Ahmed Yehia Gad

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.

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.

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.

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.

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.

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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.

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.

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.

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.

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.

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.

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The retarded development of parthenote embryo could be due to aberrant epigenetic imprinting, which may disrupt many aspects and lead to conceptus demise. The present work was conducted to: 1) compare the development of in vitro produced (IVP) and parthenogenetically developed (P) buffalo embryos from the 2-cell to blastocyst stage; 2) investigate the global gene expression profile and search for new candidate transcripts differing between IVP and P buffalo blastocyst using cDNA microarray analysis (validated by Real Time PCR); 3) follow the particular expression patterns of PLAC8 and OCT4 genes at five different stages of preimplantation development by Real Time PCR; and 4) study the expression of CDX2 at the blastcocyst stage. Cleavage rate was higher (P < 0.05) in P than IVP buffalo embryos, while, progression to blastocyst and number of cells per blastocyst were lower (P < 0.05) in P than IVP blastocysts. Microarray analysis indicate that 56 differentially expressed genes between the two groups, of which 51 genes (91.07%) were up-regulated, and five genes were downregulated in IVP blastocyst, using 1.4 fold-changes as a cutoff. Differentially expressed genes are related to translation, nucleic acid synthesis, cell adhesion and placentation. Validation of candidate genes revealed that the transcript abundance of PTGS2, RPS27A, TM2D3, PPA1, AlOX15, RPLO and PLAC8 were downregulated (7/8) in parthenote blastocyst compared to the IVP blastocyst. PLAC8 gene expression was higher (P < 0.05) at 2-cell, morula and blastocyst stages in IVP embryos compared with parthenote embryos. The OCT4 gene expression was higher (P < 0.05) in 2-cell, 4-cell, 8-cell and blastocysts produced in vitro. In conclusion, the retarded development of parthenogenetic buffalo embryos could be due to downregulation of genes related to translation, nucleic acid synthesis, cell adhesion, and placental development. The low expression of PLAC8 and OCT4 during the different stages of development may be responsible, in part, to the failure of development of parthenote buffalo embryos.

Ovarian stimulation is a routine procedure in assisted reproduction to stimulate the growth of multiple follicles in naturally single-ovulating species including cattle and humans. The aim of this study was to analyze the changes induced in the endometrial transcriptome associated with superovulation in cattle and place these observations in the context of our previous data on changes in the endometrial transcriptome associated with elevated progesterone (P4) concentrations within the physiological range and those changes induced in the embryo due to superovulation. Mean serum P4 concentrations were significantly higher from day 4 to day 7 in superovulated compared with unstimulated control heifers (P < 0.05). Between-group analysis revealed a clear separation in the overall transcriptional profile of endometria from unstimulated control heifers (n = 5) compared with superovulated heifers (n = 5). This was reflected in the number of differentially expressed genes (DEGs) identified between the two groups with 795 up- and 440 downregulated in superovulated endometria. Ten times more genes were altered by superovulation (n = 1,234) compared with the number altered due to elevated P4 within physiological ranges by insertion of a P4-releasing intravaginal device (n = 124) with only 22 DEGs common to both models of P4 manipulation. Fewer genes were affected by superovulation in the embryo compared with the endometrium, (443 vs. 1,234 DEGs, respectively), and the manner in which genes were altered was different with 64.5% of genes up- and 35.5% of genes downregulated in the endometrium, compared with the 98.9% of DEGs upregulated in the embryo. In conclusion, superovulation induces significant changes in the transcriptome of the endometrium which are distinct from those in the embryo.

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In mammals, the reproductive tract plays a crucial role in the success of early reproductive events and provides an optimal microenvironment for early embryonic development. However, changes in the reproductive tract environment associated with controlled ovarian hyperstimulation and the influence on the embryo transcriptome profile have not been investigated. Therefore, we investigated differences in the development rate and the transcriptome profile of bovine blastocysts developing in the reproductive tract of unstimulated or superovulated heifers.

SummaryMicroRNAs (miRNAs) are small endogenous molecules that are involved in a diverse of cellular process. However, little is known about their abundance in bovine oocytes and their surrounding cumulus cells during oocyte development. To elucidate this situation, we investigated the relative expression pattern of sets of miRNAs between bovine oocyte and the surrounding cumulus cells during in vitro maturation using miRNA polymerase chain reaction (PCR) array. Results revealed that a total of 47 and 51 miRNAs were highly abundant in immature and matured oocytes, respectively, compared with their surrounding cumulus cells. Furthermore, expression analysis of six miRNAs enriched in oocyte miR-205, miR-150, miR-122, miR-96, miR-146a and miR-146b-5p at different maturation times showed a dramatic decrease in abundance from 0 h to 22 h of maturation. The expression of the same miRNAs in preimplantation stage embryos was found to be highly abundant in early stages of embryo development and decreased after the 8-cell stage to the blastocyst stage following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in preimplantation stage embryos, in which signals were higher up to the 4-cell stage and reduced thereafter. miR-205 and miR-210 were localized in situ in ovarian follicles and revealed a spatio-temporal expression during follicular development. Interestingly, the presence or absence of oocytes or cumulus cells during maturation was found to affect the expression of miRNAs in each of the two cell types. Hence, our results showed the presence of distinct sets of miRNAs in oocytes or cumulus cells and the presence of their dynamic degradation during bovine oocyte maturation.

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