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Ministry of Science and Higher Education

National Leading Science Centre (KNOW)

Title: Scientific Consortium „Healthy Animal – Safe food”
IAR&FR PAS Coordinator: Prof. Dariusz Skarżyński
Decision No.: 05-1/KNOW2/2015
Period: 2015-2019
Total budget: 41 476 000 PLN
IAR&FR PAS budget: 8 295 200 PLN

Iuventus Plus

Title: The role of myofibroblasts in chronic degenerative endometritis in the mare
Coordinator: Ph.D. Anna Szóstek
Agreement No.: 0463/IPI/2015/73
Period: 2015-03-16 / 2017-03-15
Total Budget: 200 000 PLN

Title: The impact of the of transforming growth factor ß superfamily on the function of corpus luteum in mare
Coordinator: DVM, Ph.D., D.Sc. Antonio Galvao
Agreement No.: 0113/IPI/2015/73
Period: 2015-03-25 / 2017-03-24
Total Budget: 180 000 PLN

Diamentowy Grant

Title: Embryonic/endometrial exosomes as a new component of embryo-maternal interaction in the pig
Coordinator: Joanna Najmuła
Agreement No.: 0041/DIA/2014/43
Period: 2014-09-15 / 2016-09-14
Total Budget: 170 555 PLN

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Methods and analysis

  • examination of the level of phytochemicals metabolites in biological fluids and tissues of organisms;
  • analysis of metabolic biomarkers upon plant food intake;
  • bioavailability study of food compounds and determination of pharmacokinetic parameters;
  • analysis of plant metabolome;
  • qualitative and quantitative analysis of phytocompounds.

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Equipment

  1. micro-UHPLC gradient system with autosampler (LC200, Eksigent) coupled with TripleTOF 5600+ mass spectrometer (AB SCIEX, USA);
  2. micro-UHPLC gradient system with autosampler (LC200, Eksigent) coupled with QTRAP 5500 mass spectrometer (AB SCIEX, USA);
  3. HPLC gradient system with autosampler (LC-20) coupled with SPD-M20A DAD detector (Shimadzu, Japan);
  4. HPLC gradient system with autosampler (LC-10, Shimadzu, Japan) coupled with DAD detector (SPD-M10AVP, Shimadzu, Japan) and mass spectrometer (LCMS- QP8000α, Shimadzu, Japan);
  5. HPLC gradient system with autosampler (LC-10, Shimadzu, Japan) coupled with UV-Vis detector (SPD-10A, Shimadzu, Japan) and fluorescence detector (RF-10AXL, Shimadzu, Japan);
  6. HPLC gradient system with autosampler (LPG-3400M, WPS-3000TSL, Dionex, USA) coupled with multi-channel electrochemical detector (5600A, ESA, USA) and fluorescence detector (FLD-3400RS, Dionex, USA);
  7. HPLC isocratic system (LC-10, Shimadzu, Japan) coupled with two electrochemical detectors (ICA-3000, TOA, Japan and ED40, Dionex, USA);
  8. gas chromatograph (7890A, Agilent, USA) with MS and FID detectors;
  9. software for pharmacokinetic calculation Biokinetica.

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Methods and analysis

  1. Immunoenzymatic (ELISA) and immunoizotopic (RIA) determination of hormone concentrations:
    • protein hormones (LH, PRL) and receptors LH/hCG with iodine (J-125)-labelled preparation,
    • iodination of protein hormones (LH, PRL) and cytokine (IL-1β, IL-6, TNF-α),
    • steroid hormones (P4, A4, T, E1, E2, cortisol),
    • prostaglandins (PGE2, PGF2α) and metabolite of PGF2α – PGFM,
    • Oxytocin and endothelins using preoxidase (HRP)- or biotin conjugated hormones
  2. Analysis of critical parameters and blood gasometry:

    Analysis of gasometry and oxymetry parameters as well as electrolytes, with application of analyser of critical parameters, RAPIDpoint 405 (SIEMENS):

    • gasometry : pH, pCO2, pO2,
    • electrolytes : NA+, K+, Ca++, Cl-,
    • glucose, HCT,
    • oxymetry: sO2, FO2Hb, FCOHb, FHHb, FMetHb, THb,

    together with a number of counted derivative parameters and many parameters of derivatives.

  3. Morphometric blood analysis:

    • Double-track measurement of leucocytes in two independent channels
    • Basic and expanded morphologic analysis of cattle, sheep, horse, cat, dog, rabbit, guinea pig, rat and mouse blood, using of hematologic analyzer ADVIA 2120i (SIEMENS).

    Desired volume of blood sample foranalysis is 157 µl.

