Search results for: hatching
Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 52

Search results for: hatching

52 Influence of IL-1β on Hamster Blastocyst Hatching via Regulation of Hatching Associated Proteases

Authors: Madhulika Pathak, Polani Seshagiri, Vani Venkatappa

Abstract:

Blastocyst hatching is an indispensable process for successful implantation. One of the major reasons for implantation failure in IVF clinic is poor quality of embryo, which are not development/hatching-competent. Therefore, attempts are required to develop or enhance the culture system with a molecule recapitulating the autocrine/paracrine factors containing the environment of in-vivo endometrial milieu. We have tried to explore the functional molecules involved in the hamster hatching phenomenon. Blastocyst hatching is governed by several molecules that are entwined and regulate this process, among which cytokines are known to be expressed and are still least explored. Two of such cytokines we have used for our study are IL-1β and its natural antagonist IL-1ra to understand the functional dynamics of cytokines involved in the hatching process. Using hamster, an intriguing experimental model for hatching behavior, we have shown the mRNA (qPCR) and protein (ICC) expression of IL-1β, IL-1ra and IL-1 receptor type 1 throughout all the stages of morula, blastocyst and hatched blastocyst. Post-asserting the expression, the functional role is shown by supplementation studies, where IL-1β supplementation showed enhancement in hatching level (IL-1β treated: 84.1 ± 4.2% vs control: 63.7 ± 3.1 %, N=11), further confirmed by the diminishing effect of IL-1ra on hatching rate (IL-1ra treated: 27.5 ± 11.1% vs control: 67.9 ± 3.1%). The exogenous supplementation of IL-1ra decreased the survival rate of embryos and affected the viability in dose-dependent manner, establishing the importance of IL-1β in blastocyst cell survival. Previously, the cathepsin L and B were established as the proteases that were involved in the hamster hatching process. The inducing effect of IL-1β was correlated with enhanced mRNA level, as analyzed by qPCR, for both CAT L (by 1.9 ± 0.5 fold) and CAT B (by 3.5 ± 0.1) fold which was diminished in presence of IL-1ra (Cat L by 88 percent and Cat B by 94 percent. Moreover, using a specific fluorescent substrate-based assay kit, the enzymatic activity of these proteases was found to be increased in presence of IL-1β (Cat L by 2.1 ± 0.1 fold and CAT B by 2.3 ± 0.7 fold) and was curtailed in presence of IL-1ra. These observations provide functional insights with respect to the involvement of cytokines in the hatching process. This has implications in understanding the hatching biology and improving the embryo development quality in IVF clinics.

Keywords: Blastocyst, Cytokines, Hatching, Interleukin

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51 Hatching Rhythm, Larval Release of the Rocky Intertidal Crab Leptoduis exaratus (Brachyura: Xanthidae) in Kuwait, Arabian Gulf

Authors: Zainab Al-Wazzan, Luis Gimenez, Lewis Le Vay, Manaf Behbehani

Abstract:

The hatching rhythm and larval release patterns of the rocky shore crab Leptoduis exaratus was investigated in relation to the tidal cycle, the time of the day, and lunar cycle. Ovigerous females were collected from rocky shores at six sites along the Kuwait coastline between April and July of 2014. The females were kept separated in aquaria under a natural photoperiod cycle and the pattern of larval release was monitored in relation to local tidal and dial cycles. Larval release occurred mostly during the night time, and was highly synchronized with neap tides that followed full moon; at the end of the hatching period, significant larval release occurred also during spring tides. Time series analysis showed a highly significant autocorrelation and the periodicity at a peak of 14-15 days. The cross-correlation analysis between hatching and the daily low tide level suggests that larvae are released about a day before neap tide. Hatching during neap tides occurred early in the night at times of the expected ebb tide. During spring tide period (late in the season), larval release occurred later during night at tides of the ebb tide. The results of this study indicated a strong relationship between the tidal cycle, time of the day and the hatching rhythm of L. exaratus. In addition, the results suggest that water level in the intertidal zone is also playing a very important role in determining the time of the hatching. Hatching and larval release synchronize with the preferred larval environmental conditions to prevent exposing larvae to physiological or environmental stress during their early larval stages. It is also an important factor in determining the larval dispersal.

Keywords: brachyura, hatching rhythm, larvae, Kuwait

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50 Post-Hatching Development of the Cloacal Bursa in Chicken

Authors: Fatimah A. Alhomaid

Abstract:

A total of 40 one day-old LSL chicks (Lohman Selected Loghorn) were used in this study. In 20 days-old chicks, the bursa was formed of mucosa, musculosa and serosa. Its lamina propria was lymphoid in nature. After hatching, the bursa continued to grow and became fully developed at the 30th day post- hatching. It appeared as a blind sac. Its lumen was occupied by 12-13 mucosal folds. Each fold was lined by tall columnar or pseudo- stratified columnar epithelium. Its core was made of lamina propria infiltrated by a large number of lymphoid follicles. Most follicles possessed an outer corona surrounding a germinal center. At the age of 6 weeks physiological regression of the bursa was observed. The lymphoid follicles were decreased in size, the lymphocytes were depleted and the interfollicular stroma became obvious, thicker and more fibrous. Fibrosis of the lymphoid follicles was frequently seen in some sections at the age of 30 weeks.

Keywords: Bursa of fabricius, lymphocytes, cloacal Bursa

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49 Beak Size and Asynchronous Hatch in Broiler Chicks

Authors: Mariana Thimotheo, Gabriel Carvalho Ripamonte, Marina De Almeida Nogueira, Silvia Camila Da Costa Aguiar, Marcelo Henrique Santana Ulian, Euclides Braga Malheiros, Isabel Cristina Boleli

Abstract:

