Reproductive biotechnology and Cryobiology of gametes

  • Main research topics

    Head of Department: Prof. Dr. Maria Georgieva Ivanova, das

    Main research topics

    The Department of Reproductive Biotechnologies and Cryobiology of Gametes has a long history that is closely related to the development of the Institute since its creation. The scientific and practical subject of the department is the founder of the Institute’s theme and proudly takes place as a leader among the academic community in the field of biology and immunology of reproduction of animals and humans.

    In the first years of its development, the scientific theme of the department has been mainly associated with research on the biotechnology for artificial insemination in animals. These scientific developments have contributed to enrich and improve the gene pool in Bulgaria. The department has developed and implemented for the first time in practice different methods, media and technological approaches for long-term storage of gametes. As a result, the practical use of the reproductive potential of breeding animals has achieved high economic efficiency and the first cryo-banks for valuable and elite breeding stock have been created.

    Today’s scientific research in the department is focused in two main directions: “Breeding of farm animals, biology and biotechnology of reproduction” and “Physiology of animals and humans”. At present a large part of the research is associated with the study of the problems of the reproductive cryobiology, in light of contemporary biological, structural and functional analysis of the gametes, in order to regulate and improve the fertility and fecundity of the animals.

    Currently, the scientific research of the department focuses on one of the priority programs of the Bulgarian science, related to improving the quality of life and living nature, namely: Biotechnology, food and health. The main objective is to contribute to the rapid genetic progress and efficiency of reproduction in farm animals. The main research approaches in the department are related to studies that have the potential to turn into practical applications, both at home and abroad.

    • Innovative studies for the development and integration of applied research with business interest in the field of reproductive biotechnology and cryobiology of stem cells, gametes and embryos.
    • Establishment of joint research teams of scientists from the Department and lecturers from universities and promoting new opportunities to work with the modern equipment, property of the Institute.
    • Involvement of young scientists (graduates and doctoral students) in training to increase the capacity of the research collectives.
    • Development and application for funding of national and European programmes, including the Framework Programmes for Research and the Structural Funds.

    The main scientific direction of the department is: “Environmental factors and cell markers responsible for the structural and functional status of gametes and cells during in vitro storage and cryopreservation in relation to the optimization of biotechnology.” This direction is based on the scientific and applied interest to improve the existing biotechnologies, to increase the economic efficiency of raising and breeding of poultry and livestock, and to provide new scientific works for use in biology and regenerative medicine. The main research problems are consistent with the scientific interests of the staff of the Department, with the funded research projects currently in development, and with the perspective to contract new financing projects. In the last three years in the Department of Reproductive Biotechnologies and Cryobiology of Gametes” two PhD and one DsC dissertations had been defended.

    Basic research problems

    • Development of models and methods for improvement of the cryo-techniques and biotechnologies for storage of gametes, related to conservation and sustainable development of indigenous and endangered animal species;
    • Studies on environmental factors and cell markers responsible for the cryo-tolerance and cryo-resistance of the semen during in vitro storage;
    • Studies on cellular, sub-cellular and molecular level on the biological role of the media and biologically active substances in relation to the protection of the structure and function of gametes.
    • Isolation and characterization of seminal plasma proteins and identification of their role in the mechanism of protection of the gametes.
    • Cryo-biological research on embryonic stem cells.
    • Comparative studies on methods for isolation, cultivation, differentiation and cryopreservation of hematopoietic stem cells.
    • 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. RStefanov, 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. MSabev, 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. PTodorov, 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/.
      • Research grants

        СС-807/98, Ministry of Education and Science. In vitro stimulation of the vitality and the fertilization capacity of mammalian spermatozoa. Project leader: Assoc. Prof. Hussein Al-Hanak

        МУ-К-1201/02, Ministry of Education and Science. Smoking and reproduction: influence of cigarette smoke on the membrane integrity of human spermatozoa. Project leader: Res. Assoc. A. Belcheva.

        МУ-Б-1410/04, Ministry of Education and Science. Isolation of Annexin V as a biological marker of cellular apoptosis. Project leader: Res. Assoc. A. Kukov.

        CC-1303/03, Ministry of Education and Science. Selenium diet as a tool for stimulation of the vitality and increment of the fertilization capacity of boar spermatozoa. Project leader: Assoc. Prof. Ch. Vaisberg.

        Б-1522/05, Ministry of Education and Science. Cryobiological studies on human fetal liver cells. Project leader: Res. Assoc. P. Todorov.

        CC- 807/98, Ministry of Education and Science. In vitro stimulation of the vitality and fertilization capacity of mammalian spermatozoa. Project leader: Assoc. Prof. M. Sabev.

        КБ-МУ-1/01, Ministry of Education and Science. Cryobiological studies on human ovarian tissue. Project leader: Res. Assoc. P. Todorov

        22/03, Medical University, Sofia. Morphological and functional parameters of human ovarian tissue after different methods of cryopreservation. Project leader: Res.Assoc. P. Todorov

        Experimental Bee Breeding Station, Agricultural Academy, 1996. Project leader: Res. Assoc. P. Todorov

        National Center for Domestic Animal Breeding and Selection, 1998. Stimulation of the vitality and enhancement of the fertilization capacity of gametes from bull brood animals by means of in vivo and in vitro selenium-containing preparations. Project leader: Assoc. Prof. Ch. Vaisberg.

        Zoological Garden, Sofia, 2003. Reproductive cycle in ursus thibetanus and ursus arctos horribilis, bred under zoo conditions. Project leader: Assoc. Prof. M. Ivanova – Kicheva.

        Cooperative program for research and development with Obst. Gyn. Center “Dimitrov”, 2001. Project leader: Res. Assoc. P. Todorov

        2001-2004, Stimulation of the vitality and fertilization capacity of spermatozoa from mammals in vivo and in vitro and antioxidant protection, sponsored by interacademical exanche with Academy of Sciences and Technologies of Vietnam. Project leader for IBIR: Prof. Z. Zachariev.

        2004-2006, Technology development of the fertility of stock breeds, sponsored by interacademical exanche with Academy of Sciences and Technologies of Vietnam. Project leader for IBIR: Prof. Z. Zachariev and Res. Assoc.R. Stefanov.

        2006-2008, Characteristics of the phospholipid assimetry in the plasma membrane and DNA-fragmentation of spermatozoa during cryopreservation, sponsored by interacademical exanche with University of Warmia and Masury, Poland. Project leader for IBIR: Assoc. Prof. M. Ivanova-Kicheva.

        1995-1998, Cryopreservation of reproductive tissue, sponsored by interacademical exanche with Academy of Sciences of Ukraine. Project leader for IBIR: Res.Assoc. P. Todorov

        1998-2001, Cryopreservation of gametes and embryos, sponsored by interacademical exanche with Academy of Sciences of Ukraine. Project leader for IBIR: Res.Assoc. P. Todorov

        1998-2001, Cooperative program for research and implementation in the field of assisted reproductive technologies in animals and humans. Preparation of reproductive cells and embryos for pregnancy induction., sponsored by interacademical exanche with Academy of Sciences of Ukraine. Project leader for IBIR: Res. Assoc. P. Todorov

        2002-2005, Cryopreservation of human gametes and ovarian tissue, sponsored by interacademical exanche with Academy of Sciences of Ukraine. Project leader for IBIR: Res. Assoc. P. Todorov

        2006-2008, Cryopreservation of gametes, embryos and stem cells, sponsored by interacademical exanche with Academy of Sciences of Ukraine. Project leader for IBIR: Res.Assoc. P. Todorov

        IAEA (SISMER) C6/BUL/0101P, Preimplantation genetic diagnosis. Project leader: Res.Assoc. P. Todorov

        BG051PO 001/07/3.3–01 European Social Fund – Operative Program “Development of Human Resources” Call: Fellowship funding scheme for young scientists who carry out PhD thesis in a national firm enterprise”.

        end_of_the_skype_highlightingBV-5/2007 – RVietnam project on an international convention. Investigation of approaches for increasing reproduction in buffaloes. Leaders: Associate Professor Dr Maria Kicheva and Prof. Dimitrina Kacheva.

