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Ovine sperm DNA oxidation quantification using an 8‐OHdG immunodetection assay
Author(s) -
SoriaMeneses Pedro Javier,
JuradoCampos Alejandro,
Montoro Vidal,
Soler Ana Josefa,
Garde José Julián,
FernándezSantos María del Rocío
Publication year - 2019
Publication title -
reproduction in domestic animals
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.546
H-Index - 66
eISSN - 1439-0531
pISSN - 0936-6768
DOI - 10.1111/rda.13533
Subject(s) - sperm , dna , dna oxidation , chemistry , andrology , microbiology and biotechnology , biology , dna damage , biochemistry , medicine
Our aim was to optimize 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) immunodetection in order to detect DNA damage caused by oxidative stress that may not be detected by other DNA integrity analysis techniques, especially due to the high compaction of DNA in ruminants. Semen samples from 6 rams were cryopreserved. After thawing, samples were subjected to the DNA oxidation quantification using an 8‐OHdG immunodetection assay by flow cytometry. We have evaluated two different incubation times (30 min vs . overnight) at 4°C of the primary antibody (monoclonal anti‐8‐OHdG antibody). We have also compared the results of this technique with the sperm chromatin structure assay (SCSA ® ). The analysis revealed that there were no significant differences ( p > .05) between different incubation times. However, overnight incubation seems to cause more non‐specific binding of the secondary antibody. Significant differences ( p < .05) between subjects and oxidation controls (8 M H 2 O 2 /800 μM FeSO 4 •7H 2 O) were evident. We can conclude that the 8‐OHdG immunodetection assay for DNA oxidation quantification of ram sperm can be performed subjecting sperm samples to a very high oxidative treatment.