CHARACTERIZATION OF THE BIOACTIVE PROPERTIES OF CAROTENOIDS ISOLATED FROM AN OVERPRODUCER STRAIN OF HALOPHILIC ARCHAEA

MIRÓ, MV; ZALAZAR, L.; DE CASTRO, R.E.; CESARI, A.

San Miguel de Tucumán

Simposio; XII Congreso Argentino de Microbiología General; 2017

Sociedad Argentina de Microbiología General

Archaea have evolved with distinct ecophysiological adaptations consistent with the extreme environments they inhabit, thus, their molecules may present novel characteristics. An example of these are bacterioruberins (Bctr), orange/red carotenoid pigments that exert a protective effect against the harmful effects of UV light. Due to their antioxidant capacity, carotenoids have numerous applications; however, the bioactive features of haloarchaeal Bctr have been scarcely explored. A target field for antioxidants is the protection of sperm from cryodamage. Although semen cryopreservation has contributed to theexpansion of artificial insemination, this technique did not prove to be completely efficient in species such as sheep. Cryopreservation induces oxidative stress, altering the motility and viability, increasing the influence of calcium. The aim of this investigation was to explore the bioactive properties of Bctr focusing on their protective effect on ram sperm after freezing/thawing. Bctr was extracted from a Bctr-overproducer mutant strain of the haloarchaeon Haloferax volcanii using acetone-methanol solution (1:1, v/v). The organic extract enriched in Bctr was evaporated and preserved at-20 °C protected from light and before use it was solubilized in ethanol. The antioxidant capacity of Bctr extracts with different times of storage was evaluated using the 2,2diphenyl-1-picrylhydrazyl (DPPH) radical spectrophotometrically and was also compared to that of b-carotene. It was confirmed that the antioxidant capacity of Bctr was 2.5-3.0 fold higher than that of b-carotene and it was also observed that the extracts did not decrease their antioxidant activity after several months of storage. The effect of the Bctr extracts was evaluated on different ram sperm parameters and compared to b-carotene. Once thawed and selected, sperm was incubated with different Bctr or b-carotene concentrations or the respective controls. At different incubation times, the pattern of tyrosine phosphorylated proteins was evaluated by WesternBlotting and various sperm parameters were analysed by flow cytometry including viability, mitochondrial functional status, lipid peroxidation, ROS content and DNA damage. Treatment of sperm cells with 2.5, 7, 15 and 20 μM Bctr for 120 min. significantly increased the percentage of viable cells and decreased the proportion of cells with intracellular ROS, compared to their respective controls, and the effect was similar to that observed for b-carotene. In conclusion, although fertility trials are needed, Bctr is a potential antioxidant that can be applied to ram semen to improve insemination yields. Supported by EXA-731/15-UNMdP, PIP1106-CONICET and PICT1477 (RDC); PIP 2014 0273 (AC).