The aim of the present study was to establish a reliable procedure with nocodazole treatment for the synchronous cleavage of blastomeres of bovine embryos used as nuclear donors for nuclear transfer. Sixteen-cell stage embryos derived from in vitro-maturation, fertilization and culture were used. In three initial experiments, embryos were incubated in mTCM-199 + FCS with various concentrations (0-20 mu-M) of nocodazole under 5% CO2 in air. The concentrations required to arrest the blastomeres in the mitotic phase were examined. The effects of 10 mu-M nocodazole were also examined by observation of the division rate of blastomeres after the removal of nocodazole. Ninety percent (90%) of the blastomeres were arrested in the mitotic phase when embryos were exposed to 10 and 20 mu-M nocodazole. Exposure to 10 mu-M nocodazole had the highest blastomere-cleavage rate (47%). When the exposure period to 10 mu-M nocodazole was prolonged to 36 hr, the division rate of the blastomeres decreased. Furthermore, the effects of 2 culture conditions (mTCM-199 under 5% CO2 in air vs modified synthetic oviduct fluid medium under 5% CO2, 5% O2 and 90% N2) were compared on the division rate of blastomeres of embryos exposed to 10 mu-M nocodazole for 12 hr. When the embryos were exposed to nocodazole in mSOF, the division rate of blastomeres was improved to about 60%. The blastomeres produced by this treatment condition were used as nuclear donors and the developmental potential of the reconstituted embryos was investigated. The developmental rate to the blastocyst stage was 30.1% (58/193). Five embryos were transferred to 5 recipient cows and 2 of the 5 recipients (40%) became pregnant. Subsequently, one normal calf was born.

Equine demineralized bone matrix, particle size 2 to 4 mm, was implanted SC and IM in 4 foals and 4 adult horses. The implants were removed between 5 and 8 weeks after implantation. Bone formation was induced by SC and IM implantations in all animals. The implantation site had a marked effect on the amount of bone that developed, bone being formed earlier and in greater amounts when the matrix was implanted IM. The amount of bone formed increased with increasing time after matrix implantation at both sites. Demineralized bone matrix implantation also led to formation of small amounts of chondroid tissue; this tissue was more common in IM than SC matrix implants, and increased in amount with increasing time after implantation. Formation of this chondroid tissue did not precede the formation of bone, and there was no evidence that implantation of demineralized bone matrix in horses induced endochondral ossification. Age of the host did not appear to affect the response.

The present study examined the influence of post-cleavage time of nuclear donors on the development of reconstituted embryos in bovine nuclear transfer. Blastomeres of 16-cell stage embryos derived from in vitro-maturation, fertilization and culture were used as nuclear donor source. They were treated with 10 mu-M nocodazole for 12 hr. Blastomeres that cleaved within 3 hr after the removal of nocodazole were used-for the study. Metaphase II (M-II) oocytes were used as recipient cytoplasm. IN experiment 1, donor blastomeres at 6, 11 and 15 hr after the removal of nocodazole and donor blastomeres no treated with nocodazole were transferred into ethanol-exposed and enucleated oocytes. The reconstituted embryos produced by donor blastomeres oat 6 hr after the removal of nocodazole had a significantly higher developmental rate to the blastocyst stage than those at 15 hr and the untreated groups (P<0.01). In experiment 2, blastomeres at 6 hr after the removal of nocodazole used as nuclear donors were transferred into ethanol-exposed and enucleated M-II oocytes. The reconstituted embryos with ethanol-exposed and enucleated oocytes as recipient cytoplasm had a significantly higher rate of initial-cleavage (P<0.05) and development to the blastocyst stage (P<0.01) than non ethanol-exposed and enucleated M-II oocytes. These results demonstrate that the development of reconstituted embryos was improved when cleaved donor blastomeres after the removal of nocodazole were immediately transferred (at 3-6 hr post-cleavage) into activated enucleated oocytes by exposure to ethanol.

The change of plasma prostaglandin E2 level, natural killer cell activity and tumor cell growth were assayed after transplantation of EL-4 leukemia cells in C57BL/6 mice. Plasma prostaglandin E2 level was increased in EL-4 bearing mice, but indomethacin treated mice group showed low level. The tumor-derived prostaglandin E2 inhibited the post-target binding cytolytic process of natural killer activity. Indomethacin inhibited the growth of prostaglandin secreting EL-4 solid tumor

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 study has stated that heifer replacements, from the date of birth to 18 months, got through transplantation of frozen-defrosted embryos were not inferior to and in many cases superior to the heifers of their age but got through traditional reproduction. Transplant first-calf heifers have a high level of milk productivity which amounts to 7536,10 kilos, with 3,62% of fat and 3,23% of protein. Thus they provide a good selection material for new generation champions to be chosen among them and for being used as mothers of bulls for service

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 %