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2020
Gebremedhn, S., A. Gad, H. S. Aglan, J. Laurincik, R. Prochazka, D. Salilew-Wondim, M. Hoelker, K. Schellander, and D. Tesfaye, "Extracellular vesicles shuttle protective messages against heat stress in bovine granulosa cells", Scientific Reports, vol. 10, no. 1: Nature Publishing Group, pp. 1–19, sep, 2020. AbstractWebsite

Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS.

Gad, A., J. M. Sánchez, J. A. Browne, L. Nemcova, J. Laurincik, R. Prochazka, and P. Lonergan, "Plasma extracellular vesicle miRNAs as potential biomarkers of superstimulatory response in cattle", Scientific Reports, vol. 10, no. 1: Nature Publishing Group UK, pp. 1–16, dec, 2020. AbstractWebsite

The ability to predict superstimulatory response would be a beneficial tool in assisted reproduction. Using small RNAseq technology, we profiled extracellular vesicle microRNA (EV-miRNA) abundance in the blood plasma of heifers exhibiting variable responses to superstimulation. Estrous synchronized crossbred beef heifers (n = 25) were superstimulated and blood samples were collected from each heifer on Day 7 of consecutive unstimulated (U) and superstimulated (S) cycles. A subset of high (H) and low (L) responders was selected depending on their response to superstimulation and EV-miRNA profiles were analysed at both time-points in each heifer. Approximately 200 known miRNAs were detected in each sample with 144 commonly detected in all samples. A total of 12 and 14 miRNAs were dysregulated in UH vs. UL and in SH vs. SL heifers, respectively. Interestingly, miR-206 and miR-6517 exhibited the same differential expression pattern in H compared to L heifers both before and after superstimulation. Pathway analysis indicated that circadian rhythm and signaling pathways were among the top pathways enriched with genes targeted by dysregulated miRNAs in H vs. L responding heifers. In conclusion, heifers with divergent ovarian responses exhibited differential expression of plasma EV-miRNAs which may be used as a potential biomarker to predict superstimulation response.

Ahmed, D. A. - E. R., N. Ghanem, S. M. Dessouki, M. S. Faheem, A. Y. Gad, and A. H. Barkawi, "Developmental Competence of Buffalo Oocytes Cultured Under Different Oxygen Tensions after Selection with Brilliant Cresyl Blue", World's Veterinary Journal, vol. 10, issue 2, pp. 246-253, 2020. Abstract
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Ashour, G., A. Gad, A. K. Fayed, N. A. Ashmawy, and A. El-Sayed, "Evaluation of Growth Performance, Blood Metabolites and Gene Expression Analysis in Egyptian Sheep Breeds, in Relation to Age", World’s Veterinary Journal, vol. 10, issue 4, pp. 18-29, 2020. 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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Gebremedhn, S., A. Ali, A. Gad, R. Prochazka, and D. Tesfaye, "Extracellular Vesicles as Mediators of Environmental and Metabolic Stress Coping Mechanisms During Mammalian Follicular Development", Frontiers in Veterinary Science, vol. 7, pp. 961, 2020. AbstractWebsite

Extracellular vesicles are evolutionarily conserved nano-sized phospholipid membraned structures and released from virtually all types of cells into the extracellular space. Their ability to carry various molecular cargos (mRNA, miRNA, proteins, and lipids) from one cell to the other to exert functional impact on the target cells enables them to play a significant role in cell to cell communication during follicular development. As the molecular signals carried by extracellular vesicles reflect the physiological status of the cells of origin, they are expected to mediate any effect of environmental or metabolic stress on the follicualr cells and the growing oocyte. Recent studies have evidenced that reproductive cells exposed to various environmental stressors (heat and oxidative stress) released extracellular vesicles enriched with mRNA and miRNA associated with stress response mechanisms. Moreover, the metabolic status of post-calving cows could be well-reflected in the follicular extracellular vesicle's miRNA profile, which signified the potential role of extracellular cellular vesicle molecular signals in mediating the effect of metabolic stress on follicular and oocyte development. In the present review, the potential role of extracellular vesicles in mediating the effect of environmental and metabolic stress in various reproductive cells and oocytes are thoroughly discussed Moreover, considering the importance of extracellular vesicles in shuttling protective or rescuing molecular signals during stress, their potential usage as means of targeted delivery of molecules to mitigate the effect of stress on oocytes are addressed as the focus of future research.

Gad, A., L. Nemcova, M. Murin, V. Kinterova, J. Kanka, J. Laurincik, M. Benc, L. Pendovski, and R. Prochazka, "Global transcriptome analysis of porcine oocytes in correlation with follicle size", Molecular Reproduction and Development, vol. 87, issue 1, no. July: John Wiley {&} Sons, Ltd, pp. 102-114, 2020. AbstractWebsite

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2019
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.

