Developmental competence of equine oocytes after ICSI : Implications on technical, morphological and cellular aspects

Using equine intracytoplasmic sperm injection (ICSI) procedure, the present study was performed, (i) to study the effect of assisted oocyte activation with calcium ionophore A23187 on developmental competence of oocytes, (ii) to re-evaluate the effect of cumulus morphology on meiotic and developmental competence of oocytes, (iii) to investigate the effect of glucose-6-phosphate dehydrogenase (G6PD) activity on meiotic and developmental competence of oocytes and (iv) to analyze zona pellucida (ZP) properties of equine oocytes of different quality and maturational status, quantitatively by polarization light microscopy. One hour after ICSI, injected oocytes were categorized into two groups: those treated with calcium ionophore A23187 and non-treated group. Cleavage and blastocyst rate were significantly (PDie Entwicklungskompetenz equiner Eizellen nach ICSI: technische, morphologische und zelluläre Aspekte inbegriffen Die vorliegende Studie untersucht den Effekt der assistierten Eizellaktivierung mit Calcium Ionophor A23187 equiner Embryonen nach der intrazytoplasmatischen Spermainjektion (ICSI) auf die Entwicklungskompetenz. Es wurde eine Re-evaluierung des Einflusses der Kumulusmorphologie, der Glucose-6-Phosphat-Dehydrogenase (G6PD) Aktivität und der Beschaffenheit der Zona pellucida mit Hilfe der Polarisationslichtmikroskopie im Hinblick auf die präimplantative Entwicklungskompetenz durchgeführt. Eine Einteilung in zwei Gruppen wurde eine Stunde nach ICSI vorgenommen, wobei nur eine Gruppe mit Calcium- Ionophor A23187 behandelt wurde. Die mit Calcium-Ionophor A23187 behandelten Eizellen zeigten signifikant höhere Teilungs- und Blastozystenraten (P < 0,05) als jene, die nicht behandelt wurden. Die Kumulusmorphologie der ungereiften Eizellen (expandierter Kumulus (Ex) vs. kompaktierter Kumulus (Cp)) zeigte ebenfalls signifikante Unterschiede (P<0,05) in der Maturationsrate und der Blastozystenrate nach ICSI. Zur Messung der G6PD Aktivität der Eizellen wurden diese mit Brilliant Cresyl Blue (BCB) gefärbt. Der prozentuale Anteil der Eizellen mit einer geringeren G6PD-Aktivität und blauem Cytoplasma war in der Gruppe der Ex Eizellen signifikant höher (P<0,01) als in der Gruppe der Cp Eizellen. Des Weiteren wurden signifikant höhere (P<0,05) Maturations- und Entwicklungsraten der BCB+ Eizellen beobachtet verglichen mit Eizellen mit hoher G6PD Aktivität. Mit Hilfe der Polarisationslichtmikroskopie wurde die Zona pellucida der Eizellen mit unterschiedlicher Entwicklungskompetenz (Ex vs. Cp und BCB+ vs. BCB-) und in verschiedenen Reifungsstadien (Immature Eizellen und mature Eizellen mit und ohne Polkörper) vermessen. Unsere Ergebnisse zeigen, dass die Ex Eizellen verglichen mit den Cp Eizellen und die BCB+ Eizellen verglichen mit den BCB- Eizellen eine signifikant (P<0,05) dickere Zona pellucida und eine höhere Doppellichtbrechungsintensität aufweisen. Zusätzlich hatten die Eizellen, die nach der in vitro Maturation keinen Polkörper ausschleusten, eine signifikant (P<0,05) dickere Zona pellucida und eine signifikant (P<0,05) höhere Doppellichtbrechungsintensität als immature Eizellen und Eizellen, die einen Polkörper ausschleusten. Zusammenfassend zeigte sich dass sich die Aktivierung der Eizellen nach ICSI mit Calcium-Ionophor A23187 äußerst positiv auf die Entwicklung der equinen Embryonen auswirkt. Kumulusmorphologie wie auch G6PD Aktivität sind verlässliche Indikatoren für die Entwicklungskompetenz. Ebenfalls könnten die Dicke und die Struktur der Zona pellucida, gemessen an Hand der Doppellichtbrechungsintensität, als neue Methode zur Bestimmung des Entwicklungspotentials equiner Eizellen dienen

