Search results for: zebrafish embryos

Authors: Manjunatha Bangeppagari, Lee Sang Joon

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Recently, graphene-related nanomaterials are receiving fast-increasing attention with augmented applications in various fields. Especially, graphene-related materials have been widely applied to the biomedical field in the past years. In the present study, we evaluated the adverse effects of pristine graphene (pG) in zebrafish (Danio rerio) embryos in various aspects, such as mortality rate, heart rate, hatching rate, cardiotoxicity, cardiovascular defect, cardiac looping, apoptosis, and globin expression. For various trace concentrations of pG (1, 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μg/L), early life-stage parameters were observed at 24, 48, 72, and 96 hpf. As a result, pG induces significant developmental defects including yolk sac edema, pericardial edema, embryonic mortality, delayed hatching, heartbeat, several morphological defects, pericardial toxicity, and bradycardia. Moreover, the exposure to pG was found to be a potential risk factor to the cardiovascular system of zebrafish embryos. However, further study on their properties which vary according to production methods and surface functionalization is essentially required. In addition, the possible risks of pG flakes to aquatic animals, and public health should be evaluated before releasing them to the surrounding environment.

Keywords: apoptosis, cardiovascular toxicity, globin expression, pristine graphene, zebrafish embryos

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Authors: Titis Indah Adi Rahayu

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Angiogenesis is the process of generating new capillary from pre-existing blood vessels. VEGFA is a major regulator in angiogenesis that binds and activates two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1/KDR) which regulate pathological and physiological angiogenesis. Disruption of VEGFA and VEGFR2 regulation lead to many diseases. The study of α-Mangostin (derivate of xanthone) as anti-oxidant and anti inflammation has been explored recently and both of them have relation to vasculature however the effect of α-Mangostin in blood vessel formation in healthy tissue in vivo has not been studied. Zebrafish is a powerful model in studying angiogenesis and shared many advantages that is a viable whole animal model for screening small molecules that affect blood vessel formation. Therefore the aim of this study is to evaluate angiogenic activity of α-Mangostin in healthy tissue in vivo in zebrafish embryo in relation of patterning blood vessel. Blood vessel patterning is highly characteristic in the developing of zebrafish embryo and the subintestinal vessel (SIV) can be stained and visualized microscopically as a primary screen for α-Mangostin that effect angiogenesis. The zebrafish embryos are divided into 2 groups. Group one consists of the zebrafish embryos at 1 dpf for 4 days which are tested to α-Mangostin in several concentration 2 µM, 4 µM, 6 µM, 8 µM and 10 µM whereas in group two the zebrafish larva at 4 dpf are exposed to α-Mangostin 1,75 µM, 2,3 µM, 2,9 µM, 3,8 µM dan 5 µM for 2 days. DMSO is served as a control for each group. The level expression of vegfa and vegfr2 are observed quantitatively using real time q-PCR and patterning of SIV are then analized via alkaline phospatase staining. Result shows that the level expression of vegfa and vegfr2 is repressed quantitatively as shown in real time q-PCR in the group of 1-4 days of α-Mangostin exposure where it is increased in the group of 4-6 days of α-Mangostin exposure. The result is then compared to alkaline phospatase staining of SIV using stereo microscope. It indicates that α-Mangostin does not disturb the patterning of SIV formation in zebrafish.

Keywords: angiogenesis, Danio rerio, α-Mangostin, SIV, vegfa, vegfr2

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Authors: Shubham Dwivedi, Raghavender Medishetti, Rita Rani, Aarti Sevilimedu, Pushkar Kulkarni, Yogeeswari Perumal

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Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder of early onset, characterized by impaired sociability, cognitive function and stereotypies. There is a significant urge to develop and establish new animal models with ASD-like characteristics for better understanding of underlying mechanisms. The aim of the present study was to develop a cost and time effective zebrafish model with quantifiable parameters to facilitate mechanistic studies as well as high-throughput screening of new molecules for autism. Zebrafish embryos were treated with valproic acid and a battery of behavioral tests (anxiety, inattentive behavior, irritability and social impairment) was performed on larvae at 7th day post fertilization, followed by study of molecular markers of autism. This model shows a significant behavioural impairment in valproic acid treated larvae in comparison to control which was again supported by alteration in few marker genes and proteins of autism. The model also shows a rescue of behavioural despair with positive control drugs. The model shows robust parameters to study behavior, molecular mechanism and drug screening approach in a single frame. Thus we postulate that our 7 days zebrafish larval model for autism can help in high throughput screening of new molecules on autism.

Keywords: autism, zebrafish, valproic acid, neurodevelopment, behavioral assay

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Authors: Jisun Jun, Eun Ko, Sooim Shin, Kitae Kim, Moonsung Choi

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Diabetes is a metabolic disease characterized by hyperglycemia, insulin resistant, mitochondrial dysfunction. Diabetes is associated with the development of diabetic retinopathy resulting in worsening vision and eventual blindness. In this study, eyes were enucleated from glucose-immersed zebrafish which is a good animal model to generate diabetes, and then mitochondria were isolated to evaluate activities of mitochondrial electron transfer complexes. Surprisingly, the amount of isolated mitochondria was increased in eyes from glucose-immersed zebrafish compared to those from non-glucose-immerged zebrafish. Spectrophotometric analysis for measuring activities of mitochondrial complex I, II, III, and IV revealed that mitochondria functions was even enhanced in eyes from glucose-immersed zebrafish. These results indicated that 3 days or 7 days glucose-immersion on zebrafish to induce diabetes might contribute metabolic compensatory mechanism to restore their mitochondrial homeostasis on the early stage of diabetes in eyes.

