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Ing. Jaromír Vašíček, PhD.
Identification number: 49883

Final thesis

Basic information

Basic information about a final thesis

Type of thesis: Dissertation thesis
Thesis title:
Animal cell magnetic separation
Written by (author): Ing. Jaromír Vašíček, PhD.
Department of Biochemistry and Biotechnology (FBFS)
Thesis supervisor:
prof. Ing. Peter Chrenek, DrSc.
Opponent 1:
Opponent 2:
prof. MVDr. Igor Valocký, CSc.
Opponent 3:
prof. Dr hab. Marek Bednarczyk
Final thesis progress:
Final thesis was successfully defended.

Additional information

Additional information about the final thesis follows. Click on the language link to display the information in the desired language.

Language of final thesis:

Slovak        English

Title of the thesis:
Animal cell magnetic separation
Summary:The main aim of this thesis was the magnetic separation of two different animal cell types using MACS technique and different magnetic separation strategies (depletion and positive selection). First part of the thesis was focused on the depletion of annexin V-positive rabbit spermatozoa from either heterospermic (E1) or individual buck (E2) semen in order to enhance insemination dose quality by the elimination of apoptotic spermatozoa. MACS sorting efficiency was assessed basing on the quality of separated rabbit spermatozoa. Sorted spermatozoa were evaluated in vitro in terms of their concentration and motility using computer-assisted sperm analysis (CASA), in terms of the sperm head membranes morphology using transmission electron microscopy (TEM) and in terms of plasma membrane integrity (annexin V/PI/DAPI) using fluorescence microscopy. Magnetic separated spermatozoa were then used for the artificial insemination of hormonally treated does for the purposes of conception rate enhancement, and thus to improve the efficiency of broiler rabbit breeding. In the second part of the thesis, different clones of anti-CD34 monoclonal antibodies (AC136, 581 and 8G12) and polyclonal anti-rabbit CD34 antibody and anti-CD133 monoclonal antibodies (AC133 and 293C3) were used in order to find proper marker for the flow-cytometric detection of rabbit hematopoietic stem cells. The FITC-conjugated clone AC136 chosen on the basis of flow-cytometric analysis were then used for the enrichment of CD34 positive rabbit and human cells using indirect immunomagnetic labeling and Anti-FITC microbeads. The CASA analysis after MACS sperm sorting revealed that proportion of apoptotic spermatozoa in the semen of New Zealand White bucks varied from 7 to 20%. The electron microscopy (TEM) revealed that MACS treatment might eliminate spermatozoa with membrane damages and released acrosomal matter. However, the MACS separation (in both E1 and E2) did not affect the reproductive parameters of rabbit does. The use of MACS technique for the isolation of rabbit hematopoietic stem cells is also doubtful, since the enrichment of rabbit CD34+ cells after sorting was relatively low in comparison to the human samples. On the other hand the sorting efficiency of rabbit CD34 as evaluated using Labeling Check Reagent was comparable to human samples. The reason for this might be dim expression of human CD34 antibody on the rabbit mononuclear cells. Therefore, the applied antibodies in this study might be suitable for detection of rabbit CD34+ HSC but not ideal for sorting these cells.
Key words:
rabbit, spermatozoa, artificial insemination, HSCs, MACS

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