Islam M. Saadeldin

Effect of different culture media on the temporal gene expression in the bovine developing embryos

We have previously shown that the in vitro embryonic development and the yield of viable calves were increased by using a two-step chemically defined medium for post-fertilization culture of bovine embryos. In this study, we explored the embryonic development and the temporal behavioral interaction of the genes involved in IFNτ gene expression and how they behave in an orchestrated manner to increase the developmental competence of IVF produced embryos by culturing in the chemically defined medium. Behavior of genes included ETS2, CDX2, GATA2, GATA3, OCT4 and NANOG was analyzed in early bovine IVF produced embryos, (from compact morulae to the blastocyst hatching stages), by semi- and relative quantitative PCR and compared between two in vitro culture (IVC) systems, two-step chemically defined medium and modified synthetic oviductal fluid (mSOF) containing 8 mg/mL, BSA. Early embryonic development was found to be better in two-step chemically defined culture system than that of mSOF as indicated by the increment of blastocyst yield, 33.1% in two-step culture system vs 18.8% in mSOF medium, and the blastocyst hatching, 52.3% in two-step culture system vs 33.5% in mSOF medium. Relative quantitative gene expression showed harmonic behavior in the two-step culture system rather than the culture in mSOF, IFNτ showed even increase throughout the embryonic development in the two-step culture medium while it decreased with blastocyst hatching in mSOF culture condition. Temporal dominance of OCT4 over all the transcription factors was found in regulation of IFNτ expression (the major factor of expression regulation but in inverse manner). However, ETS2, CDX2, GATA2 and GATA3 are potent IFNτ stimulator in cumulative manner but in case of OCT4 decrement. CDX2 directly related with IFNτ, but still under OCT4 dominance and also regulated by the subservient of OCT4 which is NANOG. In conclusion, this study confirmed our previous results about the usefulness of using the two-step chemically defined culture medium for increasing the developmental competence of IVF produced embryos and elucidated the dominance of OCT4 over the other genes implicated in regulation of IFNτ expression.

Paradoxical effects of kisspeptin: it enhances oocyte in vitro maturation but has an adverse impact on hatched blastocysts during in vitro culture

Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus-oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10(-6)M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4×10(-6)M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine-paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.

Effect of 7,8-dihydroxyflavone as an antioxidant on in vitro maturation of oocytes and development of parthenogenetic embryos in pigs.

One of the factors that impairs in vitro produced porcine embryos is the oxidative stress that is mainly caused by the imbalance between reactive oxygen species (ROS) generation and antioxidants activity, especially that of glutathione (GSH). Here, we examined the effect of 7,8-dihydroxyflavone (7,8-DHF), a kind of flavonoid antioxidant, on porcine oocyte maturation and its developmental competence. Porcine oocytes were cultured in media supplemented with 0, 1, 5 and 10 μM 7,8-DHF during both in vitro maturation (IVM) and in vitro culture (IVC) after parthenogenetic activation. Maturation of oocytes was evaluated based on first polar body (PB) extrusion and intracellular GSH level, and developmental competence was assessed through observing cleavage and blastocyst formation. In each step, the levels of intracellular GSH and ROS were assessed by fluorescence intensity, and the apoptosis-related gene expression was examined using semiquantitative RT-PCR. The group treated with 1 μM 7,8-DHF during IVM and IVC showed increased cytoplasmic maturation and reached the blastocysts stage (36.1%) at a higher rate than the other groups (24.7, 16.0 and 10.3% for 0, 5 and 10 μM, P<0.05). In that group, the intracellular GSH level was significantly increased while ROS generation was significantly decreased after IVM and IVC (P<0.05). Moreover, it showed high expression of an anti-apoptotic gene (BCL2L1) and low expression of a pro-apoptotic gene (BAK1) (P<0.05). In conclusion, treatment with 1 μM 7,8-DHF during IVM and IVC showed an anti-apoptotic effect by increasing intracellular GSH synthesis and scavenging ROS and therefore improved the developmental competence of porcine embryos.

Oct4 overexpression facilitates proliferation of porcine fibroblasts and development of cloned embryos.

Octamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency of embryonic stem cells. Recent reports have shown that Oct4 also controls cell-cycle progression and enhances the proliferation of various types of cells. As the high proliferation of donor fibroblasts is critical to the production of transgenic pigs, using the somatic cell nuclear transfer technique, we analysed the effect of Oct4 overexpression on the proliferation of porcine fibroblasts and embryos. Porcine endogenous Oct4 cDNA was cloned, sequenced and inserted into an expression vector. The vector was transfected into porcine fibroblasts, and a stable Oct4-overexpressed cell line was established by antibiotic selection. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells. Real-time RT-PCR results showed that p16, Bcl2 and Myc were upregulated in Oct4-overexpressed cells. Somatic cell nuclear transfer was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared with that of wild-type cloned embryos. The cleavage and blastocyst formation rates were improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as day 5 in culture, while blastocysts were observed from day 6 in wild-type cloned embryos. In conclusion, the overexpression of Oct4 enhanced the proliferation of both porcine fibroblasts and embryos.

Production of porcine cloned embryos derived from cells conditionally expressing an exogenous gene using Cre- loxP

It is increasingly evident that conditional gene expression in pigs is necessary to make transgenic models. In this study, we investigated conditional expression in porcine fetal fibroblasts using Cre-loxP recombination, a system that has had limited application in large animals to date. Transformed fibroblasts were reprogrammed in enucleated oocytes to support further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with a plasmid that contained a red fluorescent protein marker (pCALNL-DsRed) and a floxed neomycin-resistance gene. Cells were selected with 750 μg/ml neomycin for 2 weeks following transfection but did not express DsRed after visualization under a fluorescence microscope. Expression was achieved only after transient transfection with plasmid DNA that expressed the Cre recombinase enzyme. The cells that expressed DsRed were used for somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%) were seen to have cleaved. Six blastocysts developed after SCNT and expressed DsRed. Deletion of the floxed neomycin-resistance gene was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in cloned blastocysts. This study demonstrated that Cre-loxP recombination can be conducted successfully in miniature pig fibroblasts and that the sequentially transformed cells can develop to the pre-implantation embryo stage via SCNT.

Altered cell cycle gene expression and apoptosis in post-implantation dog parthenotes.

Mature oocytes can be parthenogenetically activated by a variety of methods and the resulting embryos are valuable for studies of the respective roles of paternal and maternal genomes in early mammalian development. In the present study, we report the first successful development of parthenogenetic canine embryos to the post-implantation stage. Nine out of ten embryo transfer recipients became pregnant and successful in utero development of canine parthenotes was confirmed. For further evaluation of these parthenotes, their fetal development was compared with artificially inseminated controls and differentially expressed genes (DEGs) were compared using ACP RT-PCR, histological analysis and immunohistochemistry. We found formation of the limb-bud and no obvious differences in histological appearance of the canine parthenote recovered before degeneration occurred; however canine parthenotes were developmentally delayed with different cell cycle regulating-, mitochondria-related and apoptosis-related gene expression patterns compared with controls. In conclusion, our protocols were suitable for activating canine oocytes artificially and supported early fetal development. We demonstrated that the developmental abnormalities in canine parthenotes may result from defective regulation of apoptosis and aberrant gene expression patterns, and provided evidence that canine parthenotes can be a useful tool for screening and for comparative studies of imprinted genes.

Blastomeres aggregation as an efficient alternative for trophoblast culture from porcine parthenogenetic embryos

Zona pellucida free (ZPF) oocytes were cultured after electrical activation to allow blastomeres aggregation and compared to ZP intact (ZPI) oocytes. In feeder‐dependent conditions, the trophoblast attachment and primary outgrowths were significantly higher in ZPF than in ZPI groups. In feeder‐free conditions, trophoblast attachment and typical morphological trophoblast primary outgrowths were observed in ZPF group. The primary colonies derived from the ZPF embryos in both culture conditions were able to establish secondary and tertiary colonies and showed mRNA expression of CDX2, TEAD4 and KRT8 as trophoblast markers, while outgrowths from the ZPI embryos could not grow beyond primary colonies.

