Acquisition of embryos in pigs

Summary

The acquisition of embryos in pigs requires the collection of embryos after super-expulsion of treated sows. At present, the international general embryos are generally divided into four grades: A, B, C and D. Grade A: the embryo development stage is consistent with the age of the embryo, the embryo has complete morphology, clear outline, spherical shape, uniform size of dividing sphere, compact structure, moderate color tone and transparency, no free cells or very few cells, and the proportion of degenerate cells is less than 10%. grade B: the embryo development stage is basically consistent with the age of the embryo, the outline is clear, the size of dividing sphere is basically the same. Grade B: the embryo development stage is basically consistent with the embryonic age, clear outline, the size of the dividing sphere is basically consistent, the color tone and transparency are good, some free cells and vesicles can be seen, and the percentage of degenerated cells is 10%-30%. grade C: the embryo development stage is not quite consistent with the embryonic age, unclear outline, darkened color tone, loose structure, more free cells and vesicles, and the percentage of degenerated cells is 30-50%. grade D: there are fragments of eggs and cells with no organization, and the percentage of degenerated cells is about 75%.

Operation method

Acquisition of embryos in pigs

Materials and Instruments

Equipment:
① syringe
② sterile surgical instruments
③ bovine oviduct or embryo recovery needle
④ Petri dish
⑤ 1.5 ml centrifuge tube; refrigerator
⑥ microscope, differential differential interference (DLC) inverted microscope
Reagents:
①Materials: pig
②PG, PMSG, hCG
③8% chloral hydrate (containing 5% MgSO
4
③8% chloral hydrate (containing 5% MgSO 4)
③8% chloral hydrate (with 5% MgSO 4)
⑤DPBS＋0.3% BSA
⑥DPBS＋10% FCS (Dulcimer phosphate buffer with 2% newborn bovine serum)

Move

The basic process of embryo acquisition in pigs can be divided into the following steps:

I. Super exclusion treatment 1. The selected sows are injected with PG 0.2 mg and PMSG 1000 U (can be increased or decreased according to the weight of the sows, 15-18 U/kg) intramuscularly on the 16th to 17th day of the estrous cycle, PG 0.2 mg intramuscularly 48 hours later, and hCG 500 U intramuscularly half an hour after the first mating.

2. Another method of super-excretion is to use PMSG 1000 U, and intramuscular injection of hCG 500 U within half an hour after the first mating. The first cleavage of the egg occurs 36 to 48 hours after fertilization (i.e., 36 to 48 hours of gestation; the first fertilization is designated as day 0), and on the third day of gestation, the embryo develops to the stage of 4-6 cells and reaches the oviducts and the horns of the uterus.

It reaches the hatching blastocyst stage on day 6 to 7. In order to avoid collecting hatching blastocysts, it is preferable to collect pig embryos on the 5th to 6th day of gestation, and mulberry embryos and blastocysts can usually be collected at this stage.

Collection of embryos 1. Inject 2.5% sodium pentobarbital 30-50 ml into the ear vein of the sow, and anesthetize it with appropriate amount (20-40 ml) of 8% chloral hydrate (containing 5% MgSO,).

2. The sows were placed in supine position on the surgical frame, and an 8-10 cm incision was made along the mid-abdominal line at the penultimate pair of nipples on the abdomen with a scalpel, and the uterine horns, ovaries, and oviducts were pulled out from the incision, and the number of ovulation points on each ovary was observed and recorded.

Insert a bovine oviduct or embryo retrieval needle (homemade) from the uterine horn 1/2 to 2/3 from the tip toward the uterine horn and fix it.

3. Use a syringe to aspirate 60 ml of ovulation fluid (Duchenne phosphate buffer with 2% newborn bovine serum), insert the needle from the tip of the uterine horn of the uterine tube, and slowly inject the ovulation fluid and a little bit of air, and then collect the ovulation fluid containing embryos in a 50 ml centrifuge tube with a cap.

4. Flush the other uterine horn in the same way. During the egg flushing operation, use 37 ℃ DPBS + 0.3% BSA egg flushing solution for egg flushing.

5. The flushed embryos were put into a solution of DPBS + 10% FCS at 37 ℃ for quality identification and classification of embryos.

6. Select embryos of different developmental stages for freezing and preservation.

Identification of embryos After the flushed egg solution was left to stand for 10 minutes, the lower layer of liquid was aspirated into a petri dish and the embryos were examined under a solid microscope, and the detected embryos were observed under a differential differential interference (DLC) inverted microscope. Distinguish unfertilized eggs from embryos at all stages of development.

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