Keywords: diabetes, glucose immersion, mitochondrial complexes, zebrafish

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Authors: Kinnsley Travis, Camerron M. Crowder

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Mapk8ip3 (Mitogen-Activated Protein Kinase 8 Interacting Protein 3) is a gene that codes for the JIP3 protein, which is a part of the JIP scaffolding protein family. This protein is involved in axonal vesicle transport, elongation and regeneration. Variants in the Mapk8ip3 gene are associated with a rare-genetic condition that results in a neurodevelopmental disorder that can cause a range of phenotypes including global developmental delay and intellectual disability. Currently, there are 18 known individuals diagnosed to have sequenced confirmed Mapk8ip3 genetic disorders. This project focuses on examining the impact of a subset of missense patient variants on the Jip3 protein function by overexpressing the mRNA of these variants in a zebrafish knockout model for Jip3. Plasmids containing cDNA with individual missense variants were reverse transcribed, purified, and injected into single-cell zebrafish embryos (Wild Type, Jip3 -/+, and Jip3 -/-). At 6-days post mRNA microinjection, morphological, behavioral, and microscopic phenotypes were examined in zebrafish larvae. Morphologically, we compared the size and shape of the zebrafish during their development over a 5-day period. Total locomotive activity was assessed using the Microtracker assay and patterns of movement over time were examined using the DanioVision assay. Lastly, we used confocal microscopy to examine sensory axons for swelling and shortened length, which are phenotypes observed in the loss-of-function knockout Jip3 zebrafish model. Using these assays during embryonic development, we determined the impact of various missense variants on Jip3 protein function, compared to knockout and wild-type zebrafish embryo models. Variants in the gene Mapk8ip3 cause rare-neurodevelopmental disorders due to an essential role in axonal vesicle transport, elongation and regeneration. A subset of missense variants was examined by overexpressing the mRNA of these variants in a Jip3 knock-out zebrafish. Morphological, behavioral, and microscopic phenotypes were examined in zebrafish larvae. Using these assays, the spectrum of disorders can be phenotypically determined and the impact of variant location can be compared to knockout and wild-type zebrafish embryo models.

Keywords: rare disease, neurodevelopmental disorders, mrna overexpression, zebrafish research

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Authors: Venoos Iman, Suzita Mohd Noor, Syam Mohan, Mohamad Ibrahim Noordin

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Girinimbine, a carbazole alkaloid was isolated from the stem bark and root of Murraya koenigii and its structure and purity was identified by HPLC and LC-MS. Here we report that Girinimbine strongly inhibit angiogenesis activity both in vitro and in vivo. MTT result showed that girinimbine inhibits cell proliferation of the HUVECS cell line in vitro. Result of endothelial cell invasion, migration, tube formation and wound healing assays also demonstrated significant time and does dependent inhibition by girinimbine. Moreover, girinibine mediates its anti-angiogenic activity through up- and down-regulation of angiogenic and anti-aniogenic proteins. Furthermore, anti-angiogenic potential of girinimbine was evidenced in vivo on zebrafish model. Girinimbine inhibited neo-vessels formation in zebrafish embryos during 24 hours exposure time. Together, these results demonstrated for the first time that girinimbine could effectively suppress angiogenesis and strongly suggest that it might be a novel angiogenesis inhibitor.

Keywords: anti-angiogenic, carbazole alkaloid, girinimbine, zebrafish

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Authors: Rita Emília Szabó, Róbert Polanek, Tünde Tőkés, Zoltán Szabó, Szabolcs Czifrus, Katalin Hideghéty

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Purpose: Several feature of zebrafish are making them amenable for investigation on therapeutic approaches such as ionizing radiation. The establishment of zebrafish model for comprehensive radiobiological research stands in the focus of our investigation, comparing the radiation effect curves of neutron and photon irradiation. Our final aim is to develop an appropriate vertebrate model in order to investigate the relative biological effectiveness of laser driven ionizing radiation. Methods and Materials: After careful dosimetry series of viable zebrafish embryos were exposed to a single fraction whole-body neutron-irradiation (1,25; 1,875; 2; 2,5 Gy) at the research reactor of the Technical University of Budapest and to conventional 6 MeV photon beam at 24 hour post-fertilization (hpf). The survival and morphologic abnormalities (pericardial edema, spine curvature) of each embryo were assessed for each experiment at 24-hour intervals from the point of fertilization up to 168 hpf (defining the dose lethal for 50% (LD50)). Results: In the zebrafish embryo model LD50 at 20 Gy dose level was defined and the same lethality were found at 2 Gy dose from the reactor neutron beam resulting RBE of 10. Dose-dependent organ perturbations were detected on macroscopic (shortening of the body length, spine curvature, microcephaly, micro-ophthalmia, micrognathia, pericardial edema, and inhibition of yolk sac resorption) and microscopic (marked cellular changes in skin, cardiac, gastrointestinal system) with the same magnitude of dose difference. Conclusion: In our observations, we found that zebrafish embryo model can be used for investigating the effects of different type of ionizing radiation and this system proved to be highly efficient vertebrate model for preclinical examinations.

Keywords: ionizing radiation, LD50, relative biological effectiveness, zebrafish embryo

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Authors: Hwang Kwon

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Objective: To assess the efficacy of embryo transfer (ET) of accumulated vitrified embryos and compare pregnancy outcomes between ET of thawed embryos following accumulation of vitrified embryos (frozen ET) and ET of fresh and thawed frozen embryos following accumulation of vitrified embryos (fresh ET + frozen ET). Study design: Patients were poor ovarian responders defined according to the Bologna criteria as well as a subgroup of women whose previous IVF-ET cycle through controlled ovarian stimulation (COS) yielded one or no embryos. Sixty-four frozen ETs were performed following accumulation of vitrified embryos (ACCE )(ACCE Frozen) and 51 fresh + frozen ETs were performed following accumulation of vitrified embryos (ACCE Fresh + Frozen). Positive βhCG rate, clinical pregnancy rate, ongoing pregnancy rate, and good quality embryos (%, ±SD) were compared between two groups. Results: There were more good quality embryos in the ACCE Fresh + Frozen group than in the ACCE Frozen group: 60±34.7 versus 42.9±28.9, respectively (p=0.03). Positive βhCG rate [18/64(28.2%) vs. 13/51(25.5%); p=0.75] and clinical pregnancy rate [12/64 (18.8%) vs. 11/51 (10.9%); p=0.71] were comparable between the two groups. Conclusion: Accumulation of vitrified embryos is an effective method in patients with poor ovarian response who fulfill the Bologna criteria. Pregnancy outcomes were comparable between the two groups.

