• Institute of Biology and Immunology of Reproduction
      "Acad. Kiril Bratanov"

      • Scientific achievements

        Most important scientific achievements

        • Species differences in the distribution of phosphatidylserine in the plasma membrane of spermatozoa of bull, ram, boar and dog have been found in the process of cryopreservation. Higher level of externalization of phosphatidylserine in the plasma membrane has been observed in spermatozoa of boars and rams. Externalization of phosphatidylserine with specific distribution pattern on the membrane depending on the cell functional status has been found after capacitation as well as after induced apoptosis in ram spermatozoa. A. Belcheva, M. Ivanova – Kicheva, K. Alexandrova, D. Kacheva, R.Baikushev, D. Petrova /2000/; M. Ivanova-Kicheva, A. Dimitrov, I. Nikolov, D. Daskalova, M. Petrov /2005/. After capacitation the spermatozoa with retained mitochondrial potential have been shown to be significantly more in quantity than the cells after induced apoptosis. Ivanova-Kicheva M., A. Kukov, D. Daskalova, E. Solenkova /2007/.
        • Annexin V from swine placenta has been isolated by the employment of automatic chromatographic system BioPilot (BioRad) and subsequent gel-filtration on DEAE sepharose, Superdex 200 and Mono S columns. E. Solenkova, M. Ivanova-Kicheva, A. Kukov, D. Daskalova, J. Strzezek, P. Wysocky /2008]. The obtained protein has been characterized, liophylized and method have been set up for its use to elaborate expression of phosphatidylserine on spermatozoa after cryopreservation. Ivanova-Kicheva M., A. Dimitrov, I. Nikolov, D. Daskalova, M. Petrov /2005/.
        • Expression cloning studies of the Annexin V gene in E.coli have been performed. Annexin V gene has been sequenced based on the human sequences of annexin V, published in the GeneBank. Specific primers for the annexin V gene have been obtained and total RNA preparations from swine placenta have been examined by means of TRIzol®Reagent (Invitrogen, 15596-026).
        • Research on the oxidant/antioxidant balance in bull and ram sperm during in vitro storage and cryopreservation showed prevalence of oxidant species. Positive correlation between motility and activity of the antioxidant enzymes SOD and catalase has been demonstrated. R. Stefanov, M. Angelova, Tz. Stefanova, M. Sabev, P. Dolashka, W. Voelter, Z. Zahariev /2004/. It has been found that exogenously added enzyme preparation HLSOD isolated from H. lutea to the storage media enhances the activity of NADH-TR and protects the fertilization capacity of spermatozoa. Subev M., R. Stefanov, Z. Zahariev /2005/. Based on the obtained results methods have been set up for the application of the HLSOD in the technology of sperm storage in bulls and rams. Ivanova-Kicheva M, M. Angelova, D. Daskalova, R. Stefanov, M. Subev, I. Nikolov, Trin Thi Kim Thoa, C. Chemshirova, M. Petrov /2007/.
        • Investigations have been carried out of the effect of selenium and originally synthesized methylxantines on the functional parameters of male gametes. Ch. Vaisberg, M. Boychef, K. Miteva /2002/. It has been found that vephylin-malate and originally synthesized substances in Bulgaria under code 61 and 8-/bis (hydroxyethyl)-аmine/-coffein, significantly increase the motility and survival of spermatozoa under in vitro storage conditions. There is experimental evidence that above substances can be employed successfully for the improvement of male gametes motility in astenosoospermic patients, included in established programs for assisted reproduction. P. Todorov, A. Beltcheva, Y. Dimitrov /1999/.
        • It has been established that the addition of β-mercaptoethanol and the antioxidant enzyme SOD to sperm of the species Sus scrofа during in vitro storage stabilizes the oxidant-antioxidant balance and the vitality of spermatozoa.
        • Lipoprotein fraction of low density /LDL/ has been isolated from egg yolk which is thought to be involved in the formation of biocomplexes with protective action on the sperm vitality in bulls and rams. Our studies demonstrated that addition of the LDL-fraction to the cryopreservation medium according the technology of Nagase-Niva is more effective than the technology of Cassou and helps to preserve to a higher degree the biological properties and the temperature resistance of gametes. Nikolov I, M. Subev M, M. Ivanova-Kicheva , P. Rashev, V. Gerzilov, H. Hristev /2006/.
        • The influence of mono- and disaccharides as medium constituents for in vitro storage of sperm of Sus scrofa at 150С has been studied. It has been found that media containing monosaccharides exert better protective effect on the biological parameters of spermatozoa studied in comparison with disaccharides. Zagorski D., M. Sabev, N. Bobadov /1998/; Sabev M., I. Nikolov, M. Ivanova-Kicheva, M. Petrov, R. Stefanov /2007/.
