently swirl it to mix it, and take 1 ml of semen into a 1.5 ml centrifuge tube. Place the tube in a warm oven at 38 °C for 20 minutes. 10 μl of the mix was added to 990 μl of PBS for viable sperm counting. 1.5 ml tube was centrifuged at 3000 r/min for 5 minutes, supernatant was discarded, and the tube was washed twice with pre-warmed DPBS. Finally, 1 ml of pre-equilibrated 11 ml mTBM IVF sperm diluent was used to resuspend the sperm. Calculate the final dilution by counting the spermatozoa for optimal fertilization (sperm to egg ratio of 250:1).
2. Fertilization Take 50 μl of the final diluted spermatozoa and add 50 μl of the final diluted spermatozoa to each IVF 50 μl fertilization drop; leave it in the incubator for 5 hours to be fertilized; transfer the eggs to the PZM-3 wash drop, and after washing, 30-35 eggs/well were added to the PZM-3 wells.
3. Developmental Observation After 48 hours, observe and calculate the rate of egg cleavage. After 48 hours of developmental observation, the egg cleavage rate was observed and calculated, and the blastocyst rate was obtained at 144 hours (6 days observation).

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