        2009-2011 – Proteomics of seminal plasma from ram, boar and dog in relation to cryotolerance of spermatozoa of these species. Institute-funded exchange with the University of Warmia and Mazur, Poland. Head: Associate Professor Dr. Maria Ivanova-Kicheva.

        • Publications

          Scientific papers published during last 5 years.

          Papers in International peer-reviewed scientific journals

          E.Vachkova, D.Bosnakovski, P.Yonkova, N.grigorova, Zh.Ivanova, P.Todorov, G.Penchev, A.Milanova, G.Simeonova, S.Stanilova, I.Penchev Georgiev   Adipogenic potential of stem cells derived from rabbit subcutaneous and visceral adipose tissue in vitro. In Vitro Cellular and Developmental Biology-Animal, 2016.
          DOI 10.1007/s11626-016-oo48-7
          ISSN: 1071-2690    IF: 1.145

          Isachenko, V., Todorov, P., Isachenko, E., Rahimi, G., Tchorbanov, A.    Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue. PLOS ONE, 2015.
          ISSN: 1932-6203                IF:3.534

          Gradinarska D, Ivanova M, Hristova M, Lazov K, Daskalova D.   Size exclusion chromatography of seminal plasma from buffalo bull (bubalus bubalis) ejaculates with good and poor cryotolerance of gametes. Collection of articles from International Scientific-Practical Conference “Modern trends in reproduction of lifestock animals”, Penza State Agricultural Academy, 37 – 43, 2015.
          ISBN: 978-5-94338-734-0

          V.Milkov, K.Miteva, G.Pironcheva, T.Daneva, V.Georgiev   Osteocalcin response to calcium load test in patients with hypercalciuria. Acta Endocrinologica (Buc), 10, 4, 570-576, 2014.
          ISSN: 1841-0987       IF: 0.268

          Sainova I., Vavrek I., Atanassov I., Lazov K., Pavlova V., Pashova Sh., Zasheva D., Alexander I., Manchev St., Miteva K., Nikolova E., Urlaub H., Daneva T. Experimental rodent and human models for balanced activity of oncogenes and tumor-suppressor genes and their protein products. Prime Research on Medicine (PROM) Vol. 2,3, 505-512, 2012. 
          ISSN: 2315-5302

          R.Stefanov, M.Sabev, M.Ivanova-Kicheva   Protective effect of the antioxidant enzyme on sperm from Bubalus bubalis type after cryoconservation. Biology and Animal Husbandry, 27, 3, 943-949, 2011.
          ISSN 1450-9156

          Gerzilov V., D.Kazachka, S.Jeleva, P.Petrov, M.Sabev, I.Nikolov   Effect of cryoprotectants on the mobility and the morphological integrity of muscovy spermatozoa. Zeszyty Naukowe Uniwersytetu Przyrodniczego we Wroclawiu. Biologia i Hodowla Zwierzat, 580, LXII, 157-165, 2011.
          ISSN 1897-8223

          D.Yosifov, P.Todorov, M.Zaharieva, K.Georgiev, B.Pilicheva, S.Konstantinov, M.Berger   Erucylphospho-N,N,N-trimethylpropylammonium (erufosine) is a potential antimyeloma drug devoid of mylitoxicity. Carcer Chemother.Pharmacol., 67,13-25, 2011.
          ISSN: 0344-5704; IF 2.654

          Papers in National (Bulgarian) scientific journals

          Todorov, P, Petrova, N, Mihova, A, Guenova, M, Arabadzhiev, B, Hristova, E.    The Female Age Influences the Expression of Pluripotent Stem Cells Markers in Human Ovarian Cells. Comptes rendus de l’Académie bulgare des Sciences, 68, 1, 65 – 70, 2015.
          ISSN: 1310-1331 IF:0.284

          Todorov, P., Hristova, E., Atanasova, V., Dimitrov, Y.    First Case of Pregnancy and Life   Birth after Piezo   ICSI in Bulgaria. Comptes rendus de l’Academie bulgare des Sciences, 68, 4, 475 – 478, 2015.
          ISSN: 1310-1331 IF:0.284

          Gradinarska D, Ivanova M, Daskalova D, Lazov K, Chervenkov M.   Comparative analysis of the capacitation ability of fractionaly obtained canine spermatozoa. Journal of Mountain Agriculture on the Balkans, 18, 3, Research Institute of Mountain Stockbreeding and Agriculture, Troyan, 412 – 425, 2015.
          ISSN: 1311-0489

          Kirilova I, Ivanova M, Daskalova D, Gradinarska D, Kukov A, Dimitrov P, Hristova E.   The role of antioxidants and biologically active substances on the motility and speed parameters of buffalo bull spermatozoa after cryopreservation. Bulgarian Journal of Agricultural Science, 21, 1, 209 – 214, 2015.
          ISSN: 1310-0351

          Д.Даскалова, И.Кирилов, Д.Градинарска, К.Лазов, М.Иванова   Биоконформационни взаимодействия между липопротеини от яйчен жълтък и протеини от семинална плазма и ролята им върху биологичните параметри на сперматозоиди от нерез при нискотемпературно съхранение на 4С. Journal of Mountain Agriculture on the Balkans, V.17, 5, 1087-1103, 2014.
          ISSN: 1311-0489

          D.Daskalova, P.Wysocki, D.Gradinarska, E.Hristova, M.Kicheva-Ivanova    Differences between seminal plasma proteins from boar semen with high and low cryptolerance of the spermatozoa. Comp.tRend.Acad.Bulg.Sci., 67, 12, 1663-1668, 2014.
          ISSN: 1310-1331       IF: 0.284

          D.Daskalova, A.Kukov, I.Kirilova, M.Ivanova-Kicheva   Protein analysis of boar seminal plasma proteins with protective effect during low temperature storage of spermatozoa.  Biotechnology and Biotechnological Equipment, 28, 4, 716-720, 2014.
          ISSN: 1310-2818       IF: 0.300

          М.Иванова   Репродуктивни биотехнологии и криобиология на гаметите. Списание на БАН, 127, 1, 14-17, 2014.
          ISSN: 0007-3989 

          И.Кирилова,  Д.Градинарска, Д.Даскалова, М.Христова, М.Иванова   Анализ на взаимодействията между протеини от семинална плазма и липопротеини от яйчен жълтък и тяхната роля при нискотемпературно съхранение на сперматозоиди от бивол. Journal of Mountain Agriculture on the Balkans, V.17, 5, 1116-1130, 2014.
          ISSN: 1311-0489

          И.Кирилова, М.Иванова, А.Куков, П.Димитров, Д.Даскалова   Хроматографски анализ на протеини от спермална плазма на бивол. Сборник доклади от научна конференция „Традиции и съвременност във ветеринарната медицина”, Лесотехнически университет, София, 2012 (отпечатано 2014).