Gad, A., L. Nemcova, M. Murin, J. Kanka, J. Laurincik, M. Benc, L. Pendovski, and R. Prochazka, "MicroRNA expression profile in porcine oocytes with different developmental competence derived from large or small follicles", Molecular Reproduction and Development, vol. 86, issue 4: John Wiley {&} Sons, Ltd, pp. 426–439, feb, 2019. AbstractWebsite

Oocyte developmental competence is acquired during folliculogenesis and regulated by complex molecular mechanisms. Several molecules are involved in these mechanisms, including microRNAs (miRNAs) that are essential for oocyte‐specific processes throughout the development. The objective of this study was to identify the expression profile of miRNAs in porcine oocytes derived from follicles of different sizes using RNA deep sequencing. Oocytes were aspirated from large (LO; 3–6 mm) or small (SO; 1.5–1.9 mm) follicles and tested for developmental competence and chromatin configurations. Small RNA libraries were constructed from both groups and then sequenced in an Illumina NextSeq. 500. Oocytes from the LO group exhibited higher developmental competence and different chromatin configuration compared with oocytes from the SO group. In total, 167 and 162 known miRNAs were detected in the LO and SO groups, respectively. MiR‐205, miR‐16, miR‐148a‐3p, and miR‐125b were among the top 10 highly expressed miRNAs in both groups. Eight miRNAs were differentially expressed (DE) between both groups. Target gene prediction and pathway analysis revealed 46 pathways that were enriched with miRNA‐target genes. The oocyte meiosis pathway and signaling pathways including FoxO, PI3K‐Akt, and cAMP were predictably targeted by DE miRNAs. These results give more insights into the potential role of miRNAs in regulating the oocyte development.

2018
Gad, A., S. Abu Hamed, M. Khalifa, A. Amin, A. El-Sayed, S. A. Swiefy, and S. El-Assal, "Retinoic acid improves maturation rate and upregulates the expression of antioxidant-related genes in in vitro matured buffalo (Bubalus bubalis) oocytes", International Journal of Veterinary Science and Medicine, vol. 6, issue 2: Elsevier, pp. 279–285, sep, 2018. AbstractWebsite

Retinoic acid, vitamin A metabolite, plays a role in oocyte development and maturation in different ways including gene expression alteration and/or prohibiting oxidative stress. The objective of this study was to examine the effect of 9-cis-retinoic acid (9-cisRA) on the quality and maturation rate of buffalo oocytes. Cumulus-oocyte complexes (COCs, n = 460) were collected from ovaries of slaughtered buffalos. Varying concentrations of 9-cisRA (0, 5, 50, and 200 nM) were added to the maturation medium, and the following parameters were analyzed: (i) maturation and cleavage rates, (ii) mitochondrial activity and reactive oxygen species (ROS) levels, (iii) expression level of antioxidant-related genes (PRDX1, SOD1, CAT, HOMX1, and GPX4) using RT-qPCR. Maturation rate was significantly improved in 5 nM 9-cisRA oocyte group (95.8{%}, P {\textless} .05) compared to control and other treatment groups (86.7{%} in control group). The same oocyte group exhibited significantly higher mitochondrial membrane potential activity and lower ROS accumulation level compared to other treatment groups. Antioxidant-related genes were up-regulated in oocytes matured with 5 or 50 nM 9-cisRA compared to control and 200 nM 9-cisRA groups. In contrast, 200 nM of 9-cisRA showed a clear down-regulation for antioxidant-related genes except for PRDX1. In conclusion, supplementation of 9-cisRA with a lower concentration (5 nM) to the buffalo oocytes maturation media promotes maturation rate through a protection mechanism that maintains adequate levels of antioxidant-related transcripts and improves mitochondrial activity. However, 9-cisRA has no significant effect on the cleavage rate of the treated oocytes.

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.

El-Rashidy, A., G. Waly, A. Gad, J. A. Roether, J. Hum, Y. Yang, R. Detsch, A. Hashem, I. Sami, W. G. H. H. Goldmann, et al., "Antibacterial activity and biocompatibility of zein scaffolds containing silver-doped bioactive glass", Biomedical Materials, 2018. AbstractWebsite