Beak plays a fundamental role in the hatching process of the chicks, since it is used for internal and external pipping. The present study examined whether the size of the beak influences the birth period of the broiler chicks in the hatching window. It was analyzed the beak size (length, height and width) of one-hundred twenty nine newly hatched chicks from light eggs (56.22-61.05g) and one-hundred twenty six chicks from heavy eggs (64.95-70.90g), produced by 38 and 45 weeks old broiler breeders (Cobb 500®), respectively. Egg incubation occurred at 37.5°C and 60% RH, with egg turning every hour. Length, height and width of the beaks were measured using a digital caliper (Zaas precision - digital caliper 6", 0.01mm) and the data expressed in millimeters. The beak length corresponded to distance between the tip of the beak and the rictus. The height of the beak was measured in the region of the culmen and its width in the region of the nostrils. Data were analyzed following a 3x2 factorial experimental design, being three birth periods within the hatching window (early: 471.78 to 485.42h, intermediate: 485.43 to 512.27h, and late: 512.28 to 528.72h) and two egg weights (light and heavy). There was a significant interaction between birth period and egg weight for beak height (P < 0.05), which was higher in the intermediate chicks from heavy eggs than in the other chicks from the same egg weight and chicks from light eggs (P < 0.05), that did not differ (P > 0.05). The beak length was influenced only for a birth period, and decreased through the hatch window (early < intermediate < late) (P < 0.05). The width of the beaks was influenced by both main factors, birth period and egg weight (P < 0.05). Early and intermediate chicks had similar beak width, but greater than late chicks, and chicks from heavy eggs presented greater beak width than chicks from light eggs (P < 0.05). In sum, the results show that chicks with longer beak hatch first and that beak length is an important variable for hatch period determination mainly for light eggs.

Keywords: beak dimensions, egg weight, hatching period, hatching window

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48 Egg Hatching Inhibition Activity of Volatile Oils Extracted from Some Medicinal and Aromatic Plants against Root-Knot Nematode Meloidogyne hapla

Authors: Anil F. Felek, Mehmet M. Ozcan, Faruk Akyazi

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Volatile oils of medicinal and aromatic plants are important for managing nematological problems in agriculture. In present study, volatile oils extracted from five medicinal and aromatic plants including Origanum onites (flower+steam+leaf), Salvia officinalis (leaf), Lippia citriodora (leaf+seed), Mentha spicata (leaf) and Mentha longifolia (leaf) were tested for egg hatching inhibition activity against root-knot nematode Meloidogyne hapla under laboratory conditions. The essential oils were extracted using water distillation method with a Clevenger system. For the homogenisation process of the oils, 2% gum arabic solution was used and 4 µl oils was added into 1ml filtered gum arabic solution to prepare the last stock solution. 5 ml of stock solution and 1 ml of M. hapla egg suspension (about 100 eggs) were added into petri dishes. Gum arabic solution was used as control. Seven days after exposure to oils at room temperature (26±2 °C), the cumulative hatched and unhatched eggs were counted under 40X inverted light microscope and Abbott’s formula was used to calculate egg hatching inhibition rates. As a result, the highest inhibition rate was found as 54% for O. onites. In addition, the other inhibition rates varied as 31.4%, 21.6%, 23.8%, 25.67% for the other plants, S. officinalis, M. longifolia, M. spicata and L. citriodora, respectively. Carvacrol was found as the main component (68.8%) of O. onites followed by Thujone 27.77% for S. officinalis, I-Menthone 76.92% for M. longifolia, Carvone 27.05% for M. spicata and Citral 19.32% for L. citriodora.

Keywords: egg hatching, Meloidogyne hapla, medicinal and aromatic plants, root-knot nematodes, volatile oils

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47 Ecotoxicity Evaluation and Suggestion of Remediation Method of ZnO Nanoparticles in Aqueous Phase

Authors: Hyunsang Kim, Younghun Kim, Younghee Kim, Sangku Lee

Abstract:

We investigated ecotoxicity and performed an experiment for removing ZnO nanoparticles in water. Short-term exposure of hatching test using fertilized eggs (O. latipes) showed deformity in 5 ppm of ZnO nanoparticles solution, and in 10ppm ZnO nanoparticles solution delayed hatching was observed. Herein, chemical precipitation method was suggested for removing ZnO nanoparticles in water. The precipitated ZnO nanoparticles showed the form of ZnS after addition of Na2S, and the form of Zn3(PO4)2 for Na2HPO4. The removal efficiency of ZnO nanoparticles in water was closed to 100% for two case. In ecotoxicity evaluation of as-precipitated ZnS and Zn3(PO4)2, they did not cause any acute toxicity for D. magna. It is noted that this precipitation treatment of ZnO is effective to reduce the potential cytotoxicity.

Keywords: ZnO nanopraticles, ZnS, Zn3(PO4)2, ecotoxicity evaluation, chemical precipitation

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46 The Effect of Acute Toxicity and Thyroid Hormone Treatments on Hormonal Changes during Embryogenesis of Acipenser persicus

Authors: Samaneh Nazeri, Bagher Mojazi Amiri, Hamid Farahmand

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Production of high quality fish eggs with reasonable hatching rate makes a success in aquaculture industries. It is influenced by the environmental stimulators and inhibitors. Diazinon is a widely-used pesticide in Golestan province (Southern Caspian Sea, North of Iran) which is washed to the aquatic environment (3 mg/L in the river). It is little known about the effect of this pesticide on the embryogenesis of sturgeon fish, the valuable species of the Caspian Sea. Hormonal content of the egg is an important factor to guaranty the successful passes of embryonic stages. In this study, the fate of Persian sturgeon embryo to 24, 48, 72, and 96-hours exposure of diazinon (LC50 dose) was tested. Also, the effect of thyroid hormones (T3 and T4) on these embryos was tested concurrently or separately with diazinon LC 50 dose. Fertilized eggs are exposed to T3 (low dose: 1 ng/ml, high dose: 10 ng/ml), T4 (low dose: 1 ng/ml, high dose: 10 ng/ml). Six eggs were randomly selected from each treatment (with three replicates) in five developmental stages (two cell- division, neural, heart present, heart beaten, and hatched larvae). The possibility of changing T3, T4, and cortisol contents of the embryos were determined in all treated groups and in every mentioned embryonic stage. The hatching rate in treated groups was assayed at the end of the embryogenesis to clarify the effect of thyroid hormones and diazinon. The results indicated significant differences in thyroid hormone contents, but no significant differences were recognized in cortisol levels at various early life stages of embryos. There was also significant difference in thyroid hormones in (T3, T4) + diazinon treated embryos (P˂0.05), while no significant difference between control and treatments in cortisol levels was observed. The highest hatching rate was recorded in HT3 treatment, while the lowest hatching rate was recorded for diazinon LC50 treatment. The result confirmed that Persian sturgeon embryo is less sensitive to diazinon compared to teleost embryos, and thyroid hormones may increase hatching rate even in the presence of diazinon.