          А.Куков, И.Сираков, М.Червенков, Р.Попова, Ст.Алексиев, Д.Даскалова, И.Кирилова, А.Колев   Първични изследвания на три застрашени от изчезване породи овце от западна България. Аграрни науки, 5, 13, 67-70, 2013.
          ISSN 1313-6577

          D.Daskalova, A.Kukov, M.Kicheva  The role of separated lipoproteins from egg yolk on boar spermatozoa during storage at 4ºC. Compt.Rendus Acad.Bulg.Sci., 65, 6, 2012. 
          ISSN 1310-1331. IF : 0.210

          M.Ivanova-Kicheva, G.Dimov, A.Kukov, D Daskalova, V.Gerzilov    Impact of separated seminal plasma proteins on the motility, integrity of plasma membrane and velocity parameters of ram spermatozoa during low temperature preservation. Compt.Rendus Acad.Bulg.Sci., 65,  7, 2012.
          ISSN 1310-1331 . IF : 0.210

          Тодоров П., Христова Е., Димитров Р, Димитров Й.   Методи за активация на човешки ооцити. Ембриология, 7, 1, 22-27, 2012.
          ISSN: 1312-7349

          E.Hristova   Mesenchymal stem cells: Biological properties and clinical perspectives. Embryology, 7, 1, 16-21, 2012.

          Г.Димов, М.Иванова (Кичева), Л.Танев   Генетично усъвършенстване на породите овце в България чрез прилагане на иновативен продукт за оптимизиране на биотехнологията при in vitro съхранение на мъжки гамети. Животновъдни науки, 48, 2, 14-25, 2011.
          ISSN: 0514-7441

          Г.Димов, Н.Ранчев, М.Иванова (Кичева), Л.Танев   Информационна система за управление на селекционния процес в овцевъдството ИСОВ. Сборник доклади от научната конференция „Традиции и съвременност във ветеринарната медицина”, Лесотехнически университет, София, 2010, Издателска къща при ЛТУ, София, 2011.
          ISSN: 1313-4337

          M.Ivanova-Kicheva, G.Dimov   Influence of selected seminal plasma proteins on mitochondrial integrity and speed parameters of ram sperm stored at low temperature. Biotechnol.& Biotechnol..Eq., 25, 3, 1-6, 2011.
          ISSN: 1310-2818; IF – 0.760

          A.Куков, С.Запрянова, Д.Даскалова, Ж.Минчева, В.Герзилов, А.Нейчева,  М.Иванова-Кичева, Г.Димов   Компютърен спермоанализ на протективната роля на семенни протеини при кочове от породата черноглава плевенска. Аграрни науки, 3, 6, 17-24, 2011.
          ISSN: 1313-6577

          A.Kukov, S.Zapryanova, D.Dascalova, M.Ivanova-Kicheva   Influence of separated seminal plasma proteins on the motility, viability and DNA integrity of ram spermatozoa stored at 50 C. 2nd  Conference of the Balkan Network for the Biotechnology in Animal Reproduction. Proceeding, Sofia, 24-25 March 2011.
          ISSN: 1314-3085

          Р.Стефанов, К.Лазов, М.Събев, К.Митева, Х.Христев   Виабилитет на спермазоиди от вида ovis aries след сепариране на спермалната плазма. Сборник доклади от научната конференция „Традиции и съвременност във ветеринарната медицина”, Лесотехнически университет, София, 2010, Издателска къща при ЛТУ, София, 2011.
          ISSN: 1313-4337

          R.Stefanov, M.Sabev, K.Miteva   Influence of the enzyme superoxide dismutase isolated from the fungal strain humicola lutea 103 on the content of carbonyl groups in the intracellular proteins upon in vitro cryopreservation of ram sperm. 2nd  Conference of the Balkan Network for the Biotechnology in Animal Reproduction. Proceeding, Sofia, 24-25 March 2011.
          ISSN: 1314-3085

          R.Stefanov, M.Sabev, M.Ivanova-Kicheva, A.Kukov, G.Anev, K.Lazov, K.Miteva   Studies on the fertility of sheep after artificial insemination with storied semen in hypothermic conditions. Compt.Rendus Bulg.Acad.Sci., 64, 5, 757-762, 2011
          ISSN: 1310-1331; IF – 0.219

          П.Тодоров, Е.Христова,Р.Димитров, Б.Биволарски, А.Златков   Влияние на новосинтезирани вещества от групата на метилксантините върху мотилитета на мъжки гамети. Ембриология, 6, 1, 19-25, 2011.

          E.Isachenko, V.Isachenko, P.Todorov, M.Schulz, J.Risopatron, R.Kreinberg, R.Sanchez   Improvement of human spermatozoa cryopreservation technique: comparative investigation of influence of aseptic vitrification and conventional freezing on the sperm function. Ембриология, 6, 1, 3-12, 2011.

           

          Popular Science, Current Affairs publications and Educational publications

          Books and brochures

          Т.Маринков, Г.Ангелов, Б.Аминков, М.Люцканож, М.Жиков, Д.Илиев, Г.Георгиев, С.Йорданов, Д.Гиргинов, Н.Тодоров, Т.Тодоров, М.Иванова, А.Димитрова, Н.Механджийски, А.Панков, И.Райчев, Т.Мехмедов, К.Христов, Р.Пепович, И.Пеев, В.Пиралков   Наръчник на ветеринарния лекар, стартиращ собствен бизнес. ЛТУ, София, 2012. 
          ISBN:  978-954-2910-15-2

          Николов И., Ж. Байчев, М. Събев, Д. Казачка, Р. Стефанов, П. Първанов   Биологичен контрол и съхраняване на сперма от селскостопански  разплодници.  Издателство „Виденов и син”, София, 2012. 
          ISBN: 978-954-8319-57-7.

          Събев М., Ж. Байчев, И. Николов, Р. Стефанов, С. Манчев   Справочник по размножаване при селскостопански животни и репродуктивни биотехнологии.  Издателство „Виденов и син” София, 2012. 
          ISBN: 978-954-8319-60-7.

          М.Семков, М.Иванова-Кичева, Б.Герзилов   Размножаване на домашните животни. Акад. изд. на Аграрния университет, Пловдив, 2011.
          ISBN: 978-954-517-113-0

           

          Previous publications

          Papers in National (Bulgarian) scientific journals

          1. Иванов Ц., Иванова Т., Стоилов Н., Тодоров П., Тодоров Я., Събев М. Влияние на условията на съхранение на мъжки гамети от Apis Mellifera L. върху техните биологични качества. Животновъдни науки, 2005, 2: 46-50.

          2. Todorov P., Konakchieva R., Dimitrov J., Chub N., Petrenko Y.  Vitrification of human embryonic stem cells. Comptes rendus de l’Academie bulgare des Sciences, 2005, 58, 9:1095-1100.

          3. Анев Г., И. Николов, Р. Стефанов. Изследване върху половите рефлекси и спермопродукцията на кочове. Животновъдни науки, 2005, 5, 149-152.

          4. Събев М., Р. Стефанов, З. Захариев. Влияние на екзогенно добавен антиоксидант към сперма от коч върху мотилитета и клетъчния метаболизъм на мъжки гамети. Животновъдни науки, 2005 5, 153-155.

          5. Угринов, К., И. Минков, К. Митева, С.Златарев, Ч. Вайсберг. Изследване на АТФ-азната активност в митохондрии,сперматозоиди и семенна течност от коч преди и след криоконсервация. Животновъдни науки,2005, XLII. 3, 51-53.

          6. Вайсберг, Ч., Л. Железарски, К. Митева, Т. Машиах, Т. Чаушев. Субстратна активност и имунодетекция на глутатион пероксидаза в сперма от нерез и бик. Животновъдни науки 20055, 188-190.

