Development of an analytical method to determine malondialdehyde as an oxidative marker in cryopreserved bovine semen

YONNY, M. E.; REINERI, P.; PALMA, G.; NAZARENO M.A.

ANALYTICAL METHODS

ROYAL SOCIETY CHEMISTRY

Lugar: CAMBRIDGE; Año: 2015 vol. 7 p. 6331 - 6338

1364-5528

Frozen sperm is widely used in artificial insemination of cattle as well as other animal species. As a consequence of the freezing and thawing processes of semen, an excess of reactive oxygen species (ROS) are formed. ROS produce oxidative damage in sperm cells affecting both motility and fertility. Malondialdehyde (MDA) is one of the most recognized biomarkers of the advanced oxidative status. MDA was analyzed after its condensation reaction with thiobarbituric acid (TBA); however, other molecules can also react with TBA. In order to determine specifically MDA-TBA2 condensation product in cryopreserved bovine semen, a sensitive, and selective separative strategy was developed using high performance liquid chromatography (HPLC) with diode array detection (DAD). This is the first report of MDA determination in bovine semen by a separative method. Different methodological approaches were assayed. Treatment A directly measured total MDA through acidic hydrolysis combined with TBA condensation in a single step. Treatment B evaluated separately TBA condensation product of free MDA and protein bound MDA after its releasing with alkaline hydrolysis. The highest concentration of MDA was detected following treatment A. An HPLC method was developed and validated comparing with the traditional spectrophotometric method. The detection and quantification limits were 0.034 µM and 0.086 µM. The DAD response was linear in the range between 0.086 and 9.1 µM. The recovery was 91%. The intra and interday relative standard deviations were 3.7 and 3.8%, respectively. The HPLC proposed method was markedly more sensitive and more specific than the traditional spectrophotometric one.