Effect of PLGA-IB on ICAM-1 and VCAM-1 expression in a mice adhesion model

Background: In this study, we compared the effect of ibuprofen (IB) while incorporating by Poly Lactic-co-Glycolic Acid (PLGA) nanofiber on expression of adhesion molecules ICAM-1 and VCAM-1 in a mice adhesion model.Materials and Methods: Using an adhesion model were induced in mice, PLGA-IB and PLGA membranes and IB were sutured between the abdominal wall and peritoneum after surgical operation to reveal the best membrane for prevention of postoperative adhesion bands by comparison of ICAM-1 and VCAM-1 expression.Results: Compared with other groups, PLGA-IB showed a greater ability to reduce ICAM-1 and VCAM-1 expression.Conclusion: These results suggested that in considering the FDA approved polymers, PLGA-IB could be introduced as a potential candidate for prevention of abdominal post-surgery inflammation and adhesion band formation after surgeries

Lecithin nanoparticles enhance the cryosurvival of caprine sperm

P. 38-44This study was designed to compare the effects on goat spermatozoa cryosurvival of nano-lecithin-based (NL), lecithin-based (L) and egg yolk-based (EY) extenders. Ejaculates were collected from four fertile goats using artificial vagina and diluted with nine extenders. NL and L were tested at concentrations 1%, 2%, 3% and 4% (w/v), versus 15% (v/v) egg yolk-based extender. Overall, sperm quality (higher motility, viability and HOST, and lower apoptosis) was higher for NL than for L treatments (P < 0.05 for most cases, except for 1%). NL at 1% and especially at 4% showed lower motility and viability than 2% or 3% NL. NL at 2% achieved a better performance (P < 0.05) than EY for VCL (131.5 ± 1.3 vs. 120.3 ± 1.9 μm/s), VSL (43.9 ± 1.5 vs. 35.8 ± 1.4 μm/s), LIN (35.7 ± 0.6 vs. 29.3 ± 0.8%), WOB (47.0 ± 0.5 vs. 43.9 ± 0.9%) and viability (66.4 ± 1.7 vs. 52.7 ± 1.9%). Late apoptotic spermatozoa were also lower in 2% NL compared to EY (16.0 ± 0.5 vs. 26.3 ± 1.1%, P < 0.001). EY and 2% NL were compared in an IVF trial, with no significant differences in cleavage (68.8 vs. 70.8%) or blastocyst ratios (21.3 vs. 20.8%). In conclusion, using 2% nanolecithin in semen dilution could improve sperm cryosurvival of goat.S

Efficacy of dietary supplementation of nanoparticles-chromium, chromium-methionine and zinc-proteinate, on performance of Japanese quail under physiological stress

The effects of dietary Nanoparticles-Chromium, Chromium methionine, and Zinc-proteinate on performance, blood characteristics and meat quality of physiologically stressed quails were studied. The stress was induced by adding dexamethasone (0.6 mg/kg BW) to diet when quails were between 17 and 22 days of age. Three hundred one-day chicks were randomly assigned to five groups comprising four replicates of 15 birds: negative control diet (no-stress, no-additive), positive control diet (stress, no-additive), and stress additive diets which included three diets containing 800 µg/kg of Nano-Cr, 800 µg/kg Cr-met, and 60 mg/kg Zn-pro. Performance and hematological parameters were affected by physiological stress (p < .05), but were not affected by dietary Nano-Cr, Cr-met or Zn-pro. During the stress period, feed consumption was improved by Zn-pro supplementation. By the end of the experiment, the ultimate pH value of the muscle in stressed birds, which were fed on the Cr-met diet, was higher than those treated with NC, but pH values were not affected by the treatments. Diets containing Nano-Cr and Zn-pro caused a reduction in malondialdehyde concentration in the thigh muscle. In summary, 800 µg/kg of Nano-Cr and 60 mg/kg of Zn-pro can reduce the negative effects of stress on meat quality.Highlights Dietary dexamethasone induced physiological stress in quails. The supplementation of 60 mg/kg of Zinc proteinate had positive effect on feed intake of birds under physiological stress. Dietary Zinc proteinate and Nanoparticles-Chromium increased meat oxidative stability in birds which experienced physiological stress

A two-step feeding of calcium salts of fish oil improves reproductive performance in Holstein cows

