SU1551315A1 - Method of freezing embrios of hens - Google Patents

Abstract

The invention relates to biology and can be used in cryobiology, genetics and cell engineering. The aim of the invention is the viability of embryos. According to the invention, the embryo is used at the end of blastula-onset stage of the gastrula, which is additionally separated from its own yolk membrane, kept for 5-10 minutes in an 8-12% solution-α propylene glycol, then cooled at a rate of 50-60 deg / min before the start of the crystallization process, incubated for 25-35 s and frozen to - 18-22 ° C at a speed of 30-35 deg / min and to -79 ° C at a speed of 95-105 deg / min. After thawing, incubated in saline for 5-10 minutes and transplanted for cultivation on a substrate, which is used as a yolk shell. 6 tab.

Description

The invention relates to the field of biology and can be used in cryobiology, genetics and cell engineering.

The purpose of the invention is to increase the viability of embryos.

The method is carried out in the following sequence.

The objects of research are embryos at the end of blastula stage — the onset of the gastrula, the source of which is freshly carried eggs of the Rhode Island breed.

In order to isolate the embryo, a ring of filter paper is placed on the yolk placed in the Petri dish with the blastodisk upwards, with a tendency for the blastodisc to appear in the center of the ring. The pancreatic membrane is injected with saline in an amount up to 5 ml (0.9% sodium chloride solution, Ringer's, Howard, Tyrode, etc. solutions are used as saline). Then cut the yolk membrane on the outer edge of the ring and transfer the drug to another Petri dish with saline. Oscillating movements in solution wash the embryo from the yolk membrane. Separated in this way from its own

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The yolk membrane of the embryo is pipetted (transferred to another Petri dish with 8-2% oL -propylene glycol in Howard solution, kept for 5-10 minutes, then transferred to a freezing container, for example an aluminum cylinder 11 mm in diameter and high 2 mm.

The embryo in the container in the specified B-12% solution of the cryoprotectant is cooled at a rate of 50-60 degrees / min from the beginning of the crystallization process, kept for s and frozen to (-18) - (-22) ° С at a speed of 30-35 deg / min and up to -79 ° C at a speed of 95-105 deg / min. Speed (cooling is controlled using an inspection device, for example, a thermocouple connected to an LKS-003 recording potentiometer. Frozen embryos are stored using solid carbon dioxide as a refrigerant. Thawing is carried out by placing containers with embryos in a solution ( Howard is at 18-22 ° C. After that, the embryo is kept for 5-10 minutes in saline solution (Howard's solution) to wash it away from the cryoprotectant. Before thawing the embryo: prepare the yolk membrane. they are separated from the yolk (food, unfertilized, ia are also used for any other cheap eggs). | For this, they put a ring of filter paper on the yolk, the inside diameter of which should be 2–3 mm greater than the outer diameter of the chamber used for cultivation. saline is injected in an amount of up to 5 ml, and then the yolk membrane is cut on the outer side of the ring and transferred to a Petri dish with saline solution.

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The scraper cleans the yolk membrane from the remains of the yolk mass and is laid upside down on the culture chamber. The culture chamber is previously filled with a nutrient medium, as in the known method.

The thawed embryo pipette with the end extended to 5 mm is transferred to the yolk membrane. At the same time, it tends to ensure that the embryo lies on the yolk membrane by the dorsal side. If he lay down with the ventral side or turned, a thin glass rod leads him to the desired position. The residual saline solution around the embryo is then removed with a thin pipette. After this, the embryo is cultivated in a known manner. The first control over the development of the embryo was carried out after 12 hours of cultivation.

The description contains data justifying the limiting limits of the duration of exposure of eambrions in a cryoprotectant solution before freezing and in saline solution after thawing, as well as cooling mode, exposure during the period of crystallization and freezing to -79 C.

The resistance of embryos to freezing also depends on the duration of their exposure during the crystallization process. With an exposure for 25-35 with 25-26% of embryos, viability is maintained, while with an exposure of less than 25 s, the number of viable is 3-5%, and more than 35 s - 15-18%.

The viability,% of the total number of conserved embryos, depending on the duration of the contact before freezing with the cryoprotector in various concentrations, is given in Table. one.

Table 1

thirty

35

40

1 2 3

c

5 6 7

The viability of deconserved embryos,% of their total number, depending on the cooling rate prior to the start of the crystallization process, is given in Table. 2

Table 2

The yield,%, of viable embryos, depending on the speed of freezing from (-18) - (- 22) ° C to -79 ° C, is given in Table. B.

Table k

Attachment of embryos to the yolk membrane, depending on the duration of their exposure to salt

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Continuation of table 2

50 60 65 70

26

25 20 18

The viability of deconserved embryos,% of their total number, depending on the speed of their freezing from the beginning of the crystallization process to (-18) - (- 22) ° C is given in Table. 3

Table 3

the solution after thawing is given in table. five.

Table 5

In tab. 6 shows the comparative test data on the freezing of chicken embryos by known and proposed methods. They indicate a significant advantage of the proposed method over the known.

Claims (1)

Invention Formula The method of freezing kyp embryos, including the separation of embryos from the yolk, washing from the yolk cannula, aging in a cryoprotector solution, cooling (and, in order to increase the viability of the embryos, embryos at the end of blastula stage - beginning of the gastrula, complement Table 6 separation from the yolk membrane is conveniently carried out, an 8–12% solution of oЈ-propylene glycol is used as a cryoprotector, incubated for 5–10 min, cooling is carried out in three stages: first with a speed of 50–60 deg / min before crystallization, in which it is kept from, and then at a speed of 30-35 deg / min to (-18) - (- 22) ° С and at a speed of 95,105 deg / min to -79 ° С.