Abstract Composite 3D scaffolds combining natural polymers and bioceramics are promising candidates for bone tissue engineering (BTE). Zein, as a natural plant protein, offers several advantages, including biocompatibility, adequate strength properties, and low/no immunogenicity; however, it lacks bioactivity. Thus, composite zein:bioactive glass (BG) scaffolds are proposed as promising candidate for BTE applications, with silver-doping of bioactive glass providing an antibacterial effect against possible post-implantation infection. Therefore, the aim of this study was to investigate the in vitro antibacterial properties, biocompatibility, bioactivity and compressive strength of zein scaffolds containing silver-doped bioactive glass. BG nanoparticles, undoped and Ag-doped, were fabricated using the sol-gel method. 3D composite zein:BG scaffolds, containing 20 wt.% BG, were prepared and their antibacterial activity against E. coli and S. aureus was assessed using the disk diffusion assay. Human osteoblast-like MG-63 cells were used to evaluate the in vitro biocompatibility of the prepared scaffold groups. In addition, the compressive strength of the scaffolds was determined using uniaxial compression strength testing and the scaffold interconnected porosity was measured using helium pycnometer. Disc diffusion assay showed that only zein scaffolds containing Ag-doped sol-gel BG are antibacterially positive against E. coli and S. aureus. Pure zein scaffolds and zein scaffolds containing sol-gel-derived BG showed no negative influence on the growth of MG-63 cells, as evident by the cells’ ability to survive, proliferate, and function on these scaffolds. Moreover, incorporating sol-gel-derived BG into zein scaffolds at zein:BG of 80:20 ratio showed bioactive properties with adequate porosity without affecting the scaffolds’ compressive strengths, which was similar to that of trabecular bone, suggesting that the new composites have potential for BTE applications in non-loaded bearing areas.

Abdelatty, A. M., M. E. Iwaniuk, S. B. Potts, and A. Gad, "Influence of maternal nutrition and heat stress on bovine oocyte and embryo development", International Journal of Veterinary Science and Medicine: Elsevier, 2018. Abstract
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El-Rashidy, A. A., G. Waly, A. Gad, A. A. Hashem, P. Balasubramanian, S. Kaya, A. R. Boccaccini, and I. Sami, "Preparation and in vitro characterization of silver-doped bioactive glass nanoparticles fabricated using a sol-gel process and modified Stöber method", Journal of Non-Crystalline Solids, vol. 483: North-Holland, pp. 26-36, 2018. Abstract
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2017
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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2016
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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2015
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.

El-Rashidy, A. A., A. Gad, A. E. - H. G. Abu-Hussein, S. I. Habib, N. A. Badr, and A. A. Hashem, "Chemical and Biological Evaluation of Egyptian Nile Tilapia (Oreochromis Niloticas) Fish Scale Collagen", International Journal of Biological Macromolecules, vol. 79: Elsevier, pp. 618–626, 2015. Abstract

Collagen is considered to be one of the most useful biomaterials with different medical applications. However, collagen properties differ from one source to another. The aim of this study was to extract, purify, characterize and perform preliminary biological evaluation of type I collagen from scales of Egyptian Nile Tilapia. Pepsin-solubilized collagen (PSC) was successfully prepared from Nile Tilapia fish scale waste. Lyophilized collagen was dissolved in dilute HCl to form acidic collagen solutions (ACS) which was neutralized to form gel. To confirm the biocompatibility of the produced gel, baby hamster kidney (BHK-21) fibroblast cells were seeded onto a 3D collagen gel (0.3% and 0.5%, w/v). The results of an SDS-PAGE test showed that the extracted collagens were type I collagen, with α chain composition of (α1)2α2. Thermal analysis showed that the denaturation temperature was 32 °C. X-ray diffraction (XRD) analysis and Fourier-transform infrared spectra (FTIR) showed that the extracted collagen had a triple helix structure. Active proliferation of BHK-21 cells with no signs of toxicity was evident with both collagen gel concentrations tested. The results show that Nile Tilapia scales can be an effective source of collagen extraction that could be used as a potential biomaterial in biomedical applications.

Gad, A., U. Besenfelder, V. Havlicek, M. Hölker, F. Rings, I. Dufort, M. A. Sirard, K. Schellander, and D. Tesfaye, "In vitro Culture During Oocyte Maturation and Fertilization Influences The Treanscriptome Profiles of Bovine Blastocysts", Reproduction, Fertility and Development, vol. 27, no. 1: CSIRO PUBLISHING, pp. 186–187, 2015. Abstract

