The in vitro differentiative potential of mouse parthenogenetic (PG) embryonic stem (PGES) cells were investigated in the formation of embryoid bodies (EBs). EBs derived from PGES cells retarded in growth and showed restricted differentiation compared to their fertilized counterpart. In chimeric EBs from the aggregation of PGES and fertilized ES cells, morphological examination revealed that PGES cells were reduced in their population and distributed in endodermal layer as culture periods proceeded. These findings were comparable to those in aggregation chimeras of fertilized and PG embryos, and suggest that the differentiation of PGES cells in vitro is restricted in the formation of EBs

Murine parthenogenetic embryonic stem (ES) cell lines expressing lac zeta reporter gene were isolated after co-transfection with lac zeta reporter gene (pENL) and neoR gene (pSTneo) to TMA-48P cell line of 129/Sv origin. Karyotype analyses showed that all of four transfected cell lines examined contained 41 chromosomes with trisomy 8. Bacterial neoR transgene required for G418 selection were integrated into several chromosomes including chromosome 8. Histological studies of teratomas formed in syngenic mice and embryoid bodies grown in vitro showed that the differentiative potential remained almost identical in chromosomally normal parental cell line and its derivative cell lines trisomic for chromosome 8

Ovulated mouse oocytes denuded of their cumulus cells, were vitrified in a solution containing 7 M ethylene glycol as the sole cryoprotectant using one or two steps of exposure before vitrification and were diluted in 1 M sucrose solution in 5 or 10 min after warming. The results proved that the viability of oocytes are detrimentally affected by exposure to the vitrification solution even without vitrification. At 5 min dilution time, the two-step exposure was superior to the one-step in terms of the post-warming recovery rate of vitrified oocytes with normal morphology and their subsequent development to the blastocyst stage (p0.001) after fertilization in vitro. At 10 min dilution time, no significant difference between one or two-step exposure was found. The effect of the addition of 0.5 M sucrose to the vitrification solution was also determined and did not result in a significant improvement in the viability of oocytes vitrified in one-step and diluted for 10 min. In conclusion, the results in this study indicate that oocytes can be vitrified with 7 M ethylene glycol as the sole cryoprotectant in the vitrification solution, and that the recovery of normal oocytes after one-step exposure in the vitrification solution can be improved by 10 min dilution time. However, the improvement in the recovery rate of oocytes with normal morphology and their subsequent developmental in vitro was not improved by the addition of 0.5 M sucrose to the vitrification solution

Methods of increasing the efficiency of semen fertilizing capacity in vitro with application of hormonal and biophysical methods of influence, as well as the determination of metabolic criterion of spermium viability were studied in the conditions of the republic of Belarus. Research object was frozen-deiced sperm of cattle. Research results showed that entering of 50 mkg/ml of prostaglandin into capacitation media increased the breaking level on 2,3-3,1%, but at the same time there was the decreasing of embryo output at the pre-implantation stages. Increasing of estrophan concentration in media for capacitation up to 100 mkg/ml, and its addition into media for fertilization made it possible to increase the embryo output at the stage of morula-blastocyte up to 1,6-18,5%. Application of 8mg/ml of caffeine as a capacitation agent for the preparation of cattle sperm for fertilization in vitro made it possible to obtain embryo output of 16,7% with breaking level of 25,9%. Influence of directed polarized light on semen after its maturation was more efficient in comparison with the influence on it just after swim-up procedure; the output of pre-implantation embryos was 16,7% against 12,9%. Application of laser radiation made it possible to get 14,7 % of morula-blastocytes. Intensity of sperm breath (0,41-0,63 tg), intensity of lipid peroxidation (0,88-0,97 conditional units /10E6 cells), intracellular content of adenosine triphosphate (0,97-1,60 Nm/10E6 cells) and membrane potential (33-35 uV) in the conditions of matured in vitro oocyte made it possible to get 16,4-17,3% of pre-implantation embryos with the breaking level of 39,8-42,3%