Keywords: Persian sturgeon, diazinon, thyroid hormones, cortisol, embryo

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45 Tetraploid Induction in the Yellowtail Tetra Astyanax altiparanae

Authors: Nivaldo Ferreira do Nascimento, Matheus Pereira-Santos, Nycolas Levy-Pereira, José Augusto Senhorini, George Shigueki Yasui, Laura Satiko Okada Nakaghi

Abstract:

Tetraploid individuals, which could produce diploid gametes, can be used for production of 100% triploid fish. Therefore, the aim of this study was to develop a tetraploidization protocol for A. altiparanae. We tested the effect of heat shock (40 °C; 2 min) at 16, 18, 20, 22, 24 and 26 minutes post fertilization (mpf). Untreated eggs were used as control. After hatching, ploidy status of the larvae was checked by flow cytometry. No difference were observed for the hatching rate between all treatments (P = 0.5974). However, we observed an increase in the larval abnormality in the heat shock treatments, in special at 22 (82.17 ± 6.66%) 24 (78.31 ±7.28%) and 26 mpf (79.01 ± 7.85%) in comparison with the control group (12.87 ± 4.46%). No tetraploid was observed at 16 and 18 mpf. The higher number of tetraploid individuals (52/55) was observed at 26 mpf. Our results showed that high percentages of tetraploids are obtained by heat shock (40°C; 2min) at 26 mpf, which could enable the mass production of triploid individuals in A. altiparanae.

Keywords: chromosome manipulation, polyploidy, flow cytometry, tetraploidization

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44 Impact of Propolis on Cryopreservation of Arctic Charr (Salvelinus alpinus) Sperm

Authors: K. A. El-Battawy, E. Brannas

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Cryopreservation of sperm causes damages and adversely affected sperm motility and viability resulting in lower hatching rates. The aim of this study is to determine whether propolis has potential protective effect on cryopreservation and fertilization ability of spermatozoa of Salvelinusalpinus. The extenders were prepared by using simple glucose solution (0.3 M glucose) to which 10% Me2SO added with different levels of propolis (0.4, 0.8 and 1 mg/ ml) and 10% egg yolk (as a control without propolis). The pooled semen samples diluted at the ratio of 1:3 by the extenders were subjected to cryopreservation. The percentage and duration of motility and fertilization tests of cryopreserved sperm samples have been done immediately after thawing and compared with control and fresh semen. The extenders containing propolis showed higher percentage motility and motility duration than control group (P < 0.05). Especially the group II (0.8 mg/ ml propolis) and the group III (1 mg/ ml propolis) showed significant positive effects on both post thaw motility and hatching ability. In conclusion, this study confirms that the propolis is an appropriate cryoptrotective agent in fish semen and it maintained the integrity of the spermatozoa during the cryopreservation process.

Keywords: propolis, arctic charr, semen, cryopreservation

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43 Effects of Gamma Radiation on Tomato Leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

Authors: Akın Kuyulu, Hanife Genç

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In present study, it was aimed to evaluate the gamma radiation impacts on tomato leaf miner at different biological stages. The laboratory colony of tomato leaf miner was used to set up the experiments. Different biological stages of the insects (eggs, 4th instars and pupae) were irradiated using Cobalt-60 at doses of 0 (control), 100 Gray (Gy), 200 Gy, 300 Gy and 400 Gy in Cos-44HH-N source, at dose rate of 480 Gy/h. After irradiation, the eggs were incubated until hatching; the mature larvae were reared to complete their developments. Adult emergences from irradiated pupae were also evaluated. The results showed that there were no egg hatching at all tested irradiation doses. Although, the pupal percentages of irradiated mature larvae were 54%, 15% and 8% at doses of 100 Gy, 200 Gy and 300 Gy respectively, there were no adult emergences from irradiated mature larvae. On the other hand, the adult emergences were observed from irradiated pupae, decreased as radiation doses increased along with malformed adult appearance. Male and female individuals were out crossed with laboratory reared adults. Fecundity was correlated with radiation doses.

Keywords: irradiation, tomato, tomato leafminer, Tuta absoluta

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42 Morphometric Study of the Eggs of Pheasant Eggs Phasianus colchicus (Aves, Phasianidae)

Authors: S. Zenia, A. Menasseria, A. E. Kheidous, F. Larinouna, A. Smai, H. Saadi, F. Haddadj, A. Milla, F. Marniche

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Pheasant, is a bird of great ornamental value through the beauty of its form and colors, it is among the most popular birds. The present study was conducted in an experimental breeding. The objective of this work is to know the quality of the eggs of this bird. A total of 938 eggs were collected. To deepen the knowledge about the characteristics of external shell quality, biometric parameters were studied, among them we find the weight with a mean value of 29.2± 2, 24 g. Egg length (mm) and egg width (mm) mean value are respectively 43.01 ± 1,84 cm and 34.05 ± 1,44cm. The volume and shape index of eggs obtained are respectively 25,63±2,88cm3 and 79.00 ± 3%, shell index which recorded an average of 68%. Water loss recorded is 13%. Note that all these parameters and others may influence hatching. The analysis of variance applied for the comparison of egg weight shows that there is no significant difference in the same form factor (P> 0.05). Otherwise, the comparison test used shows a significant difference with P <0.05 for length, width, volume, density, indices of shell and water loss of eggs between the different. Indeed, several factors may explain the difference as the absence of sorting eggs during incubation and other factors that will be exposing later.

Keywords: analysis of variance, egg, hatching, morphometry of eggs Phaisan (Phasianus colchicus.L.)

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41 Histogenesis of the Stomach of Pre-Hatching Quail: A Light and Electron Microscopic Study

