The method of determining the viability of embryos

 

(57) Abstract:

The method of determining the viability of embryos relates to agriculture and can be used for embryo transfer in farm animals. The essence of the invention lies in the fact that the quality of the embryo is evaluated twice: before freezing, after freezing and thawing, and by comparing the results of these assessments determine the viability of the embryo. It considered viable embryos, which did not change its assessment in the process of cryopreservation. The method improves the accuracy of determining the viability of the embryos during freezing and the degree of change in their quality and to predict the results of engraftment after non-surgical transplantation. The method also enables the use of embryos with score 3 points for freezing. table 2.

The invention relates to agriculture, in particular biotechnology, and can be used for embryo transfer in farm animals.

To ensure high efficiency of the method embryo transfer in breeding programs requires a high degree of accuracy to predict prize is, however, simple methods of determining the quality of the embryos and predict their survival.

There are methods of assessing the quality of embryos in vitro, which involve the use of dyes detecting enzyme activity (fluorescein-diacetate) (2) or a special label that determines the damage of the membranes when the defective condition of embryonic cells (3). However, these methods are not without drawbacks, as additional manipulation of the embryos in the process of evaluation can cause a reduction or complete loss of the ability to further develop, in addition, they require additional costs for reagents and efforts of specialists.

Closer to the present invention is a method of temporary cultivation of embryos in vitro in the laboratory for more accurate quality evaluation by morphological criteria (1). The assessment of embryo performed under a stereo microscope with an increase in 60-80 times, given the stage of development of the embryo, according to her chronological age, determine the morphology of embryos and, based on these criteria, the quality of the embryo and its suitability for further use. The cultivation is carried out in a thermostat at a temperature of 37oC for 24-48 hours. Then conduct a second assessment of their viability under the microscope. Emb who have given of the method include the additional costs of time and prolonged embryo outside the body, that can cause damage to its structures and decreased viability.

The aim of the invention is to improve the accuracy of determining the viability of bovine embryos.

This objective is achieved in that after morphological evaluation of the extracted embryos they can be frozen to a temperature of minus 196oC, then thawed and re morphological assessment of their quality. Thus take into account the change in scoring after thawing compared with the assessment before freezing.

The essence of the method lies in the fact that viable to include those embryos that are in the process of freezing and thawing has not lowered its assessment.

The proposed method improves the accuracy of determining the viability of the embryos during freezing and allows for a degree of change in their quality and to predict the results of engraftment after non-surgical transplantation.

However, this method can improve the efficiency of the method embryo transfer when breeding animals of outstanding genotypes, because it allows us to use all embryos, including the germ rated 3 points is subject to culling.

Due to the fact that a re-evaluation of embryo quality is carried out after thawing, the viability of embryos can be defined directly in front of transfers at a convenient time, excluding long process of cultivation outside the body (24-48 hours). In addition, the embryo is frozen may is a long time without losing its viability.

Because the definition of viability in the proposed method does not depend on the transition from one stage of development to another, and the method allows to determine the viability of embryos at any stage, including the late blastocyst, the accuracy of the proposed method is much higher compared to the prototype.

The proposed method is as follows: cattle embryos at the age of 7-8 days after surgical extraction is assessed according to the stage of development and morphological criteria on a 4-point scale ("instructions for transplantation of bovine embryos", M, 1987). Then freeze them in an environment Dulbecco with 20% fetal calf serum using as a cryoprotectant 1.5 M solution of glycerin in a programmable freezer, EMBI ("guidelines p is thawed, remove cryoprotector and conduct a re-evaluation by morphological criteria. On the basis of morphological structure, stage of development and compliance with its age embryos are divided into categories (points):

- excellent (5) embryo ideal, spherical, symmetrical, with cells of the same size, color and density, stage of development corresponds to the age;

- good (4) - the embryo has minor flaws such as a few blastomeres separated from the total mass in perivitelline space, irregular cell mass, multiple vacuoles, stage corresponds to the age;

satisfactory (3) - morphological structure has serious remarks: presence destroyed blastomeres, strong vacuolization, heterogeneous staining, low density cellular mass, the presence of many cells in perivitelline space behind the development of up to 24 hours;

- bad (2) - numerous displaced from the total mass of blastomeres, disruption of intercellular contacts, degenerated cells, cells of different sizes, many large vacuoles and granules, retarded the development of more than 24 hours. Such embryos are discarded. In accordance with the degree of quality deterioration ambrina is="ptx2">

The invention is illustrated by the following example.

In the farms of the Moscow region transplant embryos, non-surgical method of animal-recipients. The recipients were selected heifers slucero age (17-20 months.) with a live weight of 360-380 kg groups of animals were formed by the method of group-analogues. All transfers and manipulation of embryos carried out by the same experts. One group of recipients transplanted embryos, did not alter its quality assessment after freezing-thawing. The second group of heifers transplanted embryos after thawing reduced the score by 1 point, to a third group of animals transplanted embryos with reduced quality more than 1 point (PL. 2). At a constant quality embryos after cryopreservation their accepted equaled 56.9%, whereas the decline in the quality of 1 scores were springing 43.1% of recipients, but at a lower quality than 1 point caught only 29.3% of the embryos. That is, at lower scoring after freezing-thawing viability of embryos also decreased 13.8% and 27.6%, respectively.

From the data obtained it follows that the viability of the embryos is inversely proportional to the degree of reduction of their cachette the settling down of embryos decreased by a statistically significant amount.

The method of determining the viability of bovine embryos, including two estimates of embryo quality, separated by a time interval, and determining its viability by comparing the results of these assessments, characterized in that, to improve the accuracy of the method, the quality of the embryo re-evaluate after freezing and thawing, and referred to a viable embryos, which did not change its assessment in the process of cryopreservation.

 

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FIELD: medicine.

SUBSTANCE: method involves using product containing spermatozoa treated by drying with freezing to humidity level of 1% and having injured membrane or spermatozoon head so that spermatozoon nucleus retains its genetic validity enough for fertilization. The spermatozoon heads fertilize an isolated oocyte after rehydration and microinjection being done. The retained genetic integrity is enough for fertilizing an oocyte and producing living descendants. Method involves collecting living mature spermatozoa, making spermatozoa suspension in special purpose physiological medium, freezing the spermatozoa suspension for producing frozen spermatozoa, drying the frozen spermatozoa or spermatozoa heads in vacuum to humidity level of 1%, making rehydration of spermatozoa or spermatozoa heads with injured membrane and selecting those retaining nucleus of genetic integrity. The selected spermatozoa or spermatozoa heads are used for fertilizing isolated oocytes with living descendants being produced.

EFFECT: enhanced effectiveness in producing living descendants.

39 cl, 5 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: method involves using product containing spermatozoa treated by drying with freezing to humidity level of 1% and having injured membrane or spermatozoon head so that spermatozoon nucleus retains its genetic validity enough for fertilization. The spermatozoon heads fertilize an isolated oocyte after rehydration and microinjection being done. The retained genetic integrity is enough for fertilizing an oocyte and producing living descendants. Method involves collecting living mature spermatozoa, making spermatozoa suspension in special purpose physiological medium, freezing the spermatozoa suspension for producing frozen spermatozoa, drying the frozen spermatozoa or spermatozoa heads in vacuum to humidity level of 1%, making rehydration of spermatozoa or spermatozoa heads with injured membrane and selecting those retaining nucleus of genetic integrity. The selected spermatozoa or spermatozoa heads are used for fertilizing isolated oocytes with living descendants being produced.

EFFECT: enhanced effectiveness in producing living descendants.

39 cl, 5 dwg, 1 tbl

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