Keywords: accumulation of embryos, frozen embryo transfer, poor responder, Bologna criteria

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Authors: Sara Ignoto, Elena M. Scalisi, Carmen Sica, Martina Contino, Greta Ferruggia, Antonio Salvaggio, Santi C. Pavone, Gino Sorbello, Loreto Di Donato, Roberta Pecoraro, Maria V. Brundo

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The new fifth generation technology (5G), which should favor high data-rate connections (1Gbps) and latency times lower than the current ones (<1ms), has the characteristic of working on different frequency bands of the radio wave spectrum (700 MHz, 3.6-3.8 GHz and 26.5-27.5 GHz), thus also exploiting higher frequencies than previous mobile radio generations (1G-4G). The higher frequency waves, however, have a lower capacity to propagate in free space and therefore, in order to guarantee the capillary coverage of the territory for high reliability applications, it will be necessary to install a large number of repeaters. Following the introduction of this new technology, there has been growing concern in recent years about the possible harmful effects on human health and several studies were published using several animal models. This study aimed to observe the possible short-term effects induced by 5G-millimeter waves on heartbeat of early life stages of Danio rerio using DanioScope software (Noldus). DanioScope is the complete toolbox for measurements on zebrafish embryos and larvae. The effect of substances can be measured on the developing zebrafish embryo by a range of parameters: earliest activity of the embryo’s tail, activity of the developing heart, speed of blood flowing through the vein, length and diameters of body parts. Activity measurements, cardiovascular data, blood flow data and morphometric parameters can be combined in one single tool. Obtained data are elaborate and provided by the software both numerical as well as graphical. The experiments were performed at 27 GHz by a no commercial high gain pyramidal horn antenna. According to OECD guidelines, exposure to 5G-millimeter waves was tested by fish embryo toxicity test within 96 hours post fertilization, Observations were recorded every 24h, until the end of the short-term test (96h). The results have showed an increase of heartbeat rate on exposed embryos at 48h hpf than control group, but this increase has not been shown at 72-96 h hpf. Nowadays, there is a scant of literature data about this topic, so these results could be useful to approach new studies and also to evaluate potential cardiotoxic effects of mobile radiofrequency.

Keywords: Danio rerio, DanioScope, cardiotoxicity, millimeter waves.

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Authors: Elena Maria Scalisi

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Currently, photodegradation by nanoparticles (NPs) is a common solution for wastewater treatment. Nanoparticles are efficient for removing organic and inorganic pollutants, heavy metals from wastewater and killing microorganisms through environmentally friendly. In this context, the major representative of photocatalytic technology for industrial wastewater treatment are TiO₂ nanoparticles (TiO₂-NPs). TiO₂-NPs have a strong catalytic activity that depends to their physicochemical properties. Thanks to their small size (between 1-100 nm), nanoparticles occupy less volume, then their surface area increases. The increase in the surface-to-volume ratio results in the increase of the particle surface energy, which improve their reactivity potential. However, these unique properties represent risks to the ecosystems and organisms when unintentionally TiO₂-NPs are release into the environment and absorbed by living organisms. Several studies confirm that there is a high level of interest concerning the safety of TiO₂-NPs in the aquatic environment, furthermore, ecotoxicological tools are useful to correctly evaluate their toxicity. In the current study, we aimed to characterize potential toxic effects of TiO₂-NP suspension to zebrafish during embryo-larval stages to evaluate parameters such as survival rates, malformation, hatching, the overall length of the larvae heartbeat, and biochemical biomarkers that reflect the acute toxicity and sublethal effects of TiO₂-NPs. Zebrafish embryos were exposed to titanium dioxide nanoparticles (TiO₂-NPs at 1mg/L, 2mg/L, and 4mg/L) from fertilization to the free swimming stage (144hpf). Every day, we recorded the toxicological endpoints, moreover, immunohistochemical analysis has been performed at the end of the exposure. In particular, we have evaluate the expression of the following biomarkers: Heat Shock Protein 70 (HSP70), Poly ADP-Ribose Polymerase-1 (PARP-1), Metallothioneins (MTs). Our results have shown that hatch ability, survival, and malformation rate were not affected by TiO₂ NPs at these exposure levels. However, TiO₂-NPs caused an increase of heartbeat and reduction of body length; at the same time, TiO₂-NPs have inducted the production of ROS and the expression of oxidative stress biomarkers HSP70 and PARP-1. Hight positivity for PARP-1 at all concentration tested was observed. As regards MT, positivity was found in the expression of this biomarker in the whole body of the embryo, with the exception of the end of the tail. Metallothioneins (MT) are biomarkers widely used in environmental monitoring programs for aquatic creatures. At the light of our results i.e. no death until the end of the experiment (144hpf), no malformation and expression of the biomarkers mentioned, it is evident that zebrafish larvae with their natural detoxification pathways are able to resist the presence of toxic substances and then they can tolerate the presence of metal concentrations. However, an excessive oxidative state can compromise cell function, therefore the uncontrolled release of nanoparticles into the environment is severe and must be constantly monitored.

Keywords: nanoparticles, embryo zebrafish, HSP70, PARP-1

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Authors: Eun Ko, Moonsung Choi, Sooim Shin

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4,4’-Dichlorodiphenyltrichloroethane (4,4’-DDT) is colorless, odorless organochlorine and known as persistent toxic organic pollutant accumulated in organs. In this study, effects of 4,4’-DDT on activities of mitochondrial electron transport chain system was analyzed. 4,4’-DDT is directly treated to isolated mitochondria from eyes of zebrafish and then activities of mitochondrial complex I, II, III, IV were measured spectrophotometrically. The reaction was proceeded immediately after adding 4,4’-DDT to examine the short-term exposing effects of persistent organic pollutant. As a result, high concentration of 4,4’-DDT treated mitochondria exhibited slightly enhanced activity in all complexes than non-treated one except complex III in male. Particularly, 4,4’-DDT was more effective on enzymatic activity in mitochondria isolated from eyes of male zebrafish. These results represented that 4,4’-DDT might temporarily induce to open up ion channel on isolated mitochondria resulting in increasing the functional activity of mitochondrial electron transport chain system.