Authors: Soha A Soliman, Yasser A Ahmed, Mohamed A Khalaf

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Although the enormous literature describing the histology of the stomach of different avian species during the posthatching development, the available literature on the pre-hatching development of quail stomach development is scanty. Thus, the current study was undertaken to provide a careful description of the main histological events during the embryonic development of quail stomach. To achieve this aim, daily histological specimens from the stomach of quail of 4 days post-incubation till the day 17 (few hours before hatching) were examined with light microscopy. The current study showed that the primitive gut tube of the embryonic quail appeared at the 4th day post incubation, and both parts of stomach (proventriculus and gizzard) were similar in structure and composed of endodermal epithelium of pseudostratified type surrounded by undifferentiated mesenchymal tissue. The sequences of the developmental events in the gut tube were preceded in a cranio-caudal pattern. By the 5th day, the endodermal covering of the primitive proventriculus gave rise to sac-like invaginations. The primitive gizzard was distinguished into thick-walled bodies and thin-walled sacs. In the 6th day, the prospective proventricular glandular epithelium became canalized and the muscular layer was developed in the cranial part of the proventriculus, whereas the primitive muscular coat of the gizzard was represented by a layer of condensed mesenchyme. In the 7th day, the proventricular glandular epithelial invaginations increased in depth and number, while, the muscularis mucosa and the muscular layer began to be distinguished. In the 8th day, the myoblasts differentiated into spindle shaped smooth muscle fibers. In the 10th day, branching of the proventricular glands began. The branching continued later on. The surface and the glandular epithelium were transformed into simple columnar type in the 12th day. The epithelial covering of the gizzard gave rise to tubular invaginations lined by simple cuboidal epithelium and the surface epithelium became simple columnar. Canalization of the tubular glands was recognized in the 14th day. In the 15th day, the proventricular surface epithelium invaginated in an concentric manner around a central cavity to form immature secretory units. The central cavity was lined by eosinophilic cells which form the ductal epithelia. The peripheral lamellae were lined by basophilic cells; the undifferentiated oxyntico-peptic cells. Entero-endocrine cells stained positive for silver impregnation in the proventricular glands. The mucosal folding in the gizzard appeared in the 15th day to form the plicae and the sulci. The wall of the proventriculus and gizzard in the 17th day acquired the main histological features of post-hatching birds, but neither the surface nor the ductal epithelium were differentiated to mucous producing cells. The current results shoed be considered in the molecular developmental studies.

Keywords: quail, proventriculus, gizzard, pre-hatching, histology

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40 Defense Priming from Egg to Larvae in Litopenaeus vannamei with Non-Pathogenic and Pathogenic Bacteria Strains

Authors: Angelica Alvarez-Lee, Sergio Martinez-Diaz, Jose Luis Garcia-Corona, Humberto Lanz-Mendoza

Abstract:

World aquaculture is always looking for improvements to achieve productions with high yields avoiding the infection by pathogenic agents. The best way to achieve this is to know the biological model to create alternative treatments that could be applied in the hatcheries, which results in greater economic gains and improvements in human public health. In the last decade, immunomodulation in shrimp culture with probiotics, organic acids and different carbon sources has gained great interest, mainly in larval and juvenile stages. Immune priming is associated with a strong protective effect against a later pathogen challenge. This work provides another perspective about immunostimulation from spawning until hatching. The stimulation happens during development embryos and generates resistance to infection by pathogenic bacteria. Massive spawnings of white shrimp L. vannamei were obtained and placed in experimental units with 700 mL of sterile seawater at 30 °C, salinity of 28 ppm and continuous aeration at a density of 8 embryos.mL⁻¹. The immunostimulating effect of three death strains of non-pathogenic bacterial (Escherichia coli, Staphylococcus aureus and Bacillus subtilis) and a pathogenic strain for white shrimp (Vibrio parahaemolyticus) was evaluated. The strains killed by heat were adjusted to O.D. 0.5, at A 600 nm, and directly added to the seawater of each unit at a ratio of 1/100 (v/v). A control group of embryos without inoculum of dead bacteria was kept under the same physicochemical conditions as the rest of the treatments throughout the experiment and used as reference. The duration of the stimulus was 12 hours, then, the larvae that hatched were collected, counted and transferred to a new experimental unit (same physicochemical conditions but at a salinity of 28 ppm) to carry out a challenge of infection against the pathogen V. parahaemolyticus, adding directly to seawater an amount 1/100 (v/v) of the live strain adjusted to an OD 0.5; at A 600 nm. Subsequently, 24 hrs after infection, nauplii survival was evaluated. The results of this work shows that, after 24 hrs, the hatching rates of immunostimulated shrimp embryos with the dead strains of B. subtillis and V. parahaemolyticus are significantly higher compared to the rest of the treatments and the control. Furthermore, survival of L. vanammei after a challenge of infection of 24 hrs against the live strain of V. parahaemolyticus is greater (P < 0.05) in the larvae immunostimulated during the embryonic development with the dead strains B. subtillis and V. parahaemolyticus, followed by those that were treated with E. coli. In summary superficial antigens can stimulate the development cells to promote hatching and can have normal development in agreeing with the optical observations, plus exist a differential response effect between each treatment post-infection. This research provides evidence of the immunostimulant effect of death pathogenic and non-pathogenic bacterial strains in the rate of hatching and oversight of shrimp L. vannamei during embryonic and larval development. This research continues evaluating the effect of these death strains on the expression of genes related to the defense priming in larvae of L. vannamei that come from massive spawning in hatcheries before and after the infection challenge against V. parahaemolyticus.

Keywords: immunostimulation, L. vannamei, hatching, survival

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39 Effect of Pristine Graphene on Developmental Toxicity in Zebrafish (Danio rerio) Embryos: Cardiovascular Defects, Apoptosis, and Globin Expression Analysis

Authors: Manjunatha Bangeppagari, Lee Sang Joon

Abstract:

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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38 Comparative Effects of Homoplastic and Synthetic Pituitary Extracts on Induced Breeding of Heterobranchus longifilis (Valenciennes, 1840) in Indoor Hatchery Tanks in Owerri South East Nigeria

Authors: I. R. Keke, C. S. Nwigwe, O. S. Nwanjo, A. S. Egeruoh

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An experiment was carried out at Urban Farm and Fisheries Nigeria Ltd, Owerri Imo State South East Nigeria between February and June 2014 to induce Brood stock of Heterobranchus longifilis (mean wt 1.3kg) in concrete tanks (1.0 x 2.0 x 1.5m) in dimension using a synthetic hormone (Ovaprim) and pituitary extract from Heterobranchus longifilis. Brood stock males were selected as pituitary donors and their weights matched those of females to be injected at 1ml/kg body weight of Fish. Ovaprim, was injected at 0.5ml/kg body weight of female fish. A latency period of 12 hours was allowed after injection of the Brood stock females before stripping the egg and incubation at 23 °C. While incubating the eggs, samples were drawn and the rate of fertilization was determined. Hatching occurred within 33 hours and hatchability rate (%) was determined by counting the active hatchings. The result showed that Ovaprim injected Brood stock eggs fertilized up to 80% while the pituitary from the Heterobranchus longifilis had low fertilization and hatching success 20%. Ovaprim is imported and costly, so more effort is required to enhance the procedures for homoplastic hypophysation.