The problem of creation of improved conditions for cattle oocyte maturation in vitro was studied in the Republic of Belarus. The objects of study were cattle oocytes and embryos of early preimplantation stages of development obtained in vitro conditions. Research results showed that application of bovine serum in complex with embryonic or fetal serum in the process of oocyte cultivation in vitro conditions made it possible to obtain 35,5-44,8% of breaking down cells and 16,1-17,2 % of embryos at the stage of morula-blastocyte. Entering of 0,02 ng/ml of gonadotropin-releasing hormone (Surfagon) into media for oocyte maturation at the cost of regulation of the processes of growing and development of oocyte-cumulus complexes made it possible to increase of yield of matured till the stage of metaphase II oocytes up to 89,3 %. At the same time, the yield of divided embryos after fertilization increased on 2,5 % in comparison with control indexes and amounted 45,2 %. Research results showed that the synthetic phytohormone epibrassinolide could be used as a biologically active factor in the process of getting early embryos in vitro in concentration of 2 x 10E-7 – 2 x 10E-9 mole/l. It made it possible to get 46,4-55,0 % of divided cells and 14,2-16,2% preimplantation embryos

Determination of the optimum parameters of cattle embryos production in vitro from high-producing cows after their slaughter on a meat-packing plant was realized in the conditions of the Republic of Belarus. Oocyte cumulus complexes of cows of black-motley breed and the conditions of their maturing were used as the object of experimental research. Oocyte cumulus complex were allocated with dissection of ovaries tissue placed into Hanks culture medium. The search and morphological estimation of quality of received oocyte cumulus complexes were realized by microscopic research. Then, the oocytes were placed into a culture medium for cell maturing in СО2-incubator at 38,5 deg C with the maximum humidity (98%), with presence of 5% СО2 under a layer of mineral oil for 24 hours. Matured oocytes were impregnated with the frozen-thawed sperm after capacitation. Oogenesis efficiency of production of pre-implantation germs in vitro was determined in accordance with the level of embryonic fission and production of viable germs. Use cumulus cells monolayer made it possible to increase the quantity of ripened oocytes on 5,2-5,7%, level of embryonic fission - on 14,0-14,6% and production of embryos - on 13,8-12,5% depending on a way of its production in comparison with groups of the cells which were cultivated without a monolayer. Thus, the research has shown the exploitability of use of cellular reproductive technologies in selection and breeding activities in cattle breeding. Their application in combination with embryo transplantation could make it possible to use the reproductive potential of high-priced oocyte donors more effectively for the genetic improvement of animal population efficiency

Investigation of the efficiency of ethylene glycol and sucrose application in the capacity of cryoprotectors for cryopreservation of cattle embryos and their thawing in the conditions of application of saline solutions (as their dissolution medium) and nutritive media with a various structure was realized in the conditions of the Republic of Belarus. Research results showed that application of ethylene glycol in concentration 1,5 M and sucrose in concentration 1,0M proved to be the most effective. Regardless of the applied media the average safety of embryos was 93,8-96%, and acceptability - 59,3-62,5%. Peculiar feature of ethylene glycol use as cryoprotector for preservation of cattle embryos was that it could be quickly absorbed by a cell and quickly deduced from it. It made it possible to realize embryo transfer immediately after thawing. Application of ethylene glycol and sucrose as cryoprotectors could considerably simplify the procedure of transplantation of the frozen-thawed embryos, practically reducing it to a procedure of artificial insemination

Efficiency of cultivation of cow preimplantation embryos and support of pluripotent properties in different nutritive media was analyzed; a method of getting early embryos in vitro for the genetically engineered purposes was developed in the conditions of the Republic of Belarus. Oocyte separation was realized in 3; 3-6; 6-9; and 9-12 hours after ovariectomy for the studying of the influence of storage period of ovaries on the output qualitative embryos. Oocyte separation was realized by bisection with adding of 1% of fetal cattle serum, 10 units/ml of gentamicin and 1 unit/ml of heparin. Oocyte-cumulus complexes maturing took place in CO2-bath with 5% of CO2 in atmosphere of maximum humidity and temperature of 38 deg C in TS-199 (Sigma) media with entering of 25 mM/l of buffer Hepes, 10 unit/ml of gentamicin and biologically active substance (20 % of fetal calf serum, 10 mg/ml of bovine serum albumin and 5 % of estrous cow serum with 10 mg/ml of bovine serum albumin). Research result showed that application of 10 mg/ml of bovine serum albumin into TS-199 media made it possible to increase the yield of cattle embryos at the preimplantation stages in vitro conditions on 19,1%, as well as to increase the fetal calf serum in quantity 15% to the nutritive media volume up to 16,0 %. Application of sodium pyruvate and calcium lactate in the process of cultivation of early cattle embryos obtained in vitro conditions made it possible to increase the yield of preimplantation embryos on 5,4 %. Specifically, the level of transformation of morulas into blastocysts was 44,4 %