Aim of study: To examine the effects of a biphasic schedule of feeding n-3 fatty acids on dairy cows.Area of the study: Isfahan, Iran.Materials and methods: 140 lactating Holstein cows were allotted at calving into two groups of 70 animals and received one of two dietary treatments: 1) saturated fatty acids (SFA, containing 80% palmitic acid) or 2) calcium salt of fish oil (CSFO, containing 16% eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA)), with an n-6/n-3 FA ratio of approximately 7 for SFA and 5 for CSFO treatments. The dietary supplements were fed to the respective groups at 240 g/head.day from 0 to 21 days in milk, and 120 g/head.day from 22 to 150 days in milk. Milk yield was recorded biweekly and milk composition was evaluated monthly. The concentration of FA in the milk and blood was determined on d-90 of the experiment in 10 cows randomly selected from each group. Reproductive indices were recorded until d-150.Main results: The CSFO supplementation did not affect average milk yield, milk composition or milk somatic cell count (SCC); however, in some weeks it decreased milk SCC (p<0.05). Plasma concentrations of palmitic acid and n-3 FA as well as milk fat concentration of EPA and DHA increased in the CSFO-fed cows (p<0.05). Feeding the CSFO decreased open days (100 vs 119 days, p<0.05), service per conception and all service conception rates (p<0.05).Research highlights: The implementation of a two-stage feeding program of n-3 FA improved reproductive variables and reduced milk SCC in dairy cows

Effect of Poly lactic-co-glycolic acid-Ibuprofen on ICAM-1 and VCAM-1 Expression in a Mice Adhesion Model

Background: In this study, we compared the effect of ibuprofen (IB) while incorporating by Poly Lactic-co-Glycolic Acid (PLGA) nanofiber on expression of adhesion molecules ICAM-1 and VCAM-1 in a mice adhesion model. Materials and Methods: Using an adhesion model were induced in mice, PLGA-IB and PLGA membranes and IB were sutured between the abdominal wall and peritoneum after surgical operation to reveal the best membrane for prevention of postoperative adhesion bands by comparison of ICAM-1 and VCAM-1 expression. Results: Compared with other groups, PLGA-IB showed a greater ability to reduce ICAM-1 and VCAM-1 expression. Conclusion: These results suggested that in considering the FDA approved polymers, PLGA-IB could be introduced as a potential candidate for prevention of abdominal post-surgery inflammation and adhesion band formation after surgerie

<i>In vitro</i> fibroblast migration by sustained release of PDGF-BB loaded in chitosan nanoparticles incorporated in electrospun nanofibers for wound dressing applications

<p>Migration of fibroblasts into wound area is a critical phenomenon in wound healing process. We used an appropriate system to fabricate an electrospun bioactive scaffold with controlled release of PDGF-BB in order to induce migration of primary skin fibroblast cells. First of all, protein-loaded chitosan nanoparticles based on ionic gelation interaction between chitosan and sodium tripolyphosphate were prepared. Then polycaprolactone electrospun fibers containing chitosan nanoparticles or PDGF-BB-loaded chitosan nanoparticles were prepared. Cellular attachment and morphology of cells seeded on scaffolds with or without PDGF-BB were evaluated by using a fluorescence microscope and scanning electron microscopy. Cells were well-oriented 72 h after seeding on the scaffolds containing PDGF-BB. The mean aspect ratio of populations on scaffold containing PDGF-BB-loaded chitosan nanoparticles was significantly greater than those on the scaffold containing chitosan nanoparticles but no PDGF-BB. Furthermore, the Arp2 gene, which is involved in cell protrusion formation, showed about three times more expression at mRNA level, in cells seeding on PDGF-BB-containing scaffold compared to cells seeding on scaffold containing only chitosan nanoparticles, using Real Time PCR test. Finally, under agarose migration assay results demonstrated that cells’ chemotaxic behavior was more toward scaffold containing PDGF-BB compared to the PDGF-BB alone or FBS group. In addition, in terms of distance, the cell mass could grow faster, in response to scaffold containing PDGF-BB compared to FBS or PDGF-BB alone; however, the number of migrating cells might be the same or significantly higher in the latter groups.</p

Isolation, characterization, and mesodermic differentiation of stem cells from adipose tissue of camel (Camelus dromedarius)