          7. Христев Хр., И. Николов, В. Герзилов, Д. Михайлова. Електрофоретичен профил на сперма от кочове след разреждане с протективни среди. Научни трудове АУ – Пловдив, т. L, кн. 3, 2005, 101-106.

          8. В. Герзилов, Б. Георгиев, И. Николов, Д. Качева. Зависимости между ключови ензими и показатели на спермопродукцията при мускусни патоци. Научни трудове – АУ-Пловдив, т. L, кн. 3, 2005, 127-130.

          9. Железарски, Л., Ч. Вайсберг, Т. Чаушев. Получаване и характеризиране на поликлонално антитяло срещу глутатион пероксидаза. Животновъднинауки,20055, 183-187.

          10. Иванов Ц., Т. Иванова, Н. Стоилов, П. Тодоров, Я. Тодоров, М. Събев. Влияние на условията на съхранение на мъжки гамети от Apis Meliffera L. върху техните биологични качества. Животновъдни науки, 2005, 2: 46-50.

          11. Иванова–Кичева М., Д. Даскалова, А. Димитров, П. Цветкова. Сигнални пътища при капацитация и апоптоза във връзка с поведението на биомолекули и структури при сперматозоиди. Андрология 2005 13: 14–22.

          12. Тодоров П. Възможности за използване на фетални клетки и тъкани за нуждите на клетъчната терапия. Репродуктивно здраве2005, 9: 6-10.

          13. Тодоров П., Конакчиева Р., Димитров Й. Ефект на  рекомбинантен ФСХ (Gonal F и Puregon) върху синтеза и секрецията на естрадиол-17 бета от човешки гранулозни клетки “ин витро”. Ендокринология2005, 3: 149-153.

          14. Иванова – Кичева, М. Петров, А. Димитров, В. Василев. Прилагане на Анексин V като маркер за определяне биологичната пълноценност на сперматозоиди при криоконсервация. Сборник от научна сесия “10 години факултет по ветеринарна медицина”, ЛУ, София , 2005, 62-71.

          15.  Вайсберг, Ч., Л. Железарски, С. Йовчев, А. Данев, Й. Марчев Подобряване качеството на сперма от нерез след добавяне на селен към хранителната дажба. Животновъдни науки, 2006, 4, 42-44.

          16. Тодоров П., Конакчиева Р., Димитров Й., Славчев Б., Новачков В. Криоконсервация на човешка овариална тъкан – нашият опит. Ембриология2006, 1: 4-8.

          17. Тодоров П. Получаване, култивиране и криоконсервация на човешки ембрионални стволови клетки. Ембриология , 2006, 2: 23-29.

          18. Тодоров П., Димитров Й., Пандева И. Криоконсервация на кръв от пъпна връв. Технологични аспекти. Акушерство и гинекология, 2006, 2:17-21.

          19. М. Шкодрова, К. Митева, Г. Бъкова и И. Минков (Влияние на 2-хидрокси 1-нафталдехидметансулфонилхидразон върху АТФ-азната активност на митохондрии,изолирани от сперматозоиди и черен дроб. Годишник на Шуменския университет “Епископ К.Преславски” т.16 В4. Природни науки, 2006, 39-49.

          20. Иванова-Кичева М., А. Димитров, И. Николов, М. Събев, В. Герзилов. Поведение на фосфатидилсерин от плазмената мембрана на сперматозоиди от коч при in vitro индуцирана апоптоза, капацитация и след криоконсервация. Аграрен Университет-Пловдив, Научни трудове, 2006,  т. LI, 87-96.

          21. Николов И., М. Събев, М. Кичева, П. Рашев, В. Герзилов, Хр. Христев. Изследвания върху протективната роля на нискомолекулна липопротеинова фракция (LDL) върху криотолерантността на сперматозоиди от коч. Аграрен Университет-Пловдив, Научни трудове, 2006, т. LI, 79-86.

          22. Събев М., И. Николов, М. Иванова-Кичева, М. Петров, П. Рашев. Влияние на захари като компоненти на разредители върху жизнеспособността на сперматозоиди от нерез при “in vitro” съхранение. В: Сб. Докл. Научна конференция  с международно участие “Традиции и съвременност във ветеринарната медицина” , ФВМ, ЛУ , София, 2006 , 197-202.

          23. Иванова-Кичева М., М. Ангелова, Д. Даскалова, Р.Стефанов, М. Събев, И. Николов, Трин Тхи Ким Тхоа, Ц.Чемширова, М. Петров. Антиоксидантна защита на сперматозоиди от биволи при криоконсервация. В: Сб. Докл. Научна конференция  с международно участие “Традиции и съвременност във ветеринарната медицина” , ФВМ, ЛУ , София, 2006 , 188-196.

          24. Христев Хр., И. Николов, М. Събев, М. Иванова-Кичева, Р. Стефанов, П. Рашев. Влияние на фактори на околната среда върху криорезистентността на сперматозоиди от еякулати на кочове получени през извънразмножителен сезон. АУ- Пловдив , Научни трудове, 2007, 52, 309-314.

          25. Куков А, Д. Даскалова, Е. Соленкова, М. Иванова-Кичева, И. Николов, М. Събев, Хр. Христев. Влияние на облъчването с ултравиолетови лъчи върху  проявата на апоптоза-свързани белези при сперматозоиди от коч. Докл. на Конференция по екопроблеми в животновъдството, АУ- Пловдив, Научни трудове, 2007, 52, 315-318.

          26. Железарски, Л., Ч. Вайсберг, Т. Чаушев, Е. Сапунджиев. Глутатион пероксидаза в мехурчести жлези и в семенна плазма от нерез. Сборник доклади от научната конференция: “Традиции и съвременност във ветеринарната медицина”, Лесотехнически университет ,София, 2006, 231-238.

          27. Вайсберг, Ч., Л. Железарски, Т. Чаушев, А. Данев, Й. Марчев. Влияние на селена върху качеството на сперма от нерез. Сборник доклади от научната конференция: “Традиции и съвременност във ветеринарната медицина”, Лесотехнически университет ,София, 2006, 239-243.

          28. Иванова – Кичева м, А. Куков, Д. Даскалова, Е. Соленкова. Роля на ултравиолетовото лъчение върху интегритета на плазмената мембрана, митохондриите и ДНК при in vitro третиране на сперматозоиди от коч. Животновъдни науки, 2007, 3; 33-39.

          29. ИвановаКичева М., А. Куков, Д. Даскалова, Е. Соленкова. Роля на ултравиолетовото лъчение върху интегритета на плазмената мембрана, митохондриите и ДНК при in vitro третиране на сперматозоиди от коч.  Животновъдни науки, 2007, 3: 33-38.

          30. Тодоров П. Човешки ембрионални стволови клетки. Медицински дневник 2007, 8: 13-16.

          31. ИвановаКичева  М. Съвременни постижения при разработването на биотехнологични програми за „ин витро” съхранение и криоконсервация на гамети. Животновъдство 2007, 4, 9-11.

          32. Dimitrov А., M. Ivanova-Kicheva, D. Daskalova, D. Kacheva, E. Solenkova  Protective role of separated seminal plasma proteins in “in vitro’ sterage of ram spermatozoa. Journal of Mountain Agriculture on the Balkans, 2007, 10: 5, 922-932.