Keywords: electron transport chain, mitochondrial function, persistent organic pollutant, spectrophotometric assay, zebrafish

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Authors: Ehsan Ahmadifar, Mohammad Ali Yousefi, Zahra Roohi

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In this study, the effects of different levels of Ergosan (control group (0), 2, 4 and 6 gr Ergosan per Kg diet) as a nutritional supplement were investigated on growth indices and stress in Zebrafish for 3 months. Larvae (4-day-old after hatching) were fed with experimental diet from the beginning of feeding until adult (adolescence) (average weight: 69.3 g, length: 5.1 cm). Different levels of Ergosan had no significant effect on rate survival (P < 0.05). The results showed that diet containing 6 gr Ergosan significantly caused the best FCR in Zebrafish (P < 0.05). By increasing the Ergosan diet, specific growth rate increased. Body weight gain and condition factor had significant differences (P < 0.05) as the highest and the lowest were observed in treatment 3 gr of Ergosan and control, respectively. The results showed that fish fed with experimental diet, had the highest resistance to environmental stresses compared to control, and the test temperature, oxygen, salinity and alkalinity samples containing 6 gr/kg, was significantly more resistance compared to the other treatments (P < 0.05). Overall, to achieve high resistance to environmental stress and increase final biomass using 6 gr/kg Ergosan in diet fish Zebrafish.

Keywords: Ergosan, stress, growth performance, Danio rerio

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Authors: Hasan Basri Jumin, Danil Endriand Basri

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Isolated protoplast from embryogenic callus of orange Jessamine (Murraya paniculata L. (Jack) cultured and maintained under growth chamber at the temperature +25oC. The parameter observed are the plating efficiency, the number of spherical embryos, heard-shaped embryos-like structure, shoot formation, and plantlets obtained. Treatment was arranged with 0.0, 0.001, 0.01, 0.1 or 1.0 mg 1-1 Naphthalene acetic acid (NAA), and 0, 300, 500 mg 1/l malt extract (ME) and 0.M sorbitol in the medium with 2.5 % sucrose. Interaction between 0.001 mg/l NAA and 500 mg/l was observed the higher percentage of planting efficiency. For embryo development from callus, the media was added to 0.0 mg/l, 0.001 mg/l, 0.01 ,mg/l, 0.1 mg/l, 1.0 mg/l NAA, and 1.0 %, 2.0 %, 3.0 %, 4.0 % sucrose. Media supplemented with 0.01mg/l NAA, and 1.0% sucrose was found to be a suitable medium for the development of spherical somatic embryos. A combination of 0.1 mg/ indole acetic acid (IAA) and 0.1 mg/l zeatin constituted the spherical somatic embryo became heart-shaped embryos-like structure. A combination between GA3 0.1 mg 1/l GA3 and 0.1 mg 1-1 zeatin is looking high, growing the heart-shaped embryos-like structure to form a shoot. Cells were developed into spherical embryos and grew into heart-shaped embryos, and then spherical somatic embryos developed into shoot formation. Sequence from single protoplast to plantlets was obtained by using a low concentration of plant growth regulator and sucrose; This recovery of single protoplast to be completed plantlets is a new technology in plant cell culture, and this could be used in genetic engineering in citrus.

Keywords: heart-shaped-embryos-like-structure, Muraya-paniculata, plant-growth-regulator, spherical- somatic-embryo, single protoplast, glucose

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Authors: Anamarel Medina-Hernandez, Teresa Ponce-Noyola, Ileana Vera-Reyes, Ana C. Ramos-Valdivia

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Somatic embryos reproduce original seed characteristics and could be implemented in biotechnological studies. Jatropha curcas L. is an important plant for biodiesel production, but also is used in traditional medicine. Seeds from J. curcas are toxic because contain diterpenoids called phorbol esters, but in Mexico exist a non-toxic variety. Therefore, somatic embryos suspension cultures from non-toxic J. curcas variety were induced. In order to investigate the characteristics of somatic embryos, a differential proteomic analysis was made between pre-globular and globular stages by 2-D gel electrophoresis. 108 spots were differentially expressed (p<0.02), and 20 spots from globular somatic embryos were sequenced by MALDI-TOF-TOF mass spectrometry. A comparative analysis of terpenoids production between the two stages was made by RP-18 TLC plates. The sequenced proteins were related to energy production (68%), protein destination and storage (9%), secondary metabolism (9%), signal transduction (5%), cell structure (5%) and aminoacid metabolism (4%). Regarding terpenoid production, in pre-globular and globular somatic embryos were identified sterols and triterpenes of pharmacological interest (alpha-amyrin and betulinic acid) but also it was found compounds that were unique to each stage. The results of this work are the basis to characterize at different levels the J. curcas somatic embryos so that this system can be used efficiently in biotechnological processes.

Keywords: Jatropha curcas, proteomics, somatic embryo, terpenoids

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Authors: Zelikha Labbani

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Since its creation in 1964 by Guha and Maheshwari in India on Datura innoxia Mill, in vitro androgenesis has become the method of choice in the production of doubled haploid in many species. However in durum wheat, the Doubled haploid plant breeding programs remained limited due to the low production of androgenetic embryos and converting them into fertile green plants. We describe here an efficient method for inducing embryos and regenerating green plants directly from isolated microspores of durum wheat.

Keywords: Durum wheat, haploid embryos, on in vitro, pretreatment

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Authors: Zelikha Labbani

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Since its creation in 1964 by Guha and Maheshwari in India on Datura innoxia Mill, in vitro androgenesis has become the method of choice in the production of doubled haploid in many species. However, in durum wheat, the Doubled haploid plant breeding programs remained limited due to the low production of androgenetic embryos and converting them into fertile green plants. We describe here an efficient method for inducing embryos and regenerating green plants directly from isolated microspores of durum wheat.