  4. Immunochemical-chemiluminescense blood analysis with application of immunochemical analyzer IMMULITE®1000 (SIEMENS):

    • Analysis of adrenal/ pituitary, sex hormone concentrations (e.g. P4, T, E2, cortisol),
    • Analysis of thyroid physiology,
    • Analysis of concentrations of growth factors,
    • Analysis of inflammatory factors (IL-1β, TNF-α, IL-10, LBP),
    • Analysis of allergy, anaemia and diabetes factors,
    • Analysis of cardiologic, infectious disease, cancer factors,
    • Drug concentrations

    Analyses may be conducted with the following material: sera, plasma and culture media.

  5. Analysis of specific immunoglobulins E using ImmunoCAP 100 apparatus:

    • Phadiatop test for differentiation of atopic and non-atopic diseases,
    • Total IgE,
    • Total IgE (low level) – for small children,
    • Specific IgE – over 500 allergies and 70 mixes,
    • Eosynophilic protein ECP – monitoring of asthma treatment,
    • tryptase – monitoring of anaphylaxis,
    • gliadins IgA and IgG – child food intolerance,
    • specific IgA reference to milk proteins,
    • specific IgG and IgG4,
    • anti-thyroid antibodies: anti-TPO, anti-TG,
    • above 22 parameters within autoimmunological diseases in IgG, IgA classes.

    Material required for analysis: 40 µl of patient serum.

  6. Qualitative and quantitative in vitro determination of human IgG and IgE with the use of Euroimmun equipment:

    • Immunoblott to determine specific allergenic IgE antibodies in human sera,
    • The panels concerning mix of 20 allergens in one strip (pollen, pollen-pediatric, food, atopic), two for insect venoms and „pediatric profile” concerning 27 the most popular allergens i.a.: white and yolk of egg, milk proteins, wheat flour, rice, soy, fruits and vegetables within food allergens, and grass and tree pollens, Dermatophagoides allergens, and animals epidermis within pollen allergens.

    Material required for analysis: 100 µl of patient sera.

  7. Detection of functional proteins (cytokines, effector proteins, receptors, cell surface markers or antibodies) released by single cells with the use of ELISpot (Enzyme-Linked ImmunoSpot Assay).

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COST

COST Action CA22131 – Supramolecular LUminescent Chemosensors for Environmental Security (LUCES).

COST Action CA21139 – 3Rs concepts to improve the quality of biomedical science (IMPROVE).

COST Action CA21149 – Reducing acrylamide exposure of consumers by a cereals supply-chain approach targeting asparagine (ACRYRED).

COST Action CA20128 – Promoting Innovation of ferMENTed fOods.

COST Action ​CA18113 – Understanding and exploiting the impacts of low pH on micro-organisms.

Completed Actions

COST Action CA16119 CellFit – In vitro 3-D total cell guidance and fitness.

COST Action FA1402 ImpARAS – Improving Allergy Risk Assessment Strategy for new food proteins.

COST Action FA1403 POSITIVe – Interindividual variation in response to consumption of plant food bioactives and determinants involved.

COST Action FA 1205 AQUAGAMETE – Assessing and improving the quality of aquatic animal gametes to enhance aquatic resources – The need to harmonize and standardize evolving methodologies, and improve transfer from academia to industry.

COST Action FA1201 EPICONCEPT – Epigenetics and Periconception Environment.

COST Action FA1005 INFOGEST – Improving health properties of food by sharing our knowledge on the digestive process

COST Action FA0702 GEMINI – Maternal Interaction With Gametes and Embryo.

COST Action FA0801 LarvaNet – Critical success factors for fish larval production in European Aquaculture: a multidisciplinary network.

COST 926 – Impact of new technologies on the health benefits and safety of bioactive plant compounds.

COST 927 – Thermally processed foods: possible health implications.

COST Action 958 – Viticulture: biotic and abiotic stress – grapevine defence mechanism and grape development.

COST Action D31 – Novel and specific anion receptors for use in sensor technology.

COST FA802 – Feed for health.

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Equipment

  1. Work station designated to immunoenzymatic analysis of hormone concentration (ELISA) consists of:
    • ELISA microplate reader (KHB)
    • Spectrophotometer working with cuvetes and microplates (Multiscan Spectrum, Thermo Scientific)
    • Automatic well washer
    • Microplate incubator (Heraeus)
  2. Work station designated to immunoisotopic analysis of hormone concentration (RIA) consists of:
    • Beta radiation counter
    • Gamma radiation counter
    • Radiochemical hood
    • Beckman centrifuge
    • Hamilton microlab
    • Laboratorial scales
    • Deep-freezer (-85°C)
  3. Chemiluminescent  analyser IMMULITE® 1000:
    • enables analysis of over 90 tests
    • enables simultaneous analysis of 5 different parameters in one sample
    • automatic dilutions for selected tests
    • efficiency 129 tests per hour
    • enables conduction of analysis in the standard and “Turbo” mode
  4. ImmunoCAP 100:
    • fully automatic providing automatic sampling, and printing results obtained
    • the max capacity of the carousel is 48 wells
    • the max time required for conduction of the analysis is 2.4 hours
    • automatic sample dilution
    • simultaneous conduction of several analysis within one sample of the blood serum
  5. EuroimmunQuantitative in vitro tests designated to determine human IgG antibodies against selected antigens (autoimmunological and infectious diseases) as well as specific IgE directed to allergens. The set consists of test stripes with pure allergens along with set of reagents. Data analysis is conducted with EURLineScan programme.
  6. ELISpot (Enzyme-Linked ImmunoSpot Assay)
    • EliScan – scan to read the spots
    • Eli Analyse – software for computer analysis enables automatic background correction, algorithms of contaminant determination, calculation of spot size, intensity, shape and color, Dual-Color analysis, statistical evaluation of replicates, analysis of microplates series.

