Rabbit embryo freezing technology

Summary

Embryo transfer in rabbits often utilizes techniques of embryo freezing, such as the transfer of frozen mulberry embryos into the oviducts of recipients after resuscitation. Embryo freezing is easy to perform and preserves the integrity of the genetic information of the biological material, including the mitochondrial genome of the cell, under hygienic and clean conditions, allowing the biological activity of the biological material to go beyond the limits of life. Rabbits have a gestation period of 1 month, and pups can be obtained quickly through embryo transfer. However, the freezing technique requires the donor female rabbit to obtain a large number of embryos, which is difficult.

Principle

Embryo freezing techniques include slow freezing and vitrification freezing. The principle of slow freezing is based on the fact that slowing down the rate of freezing will allow sufficient time for water to be released from the cells, thus avoiding the lethal intracellular ice crystallization that brings about.

The rate of freezing is determined by the size and permeability of the cells, and most mammalian cells freeze at about 1 °C/min in glycerol or dimethyl sulfoxide cryoprotectants.

The principle of vitrification freezing is based on the fact that the cryoprotectant acts as an antifreeze, lowering the freezing temperature and increasing the viscosity of the frozen sample.

The cytoplasm of the vitrified embryo turns into amorphous ice (vitrification), which, unlike the process of ice crystallization that changes from a liquid to a solid, is a "solid-liquid" state, which can be interchanged within a small temperature range.

Operation method

Rabbit embryo freezing technology

Principle

Embryo freezing techniques include slow freezing and vitrification freezing. The principle of slow freezing is based on the fact that slowing down the rate of freezing will allow sufficient time for water to be released from the cells, thus avoiding the lethal intracellular ice crystallization that brings about. The rate of freezing is determined by the size and permeability of the cells, and most mammalian cells freeze at about 1 °C/min in glycerol or dimethyl sulfoxide cryoprotectants. vitrification freezing is based on the principle that the cryoprotectant acts as an antifreeze to reduce the temperature of freezing and increase the viscosity of the frozen sample. The cytoplasm of the vitrified embryo turns into amorphous ice (vitrification), which, unlike the process of ice crystallization that changes from a liquid to a solid, is a "solid-liquid" state, which can be interchanged within a small temperature range.

Materials and Instruments

Equipment:
① syringe
②Surgical instruments
③CO
2
Incubator
④Stereomicroscope
Reagents:
①Material: Rabbit
② Follicle stimulating hormone (FSH)
③Human chorionic gonadotropin (hCG)
④B2 culture fluid
⑤ Embryo temporary storage solution (Hepes-DPBS + 20% FBS)
⑥ OPS-1 freezing solution (Hepes-DPBS + 16% FBS + 10% ethylene glycol + 10% DMSO)
(vii) OPS-II freezing solution (Hepes-DPBS + 0.6 mol/L sucrose + 8% FBS + 10% ethylene glycol + 20% DMSO)
⑧ Cryoprotection solution I (temporary storage solution: OPS-I solution = 1:1)
⑨ Cryoprotective solution II (temporary storage solution: OPS-1l liquid = 3:1)

Move

The basic process of embryo freezing in rabbits can be divided into the following steps:

I. Embryo collection

1. Sexually mature female New Zealand white rabbits (6-18 months old) were selected as embryo donors.

2. The donor rabbits were treated with supernumerary ovulation by intramuscular injection of 0.3 mg, 0.4 mg and 0.5 mg of follicle stimulating hormone (FSH) twice a day for 3 consecutive days.

3. 12 hours after the FSH treatment, the supernumerary ovulated female rabbits were mated with male rabbits in a joint cage and injected intramuscularly with 200 U of human chorionic gonadotropin (hCG).

4. 18 hours after hCC injection and mating in donor rabbits, the oviducts of the female rabbits were removed by surgical clipping, and the embryos were flushed out in the laboratory with 5 ml of fluid from the juxtapical portion of the oviducts toward the flare.

5. Egg flushing fluid was examined under a body-view microscope, and embryos were picked up, placed in B2 culture solution and incubated in a _CO2 incubator. The collected eggs were examined under the stereomicroscope to check the discharge of the second polar body of the egg and the display of the embryonic prokaryotic nucleus, so as to initially determine whether the eggs were fertilized or not.

II. In vitro culture of embryos

1. Select good quality embryos and place them in B2+2.5% FBS culture medium for culture, and check the status of embryo development. Generally, the fertilized egg will divide into 2-cells 20-22 hours after hCG injection.

At 30 to 34 hours it will divide into 4-8-cell stage, at 40-46 hours it will develop into 8-16-cell stage, at 3 days it will develop into mulberry embryo and early blastocyst, and embryos cultured for 4 days will develop into swollen blastocysts and start to hatch.

III. Vitrification and rapid freezing of embryos

1. Embryos that have developed to more than 8-cells (including mulberry embryos and blastocysts) will be used for cryopreservation and resuscitation of embryos.

2. The procedure for freezing embryos by the pull-through open-ended tube (OPS) method is to prepare the following solution:

Embryo Storage Solution (Hepes-DPBS + 20% FBS).OPS-1 Freezing Solution (Hepes-DPBS + 16% FBS + 10% ethylene glycol + 10% DMSO).

OPS-II cryoprotective solution (Hepes-DPBS + 0.6 mol/L sucrose + 8% FBS + 10% ethylene glycol + 20% DMSO).

Cryoprotective solution I (temporary storage solution: OPS-I solution = 1:1). Cryoprotection solution II (temporary storage solution: OPS-1l solution = 3:1), all solutions were preheated to 38.5 ℃ for use. 3. 3-5 embryos were stored in the cryoprotection solution.

3. 3-5 embryos were equilibrated in the staging solution for 5 minutes, then transferred to OPS-1 solution for 2 minutes, and then the embryos were washed in 3 droplets of OPS-II solution (20 seconds per droplet wash).

4. The OPS tubes were slightly warmed and then stretched into microtubes of only half the diameter, and the treated rabbit embryos were loaded into the OPS tubes in the following order: -4 mm section of OPS-II liquid → a section of air → a section of OPS-II liquid containing the embryos → a section of air → ~4 mm section of OPS-II liquid.

The mouth of the OPS tubes was plugged with a small plastic stopper and glued with polyvinyl alcohol, and the tubes were immediately immersed in liquid nitrogen for storage after the embryos were filled.

Recovery of vitrified embryos 1. Remove the vitrified OPS tubes from liquid nitrogen and place them directly into cryoprotective solution 1 preheated to 38.5 ℃. After all liquids have melted, gently blow the embryos into cryoprotective solution I. After 1 minute of equilibration, gently blow the embryos into cryoprotective solution I. After 1 minute of equilibration, gently blow the embryos into cryoprotective solution II.

2. After equilibrating for 1 minute, transfer the embryos into cryoprotective solution I and equilibrate for 5 minutes.

3. The resuscitated cells can be transferred to B2 embryo culture solution for culture, or embryo transfer can be performed immediately

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