Keywords: durum wheat, haploid embryos, on in vitro, pretreatment

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Authors: Nisha Khatik, Ramesh Joshi

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An in vitro plant regeneration system was developed via direct somatic embryogenesis from different seedling explants of an important medicinal plant Murraya koenigii (L) Spreng. Cotyledons (COT), Hypocotyle (HYP)(10 to 15 mm) and Root (RT) segments (10 to 20 mm) were excised from 60 days old seedlings as explants. The somatic embryos induction was achieved on MS basal medium augmented with different concentrations of BAP 1.33 to 8.40 µM and TDZ 1.08 to 9.82 µM. The globular embryos originated from cut ends and entire surface of the root, hypocotyle explants and margins of cotyledons within 30-40days. The percentage of somatic embryos induction per explant was significantly higher in HYP explants (94.21±5.77%) in the MS basal medium supplemented with 6.20 µM BAP and 8.64 µM TDZ. The highest rate of conversion of torpedo, heart and cotyledonary stages from globular stage was obtained in MS medium supplemented with 8.64 µM TDZ. The matured somatic embryos were transferred to the MS basal medium without PGRs. Highest 88% of the matured embryos were germinated on transfer to the PGR free medium where they grew for a further 3-4 weeks. Out of seventy six hardened plants seventy (92%) plantlets were found healthy under field conditions.

Keywords: Murraya koenigii, somatic embryogenesis, thidiazuron, regeneration, rutaceae

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Authors: Jose Carlos Tavares Carvalho, Hady Keita, Giovanna Rocha Santana, Igor Victor Ferreira Dos Santos, Jesus Rafael Rodriguez Amado, Ariadna Lafourcade Prada, Adriana Maciel Ferreira, Helison Oliveira

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Summary: As animal model, zebrafish can be a good opportunity to establish a profile of tissue alteration caused by Bothrops alternatus venom and to screen new anti-venom drugs. Objective: To establish tissue biomarkers from zebrafish injected by snake venom and elucidate the use of glucocorticoids in ophidic accidents. Materials and Methods: The Danio rerio fish were randomly divided into four groups: control group, venom group, Dexamethasone1h before venom injected group and Dexamethasone 1 h after venom injected group. The concentration of Bothrops alternatus venom was 0.13 mg/ml and the fish received 20µl/Fish. The Body weight measurement and histological characteristics of gills, kidneys, liver, and intestine were determinate. Results: Physical analysis shows necrosis accompanied by inflammation in animals receiving the Bothrops alternatus venom. Significant difference was observed in the variation of weight between the control group, and the groups received the venom (t student test, p < 0.05). The average histological alterations index of gill, liver, kidney or intestine was statistically higher in animals received the venom (t Student test, p < 0.05). The alterations were lower in the groups that received Dexamethasone 1h before and after venom injected compared to the group that received only the venom. Dexamethasone 1h before venom injected group had minor histopathological alterations. Conclusion: The organs of zebrafish may be a tissue biomarker of alterations from Bothrops alternatus venom and dexamethasone reduced the damage caused by this venom in the organs studied, which may suggest the use of zebrafish as animal model for research related to screening new drug against snake venom.

Keywords: zebrafish, snake venom, biomarker, drugs

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Authors: Daniel M. Barry

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Since more than half a century ago, attempts have been made to culture cells and embryos outside the body (in vitro or ex vivo). This was done with different culture media and in various “incubators”. In the present study two different culture media were used: a standard TCM-199 culture medium and first trimester amniotic fluid (BAF) collected sterilely from pregnant cows after slaughter. Two different culture conditions were also investigated, the standard laboratory CO2 incubator versus culturing bovine embryos in the vagina of a goat doe. Two experiments were done: Firstly the permeability of different receptacles to CO2 gas was analyzed for possible culture in the vagina. Four-well plates and straws were used to incubate TCM-199 and BAF for a period of 120 h in the presence or absence of 5% CO2 gas. The pH values were measured and recorded every 24 h. In the second experiment pre-compacted morula stage bovine embryos were cultured in the above culture media in sealed 0.25 mL straws in a standard laboratory incubator and in the vagina of a goat doe. Evaluation was done on (1) stage of development and (2) number of blastomeres after 96 h of culture. In the first experiment it was shown that the CO2 gas diffused out of the 4-well plate as well as through the wall of the straws in the absence of CO2 gas, while in the presence of CO2 the pH of both media stabilized between 7.3 and 7.5. This meant that the semen straws were permeable to CO2 gas and could therefore be used as receptacles for culturing early stage bovine embryos. In the second experiment no statistical differences (p>0.05) were found in the number of pre-compacted bovine embryos that developed to the blastocyst stage, or the hatched blastocyst stage, neither for the culture medium used, or the method of culturing in the two incubators. Neither was there any difference (p>0.05) in the number of blastomeres that developed at the blastocyst stage between the two types of incubators. The bovine embryos tended to develop more blastomeres when cultured in BAF than when cultured in TCM-199 in both the standard laboratory incubator and when using the vagina of a goat doe as an incubator.

Keywords: alternative culture, bovine embryos, vagina, bovine amniotic fluid, incubator

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Authors: I. Bochev, A. Shterev, S. Kyurkchiev

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One of the factors associated with poor success rates in human in vitro fertilization (IVF) is the suboptimal culture conditions in which fertilization and early embryonic growth occur. Co-culture systems with helper cell lines appear to enhance the in vitro conditions and allow embryos to demonstrate improved in vitro development. The co-culture of human embryos with monolayers of autologous endometrial stromal cell (EnSCs) results in increased blastocyst development with a larger number of blastomeres, lower incidence of fragmentation and higher pregnancy rates in patients with recurrent implantation failure (RIF). The aim of the study was to examine the influence of autologous endometrial stromal cell (EnSC) co-culture on day 3 embryo quality by comparing the morphological status of the embryos from the same patients undergoing consecutive IVF/Intracytoplasmic sperm injection (ICSI) cycles without and with EnSC co-culture. This retrospective randomized study (2015-2017) includes 20 couples and a total of 46 IVF/ICSI cycles. Each patient couple included had at least two IVF/ICSI procedures – one with and one without autologous EnSC co-culture. Embryo quality was assessed at 68±1 hours in culture, according to Istanbul consensus criteria (2010). Day 3 embryos were classified into three groups: good – grade 1; fair – grade 2; poor – grade 3. Embryos from all cycles were divided into two groups (A – co-cultivated; B – not co-cultivated) and analyzed. Second, for each patient couple, embryos from matched IVF/ICSI cycles (with and without co-culture) were analyzed separately. When an analysis of co-cultivated day 3 embryos from all cycles was performed (n=137; group A), 43.1% of the embryos were graded as “good”, which was not significantly different from the respective embryo quality rate of 42.2% (p = NS) in group B (n=147) with non-co-cultivated embryos. The proportions of fair and poor quality embryos in group A and group B were similar as well – 11.7% vs 10.2% and 45.2% vs 47.6% (p=NS), respectively. Nevertheless, the separate embryo analysis by matched cycles for each couple revealed that in 65% of the cases the proportion of morphologically better embryos was increased in cycles with co-culture in comparison with those without co-culture. A decrease in this proportion after endometrial stromal cell co-cultivation was found in 30% of the cases, whereas no difference was observed in only one couple. The results demonstrated that there is no marked difference in the overall morphological quality between co-cultured and non-co-cultured embryos on day 3. However, in significantly greater percentage of couples the process of autologous EnSC co-culture could increase the proportion of morphologically improved day 3 embryos. By mimicking the in vivo relationship between embryo and maternal environment, co-culture in autologous EnSC system represents a perspective approach to improve the quality of embryos in cases with elevated risk for development of embryos with impaired morphology.