Keywords: heterobranchus longifilis, ovaprim, hypophysation, latency period, pituitary

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37 Conservation of Sea Turtle in Cox’s Bazar- Teknaf Peninsula and Sonadia Island Ecologically Critical Area (ECA) of Bangladesh

Authors: Pronob Kumar Mozumder M. Nazrul Islam, M. Abdur Rob Mollah

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This study was conducted in Cox’s Bazar-Teknaf Peninsula and Sonadia Island Ecologically Critical Areas during the period of October, 2011 to June, 2013. Six species of marine turtle are found in the Indian Ocean. Among them, olive ridley (Lepidochelys olivacea) listed as endangered in the IUCN Red List of Threatened Species. Marine turtle populations in the Indian Ocean have been depleted through long-term exploitation of eggs and adults, incidental capture (fisheries bycatch) and many other sources of mortality. The specific objective of the study was to conserve the sea turtles specially the olive ridley (Lepidochelys olivacea) with a view to contribute towards protection of the turtle species from extinction and to facilitate hatching of eggs through providing protection to turtle eggs or nest through ex-situ conservation efforts. In order to achieve the desired outputs and success, a total of five turtle hatcheries were established at Pechardwip, Khurermukh, Hazompara, Bodormokam, and Sonadia Eastpara sites. In total, 31,853 eggs were collected from 260 nests and were transferred to five hatcheries. The number of eggs/nest varied from 38 to 190 with an average clutch size of 122 eggs/ nest. Hatching of eggs took place during January to June with a peak in April. Sea turtle eggs were incubated by metabolic heat and the heat of the sun. The incubation period of turtle eggs in Cox’s Bazar-Teknaf Peninsula and Sonadia Island ECAs extended from 54 to 75 days depending on the month with an average of 66 days. During study period the temperature in the ECAs varied between 10.5-34.5°C. A total of 27,937 hatchlings of turtle were produced from the five hatcheries and all the hatchlings produced were released into the sea. Hatching rates varied from 74-98 % depending on the location and months with an average of 88 %. Sea turtles spend the majority of their lives in the sea, only emerging on beaches to nest. Despite the intense conservation efforts on the beaches, some populations have still declined to the edge of extinction. So proper conservation and awareness measure should be taken for prevention of turtle extinction.

Keywords: conservation of sea turtle, Bangladesh, ecologically critical area, ECA, Lepidochelys olivacea

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36 Sensitivity of Steindachneridion parahybae Mature Oocytes versus Embryos at Low Temperature

Authors: Tais Silva Lopes, Danilo Caneppele, Elizabeth Romagosa

Abstract:

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

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35 Evaluation of Stable Isotope in Life History and Mating Behaviour of Mediterranean Fruit Fly Ceratitis capitata (Diptera: Tephidae) in Laboratory Conditions

Authors: Hasan AL-Khshemawee, Manjree Agarwal, Xin Du, Yonglin Ren

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The possibility use of stable isotopes to study Medfly mating and life history were investigated in these experiments. 13C6 glucose was incorporated in the diet of the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephidae). Treatments included labelling and unlabelled of either the media or adult sugar water. The measured started from egg hatching till the adults have died. After mating, the adults were analysed for 13C6 glucose ratio using Liquid chromatography-mass spectrometry LC-MS in two periods of time immediately and after three days of mating. Results showed that stable isotopes were used successfully for labelling Medfly in laboratory conditions, and there were significant differences between labelled and unlabelled treatment in eggs hatching, larval development, pupae emergence, survival of adults and mating behaviour. Labelling during larval development and combined labelling of larvae and adults resulted in detectable values. The label glucose in larvae stage did not effect on mating behaviour, however, the label glucose in adults’ stage was affected by mating behaviour. We recommended that it is possible to label adults of Mediterranean fruit fly C. capitata and detected the label after mating. This method offers good tools to study mating behaviour in Medfly and other types of insects and could be providing useful tools in genetic studies, sterile insect technique (SIT) or agricultural pest management. Also, we recommended using this technique in the field.

Keywords: stable isotope, sterile insect technique (SIT), medfly, mating behaviour

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34 Efficacy of Gamma Radiation on the Productivity of Bactrocera oleae Gmelin (Diptera: Tephritidae)

Authors: Mehrdad Ahmadi, Mohamad Babaie, Shiva Osouli, Bahareh Salehi, Nadia Kalantaraian

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The olive fruit fly, Bactrocera oleae Gmelin (Diptera: Tephritidae), is one of the most serious pests in olive orchards in growing province in Iran. The female lay eggs in green olive fruit and larvae hatch inside the fruit, where they feed upon the fruit matters. One of the main ecologically friendly and species-specific systems of pest control is the sterile insect technique (SIT) which is based on the release of large numbers of sterilized insects. The objective of our work was to develop a SIT against B. oleae by using of gamma radiation for the laboratory and field trial in Iran. Oviposition of female mated by irradiated males is one of the main parameters to determine achievement of SIT. To conclude the sterile dose, pupae were placed under 0 to 160 Gy of gamma radiation. The main factor in SIT is the productivity of females which are mated by irradiated males. The emerged adults from irradiated pupae were mated with untreated adults of the same age by confining them inside the transparent cages. The fecundity of the irradiated males mated with non-irradiated females was decreased with the increasing radiation dose level. It was observed that the number of eggs and also the percentage of the egg hatching was significantly (P < 0.05) affected in either IM x NF crosses compared with NM x NF crosses in F1 generation at all doses. Also, the statistical analysis showed a significant difference (P < 0.05) in the mean number of eggs laid between irradiated and non-irradiated females crossed with irradiated males, which suggests that the males were susceptible to gamma radiation. The egg hatching percentage declined markedly with the increase of the radiation dose of the treated males in mating trials which demonstrated that egg hatch rate was dose dependent. Our results specified that gamma radiation affects the longevity of irradiated B. oleae larvae (established from irradiated pupae) and significantly increased their larval duration. Results show the gamma radiation, and SIT can be used successfully against olive fruit flies.