        • Experiments have been carried out to explore the protective effect of chromatography selected proteins isolated from ram seminal plasma by spermatozoa storage manipulations at 40С as well as after cryopreservation. There has been evidence that proteins with apparent molecular weight of 30-60 kDa exert protective effect on the integrity of the cellular plasma membrane. Localization of the above proteins has been demonstrated by means of immunocytochemistry.
        • Studies on the cryoresistance of ejaculated spermatozoa of autochtone breeds of rams (cooper-red, shoumen, karakachan, local karnobat) have been carried out. It has been demonstrated that during the estrous season spermatozoa of the prevailing part of ejaculates possess good cryoresistance. Genebank of semen received from autochtone breeds of brood animals obtained during the estrous season has been set up.
        • Schedule of sexual reflexes induction during the anestrous season has been elaborated by aborigine breeds of brood animals from the species Ovis aries. It has been found that the manifestation of sexual behaviour and appearance of libido sexualis can be successfully induced by an adequate alimentary and medical treatment of the animals. This finding has contributed to the improvement of breed selection programs by making possible the effective use of the animals as sperm donors out of the breeding season. M. Sabev, I. Nikolov, M. Ivanova-Kicheva, R. Stefanov, Ts. Chenshirova, E. Baycheva, M. Popova /2006/.
        • Artificial insemination with cryopreserved semen has been successfully employed to achieve higher rate of eggs fertilization of Muscus duck. Optimal doses of the cryoprotectants have been set as follows: glycerine /6%/, ethyleneglycol /7%/, DMSO /4%/ and polyethyleneglycol /5%/. Gerzilov V., S. Dimitrov, I. Zaprianov, M. Ivanova-Kicheva, I. Nikolov /2000/.
        • It has been demonstrated that the addition of glutathione, methionine and follicular fluid to media for in vitro preparation of spermatozoa results in improvement of their biological properties. M. Sabev, D. Zagorski, B. Bobadov, K. Vlahov, E. Sapundjie, G. Atalidis /1999/. Inclusion of cytochrome C to the thawing media has increased the motility and the activity of the enzyme succinate dehydrogense. Z. Zahariev, R. Stefanov, M. Sabev, K. Miteva, I. Nikolov /2007/.
        • Research on the factors influencing the successful outcome of intrauterine insemination technologies has been performed including the development of comparative studies on cryoprotective media and regimes for cryopreservation of human semen. Todorov P., E. Arsenova, J. Dimntrov /2004/; L. Rodionov, N.Tchoob, T. Schegoleva, P. Todorov /2004/.
        • It has been found that prolongation of the cultivation period of in vitro pre-implantation embryos (up to blastocyst stage) facilitates the selection of the best embryos suitable for transfer and decreases the risk of multiple pregnancy. Y. Vladimirov, D. Tacheva, P. Todorov /2004/.
        • Methods for isolation of embryonic stem cells from human blastocysts, obtained in vitro has been elaborated and implemented in the clinical practice (Center of Obstetrics and Gynecology “Dimitrov”). Comparative cryobiological investigations on isolated colonies from human embryonic stem cells have been carried out. It has been established that stem cells retain their biological properties in a highest degree after vitrification under the protection of ethyleneglycol and Ficoll-70. P. Тоdоrоv /2006/.
        • With the aim to cryopreserve blood stem cells umbilical cord blood has been stored in stocks. Stem cells in the respective preparations have been immune phenotyped and the level of apoptosis has been investigated after thawing. It has been demonstrated, that 85% of the frozen-thawed cells have retained their vitality and their clonogenic potential. Obtained results have found application by the set-up of a stem cell – cryobank at the Hospital “St. Sofia”. Todorov P., J. Dimitrov, I. Pandeva /2006/.
        • Human fetal liver cells have been isolated, cultivated under long-term conditions and characterized (adhesive properties, proliferative potential, morphology, immune phenotypization, level of apoptosis). P. Тоdоrоv /2005/; P. Todorov, R. Konakchieva, Y. Dimitrov, V. Novachkov, D. Kyurkchiev, N. Tchoob, N. Gorohova /2010/.
        • Vitrification methods have been set for cryopreservation of human embryonic stem cells and preimplntation embryos. P. Todorov, R. Konakchieva, J. Dimitrov, N. Chub, Y. Petrenko /2005/; V. Isachenko, F. Nawroth, M. Montag, S. Yakovenko, P. Todorov, E. Isachenko /2010/.
        • Direct immersing in liquid nitrogen has set application methods for cryopreservation of oviductal epithelial fragments. Chub N., V. Grishchenko, L. Demina, P. Tododrov /2010/.
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