Keywords: autologous endometrial stromal cells, co-culture, day 3 embryo, morphological quality

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Authors: Liang Ning, Chung Hau Yin

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Angiogenesis is the process of new blood vessel development. It has been recognized as a therapeutic target for blocking cancer growth four decades ago. Vascular sprouting is initiated by pro-angiogenic factors. Vascular endothelial cell growth factor (VEGF) plays a central role in angiogenic initiation, many patients with cancer or ocular neovascularization have been benefited from anti-VEGF therapy. Emerging approaches impacting in the later stages of vessel remodeling and maturation are expected to improve clinical efficacy. TIE receptor as well as the corresponding angiopoietin ligands, were identified as another endothelial cell specific receptor tyrosine kinase signaling system. Much efforts were made to reduce the activity of angiopoietin-TIE receptor axis. Two eudesmane-type sesquiterpenes from laggera alata, namely, 15-dihydrocostic acid and ilicic acid were found with strong anti-angiogenic properties in zebrafish model. Meanwhile, the mRNA expression levels of VEGFR2 and TIE2 pathway related genes were down-regulated in the sesquiterpenes treated zebrafish embryos. Besides, in human umbilical vein endothelial cells (HUVECs), the sesquiterpenes have the ability to inhibit VEGF-induced HUVECs proliferation and migration at non-toxic concentration. Moreover, angiopoietin-2 induced TIE2 phosphorylation was inhibited by the sesquiterpenes, the inhibitory effect was detected in angiopoietin-1 induced HUVECs proliferation as well. Thus, we hypothesized the anti-angiogenic activity of the compounds may via the inhibition of VEGF and TIE2 related pathways. How the compounds come into play as the pathways inhibitors need to be evaluated in the future.

Keywords: Laggera alata, eudesmane-type sesquiterpene, anti-angiogenesis, VEGF, angiopoietin, TIE2

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Authors: D. P. N. De Silva, N. P. P. Liyanage

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Zebrafish (Danio rerio) in the family Cyprinidae, is a tropical freshwater fish widely used as a model organism in scientific research. Use of effective and economical anaesthetic is very important when handling fish. Clove oil (active ingredient: eugenol) was identified as a natural product which is safer and economical compared to synthetic chemicals like methanesulfonate (MS-222). Therefore, the aim of this study was to identify the most effective dosage of clove oil solution as an anaesthetic on mature Zebrafish. Clove oil solution was prepared by mixing pure clove oil with 94% ethanol at a ratio of 1:9 respectively. From that solution, different volumes were selected as (0.4 ml, 0.6 ml and 0.8 ml) and dissolved in one liter of conditioned water (dosages : 0.4 ml/L, 0.6 ml/L and 0.8 ml/L). Water quality parameters (pH, temperature and conductivity) were measured before and after adding clove oil solution. Mature Zebrafish with similar standard length (2.76 ± 0.1 cm) and weight (0.524 ± 0.1 g) were selected for this experiment. Time taken for loss of equilibrium (initiation phase) and complete loss of movements including opercular movement (anaesthetic phase) were measured. To detect the efficacy on anaesthetic recovery, time taken to begin opercular movements (initiation of recovery phase) until swimming (post anaesthetic phase) were observed. The results obtained were analyzed according to the analysis of variance (ANOVA) and Tukeys’ method using SPSS version 17.0 at 95% confidence interval (p<0.5). According to the results, there was no significant difference at the initiation phase of anaesthesia in all three doses though the time taken was varied from 0.14 to 0.41 minutes. Mean value of the time taken to complete the anaesthetic phase at 0.4 ml/L dosage was significantly different with 0.6 ml/L and 0.8 ml/L dosages independently (p=0.01). There was no significant difference among recovery times at all dosages but 0.8 ml/L dosage took longer time compared to 0.6 ml/L dosage. The water quality parameters (pH and temperature) were stable throughout the experiment except conductivity, which increased with the higher dosage. In conclusion, the best dosage need to anaesthetize Zebrafish using clove oil solution was 0.6 ml/L due to its fast initiation of anaesthesia and quick recovery compared to the other two dosages. Therefore clove oil can be used as a good substitute for synthetic anaesthetics because of its efficacy at a lower dosage with higher safety at a low cost.

Keywords: anaesthetics, clove oil, zebrafish, Cyprinidae

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Authors: Paula I. Buonfiglio, Carlos David Bruque, Lucia Salatino, Vanesa Lotersztein, Sebastián Menazzi, Paola Plazas, Ana Belén Elgoyhen, Viviana Dalamón