Adipose-derived stem cells are an attractive alternative as a source of stem cells that can easily be extracted from adipose tissue. Isolation, characterization, and multi-lineage differentiation of adipose-derived stem cells have been described for human and a number of other species. Here we aimed to isolate and characterize camel adipose-derived stromal cell frequency and growth characteristics and assess their adipogenic, osteogenic, and chondrogenic differentiation potential. Samples were obtained from five adult dromedary camels. Fat from abdominal deposits were obtained from each camel and adipose-derived stem cells were isolated by enzymatic digestion as previously reported elsewhere for adipose tissue. Cultures were kept until confluency and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteogenic, and chondrogenic potential. The morphology of resultant camel adipose-derived stem cells appeared to be spindle-shaped fibroblastic morphology, and these cells retained their biological properties during in vitro expansion with no sign of abnormality in karyotype. Under inductive conditions, primary adipose-derived stem cells maintained their lineage differentiation potential into adipogenic, osteogenic, and chondrogenic lineages during subsequent passages. Our observation showed that like human lipoaspirate, camel adipose tissue also contain multi-potent cells and may represent an important stem cell source both for veterinary cell therapy and preclinical studies as well

Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte

To associate glucose-6-phosphate dehydrogenase (G6PDH) activity in goat oocytes with intracellular glutathione (GSH) content, meiotic competence, developmental potential, and relative abundance of Bax and Bcl-2 genes transcripts.Purpose To associate the glucose-6-phosphate dehydrogenase (G6PDH) activity inimmature goat oocyte with intracellular glutathione (GSH) content, developmentalcompetence after parthenogenetic activation and relative abundance of Bax and Bcl-2genes transcripts using .Methods Goat oocytes were exposed to brilliant cresyl blue (BCB) staining andcategorized into BCB+ (blue cytoplasm) and BCB&minus; (colorless cytoplasm) groups. Agroup of oocytes were not exposed to BCB stain and was considered as a control.After maturation in vitro, a group of oocytes were used for determination of nuclearstatus and intracellular GSH content and another group were subjected toparthenogenetic activation followed by in vitro embryo culture.Results We found that BCB+ oocytes yielded a significantly higher maturation rate thanthe BCB&minus; and control oocytes. Moreover, BCB+ oocytes showed increased inintracellular level of GSH than BCB&minus; and control oocytes. Furthermore, BCB+ oocytesproduced more blastocysts than BCB&minus; and control oocytes. The expression of antiapoptoticgene Bcl-2 and pro-apoptotic gene Bax in mature oocyte, their surroundingcumulus cells and blastocyst was affected by BCB staining test.Conclusions These results strongly suggest that BCB+ oocytes have a higher meioticand cytoplasmic maturity (as determined by intracellular GSH content), and that BCBstaining can be used to select developmentally competent oocytes for in vitro embryoproduction.</p

Effect of Linolenic acid during in vitro maturation of ovine oocytes: embryonic developmental potential and mRNA abundances of genes involved in apoptosis

To study the effect of alpha-linolenic acid (ALA) on meiotic maturation, mRNA abundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation in ovine oocytes.Purpose To study the effect of &alpha;-linolenic acid (ALA) on meiotic maturation, mRNAabundance of apoptosis-related (Bax and Bcl-2) molecules, and blastocyst formation inovine oocytes.Methods A preliminary experiment was conducted to analyze the concentration of ALAin &quot;small&quot; (&le;2 mm) and &quot;large&quot; (&ge;6 mm) follicles using gas chromatography/massspectrometry analysis. The concentration of ALA in small and large follicles wasdetermined to be in a range of 75.4 to 125.7 &mu;M, respectively. In vitro maturation (IVM)of oocyte was then performed in presence of 0 (control), 10 (ALA-10), 50 (ALA-50),100 (ALA-100), and 200 (ALA-200) &mu;M of ALA. Meiotic maturation and mRNAabundance of Bax, and Bcl-2 genes was evaluated after 24 h of IVM. The embryoniccleavage and blastocyst formation following parthenogenetic activation were alsodetermined for each group.Results The highest concentration of ALA (ALA-200) decreased the oocyte maturationrate compared with the control group. Analysis of apoptosis-related genes in oocytesafter IVM revealed lesser transcript abundances for Bax gene, and higher transcriptabundances for Bcl-2 gene in ALA-treated oocytes as compared with the controloocytes. In term of cleavage rate (considered as 2-cell progression), we did notobserve any differences among the groups. However, ALA-100 group promoted moreblastocyst formation as compared with the control group.Conclusion Our results suggested that ALA treatment during IVM had a beneficial effect on developmental competence of ovine oocytes by increasing the blastocystformation and this might be due to the altered abundance of apoptosis-regulatorygenes.</p