Early embryonic development, the period from oocyte maturation until blastocyst formation, is the most critical period of mammalian development. It is well known that in vitro maturation, fertilization, and culture of bovine embryos is highly affected by culture conditions. However, the stage-specific effect of culture environment is poorly understood. Therefore, we aimed to examine the effect of in vitro culture conditions during oocyte maturation and fertilization on the transcriptome profile of the resulting blastocysts. Bovine oocytes were matured in vitro and then either directly transferred to synchronized recipients, fertilized, and cultured in vivo (Vitro_M), or transferred after in vitro fertilization (Vitro_F), or at zygote stage (Vitro_Z) and blastocysts were collected at Day 7 by uterine flushing. For in vivo or in vitrofertilization, the same frozen-thawed commercial bull semen has been used. Complete in vitro (IVP) and in vivoproduced blastocysts were used as controls. Gene expression patterns between each blastocyst group and in vivoproduced blastocyst group were compared using EmbryoGENE's bovine microarray (EmbryoGENE, Québec, QC, Canada) over six replicates of each group (10 blastocyst/replicate). Microarray data were statistically analysed using the Linear Models for Microarray Data Analysis (LIMMA) package under the R program (The R Project for Statistical Computing, Vienna, Austria). Results showed that, the longer the embryos spent under in vitro conditions, the higher was the number of differentially expressed genes (DEG, fold-change = 2 with adjusted P-value = 0.05) compared within vivo control group. The Vitro_M group showed the lowest number of DEG (149); in contrast IVP group represented 841, DEG, respectively compared to in vivo control group. Ontological classification of DEG showed that lipid metabolism was the most significant function influenced by in vitro maturation conditions. More than 55% of DEG in the Vitro_M group were involved in the lipid metabolism process and most of them showed down-regulation compared to in vivo control group. On the other hand, Vitro_F and Vitro_Z groups showed nearly similar numbers of DEG (584 and 532, respectively) and the majority of these genes in both groups were involved in cell-death- and cell-cycle-related functions. Pathway analysis revealed that retinoic acid receptor activation pathways were the common ones in the Vitro_M and Vitro_F groups. However, different signalling pathways were commonly dominant in the Vitro_F and Vitro_Z groups. This study provides the transcriptome elasticity of bovine embryos exposed to different environments during maturation, fertilization, and culture periods of development.

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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El-Sayed, A., G. Ashour, M. Khalifa, and A. Gad, "Vitrification assessment of buffalo (Bubalus bubalis) oocytes: morphological and molecular aspects", Egyptian J. Anim. Prod, vol. 52, pp. 1-7, 2015. Abstract
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2014
Amin, A., A. Gad, D. Salilew-Wondim, S. Prastowo, E. Held, M. Hoelker, F. Rings, E. Tholen, C. Neuhoff, C. Looft, et al., "Bovine embryo survival under oxidative-stress conditions is associated with activity of the NRF2-mediated oxidative-stress-response pathway", Molecular Reproduction and Development, no. February, pp. n/a–n/a, 2014. AbstractWebsite

In present study, we sought to examine the ability of preimplantation bovine embryos to activate the NF-E2-related factor 2 (NRF2)-mediated oxidative-stress response under an oxidative stress environment. In vitro 2-, 4-, 8-, 16-cell-, and blastocyst-stage embryos were cultured under low (5%) or high (20%) oxygen levels. The expression of NRF2, KEAP1 (NRF2 inhibitor), antioxidants downstream of NRF2, and genes associated with embryo metabolism were analysed between the embryo groups using real-time quantitative PCR. NRF2 and Keap1 protein abundance, mitochondrial activity, and accumulation of reactive oxygen species (ROS) were also investigated in blastocysts of varying competence that were derived from high or low oxygen levels. The expression levels of NRF2 and its downstream antioxidant genes were higher in 8-cell, 16-cell, and blastocyst stages under high oxygen tension, whereas KEAP1 expression was down-regulated under the same conditions. Higher expression NRF2 and lower ROS levels were detected in early (competent) blastocysts compared to their late (non-competent) counterparts in both oxygen-tension groups. Similarly, higher levels of active nuclear NRF2 protein were detected in competent blastocysts compared to their noncompetent counterparts. Thus, the survival and developmental competence of embryos cultured under oxidative stress are associated with activity of the NRF2-mediated oxidative stress response pathway during bovine pre-implantation embryo development.

2013
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.

2012
Gad, A., M. Hoelker, U. Besenfelder, V. Havlicek, U. Cinar, F. Rings, E. Held, I. Dufort, M. - A. Sirard, K. Schellander, et al., "Molecular Mechanisms and Pathways Involved in Bovine Embryonic Genome Activation and Their Regulation by Alternative In Vivo and In Vitro Culture Conditions.", Biology of reproduction, vol. 87, issue 4, pp. 1-13, 2012. Abstract

Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro (IVP) and in vivo produced blastocysts were used as controls. We compared gene-expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENE's bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates, however in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo vs. in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.

a. Gad, K. Schellander, M. Hoelker, and D. Tesfaye, "Transcriptome profile of early mammalian embryos in response to culture environment", Animal Reproduction Science, vol. 134, issue 2010: Elsevier B.V., pp. 76-83, 2012. Abstract

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Tourism