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Hearing loss (HL) is the most prevalent sensorineural disorder affecting about 10% of the global population, with more than half due to genetic causes. About 1 in 500-1000 newborns present congenital HL. Most of the patients are non-syndromic with an autosomal recessive mode of inheritance. To date, more than 100 genes are related to HL. Therefore, the Whole-exome sequencing (WES) technique has become a cost-effective alternative approach for molecular diagnosis. Nevertheless, new challenges arise from the detection of novel variants, in particular missense changes, which can lead to a spectrum of genotype-to-phenotype correlations, which is not always straightforward. In this work, we aimed to identify the genetic causes of HL in isolated and familial cases by designing a multistep approach to analyze target genes related to hearing impairment. Moreover, we performed in silico and in vivo analyses in order to further study the effect of some of the novel variants identified in the hair cell function using the zebrafish model. A total of 650 patients were studied by Sanger Sequencing and Gap-PCR in GJB2 and GJB6 genes, respectively, diagnosing 15.5% of sporadic cases and 36% of familial ones. Overall, 50 different sequence variants were detected. Fifty of the undiagnosed patients with moderate HL were tested for deletions in STRC gene by Multiplex ligation-dependent probe amplification technique (MLPA), leading to 6% of diagnosis. After this initial screening, 50 families were selected to be analyzed by WES, achieving diagnosis in 44% of them. Half of the identified variants were novel. A missense variant in MYO6 gene detected in a family with postlingual HL was selected to be further analyzed. A protein modeling with AlphaFold2 software was performed, proving its pathogenic effect. In order to functionally validate this novel variant, a knockdown phenotype rescue assay in zebrafish was carried out. Injection of wild-type MYO6 mRNA in embryos rescued the phenotype, whereas using the mutant MYO6 mRNA (carrying c.2782C>A variant) had no effect. These results strongly suggest the deleterious effect of this variant on the mobility of stereocilia in zebrafish neuromasts, and hence on the auditory system. In the present work, we demonstrated that our algorithm is suitable for the sequential multigenic approach to HL in our cohort. These results highlight the importance of a combined strategy in order to identify candidate variants as well as the in silico and in vivo studies to analyze and prove their pathogenicity and accomplish a better understanding of the mechanisms underlying the physiopathology of the hearing impairment.

Keywords: diagnosis, genetics, hearing loss, in silico analysis, in vivo analysis, WES, zebrafish

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Authors: Ayman A. Swelum, Abdullah N. Al-Owaimer, Mohamed A. Abouheif

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This study aimed to evaluate the effects of sheep breed and the number of embryos transferred on the success of multiple ovulation and embryo transfer (MOET). Sixteen Najdi and Naeimi ewes were used as donors. Multiple ovulation was achieved using equine chorionic gonadotropin (eCG). Thirty-five recipient ewes were divided into four groups: Najdi or Naeimi ewes that received either one or two embryos. After lambing, the gestation length, litter size, and sex of the lambs were recorded. The rates of pregnancy, lambing, and embryo survival were lower in the recipient Najdi than Naeimi ewes when two embryos were transferred. In contrast, the Naeimi ewes that received one embryo had a significantly lower embryo transfer success. In conclusion, the response of ewes to multiple ovulation stimulation using eCG was significantly high in Naeimi ewes (9.8±1.17). Moreover, transferring one embryo resulted in a significantly high pregnancy rate in the Najdi sheep (60%).

Keywords: embryo transfer, multiple ovulation, Najdi, Naeimi, sheep

Procedia PDF Downloads 729

Authors: Nurul Hamirah Kamsani, Mohd Hamim Rajikin, Nor Ashikin Mohamed Noor Khan, Sharaniza Abdul Rahim

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Cytoskeletal structures, in particular actin and tubulin, provide a fundamental framework in all cells, including embryos. Under influence of nicotine, the cytoskeletal organization may be subjected to oxidative stress (OS) insult and cause alteration. Tocotrienol-rich fraction (TRF) is proven to enhance fertility better than the other sub-group of Vitamin E, tocopherols (TCPs). The objective of this study was to evaluate the effects of TRF on 1) actin and tubulin of 2- and 8-cell murine embryos and 2) the regulation of reactive oxygen species (ROS)-scavenging enzymes; induced by nicotine. Twenty four female Balb/C were subjected to either subcutaneous (sc) injection of 0.9% NaCl; sc injection of 3.0 mg/kg bw/day nicotine; sc injection of 3.0 mg/kg bw/day nicotine + oral gavage (OG) of 60 mg/kg bw/day TRF; or OG of 60 mg/kg bw/day TRF for 7 consecutive days. After superovulation and mating, animals were euthanized. 2-cell developing embryos were retrieved. 50% of the retrieved embryos were visualized under confocal laser staining microscopy (CLSM) for alterations of actin and tubulin. The remaining amount of embryos was cultured in vitro until 8-cell stage followed by CLSM visualization. Blood plasma was subjected to OS assays. Plasma malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined and analysed accordingly. At both 2- and 8-cell developing stages, actin intensities were significantly reduced in the nicotine group (p<0.001). After the intervention, actin intensity was significantly increased compared to that of the nicotine group (p<0.001). The same trend was seen in tubulin at both cell stages. TRF has minimized the deleterious effects of nicotine in actin and tubulin of both 2- and 8-cell developmental stages during pre-implantation embryonic development in mice in vitro. Levels of endogenous anti-oxidative enzymes were sustained close to control accompanied by decreased levels of OS biomarker.

Keywords: actin, nicotine, pre-implantation embryos, tocotrienol rich fraction, tubulin

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Authors: Zelikha Labbani

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Durum wheat represents an attractive species to study androgenesis via isolated microspore culture in order to increase the efficiency of androgenic yield in recalcitrant species such as in induction embryogenesis. We describe here an efficient method for inducing embryos from isolated microspores of durum wheat. It is shown that this method, associated with cold alone or cold plus mannitol pretreatment, or mannitol alone of the spikes kept within their sheath leaves during different times, has significant positive effects on embryo production. The aim of this study was, therefore, to test the effect of mannitol 0,3M and cold pretreatment on the quality and quantity of embryos produced from microspore culture from wheat cultivars.