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Equipment

Semen Analysis and Cryopreservation Zone:

  • flow cytometers (Muse, Guava, and FlowSight) – devices used for sperm quality analysis, including the assessment of viability, membrane fluidity, mitochondrial potential, and the presence of reactive oxygen species (ROS). In addition, they enable the characterization of extracellular vesicles,
  • CASA system (Computer Assisted Sperm Analysis) – a computer-based system enabling precise measurement of sperm motility parameters, used, among others, to assess the impact of storage and cryopreservation procedures on semen quality,
  • NucleoCounter – enabling rapid and accurate measurement of sperm concentration and the proportion of live and dead cells,
  • micro-osmometer – a device for measuring the osmolality of fluids, both biological (semen plasma, blood plasma) and chemical,
  • IceCube 4s system – an advanced system for programmable, computer-controlled semen cryopreservation,
  • Easy Coder printer – a device for applying identification data (e.g. species names, individual IDs, dates) to straws, ensuring full traceability of samples,
  • MPP Quattro automatic machine – a system for filling and sealing cryopreservation straws,
  • straw sealer – devices for manual sealing of straws, used as an alternative to the MPP Quattro system.

Oocyte Analysis and In Vitro Embryo Culture Zone

  • Zeiss inverted microscope with fluorescence system and micromanipulators, used for survival analysis of oocytes and embryos, with dedicated MicroImage software enabling documentation and analysis of morphological parameters,
  • Zeiss stereomicroscope for the selection, classification, and isolation of oocytes,
  • Olympus CKX41 inverted microscope for the assessment of quality and developmental stage of gametes and embryos,
  • laminar flow cabinets and tri-gas incubators (CO₂, O₂, N₂) enabling long-term culture of mammalian embryos under precisely controlled environmental conditions,
  • system for aspiration of oocytes from ovarian follicles,
  • Primo Vision digital real-time embryo development monitoring system, enabling research on embryogenesis and the dynamics of cell divisions,
  • a set of tools for in-house production of microcapillaries, including a puller, microforge, and microgrinder – essential for micromanipulation procedures such as ICSI, nuclear transfer, or microinjection of genetic material.

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Methods and analysis

  • flow cytometric analyses (including assessment of cell viability, membrane fluidity, mitochondrial potential, and the presence of reactive oxygen species (ROS), as well as cytometric characterization of extracellular vesicles),
  • analysis of sperm motility parameters,
  • analysis of semen concentration,
  • semen cryopreservation,
  • in vitro embryo culture,
  • cell and tissue banking and storage.

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Methods and analysis

  • 2-D Electrophoresis – determination of isoelectric point and molecular mass, analysis of protein profiles;
  • Isolation of protein and peptides using FPLC, determination of the main physico-chemical properties of protein;
  • Measurement of sperm concentration;
  • Kinetics analysis (determination of enzyme and inhibitor concentration, measurement of the equilibrium association constant for inhibitor – proteinase complexes;
  • Electroelution of proteins from polyacrylamide gel.

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Equipment

  1. An integrated system for 2-Dimensional (2D) Electrophoresis includes:
    • System for 2D electrophoresis (IPGphor 3 IEF System – first dimension; Ethan DALTsix Large Vertical System – second dimension),
    • System for gel analysis and documentation (ImageScanner III),
    • System for picking protein spots from 2D gels (Etttan spot picker),
    • System for in-gel digestion of proteins from 2-D electrophoresis gel plugs (Ettan Digester),
    • System for spotting digested proteins onto MALDI MS targets (Ettan Spotter),
  2. Freeze-drier with accesories;
  3. VersaFluor Fluorometr;
  4. Fast Protein Liquid Chromatography System (ÄKTApurifier);
  5. Ultra low temperature freezers;
  6. Spectrophotometer (Beckman DU800) with accessories;
  7. Ultrasonicator (Sonics vibra cell);
  8. Apparatus for electroelution of proteins from polyacrylamide gels (Whole Gel Eluter Harvesting  Box).

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