Keywords: fertility, olive fruit fly, radiation, sterile insect technique

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33 Development of the Squamate Egg Tooth on the Basis of Grass Snake Natrix natrix Studies

Authors: Mateusz Hermyt, Pawel Kaczmarek, Weronika Rupik

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The egg tooth is a crucial structure during hatching of lizards and snakes. In contrast to birds, turtles, crocodiles, and monotremes, egg tooth of squamate reptiles is a true tooth sharing common features of structure and development with all the other teeth of vertebrates. The egg tooth; however, due to its function, exhibits structural differences in relation to regular teeth. External morphology seems to be important in the context of phylogenetic relationships within Squamata but up to date, there is scarce information concerning structure and development of the egg tooth at the submicroscopical level. In presented studies detailed analysis of the egg tooth development in grass snake has been performed with the usage of light (including fluorescent), transmission and scanning electron microscopy. Grass snake embryo’s heads have been used in our studies. Grass snake is common snake species occurring in most of Europe including Poland. The grass snake is characterized by the presence of single unpaired egg tooth (as in most squamates) in contrast to geckos and dibamids possessing paired egg teeth. Studies show changes occurring on the external morphology, tissue and cellular levels of differentiating egg tooth. The egg tooth during its development changes its curvature. Initially, faces directly downward and in the course of its differentiation, it gradually changes to rostro-ventral orientation. Additionally, it forms conical dentinal protrusions on the sides. Histological analysis showed that egg tooth development occurs in similar steps in relation to regular teeth. It undergoes initiation, bud, cap and bell morphological stages. Analyses focused on describing morphological changes in hard tissues (mainly dentin and predentin) of egg tooth and in cells which enamel organ consists of. It included: outer enamel epithelium, stratum intermedium, inner enamel epithelium, odontoblasts, and cells of dental pulp. All specimens used in the study were captured according to the Polish regulations concerning the protection of wild species. Permission was granted by the Local Ethics Commission in Katowice (41/2010; 87/2015) and the Regional Directorate for Environmental Protection in Katowice (WPN.6401.257.2015.DC).

Keywords: hatching, organogenesis, reptile, Squamata

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32 Embryotoxicity of Nano-Iron Oxide (Fe2O3) to Bio-Indicator of Pollution of Land Helix Aspersa

Authors: S. Besnaci, S. Bensoltane, H. Locif, S. Saadi

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To validate an ecotoxicological approach to assessing toxicological effects caused by the oxide powder of nano-iron Fe2O3, we searched in the ecotoxicology laboratory cell bodies bio accumulators and bio-indicators of soil pollution the snail Helix aspersa. In this study, we evaluated the toxicity of nano Fe2O3 during a very sensitive phase of development H.aspersa (embryonic stage). During embryonic development, we observed in treated with various concentrations of nano Fe2O3 (1.25 g/l, 1.5 g/l, and 2 g/l) compared to control, the deformation of the membrane of the egg and accumulation of this molecule at the rear of the egg proven by the photographs, as with the influence on the hatching percentage.

Keywords: eggs, embryotoxicity, Fe2O3, Helix aspersa, nanoparticles

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31 Bioprophylaxis of Saprolegniasis in Incubated Clarias gariepinus Eggs Using Pyocyanin Extracted from Pseudomonas aeruginosa

Authors: G. A. Oladosu1, P. O. Ogbodogbo, C. I. Makinde1, M. O. Tijani, O. A. Adegboyega

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Saprolegniasis is a major pathogenic infection that contributes significantly to poor hatching rates in incubated fish eggs in the Africa catfish hatchery in Nigeria. Malachite green known to be very effective against this condition has been banned because it is carcinogenic. There is, therefore, the need for other effective yet safer methods of controlling saprolegniasis in incubated fish eggs. A total of 50 ml crude, chloroform extract of pyocyanin from which solvent was removed to attain 30 ml, having a concentration of 12.16 ug/ml was produced from 700 ml broth culture of Pseudomonas aeruginosa isolated from a previous study. In-vitro susceptibility of the fungus was investigated by exposing fungal infected eggs to two different time-concentration ratios of pyocyanin; 0.275 ug/ml and 2.75 ug/ml for 1 and 24 hours, and 5 mg/L malachite green as positive control while normal saline was the control. The efficacy of pyocyanin was evaluated using the degree of mycelial growth inhibition in different treatments. Fertilized Clarias gariepinus eggs (between 45 to 64 eggs) were then incubated in 20 ml of medium containing similar concentrations of pyocyanin and malachite green, with freshwater as a control for 24 hours. Hatching rates of the incubated eggs were observed. Three samples of un-hatched eggs were taken from each medium and observed for the presence of fungal pathogens using microscopy. Another batch of three samples of un-hatched eggs from each treatment was also inoculated on Sabourand dextrose agar (SDA) using Egg-Agar Transfer Technique to observe for fungal growth. Mycelial growth was inhibited in fungal infected eggs treated with 2.75 ug/ml for 24 hrs and the 5 mg/L malachite green for both 1 hr and 24 hrs. The mortality rate was 100% in fertilized C. gariepinus eggs exposed for 24 hrs to 0.275 and 2.75 ug/ml of pyocyanin. The mortality rate was least in malachite green followed by the control treatment. Embryonic development was observed to be arrested in the eggs treated with the two pyocyanin concentrations as they maintain their colour but showed no development beyond the gastrula stage, whereas viable eggs in the control and malachite green treatments developed fully into healthy hatchlings. Furthermore, microscopy of the un-hatched eggs revealed the presence of a protozoan ciliate; Colpidium sp, (Tetrahymenidae), as well as a pathogenic fungus; Saprolegnia sp. in the control but not in the malachite green and pyocyanin treatments. Growth of Saprolegnia sp was also observed in SDA culture of un-hatched eggs from the control, but not from pyocyanin and malachite green treated eggs. Pyocyanin treatment of incubated eggs of Clarias gariepinus effectively prevented fungal infection in the eggs, but also arrested the development of the embryo. Therefore, crude chloroform extract of pyocyanin from Pseudomonas aeruginosa cannot be used in the control of Saprolegniasis in incubated Clarias gariepinus eggs at the concentration and duration tested in this study.

Keywords: African catfish, bioprophylaxis, catfish embryo, Saprolegniasis

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30 Toxicity Evaluation of Reduced Graphene Oxide on First Larval Stages of Artemia sp.