          33. Соленкова Е., Д. Даскалова, А. Куков, М. Иванова-Кичева, В. Георгиев. Влияние на криоконсервацията върху морфологията и фосфолипидната асиметрия на плазмената мембрана при сперматозоиди от коч. Journal of Mountain Agriculture on the Balkans. 2008, 11: 6; 982-999.

          34. Тодоров П., Петрова Н., Гуленова Д., Михова А, Димитров Й. Криоконсервация на хематопоетични стволови клетки от пъпна връв – нашият опит. Ембриология, 2008, 1: 3-8.

          35. Stefanov R., M. Sabev, D. Kazatchka, I. Nikolov, K. Miteva.Studies of the effect of antioxidants mercaptoethanol and melatonin on boars sperm on in vitro conditions of preservation. Journal of Mountain Agriculture on the Balkans, 2008, 11, 6, 1000-1011.

          36. Николов И., М. Събев, Р. Стефанов, М. Иванова-Кичева, Х. Христев, П. Рашев. Протективна роля на LDL-фракция от яйчен жълтък при различни технологии за криоконсервация на сперматозоиди от вида Ovis aries. В: Сб. Доклади от Научна Конференция “Традиции и съвременност във ветеринарната медицина”, Лесотехнически университет, Факултет по ветеринарна медицина, София, 2007, 218-223.

          37. Събев М., Р. Стефанов, М. Ангелова, И. Николов, Д. Казачка, М. Иванова-Кичева, П. Рашев. Протективна роля на антиоксиданти върху сперматозоиди от вида Sus scofa в условия на in vitro съхранение. В: Сб. Доклади от Научна Конференция “Традиции и съвременност във ветеринарната медицина”, Лесотехнически университет, Факултет по ветеринарна медицина, София, 2007, 224-229.

          38. Даскалова Д., А. Димитров, Е. Соленкова, М. Иванова-Кичева, Р. Стефанов, М. Петров. Влияние на сепарирани спермално плазмени протеини върху преживяемостта на сперматозоиди от коч при съхранение на 4º С. В: Сб. Доклади от Научна Конференция  “Традиции и съвременност във ветеринарната медицина”, Лесотехнически университет, Факултет по ветеринарна медицина, София, 2007, 211-217.

          39. Кичева, М. Ангелова, Д. Даскалова, Р. Стафанов, М. Събев, И. Николов, Трин тхи Ким Тхоа, Ц. Чемширова, М. Петров. Антиoксидантна защита на сперматозоиди от биволи при криоконсервация. Сборник доклади от научна конференция „Традиции и съвременност във ветеринарната медицина”, Лесотехнически Университет, Факултет по Ветеринарна медицина, София, 2007, 188-196.

          40.  Куков А, Д. Даскалова, Е. Соленкова, М. Иванова-Кичева, И. Николов, М. Събев, Хр. Христев. Влияние на облъчването с ултравиолетови лъчи върху  проявата на апоптоза-свързани белези при сперматозоиди от коч. Докл. на Конференция по екопроблеми в животновъдството, АУ- Пловдив, Научни трудове, 2007, 52, 315-318.

          41. Събев М., И. Николов, Р. Стефанов,  К. Митева, К. Лазов. Технологични режими при изкуствено осеменяване и въздействие върху фертилитета при свине. Доклади от Юбилейна научна конференция “85 години Ветеринарномедицинско образование”. Лесотехнически университет, факултет по ветеринарна медицина, София, 2009, 58-61.

          42. Ivanova – Kicheva M., А. Kukov, D Daskalovа, R Stefanov, G Mincheva, C Gerzilov. Behavior of phosphatidylserine and intracellular structures as markers for functionality in “in vitro” storage of ram spermatozoa. Agricultural Sciences, 2010, II, 3,  43-50

          43. Тодоров П.Витрификацията – ефективен метод за криоконсервация на човешки ооцити. Ембриология 2010, 1: 13-17.


           Papers in International peer-reviewed scientific journals

          44. Maria Ivanova-Kicheva, Alexandur Dimitrov, Ivan Nikolov, Denitza Daskalova, Antoaneta Belcheva. Analysis of plasma membrane integrity by using of fluorescein labeled Annexin V and its relationship to other ram sperm characteristics after cryopreservation. Journal Central Europian Agriculture, 2005 ,6: 3, 23-31.

          45. Vaisberg Ch., L. Jelezarsky, B. Dishlianova, T. Chaushev. Activity, substrate detection and immunolocalization of glutathione peroxidase (GPx) in bovine reproductive organs and semen. Theriogenology, 2005, 64, 416-428.

          46. Maria Ivanova-Kicheva, Alexander Dimitrov, Ivan Nikolov, Denitza Daskalova, Antoaneta Belcheva. Detection of plasma membrane phoshatidylserine translocation in ram spermatozoa  after in vitro treating with dexametazone. Journal Central Europian Agriculture, 2005, 6: 3, 69-77.

          47. Чуб Н.Н., Грищенко В.И., Демина Л.Г. Тодоров П.Т. Влияние субнизких температур на морфофункциональную сохранност овариалной ткани человека. Problems of Cryobiology 2005, 15, 3: 250.

          48. Ivanova-Kicheva Maria, Alexandur Dimitrov, Ivan Nikolov, Denitza Daskalova, Malcho Petrov. Analysis of plasma membrane integrity by using of fluorescent labeled Annexin V and its relationship to other ram sperm characteristics after cryopreservation. Journal Central European Agriculture, 2005, 6: 3, 331-336.

          49. IvanovaKicheva Maria, Alexander Dimitrov, Ivan Nikolov, Denitza Daskalova, Antoaneta Belcheva. Detection of plasma membrane phosphatidylserine translocation in ram spermatozoa after in vitro treating with dexamethazone. Journal Central European Agriculture, 2005, 6: 3, 337-342.

          50. Ivan Nikolov, Milko Sabev, Maria Ivanova-Kicheva, Tzvetanka Chemshirova, Emilia Baycheva, Mariola Popova. Stimulation of sexual reflexes at aboriginal ram breeds in non – mating season. Journal Central European Agriculture, 2005, 6: 4, 521-526.

          51.  Stefanov R, E. Krumova, M. Angelova., P. Dolashka, W. Voelter, Z. Zachariev. Artificial insemination of sheep and cow with semen treated by Cu/Zn-superoxide dismutase from the fungal stray, Humicola lutea 103., WJZoology, 2006, 1: 1, 36-39.

          52. Zahariev Z., R.Stefanov, M. Sabev, K. Miteva, I. Nikolov.. Cytochrome c affects the viability and fertility of bull semen. American-Eurasian J.Agric.& Envivor Sci, 2006, 1: 3, 34-37.

          53. Sabev M, Nikolov I, Ivanova-Kicheva M, Stefanov R, Chenshirova Ts, Baycheva E, Popova M. Cryopreservation of ram sperm from autochthonous breeds during a non-mating season. Journal Central European Agriculture, 2006, 7: 4 677-682.

          54. Todorov P.,Konakchieva R, Dimitrov Y., Novachkov V., Kyurkchiev D., Tchoob N., Gorohova N. Morphological and fuctional assessment of human fetal liver cells in long-term culture. Problems of Cryobiology,  2006, 2: 201-206.

          55. Zahariev Z., R. Stefanov, M. Sabev, K. Miteva, I. Nikolov. Cytochrome C Affects The Viability and Fertility of Bull Semen. American-Eurasian J. Agric. and Environ Sci., 2007, 2: 1, 48-50.

          56. Isachenko V., Nawroth F., Montag M., Yakovenko S., P.Todorov,Isachenko.E. Vitrification of pronuclear embryos: Research ground of aseptic technology and application for oocytes and blastocysts. Embryology, 2007, 2: 1, 3-13.

