Method for embryo transfer in sheep

FIELD: biotechnology.

SUBSTANCE: the present innovation deals with individual matching donor sheep to recipient sheep at similar antigenic composition of blood types being correspondent to the value of antigenic similarity index being ra=0.51-1.00, where ra - antigenic similarity index. Moreover, the mentioned antigenic similarity index should be calculated by the following formula: where S - the number of similar antigens in a donor sheep and in a recipient sheep, n1 - the number of detected antigens in a donor sheep, n2 - the number of detected antigens in a recipient sheep. The present innovation enables to increase the level of adaptability of transferred ovine embryos by 25%.

EFFECT: higher efficiency of embryo transfer.

3 ex, 4 tbl

 

The technical field to which the invention relates.

The invention relates to the field of biotechnology of farm animals, in particular to embryo transfer in sheep.

The level of technology

Method of transplantation of embryos from cows less productive at the present time is one of the most common biotechnological techniques. This advanced technique has been successfully used in the intensification of reproduction of sheep. At the same time, a significant limiting factor is low (from 20 to 60%) accepted embryos after implantation in the body of the recipient.

Known methods and techniques to enhance the engraftment of early embryos in sheep during their transplant.

With the aim of enhancing the engraftment of transplanted embryos is recommended to enter at the top of the horns of the uterus of the recipient (Ernst L.K. Transplantation of embryos of farm animals / Lkist, Neergheen. - M.: Agropromizdat, 1989. - S-129).

It is also known that the successful settling down transplanted embryos in sheep depends on the stage of their development and synchronization of the estrous cycle in the recipient with the stage and the age of the embryo. Good results can be an exact match of the estrous cycle of the recipient with the age of the embryo (Beeham D., Sreenar J.M. Embrio transfer in farm animals Ed.K.J.Betteridge, Canada, Department of agricul. - 1977. - Monograph. 16. - P.16).

The best results engraftment get when transplanting recipients not 1, but several embryos (K. Kasymov embryo transfer in sheep / Kosimov, Galimzyanov, Nawano etc. // Zootechnics. - 1994. No. 8. - P.29-30). In addition, as a test of the high engraftment use a classification assessment of the suitability of embryos for transplantation (Sergeev, NI Bioengineering embryo transfer / Niedergeses, Sautepan // Sheep. - 1987. No. 2. - P.36-40).

There is a method of selecting horses that are resistant to grazing the clamp and its associated diseases, which includes the determination of the genetic parameters of blood, the genetic parameters of blood use polymorphic proteins and enzymes of the blood and the degree of heterozygosity, while sustainable animal is determined by the presence of phenotype serum arylesterase HB, level of heterozygosity 45-100% and the absence of transferrin phenotype FF (RF Patent No. 2212791, class. And 01 To 67/02, 12 Q 1/44).

The closest biotechnological solution to the proposed method (prototype), from among known methods for enhancing the engraftment of transplanted from donor to recipient embryos method is based on the definition of blood groups in cows-donors, recipients, bulls make the her and matching them so that to their antigenic spectrum (blood group) as little as possible was different from the antigenic spectrum of the embryo (Sukhova THAN the Accepted grafts cattle with regard to immune status manufacturers, donors, manufacturers / N.O. Sukhova, Wedplan, Vastaava etc. // Dokl. RAAS. - 1992. No..11-12. - P.27-30.; Verevochki PS Immunogenetics in breeding cattle / Psservice, Net - Kuibyshev, 1988. - 104 S.).

Significant disadvantages of the known methods is the low number obtained at the optimum stage of development of early and late blastocyst embryos due to the asynchrony of ovulation in the ovaries, the complexity of their vital evaluation, the wide variability in performance transplantation because of the complexity of influencing factors, the expenditure of a large number of embryos from high value animals to obtain acceptable results, which significantly reduces their effectiveness.

One of the reasons for the low engraftment of transplanted embryos may be immune and genetic incompatibility between donor and recipient. When embryo transfer is still unclear immunogenetic interaction system "embryo-animal-recipient", which, in some cases, successful implantation and conflict-free development of transplant in organisms is e priemysel mother, in other cases lead to its rejection. Identifying factors such mechanism could significantly improve the efficiency and effectiveness of the method embryo transfer.

Disclosure of inventions

The task of the invention is to develop a method that provides a higher level of engraftment of transplanted early embryos after their transplantation from sheep donor to the recipient.

The technical result that can be achieved using the proposed method, is to increase the level of engraftment of transplanted early embryos after their transplantation from sheep donor to the sheep-recipients.

The technical result is achieved by using method of embryo transfer in sheep, including individual selection of sheep donor to the sheep recipients, with individual selection carried out with similar antigenic composition of the blood groups, corresponding to the index value antigenic similarities from rand= from 0.51 to 1.00.

The method of selection of sheep-donor embryos to the sheep-recipients is as follows.

The selection of the elite herd of donor embryos and less productive sheep-recipients is carried out by any of known methods (instruction technology organizations on artificial insemination and transpl is ncacii embryos of farm animals. M., 2000. - 174 C.; Ernst L.K. Transplantation of embryos of farm animals / Lkist, Neergheen. - M.: Agropromizdat, 1989. - S-129.).

Then they held immunogenetic determination of blood groups in the immunological reaction of hemolysis and agglutination using monospecific immunodiagnostics with the subsequent index calculation antigenic similarity (IAS, rand).

The index calculation antigenic similarities conducted according to the following formula:

where S is the number of similar antigens in sheep donor and sheep-recipient;

n1- the number of identified antigens in sheep donor;

n2- the number of identified antigens in sheep recipient.