Authors: Roberta Pecoraro

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The focus of this work was to investigate the potential toxic effect of titanium dioxide-reduced graphene oxide (TiO₂-rGO) nanocomposites on nauplii of microcrustacean Artemia sp. In order to assess the nanocomposite’s toxicity, a short-term test was performed by exposing nauplii to solutions containing TiO₂-rGO. To prepare titanium dioxide-reduced graphene oxide (TiO₂-rGO) nanocomposites, a green procedure based on solar photoreduction was proposed; it allows to obtain the photocatalysts by exploiting the photocatalytic properties of titania activated by the solar irradiation in order to avoid the high temperatures and pressures required for the standard hydrothermal synthesis. Powders of TiO₂-rGO supplied by the Department of Chemical Sciences (University of Catania) are indicated as TiO₂-rGO at 1% and TiO₂-rGO at 2%. Starting from a stock solution (1mg rGO-TiO₂/10 ml ASPM water) of each type, we tested four different concentrations (serial dilutions ranging from 10⁻¹ to 10⁻⁴ mg/ml). All the solutions have been sonicated for 12 min prior to use. Artificial seawater (called ASPM water) was prepared to guarantee the hatching of the cysts and to maintain nauplii; the durable cysts used in this study, marketed by JBL (JBL GmbH & Co. KG, Germany), were hydrated with ASPM water to obtain nauplii (instar II-III larvae). The hatching of the cysts was carried out in the laboratory by immersing them in ASPM water inside a 500 ml beaker and keeping them constantly oxygenated thanks to an aerator for the insufflation of microbubble air: after 24-48 hours, the cysts hatched, and the nauplii appeared. The nauplii in the second and third stages of development were collected one-to-one, using stereomicroscopes, and transferred into 96-well microplates where one nauplius per well was added. The wells quickly have been filled with 300 µl of each specific concentration of the solution used, and control samples were incubated only with ASPM water. Replication was performed for each concentration. Finally, the microplates were placed on an orbital shaker, and the tests were read after 24 and 48 hours from inoculating the solutions to assess the endpoint (immobility/death) for the larvae. Nauplii that appeared motionless were counted as dead, and the percentages of mortality were calculated for each treatment. The results showed a low percentage of immobilization both for TiO₂-rGO at 1% and TiO₂-rGO at 2% for all concentrations tested: for TiO₂-rGO at 1% was below 12% after 24h and below 15% after 48h; for TiO₂-rGO at 2% was below 8% after 24h and below 12% after 48h. According to other studies in the literature, the results have not shown mortality nor toxic effects on the development of larvae after exposure to rGO. Finally, it is important to highlight that the TiO₂-rGO catalysts were tested in the solar photodegradation of a toxic herbicide (2,4-Dichlorophenoxyacetic acid, 2,4-D), obtaining a high percentage of degradation; therefore, this alternative approach could be considered a good strategy to obtain performing photocatalysts.

Keywords: Nauplii, photocatalytic properties, reduced GO, short-term toxicity test, titanium dioxide

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29 Correlation of the Biometric Parameters of Eggs

Authors: S. Zenia, A. Menasseria, A. E. Kheidous, F. Lariouna, A. Smai, H. Saadi, F. Haddadj, A. Milla, F. Marniche

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The objective of this study was to estimate the correlation ship between different pheasant external egg quality traits. A total of 938 eggs were collected. Egg weight (g), egg length (mm), egg width (mm), volume (cm3), shape index egg, surface area and water loss were measured. The overall mean values obtained for the different variables are respectively 29.2 ± 2,24, 43.01 ± 1,84, 34.05 ± 1,44, 25.63 ± 2.88 cm3, 79.00 ± 3%, 68% and 13%. Concerning studied regressions, it was considered only the most important regressions. Those that show significant links between the different parameters studied. The ANOVA procedure was applied to estimate correlations for the examined traits. The weights of the eggs being observed before incubation and before hatching are linearly correlated with a positive correlation coefficient of order 0.75. Egg length and the weight before incubation had a good and positive correlation with a coefficient r = 0.6. However, density had high and negative correlations with egg height r = -0.78. Shape index had a good linear and negative r= - 0.71 correlation with water loss.

Keywords: correlation, egg, morphometry of eggs, analysis of variance

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28 Activity of Some Plant Extracts on the Larvae and Eggs of Culex quinquefasciatus in the Laboratory

Authors: A. A. El Maghrbi

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The control of vectors like mosquitoes based on the application of chemical insecticides but due to its adverse effect on the environment, and development of resistance by most of species of mosquitoes including vectors of important diseases. Ethanol and acetone extracts of nine species of plants (Allium tuberosum, Apium leptophylum, Carica papaya, Cymbopogon citratus, Euphorbia cotinofolia, Melia azedarach, Ocimum canum, Ricinus common, and Tagetes erecta) were tested in respect of their influence on the eggs and larvae of Culex quinquifasciatus in concentration 100, 10 and 1 mg/L. In relation to the survival of larvae, ethanol extract of O. canum and acetone extract of A.tuberosum in 100 mg/L have larvicide activity against L4 of Cx. quinquifasciatus. For hatching of eggs, ethanol and acetone extract of A.tuberosum (100 and 10 mg/L) and acetone extract of C.citratus (100 mg/L) produced reduction in the number of eggs hatched of Cx. quinquifasciatus. Our results indicate that each extract of the plant have potential to control mosquito population and suggest that further studies are needed in this field.

Keywords: Cx. quinquefasciatus, plant extract, ethanol, acetone, larvae, eggs

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27 Evaluation of Toxicity of Cerium Oxide on Zebrafish Developmental Stages