          57. Isachenko V., Todorov P., Dimitrov Y., Isachenko E. Integrity rate of pronuclei after cryopreservation of pronuclearzygotes as a criteria for subsequent embryo development and pregnancy. Human Reproduction2008, 23: 4,819-826.

          58. IvanovaKicheva M., A. Kukov, D. Daskalova, E. Solenkova, I. Nikolov, L. Todorov. Cell death differentiation in black headed rams spermatozoa, using fluorescent labeled annexin V. Journal of Central European Agriculture, 2008, 9: 2, 283-288.

          59. IvanovaKicheva, J. Strzezek, A. Kukov, P. Wysocky, E. Solenkova,  D. Daskalova, L. Todorov. Isolation of Annexin V from swine placenta to assay cell plasma membrane phosphatidylserine translocation. Comptes rendus de l’Academie bulgare des Sciences, 2008, 61: 6, 753-760.

          60. Stefanov R., M. Sabev, I. Nikolov, G. Anev, M. IvanovaKicheva, K. Miteva,K. LazovInfluence of some natural–climatic factors on the sperm production of north-east Bulgarian fine-fleece breed. Biotechnology in Animal husbandry, 2009. 25 (5-6), 695-701.

          61. Kukov A, D Daskalova, M IvanovaKicheva, R Stefanov, A Neicheva. Immunocytochemical localization of separated seminal plasma proteins on the ram’s sperm plasma membrane during in vitro preservation at 40C. Biotechnology in Animal husbandry, 2009, 25 (5-6), 925-934.

          62. Rahimi G, Isachenko V, Kreienberg R, Sauer H., Todorov P., Tawadros S, Mallmann P, Nawroth F and Isachenko E. Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation. European Journal of Obstetrics & Gynecology and Reproductive Biology 2010, 149,  63-67. IF=1,6.

          63. Todorov P., Hristova E., Konakchieva R., Michova A., Dimitrov Y. Comparative studies of different cryopreservation methods for mesenchumal stem cells derived from human fetal liver. Cell Biol. Int., 201034, 455-462. IF= 1,62.

          64. Aysegül Ilhan, Sazan Rasul, Alexander Dimitrov, Ammon Handisurya, Wolfgang Gartner, Sabina Baumgartner-Parzer, Ludwig Wagner, Alexandra Kautzky-Willer, Wolfgang Base. Plasma neuropeptide Y levels differ in distinct diabetic conditionsNeuropeptides, 2010, 44: 6, 485-489.

           

          Popular Science, Current Affairs publications and Educational publications
          Books and brochures

          1.Ж. Байчев, П. Първанов, И. Николов, М. Събев, З. Шиндарска. Изкуствено осеменяване и андрология на селскостопанските животни /Учебник за студенти по ветеринарна медицина/. Изд. Виденов и син, София, 2007, 226 стр.

          2.И. Николов.Биология и биотехнология на размножаването на сс животни. /Учебник за студенти по животновъдство/ Акад. Издание на АУ- Пловдив, 2008, 259 стр.

          3.Мария Кичева. Уелнес за всеки. Първо издание на бълг.език 2009 г. Изд. Агенция “Бизнес” АД Пловдив,  ISBN: 978-954-9392-22-7.

          4. И. Николов, П. Рашев. Ръководство за упражнение по биология на размножаването и и.о. Акад. Издание на АУ- Пловдив, 2008, 168 стр.

          5. И. Николов, М. Събев, Ж. Байчев. Наръчник по размножаване на селскостопански  животни, птици, риби, кучета и котки. Изд. Ди Макс, София, 2008, 153 стр.

