The antioxidant effects of butylated hydroxytoluene on cryopreserved goat sperm from a proteomic perspective.

At present, there are few reports about the proteomics changes provoked by butylated hydroxytoluene (BHT) supplementation on cryopreserved semen in mammals. Thus, we aimed to evaluate the effects of different concentrations of BHT on goat sperm and to investigate the proteomics changes of adding BHT to cryopreserved goat (Capra hircus) sperm. Firstly, semen samples were collected from four goats, and frozen in the basic extenders containing different concentrations of BHT (0.5 mM, 1.0 mM, 2.0 mM) and a control without BHT, respectively. After thawing, the protective effects of dose-dependent replenished BHT to the freezing medium on post-thaw sperm motility, integrities of plasma membrane and acrosome, reactive oxygen species levels were confirmed, with 0.5 mM BHT being the best (B group) as compared to the control (without BHT, C group). Afterwards, TMT-based quantitative proteomic technique was performed to profile proteome of the goat sperm between C group and B group. Parallel reaction monitoring was used to confirm reliability of the data. Overall, 2,476 proteins were identified and quantified via this approach. Comparing the C and B groups directly (C vs. B), there were 17 differentially abundant proteins (DAPs) po-tentially associated with sperm characteristics and functions were identified, wherein three were upregulated and 14 were downregulated, respectively. GO annotation analysis demonstrated the potential involvement of the identified DAPs in metabolic process, multi-organism process, reproduction, reproductive process, and cellular process. KEGG enrichment analysis further indicated their potential roles in renin-angiotensin system and glutathione metabolism pathways. Together, this novel study clearly shows that BHT can effectively improve quality parameters and fertility potential of post-thawed goat sperm at the optimal concentration, and its cryoprotection may be realized through regulation of sperm metabolism and antioxidative capability from the perspective of sperm proteomic modification.

Protective effect of butylated hydroxytoluene (BHT) against the clastogenic acitivity of cadmium chloride and potassium dichromate in hamster ovary cells

The effect of butylated hydroxytoluene (BHT), a widely used food additive, on chromosomal alterations induced by cadmium chloride (CC) and potassium dichromate (PD) in Chinese hamster ovary (CHO) cells was studied both at metaphase and anaphase-telophase. CHO cells were cultured for 15-16 h in the presence of PD (6.0, 9.0 or 12.0 mM), BHT (1.0 mg/ml), or PD plus BHT as well as CC (0.5, 1.0 and 2.0 mM), BHT or CC plus BHT for the analysis of chromosomal aberrations. To perform the anaphase-telophase test, cells were cultured in cover glasses and treated 8 h before fixation with the same chemicals. An extra dose of CC (4 mM) was used in this test. Both metal salts significantly increased chromosomal aberration frequencies in relation to untreated controls, and to DMSO- and BHT-treated cells. Post-treatment with BHT decreased the yield of chromosomal damage in relation to treatments performed with CC and PD. However, chromosomal aberration frequencies were significantly higher than those of the controls. In the anaphase-telophase test, CC significantly increased the yield of lagging chromosomes with the four doses employed and the frequency of lagging fragments with the highest dose. In combined treatments of CC and BHT, frequencies of the two types of alterations decreased significantly in relation to the cells treated with CC alone. No significant variation was found in the frequencies of chromatin bridges. Significant increases of numbers of chromatin bridges, lagging chromosomes and lagging fragments were found in cells treated with PD. The protective effect of BHT in combined treatments was evidenced by the significant decrease of chromatid bridges and lagging chromosomes in relation to PD-treated cells. Whereas BHT is able to induce chromosomal damage, it can also protect against oxidative damage induced by other genotoxicants.