Keywords: in vitro embryogenesis, isolated microspores culture, durum wheat, pretreatments, mannitol 0.3m, cold pretreatment

Procedia PDF Downloads 57

Authors: Sangyeop Shin, D. C. M. Kulatunga, S. H. S. Dananjaya, Chamilani Nikapitiya, Jehee Lee, Mahanama De Zoysa

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Saprolegniasis is one of the most devastating fungal diseases in freshwater fish which is caused by species in the genus Saprolegnia including Saprolegnia parasitica. In this study, we isolated the strain of S. parasitica from diseased rainbow trout in Korea. Morphological and molecular based identification confirmed that isolated fungi belong to the member of S. parasitica, supported by its typical fungal features including cotton-like whitish mycelium, zoospores (primary and secondary) and phylogenetic analysis with internal transcribed spacer (ITS) region. Pathogenicity of isolated S. parasitica was developed in embryo, larvae, juvenile and adult zebrafish as a disease model. Up regulation of host genes encoding ZfTnf-α, Zfc-Rel, ZfIl-12, ZfLyz-c, Zfβ-def, and ZfHsp-70 was identified in zebrafish larvae after experimental challenge of S. parasitica showing the host immune responses against the S. parasitica. Survival of the juveniles upon fungal infection might be due to the increased immune protection in the host. Investigation of antifungal susceptibility of S. parasitica with natural lawsone (2-hydroxy-1,4-naphthoquinone) revealed the minimum inhibitory concentration (MIC) and percentage inhibition of radial growth (PIRG %) as 200 µg/mL and 31.8%, respectively. Lawsone was able to change the membrane permeability, and cause irreversible damage and disintegration to the cellular membranes of S. parasitica which might have effect on fungi growth inhibition. Moreover, the mycelium exposed to lawsone (MIC level) changed the transcriptional responses of S. parasitica genes. Overall results indicate that lawsone could be a potential and novel anti-S. parasitica agent for controlling S. parasitica infection.

Keywords: host-pathogen interactions, lawsone, rainbow trout, Saprolegnia parasitica, Saprolegniasis, zebrafish

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Authors: Münüre Tanur Erkoyuncu, Mustafa Yorgancılar

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Crop improvement through genetic engineering depends on effective and reproducible plant regeneration systems. Immature embryos are the most widely used explant source for in vitro regeneration in barley (Hordeum vulgare L.). However, immature embryos require the continuous growth of donor plants and the suitable stage for their culture is also certainly limited. On the other hand, mature embryos can be procured and stored easily; they can be studied throughout the year. In this study, an effective callus induction and plant regeneration were aimed to develop from mature embryos of different barley genotypes. The effect of medium (MS₁ and MS₂), auxin type (2,4-D, dicamba, picloram and 2,4,5-T) and concentrations (2, 4, 6 mg/l) on callus formation and effect of cytokinin type (TDZ, BAP) and concentrations (0.2, 0.5, 1.0 mg/l) on green plant regeneration were evaluated in mature embryo culture of barley. Callus and shoot formation was successful for all genotypes. By depending on genotype, MS₁ is the best medium, 4 mg/l dicamba is the best growth regulator in the callus induction and MS₁ is the best medium, 1 mg/l BAP is the best growth regulator in the shoot formation were determined.

Keywords: barley, callus, embryo culture, mature embryo

Procedia PDF Downloads 326

Authors: Tais Silva Lopes, Danilo Caneppele, Elizabeth Romagosa

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Surubim-do-Paraíba, Steindachneridion parahybae is a species of South American fish in critical conditions of extinction. Researches have been developed with the objective of conserving the biological material of this species. We evaluated the cooling of mature oocytes in the cryoprotective solutions containing the following alcohols: methanol, Propylene glycol and DMSO, each at concentrations of 1M, 2M and 4M, totaling nine treatments. After being submitted to treatments, the oocytes were maintained for 120 minutes in cooling to -5.52±2.58⁰C. A sample of oocytes was submitted to negative control (NC), kept in 90% L-15 solution, and positive control (PC), fertilized and taken directly to the incubator. Fertilization and hatching rates were evaluated. In order to compare the sensitivity of oocytes to embryos of the same species, the embryos maintained as CP in the previous assay were used in the free-flow stage (about 22 hours post fertilization) and submitted to the same treatments (prepared in distilled water) and also cooled for 120 min. The evaluation was done by the hatch rate. There was no fertilization rate of the oocytes submitted to the cooling with propylene glycol; the other cryoprotectants presented values of at most 3.7% of fertilization (Methanol 1M), and no treatment completed development until hatching. The cooled embryos had a significant percentage of normal larvae in all treatments, but inversely proportional to the increase in the concentration of the alcohols. DMSO 1M was the most promising treatment for embryo cooling, with 41.7% ± 20.2 of normal larvae, while mature oocytes were highly sensitive to cold.

Keywords: cryoconservation, cooling, embryos, freezing, oocytes, south American fish

Procedia PDF Downloads 241

Authors: Uche C. Okafor, Nwanneka M. Okpokwu, Felix Nwafor, Carl E. A. Okezie

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Securidaca longepedunculata Fresen (Violet tree) belongs to the family Polygalaceae characterised by papillionaceous purplish flowers. This medicinally valued plant disappears at an alarming rate due to intensified anthropopressure particularly the unregulated manner of subterranean plant parts' collection from natural stands. Some indiscriminately harvested plants bear seeds containing both mature and immature zygotic embryos that are often discarded. Here, such seeds are collected for this experiment. Seeds were collected, washed, de-coated, and dipped in 70 % (v/v) ethanol for 30 s followed by rising in 5 % solution sodium hypochlorite, containing two drops of tween 20, for another 25 min. Mature zygotic embryos (MZEs) were excised from seeds and cultured in two basal media (MS and B5), three carbon sources (sucrose, glucose and fructose) at five concentrations (0-40 g/L) while immature zygotic embryos (iMZEs) were composed on similar basal media and carbon source supplemented with 0-2 mg/L Benzylaminopurine (BAP) and 0-2 mg/L Indole acetic acid (IAA). MZEs cultured on MS + 30g/L sucrose differed significantly from other treatments at p≤0.05 with maximum percent sprouting (85.24± 5.67 %) and shoot length (7.53±0.67 cm). MZEs culture had the maximum percent sprouting (85.24± 5.67 %) and shoot length (7.53±0.67 cm) in medium containing MS+ 30g L-1 sucrose. iMZEs on the other hand had maximum growth on MS + 40g/L sucrose supplemented with 1.5 mg/L IAA+ 1.0 mg/L BAP. This study is a geared towards creating an alternative path for the maximum production of plants in vitro, thereby, preventing the plants from disappearing.

Keywords: Gamborg's medium, Murashige and Skoog medium, Securidaca longepedunculata, zygotic embryos

Procedia PDF Downloads 156