          Publications related to the Achievements

          1. Ivanov I, T. Ivanova, N. Stoilov, Todorov P., Todorov January, Sabev M. Effect of storage conditions of the male gametes from Apis Mellifera L. on their biological properties. Animal Science, 2005, 2: 46-50.
          2. Todorov P., Konakchieva R., Dimitrov J., Chub N., Petrenko Y. Vitrification of human embryonic stem cells. Comptes rendus de l’Academie bulgare des Sciences, 2005, 58, 9:1095-1100.
          3. Anev D., I. Nikolov, R. Stefanov. Research on sexual reflexes and spermoproduction of ram. Animal Science, 2005, 5, 149-152.
          4. Sabev M., R. Stefanov, Z. Zahariev. Effect of exogenously added antioxidant to ram semen on motility and cell metabolism of male gametes. Animal Science, 2005 5, 153-155.
          5. Ugrinov, K., I. Minkov, K. Miteva, S. Zlatarev, X. Weissberg. Study of ATP activity in mitochondria, sperm and semen from the ram before and after cryopreservation. Animal Science, 2005, XLII. 3, 51-53.
          6. Weissberg, X., L. hardware, K. Miteva, T. Mashiach, T. Chaushev. Substrate activity and immunodetection of glutathione peroxidase in the semen of boar and bull. Animal Science 2005, 5, 188-190.
          7. Hristev Hr., I. Nikolov, V. Gerzilov, D. Mihaylova. Electrophoretic profile of the semen of rams after dilution with protective environments. Scientific Works AU – Plovdiv, item L, Vol. 3, 2005, 101-106.
          8. C. Gerzilov, B. Georgiev, I. Nikolov, D. Kacheva. Relationships between key enzymes and indicators of spermoproduction in Muscovy. Scientific Works – AU-Plovdiv, item L, Vol. 3, 2005, 127-130.
          9. Hardware, L., X. Weissberg, T. Chaushev. Obtaining and characterization of polyclonal antibody against glutathione peroxidase. Animal Science, 2005, 5, 183-187.
          10. W. Ivanov, T. Ivanova, N. Stoilov, P. Todorov, I. Todorov, M. Sabev. Effect of storage conditions of the male gametes from Apis Meliffera L. on their biological properties. Animal Science, 2005, 2: 46-50.
          11. Ivanova-Kicheva M., D. Daskalov, A. Dimitrov, P. Tsvetkova. Signaling pathways in kapatsitatsiya and apoptosis in relation to the behavior of molecules and structures in the sperm. Andrology, 2005 13: 14-22.
          12. Todorov P. Possibilities for using fetal cells and tissues to the needs of cell therapy. Reproductive Health, 2005, 9: 6-10.
          13. Todorov P., Konakchieva R., Dimitrov J. Effect of recombinant FSH (Gonal F and Puregon) on the synthesis and secretion of estradiol-17b in human granulosa cells in vitro. Endocrinology, 2005, 3: 149-153.
          14. Ivanova – Kicheva, M. Petrov, A. Dimitrov, V. Vassilev. Apply Anexin V as a marker for determining biological value of cryopreserved sperm. Proceedings of Scientific Session “10 Years Faculty of Veterinary Medicine, Lab, Sofia, 2005, 62-71.
          15. Weissberg, X., L. ironware, S. Yovchev, A. Danev Marchev J. Improving the quality of boar semen after adding selenium to the food ration. Animal Science, 2006, 4, 42-44.
          16. Todorov P., Konakchieva R., J. Dimitrov, B. Slavchev, C. Novachkova Cryopreservation of human ovarian tissue – our experience. Embryology, 2006, 1: 4-8.
          17. Todorov P. Getting, culture and cryopreservation of human embryonic stem cells. Embryology, 2006, 2: 23-29.
          18. Todorov P., J. Dimitrov, I. Pandeva.Cryopreservation of cord blood. Technological aspects. Obstetrics and Gynecology, 2006, 2:17-21.
          19. Shkodrova M., K. Miteva, D. and I. Minkov Bakova (Effect of 2-hydroxy-1-NMSH on ATP activity of mitochondria isolated from sperm and liver. Yearbook of Shumen University “Bishop Konstantin of Preslav” t 0.16 C4. Natural Sciences, 2006, 39-49.
          20. Ivanova-Kicheva M., A. Dimitrov, I. Nikolov, M. Sabev, C. Gerzilov. Behavior of phosphatidylserine in plasma membrane of ram sperm in in vitro induced apoptosis kapatsitatsiya and after cryopreservation. Agricultural University-Plovdiv, Scientific Works, 2006, p. LI, 87-96.
          21. I. Nikolov, M. Sabev, M. Kicheva, P. Rashev, C. Gerzilov, Ch. Hristov. Studies on the protective role of low molecular weight lipoprotein fraction (LDL) on cryotolerance of ram sperm. Agricultural University-Plovdiv, Scientific Works, 2006, p. LI, 79-86.
          22. Sabev M., I. Nikolov, M. Ivanova-Kicheva, M. Petrov, P. Rashev. Effect of sugars as components of diluents on the viability of sperm from boars with “in vitro” storage. In: Sat Till. Scientific conference with international participation “Tradition and Modernity in veterinary medicine, FVM, LU, Sofia, 2006, 197-202.
          23. Ivanova-Kicheva M., M. Angelova, D. Daskalov, R. Stefanov, M. Sabev, I. Nikolov, Thi Kim Thoa Triin, W. Chemshirova, M. Petrov. Antioxidant protection of sperm cryopreservation in buffaloes. In: Sat Till. Scientific conference with international participation “Tradition and Modernity in veterinary medicine, FVM, LU, Sofia, 2006, 188-196.
          24. Hristev Hr., I. Nikolov, M. Sabev, M. Ivanova-Kicheva, R. Stefanov, P. Rashev. Influence of environmental factors on sperm from cryorezistant ram ejaculates received in unbreeding season. AU-Plovdiv, Scientific Works, 2007, 52, 309-314.
          25. Kukov A, Daskalov E. F. Solenkova, M. Ivanova-Kicheva, I. Nikolov, M. Sabev, Ch. Hristov. Influence of ultraviolet irradiation on expression of apoptosis-related features in sperm from ram. Till. Conference ekoproblemi of livestock, AU-Plovdiv, Scientific Works, 2007, 52, 315-318.
          26. Hardware, L., X. Weissberg, T. Chaushev F. Sapoundjiev. Evaluetion of Glutathione peroxidase in bubble glands and in seminal plasma of boars. Proceedings of the conference: “Tradition and Modernity in veterinary medicine, University of Forestry, Sofia, 2006, 231-238.
          27. Weissberg, X., L. ironware, T. Chaushev, A. Danev J. Marchev. Influence of selenium on the quality of boar semen. Proceedings of the conference: “Tradition and Modernity in veterinary medicine, University of Forestry, Sofia, 2006, 239-243.
          28. Ivanova – Kicheva m Kukov A., D. Daskalov, E. Solenkova. Role of ultraviolet radiation on the integrity of the plasma membrane, mitochondria and DNA in the in vitro treatment of sperm from the ram. Animal Science, 2007, 3, 33-39.
          29. Ivanova – Kicheva M., A. Kukov, D. Daskalov, E. Solenkova. Role of ultraviolet radiation on the integrity of the plasma membrane, mitochondria and DNA in the in vitro treatment of sperm from the ram. Animal Science, 2007, 3: 33-38.
          31. Todorov P. Human embryonic stem cells. Medical journal 2007, 8: 13-16.
          32. Ivanova – Kicheva M. Recent advances in the development of biotechnology programs in vitro storage and cryopreservation of gametes. Livestock 2007, 4, 9-11.
          33. A. Dimitrov, M. Ivanova-Kicheva, D. Daskalova, D. Kacheva, E. Solenkova Protective role of separated seminal plasma proteins in “in vitro ‘storage of ram spermatozoa. Journal of Mountain Agriculture on the Balkans, 2007, 10: 5, 922-932.
          34. Solenkova F. D. Daskalov, A. Kukov, M. Ivanova-Kicheva, V. Georgiev. Effect of cryopreservation on the morphology and phospholipides asymmetry in plasma membrane of ram sperm. Journal of Mountain Agriculture on the Balkans. 2008, 11: 6, 982-999.
          35. Todorov P., N. Petrova, D. Gulenova, Mihova A, Dimitrov J. Cryopreservation of hematopoietic stem cells from umbilical cord blood – our experience. Embryology, 2008, 1: 3-8.
          36. Stefanov R., M. Sabev, D. Kazatchka, I. Nikolov, K. Miteva. Studies of the effect of antioxidants mercaptoethanol and melatonin on boars sperm on in vitro conditions of preservation. Journal of Mountain Agriculture on the Balkans, 2008, 11, 6, 1000-1011.
          37. I. Nikolov, M. Sabev, R. Stefanov, M. Ivanova-Kicheva, H. Hristov, P. Rashev. Protective role of LDL-fraction of egg yolk in different technologies for cryopreservation of Ovis aries sperm. In: Sat Reports of Scientific Conference “Tradition and Modernity in veterinary medicine, University of Forestry, Faculty of Veterinary Medicine, Sofia, 2007, 218-223.
