Microfertilization, also known as micromanipulation-assisted fertilization, is an in vitro fertilization technique developed in the 1980s. The technique involves either modifying the zona pellucida of the egg with a micromanipulator or injecting sperm or sperm cells directly into the egg to assist in the fertilization process. Currently, there are two ways of zona modification and sperm injection, the former includes zona drilling (ZD) and partial zona dissection (PZD), and the latter includes subzonal insemination (SUZI) and intracytoplasmic insemination (ICSI). ).
Operation method
Porcine microfertilization technique
Materials and Instruments
Equipment: Move The basic process of porcine in vitro fertilization can be divided into the following steps: Porcine semen was stored at 17 ℃, incubated at 39 ℃ for 20 minutes to recover sperm, centrifuged (1500 r/min, 5 minutes), the supernatant was discarded, and the precipitate was added with DPBS containing 0.1% PVA (DPBS-PVA), and centrifugation was repeated, and the supernatant was discarded, and then it was ready for use. According to the experimental design, spermatozoa were pretreated by different methods: ① Live sperm: sperm precipitate was suspended in PBS-PVA and transferred to the sperm manipulation drop. ② Sperm treated with 0.1% Triton X-100: Sperm precipitates were suspended in PBS-PVA containing 0.1% Triton X-100 and 0.3% BSA, followed by centrifugation to precipitate and suspension in PBS-PVA. (iii) Liquid nitrogen primary freezing and thawing: sperm precipitation was suspended in BTS containing 5% BSA. Place the copper mesh on the liquid nitrogen surface and freeze 100 μl of each pellet directly on the copper mesh. thaw in a water bath at 37 ℃, centrifuge, and suspend in PBS-PVA. ICSI was performed on mature porcine oocytes using a micromanipulator. The injection and fixation tubes were mounted on the micromanipulator. A 100 μl TL-HEPES drop was made in the center of a 60 mm Petri dish for egg release and microinjection. Two sets of 10 μl DPBS-PVA drops are made on either side of the drop for sperm placement. Cover with liquid paraffin. 20-30 eggs were manipulated per batch. Fix the oocyte with a fixation tube, adjust the position of the first polar body of the oocyte so that it corresponds to the position of point "6" or "12" of the clockwise hand. The injection needle penetrates through the zona pellucida from the position of point "3" and penetrates deeper in the direction of point "9", and a small amount of oocytoplasm is sucked back to make the oocytoplasmic membrane be penetrated, and then individual spermatozoa and a small amount of the operating fluid are injected into the oocytoplasm. After injection, the needle is withdrawn slowly and the oocyte is released. The injected eggs were washed three times in the embryo culture droplet and placed in an incubator for 30 minutes, and then treated with different auxiliary activation methods according to the experimental design: ① Calcium ionophore A23187: 5 μmol/L calcium ionophore activation for 5 min; ① Calcium ionophore A23187: 5 μmol/L calcium ionophore activation treatment for 5 minutes; ② Calcium ionophore A23187 + 6-DMAP: 5 μmol/L calcium ionophore activation treatment for 5 minutes, then wash out the calcium ionophore in the embryo culture droplet, transfer to a new culture droplet and incubate for 4 hours, and then treat the eggs with 1.9 mmol/L 6-DMAP for 3 hours; (iii) Electrical activation: 1.2 kV/cm, 30 microseconds, 2 direct current (DC) pulses were used. Porcine ICSI oocytes were cultured in PZM-3 embryo culture medium at 38.5 ℃, 5% CO2, 95% air and saturated humidity. According to the experimental design, ICSI oocytes were cultured in PZM-3 culture medium supplemented with 1.71 mmol/L cysteine for 0 h (control group), 4 h, 12 h and 168 h, and then transferred to PZM-3 without cysteine for further culture. The number of cleavage embryos was counted on the 2nd day, and the number of blastocysts was counted on the 7th day. ICSI oocytes were cultured for 15 hours, then transferred into 4% paraformaldehyde fixative for 45 minutes, washed in washing solution, and left in permeabilizing solution for 1 hour, after which the oocytes were stained with fluorescent light-avoidance staining in DPBS-PVA with 10 mg/L Hoechst 33342 for 10 minutes, washed in washing solution for 10 minutes, and then 10 μl of antifluorescent quencher drop was made on the slide, and the stained ICSI oocytes were transferred to the slide. The stained ICSI oocytes were transferred to the drop on the slide, covered with a coverslip and sealed with nail polish. Prokaryotic formation was observed under a fluorescence microscope. The number of embryos of each type was recorded by grouping them according to the type of prokaryotic formation, using 1 male prokaryotic + 1 female prokaryotic + 2 polar bodies (1MPN + 1FPN + 2PB) as the standard for normal fertilization. For more product details, please visit Aladdin Scientific website.
① Constant temperature water bath
② Oscillator
③ Needle puller
④ Petri dish
⑤ 1.5 ml centrifuge tube, 17 ℃ refrigerator
⑥ Microscope
Reagents:
① Material: pig
② DPBS, TritonX-100, Tween
③ PVA
④ Paraformaldehyde
⑤ Hepes
⑥ DMSO, NaOH, HCL
⑦ Mannitol
⑧ Caleium ionophore A23187
⑨ PZM-3 embryo culture medium
(a) Preparation of main reagents
1. DPBS A bottle of DPBS powder (Sigma) was dissolved in 1 L of deionized water, filtered through a 0.22 μm membrane, and placed at 4 ℃ for preparation.