Authors: Roberta Pecoraro, Elena Maria Scalisi

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Engineered Nanoparticles (ENPs) and Nanomaterials (ENMs) concern an active research area and a sector in full expansion. They have physical-chemical characteristics and small size that improve their performance compared to common materials. Due to the increase in their production and their subsequent release into the environment, new strategies are emerging to assess risk of nanomaterials. NPs can be released into the environment through aquatic systems by human activities and exert toxicity on living organisms. We evaluated the potential toxic effect of cerium oxide (CeO2) nanoparticles because it’s used in different fields due to its peculiar properties. In order to assess nanoparticles toxicity, Fish Embryo Toxicity (FET) test was performed. Powders of CeO2 NPs supplied by the CNR-IMM of Catania are indicated as CeO2 type 1 (as-prepared) and CeO2 type 2 (modified), while CeO2 type 3 (commercial) is supplied by Sigma-Aldrich. Starting from a stock solution (0.001g/10 ml dilution water) of each type of CeO2 NPs, the other concentration solutions were obtained adding 1 ml of the stock solution to 9 ml of dilution water, leading to three different solutions of concentration (10-4, 10-5, 10-6 g/ml). All the solutions have been sonicated to avoid natural tendency of NPs to aggregate and sediment. FET test was performed according to the OECD guidelines for testing chemicals using our internal protocol procedure. A number of eight selected fertilized eggs were placed in each becher filled with 5 ml of each concentration of the three types of CeO2 NPs; control samples were incubated only with dilution water. Replication was performed for each concentration. During the exposure period, we observed four endpoints (embryo coagulation, lack of formation of somites, failure to lift the yolk bag, no heartbeat) by a stereomicroscope every 24 hours. Immunohistochemical analysis on treated larvae was performed to evaluate the expression of metallothioneins (MTs), Heat Shock Proteins 70 (HSP70) and 7-ethoxyresorufin-O-diethylase (EROD). Our results have not shown evident alterations on embryonic development because all embryos completed the development and the hatching of the eggs, started around the 48th hour after exposure, took place within the last observation at 72 hours. A good reactivity, both in the embryos and in the newly hatched larvae, was found. The presence of heartbeat has also been observed in embryos with reduced mobility confirming their viability. A higher expression of EROD biomarker was observed in the larvae exposed to the three types of CeO2, showing a clear difference with the control. A weak positivity was found for MTs biomarker in treated larvae as well as in the control. HSP70 are expressed homogeneously in all the type of nanoparticles tested but not too much greater than control. Our results are in agreement with other studies in the literature, in which the exposure of Danio rerio larvae to other metal oxide nanoparticles does not show adverse effects on survival and hatching time. Further studies are necessary to clarify the role of these NPs and also to solve conflicting opinions.

Keywords: Danio rerio, endpoints, fish embryo toxicity test, metallic nanoparticles

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26 Nematocidal Effects of Laurus Nobilis Essential Oil against Gastrointestinal Nematodes.

Authors: Essia Sebai, Amel Abidi, Hayet benyeddem, Akkari Hafidh

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Herbal extracts are of particular interest to the drug industry; essential oil with significant anthelmintic activity has the potential to be used as an alternative to conventional chemical drugs. In the present study, we describe the chemical profile of Laurus nobilis essential oil (EO), the in vitro anthelmintic activity of laurel oil against Haemonchus contortus and its in vivo anthelmintic effect against the murine helminth parasite model Heligmosomoides polygyrus. The chromatographic profile of L. nobilis (EO) extracted from the leaves of L. nobilis has shown the presence of monoterpenes 1,8-cineol (Eucalyptol) (29.47%), D-Limonène (18.51%) and Linalool (10.84%) in high fractions. The in vitro anthelmintic potential was expressed by an ovicidal effect against H. contortus egg hatching with an inhibition value of 3.23 mg/mL and 87.5% of immobility of adult worms after 8 hours of exposure to 8 mg/mL of L. nobilis EO. Regarding the in vivo anthelmintic potential, L. nobilis (EO) at 2400 mg/kg completely eliminated the egg output of H. polygyrus after seven days of oral treatment, together with a 79.2% of reduction in total worm counts. Based on the obtained funding, L. nobilis EO showed promising in vitro and in vivo anthelmintic capacities against gastrointestinal parasites.

Keywords: lauris nobilis, anthelmintic, haemonchus, pylogyrus

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25 Dietary Ergosan as a Supplemental Nutrient on Growth Performance, and Stress in Zebrafish (Danio Rerio)

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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24 Efficacy of Ginger (Zingiber officinale) and a Zeolite (Hydrated Sodium Calcium Aluminosilicate) in the Amelioration of Aflatoxicosis in Broilers

Authors: Ryan Stevens, Wayne L. Bryden

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This study focused on the effects of ginger and a zeolite (toxin binder) in reducing the toxic effects of aflatoxin B1 (AFB1) in broiler chickens 7 to 49 days of age. The chicks were maintained normally until experimental diets were introduced on day 7 post-hatching. Nine hundred and thirty six, 7-d-old broiler chickens were randomly assigned to 18 treatment groups; each group had four replicates, each with 13 chickens. The experimental groups or diets had factorial combinations of the following; AFB1 0, 1 and 2 mg/kg diet, ginger 0 and 5g/kg diet, and zeolite 0, 15 and 30 g/kg diet. Diets were based on corn and soybean meal and a starter diet was fed from 1 to 14 days, a grower diet from15 to 28 days and a finisher diet was provided from day 29 until the end of the experiment. Both dietary levels of AFB1 decreased (P<0.05) body weight and feed conversion, and increased relative liver weights. Independent dietary inclusion of ginger or zeolite restored chick performance when diets contained 1mg/kg but not at 2mg/kg. Supplementation of zeolite together with ginger improved performance of birds fed contaminated diets. Interestingly, adding ginger to the control diet that was not contaminated with AFB1 improved (P<0.05) performance. Our results suggest that toxin binders and ginger can provide protection against the negative effects of AFB1 on performance of broiler chicks.

Keywords: aflatoxin, broiler, ginger, zeolite

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23 Amphibians and Water Quality: An Assessment of Diversity and Physico-Chemical Parameters of Habitats for Amphibians in Sindh, Pakistan

Authors: Kalsoom Shaikh, Saima Memon, Riffat Sultana

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Water pollution affects amphibians because they are intimately water dependent. The permeable skin makes amphibians very sensitive to the physico-chemical parameters of their aquatic environment. They spawn in water bodies where quality of water can affect the growth, development, and survival of their eggs which may die even before hatching into larvae or developing into adults due to water contamination. Considering the importance of amphibians in agriculture, food web, ecosystem and pharmaceutics as well as adverse impact of environmental degradation on them, present study was proposed to comprehensively determine the status of their diversity and habitats in Sindh province of Pakistan so as to execute monitoring for their conservation in future. Physico-chemical parameters including pH, EC (electric conductivity), TDS (total dissolved solids), T-Hard (total hardness), T-Alk (total alkalinity), Cl (chloride), CO₂ (carbon dioxide), SO₄ (sulphate), PO₄ (phosphate), NO₂ (nitrite) and NO₃ (nitrate) were analyzed from amphibian habitats using instruments and methodology of analytical grade. The results of present study after being compared with scientific data provided by different researchers and EPA (environmental protection agency), it was concluded that amphibian habitats consisted of high values of analyzed parameters except pH and CO₂. Entire study area required an urgent implementation of conservation actions for saving amphibians.

Keywords: amphibians, diversity, habitats, physico-chemical parameters, water quality, Pakistan, Sindh Province

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