          38. Sabev M., R. Stefanov, M. Angelova, I. Nikolov, D. Kazachka, M. Ivanova-Kicheva, P. Rashev. Protective role of antioxidants on Sus scofa sperm in terms of in vitro storage. In: Sat Reports of Scientific Conference “Tradition and Modernity in veterinary medicine, University of Forestry, Faculty of Veterinary Medicine, Sofia, 2007, 224-229.
          39. Daskalov E., A. Dimitrov, E. Solenkova, M. Ivanova-Kicheva, R. Stefanov, M. Petrov. Influence of separated sperm plasma proteins on survival of ram sperm during storage at 4 º C. In: Sat Reports of Scientific Conference “Tradition and Modernity in veterinary medicine, University of Forestry, Faculty of Veterinary Medicine, Sofia, 2007, 211-217
          40. Kicheva, M. Angelova, D. Daskalov, R. Stafanov, M. Sabev, I. Nikolov, Thi Kim Thoa Triin, W. Chemshirova, M. Petrov. Antioxidant protection of cryopreserved sperm from buffaloes. Proceedings of the Conference “Traditions and Modernity in veterinary medicine, University of Forestry, Faculty of Veterinary Medicine, Sofia, 2007, 188-196.
          41. Kukov A, Daskalov E. F. Solenkova, M. Ivanova-Kicheva, I. Nikolov, M. Sabev, Ch. Hristov. Influence of ultraviolet irradiation on expression of apoptosis-related features in sperm from ram. Till. Conference ekoproblemi of livestock, AU-Plovdiv, Scientific Works, 2007, 52, 315-318.
          42. Sabev M., I. Nikolov, R. Stefanov, K. Miteva, K. Lazov. Technological regimes in artificial insemination and its impact on fertility in pigs. Reports of the Jubilee Conference “85 years education in veterinary medicine. Forestry University, Faculty of Veterinary Medicine, Sofia, 2009, 58-61.
          43. Ivanova – Kicheva M., A. Kukov, D Daskalov, R Stefanov, G Mincheva, C Gerzilov. Behavior of phosphatidylserine and intracellular structures as markers for functionality in “in vitro” storage of ram spermatozoa. Agricultural Sciences, 2010, II, 3, 43-50.
          44. Todorov P. Vitrification – an effective method for cryopreservation of human oocytes. Embryology 2010, 1: 13-17.
          45. Maria Ivanova-Kicheva, Alexandur Dimitrov, Ivan Nikolov, Denitza Daskalova, Antoaneta Belcheva. Analysis of plasma membrane integrity by using of fluorescein labeled Annexin V and its relationship to other ram sperm characteristics after cryopreservation. Journal Central Europian Agriculture, 2005, 6: 3, 23-31
          46. Vaisberg Ch., L. Jelezarsky, B. Dishlianova, T. Chaushev. Activity, substrate detection and immunolocalization of glutathione peroxidase (GPx) in bovine reproductive organs and semen. Theriogenology, 2005, 64, 416-428.
          47. Maria Ivanova-Kicheva, Alexander Dimitrov, Ivan Nikolov, Denitza Daskalova, Antoaneta Belcheva. Detection of plasma membrane phoshatidylserine translocation in ram spermatozoa after in vitro treating with dexametazone. Journal Central Europian Agriculture, 2005, 6: 3, 69-77.
          48. Чуб Н.Н., Грищенко В.И., Демина Л.Г. Тодоров П.Т. Влияние субнизких температур на морфофункциональную сохранност овариалной ткани человека. Problems of Cryobiology 2005, 15, 3: 250
          49. Ivanova-Kicheva Maria, Alexandur Dimitrov, Ivan Nikolov, Denitza Daskalova, Malcho Petrov. Analysis of plasma membrane integrity by using of fluorescent labeled Annexin V and its relationship to other ram sperm characteristics after cryopreservation. Journal Central European Agriculture, 2005, 6: 3, 331-336.
          50. Ivanova-Kicheva Maria, Alexander Dimitrov, Ivan Nikolov, Denitza Daskalova, Antoaneta Belcheva. Detection of plasma membrane phosphatidylserine translocation in ram spermatozoa after in vitro treating with dexamethazone. Journal Central European Agriculture, 2005, 6: 3, 337-342.
          51. Ivan Nikolov, Milko Sabev, Maria Ivanova-Kicheva, Tzvetanka Chemshirova, Emilia Baycheva, Mariola Popova. Stimulation of sexual reflexes at aboriginal ram breeds in non – mating season. Journal Central European Agriculture, 2005, 6: 4, 521-526.
          52. Stefanov R, E. Krumova, M. Angelova., P. Dolashka, W. Voelter, Z. Zachariev. Artificial insemination of sheep and cow with semen treated by Cu/Zn-superoxide dismutase from the fungal stray, Humicola lutea 103., W. J. Zoology, 2006, 1: 1, 36-39.
          53. Zahariev Z., R.Stefanov, M. Sabev, K. Miteva, I. Nikolov.. Cytochrome c affects the viability and fertility of bull semen. American-Eurasian J.Agric.& Envivor Sci, 2006, 1: 3, 34-37.
          54. Sabev M, Nikolov I, Ivanova-Kicheva M, Stefanov R, Chenshirova Ts, Baycheva E, Popova M. Cryopreservation of ram sperm from autochthonous breeds during a non-mating season. Journal Central European Agriculture, 2006, 7: 4 677-682.
          55. TODOROV P., Konakchieva R, Dimitrov Y., Novachkov V., Kyurkchiev D., Tchoob N., Gorohova N. Morphological and fuctional assessment of human fetal liver cells in long-term culture. Problems of Cryobiology, 2006, 2: 201-206
          56. Zahariev Z., R. Stefanov, M. Sabev, K. Miteva, I. Nikolov. Cytochrome C Affects The Viability and Fertility of Bull Semen. American-Eurasian J. Agric. and Environ Sci., 2007, 2: 1, 48-50.
          57. Isachenko V., Nawroth F., Montag M., Yakovenko S., P.TODOROV, Isachenko.E. Vitrification of pronuclear embryos: Research ground of aseptic technology and application for oocytes and blastocysts. Embryology, 2007, 2: 1, 3-13
          58. Isachenko V., TODOROV P., Dimitrov Y., Isachenko E. Integrity rate of pronuclei after cryopreservation of pronuclear-zygotes as a criteria for subsequent embryo development and pregnancy. Human Reproduction, 2008, 23: 4,819-826
          59. Ivanova-Kicheva M., A. Kukov, D. Daskalova, E. Solenkova, I. Nikolov, L. Todorov. Cell death differentiation in black headed rams spermatozoa, using fluorescent labeled annexin V. Journal of Central European Agriculture, 2008, 9: 2, 283-288.
          60. Ivanova-Kicheva, J. Strzezek, A. Kukov, P. Wysocky, E. Solenkova, D. Daskalova, L. Todorov. Isolation of Annexin V from swine placenta to assay cell plasma membrane phosphatidylserine translocation. Comptes rendus de l’Academie bulgare des Sciences, 2008, 61: 6, 753-760.
          61. Stefanov R., M. Sabev, I. Nikolov, G. Anev, M. Ivanova-Kicheva, K. Miteva,K. Lazov. Influence of some natural–climatic factors on the sperm production of north-east Bulgarian fine-fleece breed. Biotechnology in Animal husbandry, 2009. 25 (5-6), 695-701.
          62. Kukov A, D Daskalova, M Ivanova-Kicheva, R Stefanov, A Neicheva. Immunocytochemical localization of separated seminal plasma proteins on the ram’s sperm plasma membrane during in vitro preservation at 40C. Biotechnology in Animal husbandry, 2009, 25 (5-6), 925-934,
          63. Rahimi G, Isachenko V, Kreienberg R, Sauer H., Todorov P., Tawadros S, Mallmann P, Nawroth F and Isachenko E. Re-vascularisation in human ovarian tissue after conventional freezing or vitrification and xenotransplantation. European Journal of Obstetrics & Gynecology and Reproductive Biology 2010, 149, 63-67 (impact factor 1,6).
          64. Todorov P., Hristova E., Konakchieva R., Michova A., Dimitrov Y. Comparative studies of different cryopreservation methods for mesenchumal stem cells derived from human fetal liver. Cell Biol. Int., 2010, 34, 455-462 (impact factor 1,62).
          65. Aysegül Ilhan, Sazan Rasul, Alexander Dimitrov, Ammon Handisurya, Wolfgang Gartner, Sabina Baumgartner-Parzer, Ludwig Wagner, Alexandra Kautzky-Willer, Wolfgang Base. Plasma neuropeptide Y levels differ in distinct diabetic conditions. Neuropeptides, 2010, 44: 6, 485-489.

          Textbooks:

          G. Baichev, P. Parvanov, I. Nikolov, M. Sabev, H. Shindarska. Artificial insemination and andrology livestock / textbook for students of veterinary medicine. Publ Videnov and son, Sofia, 2007, 226 p.

          I. Nikolov. Biology and Biotechnology of Reproduction of farm animals. A textbook for students of animal, Academic Edition AU-Plovdiv, 2008, 259 p.

          Maria Kicheva. Wellness for everyone, First Edition 2009 balg.ezik g.Izd. Agency Business AD Plovdiv, ISBN: 978-954-9392-22-7,

          Guides:

          I. Nikolov, P. Rashev. Guide Exercise Biology of Reproduction and i.o. Academic Edition AU-Plovdiv, 2008, 168 p.

          I. Nikolov, M. Sabev G. Baichev. Manual for breeding farm animals, birds, fish, dogs and cats. DJ Max Publishing, Sofia, 2008, 153 p.