Estimated index is close to zero, means the minimum genetic similarity, and, on the contrary, the index is close to unity indicates maximum similarity of genetic parameters of donors and recipients. Further manipulations to obtain and embryo transfer are carried out by a known method (K. Kasymov embryo transfer in sheep / Kosimov, Galimzyanov, Nawano etc. // Zootechnics. - 1994. No. 8. - P.29-30.), that is illustrated by the following examples.

In the breeding season were selected 6 sheep donor, characterized by elite productivity, and 36 sheep-recipients with lower productivity. Donors and p is cipients were pre-selected blood samples and defined antigens of erythrocytes. Based on the immunogenetic characteristics between them were calculated indexes antigenic similarity (IAS, rand) blood group.

Multiple ovulation in sheep donor induced by the introduction of 12-14 sexual cycle 24 mg follicle-stimulating hormone (FSH) followed by injection of 125 μg of prostaglandin PGF 2 alpha Estrofan". Synchronization of the ovulatory process in the ovaries was provided by intravenous 800 units of human chorionic gonadotropin (HCG). Embryos from donors was extracted on day 5 after insemination (day 0) and after quality assessment, were transplanted recipients. Selection of pairs "donor-recipient" carried out the largest index antigenic similarities. Thus, on the value of the IAS donor recipients were divided into 3 groups.

The implementation of the invention

Examples of a specific implementation method of embryo transfer in sheep.

Example 1. In the first group consisted of a pair of "donor-recipient"with low genetic similarity blood group (rand=0.1 to 0.5) (table 1).

Table 1

Selection of pairs of donor-recipient
AnimalsIndex antigenic similarities (rand)Antigens
AAAbBbBc BdBeBgBiCaCbDaMaR
ra=0,1-0,50
DonorAABbBeBgCbMa
Recipient0,22AbBgBiMaR
Recipient0,43AABcBeCbMa
Recipient0,13AbBgDa
DonorAbBdCa
Recipient0,14AABbBdDaMa
Recipient0,33AbBcBiCaCb
Recipient0,50BdBgCa

The effectiveness and efficiency of transplantation was determined by the level of engraftment of embryos, by number of lambs received-grafts and immunogenetic data examination of authenticity of origin (table 3, 4).

Example 2. The second group (experiment 2) entered a couple with a similar range of blood groups (ra=0,1-1,00).

Table 2

Selection of pairs of donor-recipient
AnimalsIndex antigenic similarities(rand)Antigens
AAAbBbSuBdBeBgBiCaCbDaMaR
randor =0.51 to 1.0
DonorAAAbSuBeBiCaDa
Recipient0,63AASuBiCaDaMa
Recipient0,63AAAbBbBeBiCa
Retz is pient 0,75AAAbBdBeCaDaMa
DonorBbSuBgCbMaR
Recipient0,58AABbBgMaR
Recipient0,58AbBbSuCbR
Recipient0,67SuBgMaR

The floor is built, the results are shown in table 4.

Example 3. In the third group (prototype) definition of IAS between donors and recipients and individual selection of pairs "donor-recipient" was not performed (table 4).

Conducted by comparing the immunogenetic characteristics of the triad: father-mother-donor-offspring-transplant" genetic analysis showed that all lambs received 1, 2, and 3 experimental groups were obtained from transplanted zygotes: blood transplants matched groups of sheep blood donor and sheep, semen which they were seeded. The antigens of the recipient, from which were obtained the lambs-grafts, in their blood were absent, as shown in table 3.

Table 3

Genetic examination of the reliability of the origin of the lambs-grafts
AnimalsBlood group
RAM-manufacturerAb, Bb, Bc, Bd, Bg
The ewe-donorAA, Ab, Bc, Be, Bi, Ca, Da
The ewe-recipientAA, Be, Bi, Ca, Da, Ma
Descendant-transplantAb, Bb, Bc, Bd, Bg,

The results of biotechnological experiments conducted in triplicate, indicate quite clear dependence of the rate of engraftment of embryos at transplantation and from the code, the antigenic similarity of groups of blood donors and recipients (table 4).

Table 4

The settling down of embryos in sheep after transplantation, depending on the antigenic index of similarity of donors and recipients
The antigenic index of similarity (IAS), randUsed recipientsReceived lambsThe effectiveness of transplantation, %
onlyincluding
goal.transplanted embryos
0,10-0,50 (experiment 1)1224137654,2
0,51-1,00 (experiment 2)122421111087,5
The placeholder1224155762,5

Therefore, the proposed method of selecting sheep donor and recipient largest index antigenic similarity (IAS) blood groups among them in the boundary values of ra=0,51-1,00 has a number of positive differences and significant advantages compared with prototype:

- improving embryo implantation by 25% in selecting the optimal combination of donor and recipient blood groups can significantly reduce the consumption of embryos and loss of embriodered;

- significantly (71,4%) increases the number of lambs-grafts, and therefore, the efficiency of embryo transfer as a biotechnological method.

Method of embryo transfer in sheep, including individual selection of sheep donor to the sheep recipients, characterized in that the individual selection of sheep donor to the sheep-recipients spend with similar antigenic composition of erythrocytes when the value of the index antigenic similarity rand=from 0.51 to 1.00, which is determined by the formula

where rand- the index of the antigenic similarity;

S is the number of similar antigens in sheep donor and sheep-recipient;

n1- the number of identified antigens in sheep donor;

n2- the number of identified antigens in sheep recipient.



 

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