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Sakr, O. G., A. Gad, M. Rodríguez, P. G. Rebollar, and P. Millán, "Superoxide dismutase mimics improves semen quality during chilled preservation of rabbit spermatozoa", Livestock Science, vol. 221: Elsevier, pp. 70-76, jan, 2019. AbstractWebsite

In rabbit farms, artificial insemination is usually undertaken using semen preserved around 18ºC. However, increased reactive oxygen species levels during preservation produce sperm dysfunction. Our aim was to evaluate the effect of adding superoxide dismutase (SOD) antioxidant mimics (Tempo and TempoL) to the extenders of rabbit semen on quality parameters, total lipid peroxidation (LPO) and SOD level. Ejaculates from 12 sexually mature males were diluted with an extender containing 0 (control), 0.5, 1 or 2 mM of Tempo or TempoL, respectively. Semen samples were cooled till 18ºC and stored for 0, 6 or 24 h. Sperm motility, velocity parameters, viability, plasma membrane integrity, SOD activity and level of thiobarbituric acid reactive substances (TBARS) were determined. Results showed that, in general, SOD antioxidant mimics significantly improved sperm motility compared to control after 6 and 24 h storage. Moreover, at 24 h, Tempo treatments showed motility rates higher than 80{%} whilst the control group showed the lowest motility rate among all treatments (58.5{%}, P {\textless} 0.05). Sperm viability showed no significant differences between treatments at 6 h and 24 h of storage. At 24 h, most SOD antioxidant mimics treatments significantly improved both curvilinear and mean velocity parameters, but only Tempo treatments improved linear velocity parameter. Also, SOD mimics protected sperm cells decreasing TBARS concentration at 6 and 24 h compared to 0 h. In conclusion, the addition of SOD antioxidant mimics during conservation at 18ºC and storage of semen until 24 h reduces lipid peroxidation and preserves rabbit semen quality.

Salilew-Wondim, D., E. Fournier, M. Hoelker, M. Saeed-Zidane, E. Tholen, C. Looft, C. Neuhoff, U. Besenfelder, V. Havlicek, F. Rings, et al., "Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro", PloS one, vol. 10, no. 11: Public Library of Science, pp. e0140467, 2015. Abstract
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Salilew-Wondim, D., M. Saeed-Zidane, M. Hoelker, S. Gebremedhn, M. Poirier, H. O. Pandey, E. Tholen, C. Neuhoff, E. Held, U. Besenfelder, et al., "Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation", BMC Genomics, vol. 19, issue 1, pp. 424, 2018. Abstract

BACKGROUND:
Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis.

RESULTS:
The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes.

CONCLUSION:
In vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.