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Title:SPEKTROFOTOMETRIČNO DOLOČANJE STABILNOSTI BUTANOLNIH EKSTRAKTOV RESAZURINA
Authors:ID Bogataj, Mateja (Author)
ID Brodnjak-Vončina, Darinka (Mentor) More about this mentor... New window
ID Kolar, Mitja (Comentor)
ID Zrimšek, Petra (Comentor)
Files:.pdf VS_Bogataj_Mateja_2011.pdf (619,88 KB)
MD5: A00DFE1949394AC8D042E6CABAF75CBA
PID: 20.500.12556/dkum/5ec93a0b-8993-4597-a0b4-46e6d27e6d3b
 
Language:Slovenian
Work type:Undergraduate thesis
Organization:FKKT - Faculty of Chemistry and Chemical Engineering
Abstract:Redukcijski test z resazurinom temelji na sposobnosti metabolično aktivnih semenčic, da reducirajo redoks barvilo resazurin v resorufin. V diplomskem delu smo razvili spektrofotometrično aplikacijo redukcijskega testa z resazurinom za osemenjevalne doze merjaščevega semena. Uporabili smo 54 vzorcev. Redukcijo resazurina po opravljenem testu smo sledili z merjenjem absorbance v butanolnih ekstraktih pri valovni dolžini 610 nm, ki ustreza absorpcijskemu maksimumu za resazurin ter pri valovni dolžini 575 nm, ki ustreza maksimumu absorpcije za resorufin. Ponovljivost meritev smo določili z izračunom relativnega standardnega odmika. Ponovljivost meritev absorbanc v testu pri 610 nm in 575 nm je v obeh primerih ustrezna, pri določanju ponovljivosti med testi pa smo ugotovili, da je ta ustrezna za meritve A575, medtem ko relativni standardni odmik za meritve A610 ne ustreza zahtevam za ponovljivost meritev med testi. Korelacije med rezultati testa z resazurinom in spermalnim indeksom (koncentracija semenčic v vzorcu, pomnožena s kvadratnim korenom produkta med deležem progresivno gibljivih in deležem morfološko normalnih semenčic) smo izračunali z neparametričnim Spearmanovim koeficientom korelacije rangov. Najvišjo korelacijo smo ugotovili med spermalnim indeksom in A610 v testu z dodatkom 160 μl 1,8 mM resazurina po 60 min inkubaciji. Glede na to, da je ponovljivost meritev A610 neustrezna, smo za optimalne pogoje testa določili pogoje, pri katerih je bila korelacija med spermalnim indeksom in meritvami A575 najvišja, to je pri 40 min inkubaciji in dodatku 80 ml resazurina (r=0,654; P<0,0001). Ugotavljali smo možnost merjenja absorbance butanolnih ekstraktov, hranjenih pri 4°C, tudi v dnevih po izvedbi testa. Na podlagi razsevnih diagramov s premico linearne regresije smo ugotovili, da so parne meritve, izmerjene na dan izvedbe testa in 1. dan, 4. dan in 7. dan pri A575, blizu premici enakosti. Iz diagramov absolutnih razlik je razvidno, da je več kot 95 % absolutnih razlik znotraj mej ujemanja, kar potrjuje zadovoljivo ujemanje posameznih parnih meritev. Rezultati kažejo, da so butanolni ekstrakti stabilni, zato lahko redukcijo resazurina z merjenjem A575 določimo v roku enega tedna po izvedbi testa. Uporabnost testa je zato večja, saj ga izvedemo v terenskih pogojih, kjer spektrofotometer ni na voljo na dan izvedbe testa.
Keywords:veterinarska medicina, resazurin, seme, spektrofotometrija-metode
Place of publishing:Maribor
Publisher:[M. Bogataj]
Year of publishing:2011
PID:20.500.12556/DKUM-18976 New window
UDC:579.22(043.2)
COBISS.SI-ID:15160086 New window
NUK URN:URN:SI:UM:DK:SVIG9MSE
Publication date in DKUM:27.06.2011
Views:3339
Downloads:113
Metadata:XML DC-XML DC-RDF
Categories:KTFMB - FKKT
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Secondary language

Language:English
Title:SPECTROPHOTOMETRIC DETERMINATION OF STABILITY OF RESAZURIN BUTANOL EXTRACTS
Abstract:The resazurin reduction assay depends on the ability of metabolically active cells to reduce the resazurin redox dye to resorufin. In the present study we applied a spectrophotometric application of the resazurin reduction assay to evaluate boar semen quality, using 54 semen doses. Resazurin reduction was determined with absorbance at 610 nm corresponding to absorption peak of resazurin and at 575 nm, where maximum absorbance of resorufin was established. Repeatability of resazurin reduction test is given by an intra-assay and inter-assay relative standard deviation. Intra-assay repeatability is satisfactory for measuring absorbance at 610 nm and 575 nm, whereas inter-assay RSD for A610 exceeded demands for precision. Correlations between resazurin reduction and sperm index (multiplying sperm concentration by the SQRT of percentage sperm motility multiplied by percentage of morphological normal forms) were determined using Spearman rank correlation coefficient. We observed the highest correlations of resazurin reduction with sperm index using A610 after 60 min incubation and addition of 160 μl 1,8 mM resazurin. According to unsatisfactory repeatability of measuring A610, the optimal test’s conditions were set at A575, time incubation of 40 min and addition of 80 μl 1,8 mM resazurin (r=0,654, P<0,0001). To evaluate the possibility to measure the absorbance in butanol extracts a day or even a week after the assay performing, we measured the A575 of each butanol extract on days 0, 1, 4 and 7 days after storage at 4C. On the basis of scatter plots with regression lines it’s evident that regression lines are close to the lines of equality. Measurements obtained on the day of performing the test and the measurements after 1, 4 and 7 days of storage agree; more than 95% of the differences lie within the limits of agreement showing by Bland-Altman plots. The results obtained leading to the conclusion that we can measure A575 of butanol extracts within 7 days from the day of test performing, confirming a great practical value of the method. However, the usefulness of the assay will therefore be much greater and the assay could be performed even when the spectrophotometer is at a different location or it is not available at the time of performing the test.
Keywords:veterinary medicine, resazurin, semen, spectrophotometry- methods


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