Wet feed and cold water as heat stress modulators in growing Muscovy ducklings

&NA; In an attempt to alleviate the deleterious effects of high summer temperatures, the present study investigated the effects of wet feed and cold water on the growth performance, carcass and meat quality, leg problems, physiological responses, and blood parameters of growing Muscovy ducklings. A total of 180 4‐week‐old ducklings was randomly divided into 6 experimental groups in a 3 × 2 factorial design that included 3 feed systems (AD: ad libitum dry; DW: diurnal wet; and AW: ad libitum wet) and 2 systems of water (TW: tap water; and CW: cold water). Access to wet feed and cold water affected the growth performance, dressed carcass, gizzard, meat quality (tenderness, juiciness, and susceptibility), tonic immobility, body temperature, and blood parameters [albumin: globulin (A: G) ratio and levels of glucose, alanine transferase (ALT), total antioxidant capacity (T‐AOC), and malondialdehyde (MDA)] of the ducklings but had no significant effect on plumage condition, shank length, keel bone length, leg problems, or breast blisters. The body weight (BW) of the DW group was 1.97 and 3.12% greater than that of the AD and AW groups, respectively, and the BWG of the DW group was 6.91 and 10.72% greater than that of the AD and AW groups, respectively. Therefore, providing access to wet feed and cold water is highly recommended when raising Muscovy ducks in open houses under high‐temperature conditions.

110 EFFECT OF MELATONIN TREATMENT ON LIBIDO AND ENDOCRINE FUNCTION OF DROMEDARY CAMEL BULLS OUT OF THE BREEDING SEASON

The reproductive performance of camels is poor and has remained a major obstacle to the growth of dromedary populations. The limited breeding season is one of the most important causes of the poor reproductive performance. In seasonal animals, melatonin is the chemical messenger that allows the perception of daylight length changes. Commercial melatonin products have been developed for the manipulation of seasonal breeding in animals. The present study aimed to evaluate the influence of melatonin implantation on libido, serum melatonin, and testosterone concentrations in dromedary camels during the non-breeding season (June and July). Ten camel bulls were used in the 35-day-long trial; 5 of them were implanted with 30 Melovine® implant (Ceva, Libourne, France) subcutaneously on Day 0, whereas the other 5 camel bulls remained untreated as a control. Libido was evaluated weekly in response to oestrous-induced female camels treated with oestrogen (1mL Oestrocon; oestradiol benzoate 5mgmL-1) 2 days before assessment of libido. Libido was scored as follows: 0=not interested: the male did not show any libido; 1=low interested: the male went near the female and showed low frequency of sniffing and flehmen; 2=interested: the male went near the female, it showed sniffing, flehmen, grinding of teeth/whistling, yawning; 3=high interested: the male went near the female and was very agitated, it showed sniffing, flehmen, grinding of teeth/whistling, yawning, urination, and tail raising. It stood with open legs, and poll gland secretion and neck rubbing were observed; 4=excited, like 3, but the male showed blatering and dulaa extrusion, was very excited, stood with open legs, high poll gland secretion and neck rubbing were observed. Blood samples were collected weekly. Serum melatonin and testosterone concentrations were evaluated using commercial ELISA kits. Comparisons among groups were evaluated using repeated-measures ANOVA, using SAS software (SAS Institute Inc., Cary, NC, USA). A difference was considered significant at the P<0.05 level. The results revealed that at Day 0, all camel bulls in 2 groups had no libido and there was no significant difference in the melatonin or testosterone levels in the 2 groups. The libido increased gradually in the melatonin group and reached the maximum (3-5) at week 4 and week 5. The control group had low libido (0-1) along the trial. Statistically, the libido was significantly higher in the melatonin group than control group. Additionally, testosterone levels were significantly higher in melatonin group than control group, especially in the fourth week of the present trial (565.07±33.04pgmL-1 and 458.49±25.36pgmL-1, respectively). In conclusion, melatonin implantation in the non-breeding season significantly improved the libido and the reproductive performance of dromedary camel bulls. Therefore, it may be possible to improve the reproductive efficiency of camels by extending the breeding season through treatment with melatonin during the non-breeding season.

Post-maturation zona perforation improves porcine parthenogenetic trophoblast culture

This study was designed to optimize a method to improve porcine parthenogenetic embryo hatching and trophoblast culture. Mature oocytes (D0PPA) and day 6 blastocysts (D6PPA) were perforated with a 20 μm diameter needle for assisted hatching. The two groups showed a significant difference in hatching rate and blastocyst cell doubling when compared to a non-perforated control group. D0PPA blastocysts were able to form tertiary trophoblast colonies but D6PPA and control groups were not able to grow beyond primary colonies. Quantitative real-time PCR analysis showed significant differences in BAX, BAX/BCL2L1 and HSP70-2 mRNA expression between the experimental groups.