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G
Gunawan, A., S. Sahadevan, C. Neuhoff, C. Große-Brinkhaus, A. Gad, L. Frieden, D. Tesfaye, E. Tholen, C. Looft, M. J. Uddin, et al., "RNA deep sequencing reveals novel candidate genes and polymorphisms in boar testis and liver tissues with divergent androstenone levels.", PloS one, vol. 8, issue 5, pp. e63259, 2013. Abstract

Boar taint is an unpleasant smell and taste of pork meat derived from some entire male pigs. The main causes of boar taint are the two compounds androstenone (5α-androst-16-en-3-one) and skatole (3-methylindole). It is crucial to understand the genetic mechanism of boar taint to select pigs for lower androstenone levels and thus reduce boar taint. The aim of the present study was to investigate transcriptome differences in boar testis and liver tissues with divergent androstenone levels using RNA deep sequencing (RNA-Seq). The total number of reads produced for each testis and liver sample ranged from 13,221,550 to 33,206,723 and 12,755,487 to 46,050,468, respectively. In testis samples 46 genes were differentially regulated whereas 25 genes showed differential expression in the liver. The fold change values ranged from -4.68 to 2.90 in testis samples and -2.86 to 3.89 in liver samples. Differentially regulated genes in high androstenone testis and liver samples were enriched in metabolic processes such as lipid metabolism, small molecule biochemistry and molecular transport. This study provides evidence for transcriptome profile and gene polymorphisms of boars with divergent androstenone level using RNA-Seq technology. Digital gene expression analysis identified candidate genes in flavin monooxygenease family, cytochrome P450 family and hydroxysteroid dehydrogenase family. Moreover, polymorphism and association analysis revealed mutation in IRG6, MX1, IFIT2, CYP7A1, FMO5 and KRT18 genes could be potential candidate markers for androstenone levels in boars. Further studies are required for proving the role of candidate genes to be used in genomic selection against boar taint in pig breeding programs.

H
Havlicek, V., A. Gad, S. Papp, K. Stein, F. Palm, D. Tesfaye, M. Hoelker, and U. Besenfelder, "232 EFFECT OF SUPEROVULATION PRETREATMENT ON DEVELOPMENTAL CHARACTERISTICS OF IN VITRO-FERTILIZED BOVINE EMBRYOS TRANSFERRED TO THE OVIDUCT-UTERUS ENVIRONMENT", Reproduction, Fertility and Development, vol. 28, issue 2: CSIRO PUBLISHING, pp. 247-248, 2016. Abstract
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Koncicka, M., J. Cervenka, D. Jahn, R. Sucha, P. Vodicka, A. Gad, M. Alsheimer, and A. Susor, "Expression of lamin C2 in mammalian oocytes", PloS one, vol. 15, issue 4: Public Library of Science, pp. e0229781, 2020. AbstractWebsite

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M
Mehaisen, G. M. K., A. M. Saeed, A. Gad, A. O. Abass, M. Arafa, and A. El-Sayed, "Antioxidant Capacity of Melatonin on Preimplantation Development of Fresh and Vitrified Rabbit Embryos: Morphological and Molecular Aspects.", PloS one, vol. 10, no. 10, pp. e0139814, 2015. Abstractjournal.pone_.0139814.pdfWebsite

Embryo cryopreservation remains an important technique to enhance the reconstitution and distribution of animal populations with high genetic merit. One of the major detrimental factors to this technique is the damage caused by oxidative stress. Melatonin is widely known as an antioxidant with multi-faceted ways to counteract the oxidative stress. In this paper, we investigated the role of melatonin in protecting rabbit embryos during preimplantation development from the potential harmful effects of oxidative stress induced by in vitro culture or vitrification. Rabbit embryos at morula stages were cultured for 2 hr with 0 or 10-3 M melatonin (C or M groups). Embryos of each group were either transferred to fresh culture media (CF and MF groups) or vitrified/devitrified (CV and MV groups), then cultured in vitro for 48 hr until the blastocyst stage. The culture media were used to measure the activity of antioxidant enzymes: glutathione-s-transferase (GST) and superoxide dismutase (SOD), as well as the levels of two oxidative substrates: lipid peroxidation (LPO) and nitric oxide (NO). The blastocysts from each group were used to measure the expression of developmental-related genes (GJA1, POU5F1 and Nanog) and oxidative-stress-response-related genes (NFE2L2, SOD1 and GPX1). The data showed that melatonin promoted significantly (P<0.05) the blastocyst rate by 17% and 12% in MF and MV groups compared to their controls (CF and CV groups). The GST and SOD activity significantly increased by the treatment of melatonin in fresh or vitrified embryos, while the levels of LPO and NO decreased (P<0.05). Additionally, melatonin considerably stimulated the relative expression of GJA1, NFE2L2 and SOD1 genes in MF and MV embryos compared to CF group. Furthermore, melatonin significantly ameliorated the reduction of POU5F1 and GPX1 expression induced by vitrification. The results obtained from the current investigation provide new and clear molecular aspects regarding the mechanisms by which melatonin promotes development of both fresh and vitrified rabbit embryos.

P
Prastowo, S., A. Amin, F. Rings, E. Held, S. D. Wondim, A. Gad, C. Neuhoff, E. Tholen, C. Looft, K. Schellander, et al., "Fateful triad of reactive oxygen species, mitochondrial dysfunction and lipid accumulation is associated with expression outline of the AMP-activated protein kinase pathway in bovine blastocysts", Reproduction, Fertility and Development, vol. 29, issue 5: CSIRO PUBLISHING, pp. 890-905, 2017. Abstract

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S
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., 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.

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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