2. DPBS-PVA Dissolve 0.1 g of PVA in 90 ml of deionized water by heating, after the dissolution of the PVA was cooled down to room temperature, and then 10 ml of 10-fold concentrated DPBS were added. 3. Add 10 ml of 10x concentrated DPBS.
3. Wash solution 10 ml of DPBS-PVA with 10 μl of 10% TritonX-100 DPBS-PVA and 10 μl of Tween. Set aside at 4 °C.
4. Transparence solution 9 ml of DPBS-PVA with 1 ml of 10% Triton X-100 DPBS-PVA. Set aside at 4 °C.
5. 4% Paraformaldehyde (fixative) Paraformaldehyde (fixative) 4.5 ml of deionized water was added with 0.2 g of paraformaldehyde, heated to 60-70 ℃ with stirring, and 200 μl of 1 mol/L NaOH was added with stirring until the powder was dissolved, and then 200 μl of 1 mol/L HCI was added after cooling. Finally, 0.5 ml of 10-fold concentrated DPBS was added.
6. Sμmol/ LCaleium ionophore A23187 Dissolve Caleium ionophore A23187 with DMSO and add to PZM-3 embryo culture medium at a final concentration of 5 μmol/L.
7. 1.9 mmol/L 6-DMAP Dissolve 6-DMAP with DMSO and add to PZM-3 embryo culture medium at a final concentration of 1.9 μmol/L. 8. 1.9 mmol/L 6-DMAP Dissolve 6-DMAP with DMSO and add to PZM-3 embryo culture medium at a final concentration of 1.9 μmol/L. The final concentration was 1.9 μmol/L.
8. Electroactivation solution 5.46 g of mannitol, 0.015 g of CaCl2 - 2H20, 0.002 g of MgCl2 - 6H20, 0.013 g of Hepes, were dissolved in 100 ml of deionized water, filtered through 0.22 μm membrane, and set aside at 4℃.
(II) Preparation of microscopic operation needles
(1) Preparation of injection needle: Adjust the parameters of the needle puller, and pull the capillary glass with an external diameter of 1 mm into the desired needle shape. Fix the syringe on the needle puller and break the needle at 7-9 μm OD of the glass needle. Grind the mouth of the needle tube to a 35° bevel on the needle grinder. Add some water to the grinding stone so that some water remains in the needle tube and grind the needle to a bevel. After grinding the needle, the inner and outer walls were cleaned in 2.5% HF, and then washed with deionized water. The cleaned needle is pulled out on the calciner, the glass bulb is heated first, and the tip of the glass needle is touched to the glass bulb, when the tip of the needle is slightly melted, it is pulled out quickly, a good tip of the injection needle should be short and sharp. Adjust the glass needle to the glass ball above the non-contact, heating the glass ball, the glass syringe curved area to 30 ° stop.
(2) Preparation of fixed needles: Adjust the parameters of the needle puller to pull the capillary glass with an outer diameter of 1 mm into the desired ideal needle shape. Use a grinding stone to break the needle at 120-180 μm OD of the drawn glass needle, and the mouth of the needle should be flush. Then bring the glass needle mouth close to the glass sphere and heat the glass sphere at intervals so that the needle mouth shrinks to 20 μm and the heating stops. Bend the fixation tube at 30°.
(C) Collection of oocytes
(1) Ovum extraction: Use a 10 ml syringe with a 10-gauge needle to extract COCs from follicles 2-8 mm in diameter, paying attention to the downward direction of the needle, such as lifting the piston to suck out the follicular fluid in the follicle while extracting the needle. The extracted follicular fluid should be placed in a 50 ml pointed-bottom centrifuge tube in a constant temperature water bath at 37 ℃, and try to finish the extraction within 0.5 hours.
(2) Egg washing: After egg extraction, place the centrifuge tube in a warm oven and let it settle for the first time for 10 minutes. After discarding the upper layer of follicular fluid, add TL-HEPES and wash it, shake it and let it settle in a warm oven for 3 minutes, discarding the upper layer of liquid, repeating the washing process for two times, and then pouring it into a large domestic dish after adding TL-HEPES for the third time to prepare for picking up the eggs.
Discard the upper layer of follicular fluid: use a 10 ml syringe to aspirate the upper layer quickly, and then aspirate it slowly when it is close to the sediment, in order to prevent the sediment from being drawn.
Prepare a large domestic Petri dish (with pre-scored lines on the bottom about 1 cm apart) with a small domestic Petri dish and add TL-HEPES solution to the Petri dish.
Note: Because of the large amount of TL-HEPES, it is sufficient to pour about 30 ml into the centrifuge tube when washing.
(3) Egg collection: Select COCs containing at least 3 layers of intact oocytes at 1x, and place them in the petri dish prepared above. Be careful to distinguish between dead eggs, naked eggs and eggs with irregular morphology or uneven cytoplasm.
(D) In vitro oocyte maturation culture
The selected COCs were washed with 4 drops of maturation solution and placed in a 24-well culture plate, which was fully equilibrated (3-4 hours), with 500 μl of maturation solution per well, covered with liquid paraffin, and 50 COCs were cultured per well. The incubation conditions were 38.5 ℃, 5% CO2, 95% air, saturated humidity, and the 24-well plates were labeled with the incubation time, number of eggs and incubator.
(v) Sperm preparation
(vi) Intracytoplasmic single sperm injection in oocytes
(VII) ICSI oocyte activation treatment
(viii) In vitro culture of embryos, calculation of development rate