microfertilization

Summary

Microfertilization, also known as micromanipulation-assisted fertilization, is an in vitro fertilization technique developed in the 1980s. This technique uses a micromanipulator to modify the zona pellucida of the egg, or injects sperm or sperm cells directly into the egg, assisting it to complete the fertilization process with the egg. 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). ).

Appliance

Microfertilization technology has a broad application prospect in animal fertilization biology research, livestock production, and treatment of human infertility.

Operation method

microfertilization

Materials and Instruments

Equipment:
①Fixing needle
② syringe needle
③Capillary glass tube
④ Needle puller
⑤ Needle grinder
⑥Ovipositor
⑦Cell culture box
Reagents:
①Material: animal
②Hyaluronidase
③Sperm incubation solution
④PVP
⑤Paraffin
⑥Dithiothreitol (DTT)

Move

The basic process of micro fertilization technique can be divided into the following steps:

1. Preparation of fixed needle and injection needle

(1) Preparation of injection needles: A sterilized capillary glass tube with an outer diameter of 1 mm is fixed on a needle puller, and the parameters of the needle puller are adjusted to pull out the desired needle shape. Usually, the length from one end of the needle to the beginning of thinning is 8-10 mm.

Fix the drawn needle tube on the calciner, and pull off the glass needle tube at 5-6 μm of the inner diameter of the glass needle. Then place the needle tube at an angle of 40 ° ~ 50 ° on the needle grinding instrument, add some water on the grinding stone, adjust the lift lever so that the tip of the needle and the plane of the grinding stone just contact, grinding the needle into a beveled surface.

Place the needle on the calciner to pull the tip, pull the tip of the first platinum ball, glass needle position adjustment, and then start heating the platinum ball. At this time, the glass needle slowly close, when the tip of the needle slightly melted, quickly pull away, pull out the tip of the needle should be short and sharp. The prepared needles were placed in a plastic box cleaned with 70% ethanol for storage and irradiated with ultraviolet light for 20 minutes before use.

(2) Fixed needle preparation: Sterilized capillary glass tubes with an outer diameter of 1 mm were fixed on the needle puller to pull out the desired needle shape. The glass tube was fixed on the needle puller, and the glass syringe was broken at 80-120 μm of the outer diameter.

Then the broken glass syringe close to the platinum ball, adjust the temperature so that the electric wire red, until the mouth of the needle shrunk to 30 ~ 40 μm to stop, the mouth of the needle must be smooth, when the inner diameter becomes 20-40 μm, stop heating.

2. Preparation of eggs and sperms

(1) Collection and treatment of oocytes: there are two main sources of oocytes: one is mature oocytes obtained in vivo by supernumerary ovulation; the other is ovaries collected from slaughterhouses, and immature colliculus-oocyte complexes (COCs) are extracted and cultured by in vitro maturation (IVM).

Prior to microinjection, oocytes were treated with 0.1% hyaluronidase to remove the surrounding granulosa cells and then placed in liquid paraffin-covered oocyte manipulation solution for backup. For oocytes with a high fat content, such as bovine and porcine, the oocytes can be made transparent by centrifugation so that the fat droplets in the oocytes are thrown to one side to facilitate microscopic manipulation.

(2) Preparation of sperm: Fresh or frozen sperm can be used for microinjection. Before use, several treatments are required according to the experimental design, e.g., sperm capacitation; removal of sperm tails and apexes for preparation of sperm nuclei.

Sperm incubation solution to add PVP, on the one hand, so that the swimming sperm loss of motility, easy to capture sperm, on the other hand, the sperm treated in this way is not easy to stick in the wall of the injection tube, conducive to the release of sperm; sperm incubation solution to add tritiated thiothreitol (DTT), the substance of the nucleus of the sperm of some animals have a certain role in the deconvolution of the nucleus of the sperm can improve the rate of formation of the nucleus of the male protozoa.

3. micro fertilization procedure

(1) microscopic operation, if the spermatozoa with vitality to do the injection, it is necessary to destroy the plasma membrane in the middle of its tail, so that its inactivity, this process is known as the sperm brake. This process is called sperm braking, because during normal fertilization, the plasma membrane of the spermatozoa fuses and the sperm releases a factor, sperm factor (SF), in the oocyte cytoplasm that is capable of activating the oocyte, causing fluctuations in the Ca concentration in the cytoplasm of the oocyte, thereby activating the oocyte.

The purpose of braking is to enable the spermatozoa to release SF relatively quickly after injection into the oocyte cytoplasm.After the spermatozoa are braked, they are drawn into the injection needle through the tail. For injection, the oocyte is fixed with a fixation needle so that the polar body is at the 12 o'clock position. The injection needle crosses the zona pellucida at the 3 o'clock position and penetrates deeper into the oocytoplasm in the 9 o'clock direction. a small amount of the oocytoplasm is first sucked back to make sure that the plasma membrane has been punctured, and then spermatozoa are injected into the oocytoplasm.

(2) To operate with the Piezo, first load 3-4 mm of mercury into the end of the injection needle and then load the needle into the injection device. Advance the mercury toward the tip of the needle and retain a column of air between the mercury and the liquid paraffin inside the injection needle.

Lower the injection needle into the 1st PVP droplet, spit and suck the operating fluid back and forth several times to lubricate the walls of the injection needle tube, and check the mercury beating using the pulses; if the mercury is seen to beat, it means that the Piezo electrical pulses are delivered smoothly. A PVP liquid-liquid paraffin-PVP liquid buffer column was made after the needle was loaded and the mercury was kept within the field of view of the injection.

Before injection, 8 to 10 oocytes were transferred into the operative droplet with an egg pipette, and then the injection needle was used to aspirate 1 spermatozoon in the sperm droplet into the 3rd PVP droplet, which was braked by a quick stroke of the needle tip over the tail of the spermatozoon, and subsequently the spermatozoon was aspirated from the tail.

When injecting, the pole body was adjusted to the 12 o'clock position, and the needle was inserted from the 3 o'clock position, at the same time, the Piezo pulse was used to penetrate the zona pellucida, and the sperm was pushed to the tip of the needle after continuing to insert the needle. At this time, the plasma membrane was recessed into the egg along with the tip of the needle due to the elasticity of the needle, and the tip of the needle approached to the contralateral side of the plasma membrane, and the Piezo pulse was used to penetrate the plasma membrane, and spit out the sperm, and suck back the excess liquid injected into the egg, then back off the needle gently to finish the injection.

4. Activation and in vitro culture of injected eggs

1. Activation of injected eggs In traditional ICSI, the stimulation of oocytes by the microinjection process can sufficiently activate the oocytes of human, rabbit, mouse, etc.; however, for cattle and pig, etc., the mechanical stimulation of the injection needle and sperm is not enough to activate the oocytes, and additional chemical or other stimulation is needed to activate the injected eggs, which is a prerequisite for the deconjugation of spermatozoa and the formation of male prokaryotic nuclei.

2. Cultivation of fertilized eggs The oviduct epithelial cells or granulosa cells are used as the trophoblast layer for co-cultivation with the fertilized eggs.

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