The embryotoxic and teratogenic effects of metamizole sodium.

Drug use during pregnancy is an important issue that must be investigated due to its adverse effects on maternal and foetal health. This study aimed to determine the embryotoxic and teratogenic effects of in-ovo administered metamizole (dipyrone), which can be used when needed during pregnancy and has potent analgesic, antipyretic, anti-inflammatory, and long bone (tibia and femur) effects. This study used 240 fertile eggs from Atak S breed chickens, divided into eight equal groups: control, vehicle control, and 15.62, 31.25, 62.5, 125, 250 and 500 mg/kg metamizole. The eggs were hatched on the 21st day of incubation, and the chicks' body weights and mortality rates were determined. The right and left femur and tibia bones were resected from the chicks. Anatomical reference points were determined after removing the soft tissues of the bones, and necessary morphometric measures were taken from these points with a 0.01 mm precision using digital callipers. The 100% lethal dose (LD100) was identified in the highest examined dose (500 mg/kg) in the Chicken Embryotoxicity Screening Test (CHEST)-I stage. The CHEST-II stage determined the 50% lethal dose (LD50). High-dose metamizole affected skeletal development, significantly decreasing tibia and femur lengths and corpus thicknesses and increasing mortality.

The potential of in ovo-fed amino acids to alleviate the effects of heat stress on broiler chickens: effect on performance, body temperature, and oxidative status during the finisher phase.

The aim of the current study was to investigate the potential of in ovo-fed amino acids (AA) to reduce the effects of heat stress on finishing broiler chickens. To achieve this, a total of 1,400 fertile hatching eggs were randomly distributed into 5 groups (n = 280/group) and injected with one of the following in ovo treatments on embryonic day 18: 52 µL of sterile diluent/egg (CTRL), CTRL + 1.0 mg of L-Leucine (T1), CTRL + 0.45 mg of leucine + 1.15 mg of methionine (T2), CTRL + 3.0 mg of methionine + 2.0 mg of cysteine (T3), and CTRL + 0.40 mg of leucine + 1.60 mg of methionine + 1.60 mg of cysteine (T4). After hatch, chicks were allocated according to a complete randomized block design comprising 2 thermal conditions: thermoneutral (24°C, 45% RH) and heat stress (34°C, 55-60% RH) with 5 pens/group/condition. The cyclical heat stress regimen (10 h/d) was then applied from d 29 to d 34. Compared to the CTRL group, T3 and T4 exhibited a higher BW during the starter phase (P < 0.001). T4 also had a lower feed conversion ratio (FCR) than CTRL during this same phase (P = 0.03). During the grower phase, males of all treatment groups consistently exhibited higher BW compared to the CTRL group, which was not observed among female birds (PSex × TRT = 0.005). During the finisher phase, the in ovo treatment effect on performance was not significant. However, heat-stressed birds from treatment group T3 and T4 exhibited lower facial temperatures (Pday × TRT < 0.001) as well as lower plasma (Pcondition x TRT = 0.039) and liver (Pcondition x TRT < 0.001) malonaldehyde concentrations compared to the CTRL group. In conclusion, in ovo-fed AA have the potential to modulate the effects of heat stress on finishing broiler chickens by limiting its detrimental consequences, including increased body temperature and oxidative damage.

Protective effects of ferulic acid on embryonic development by improving antioxidant function in broilers embryo of thermal manipulation.

This study aimed to elucidate the effects of broiler embryos soaked in ferulic acid (FA) solution on alleviating the negative impact of thermal manipulation (TM) on chicken embryo development and to provide a theoretical and experimental basis for applying TM and FA in the poultry feeding industry. A total of 120 broiler fertilized eggs were randomly divided into three groups: control group, TM group, and comprehensive group (TM + FA), with 40 eggs in each group. The TM group and the comprehensive group from the 7th embryonic age to the 16th embryonic age received TM for ten days, treated with a temperature of 39.5 °C and relative humidity of 65% for 18 h a day. In the comprehensive group, broiler embryos were immersed in FA solution at a concentration of 80 mg/L for 6 min at 16:00 every day from the 6th to the 8th embryo age. They were incubated continuously after being soaked until the chicks hatched. The results showed that the rates of dead embryos and weak chicks in the TM group were significantly higher than those in the control group and comprehensive group. Chick body temperatures of the TM group and comprehensive group were significantly lower than those of the control group. The heart weights of the TM group and comprehensive group were significantly lower than those of the control group, and the leg weights of the TM group were significantly decreased compared with those of the control group and comprehensive group. The SOD activity of serum in the comprehensive group was significantly higher than that in the control group and TM group, while the CAT activity of serum in the comprehensive group and control group was significantly higher than that in the TM group; however, there was no difference between the comprehensive group and control group. The activities of SOD and CAT in the liver were significantly higher than those of the TM group; however, the MDA content of the liver in the comprehensive group and control group was significantly lower than that of the TM group. The gene expression of Nrf2 and SOD in the comprehensive group and TM group was significantly higher than that in the control group; however, there was no significant difference between the comprehensive group and TM group. Soaking broiler embryonic eggs in an FA solution can improve the antioxidant capacity of the liver by upregulating Nrf2-Keap1 signal pathway-related gene expression. FA can effectively alleviate the side effects of TM on chicken embryos and does not impact the effects of TM.

Determination of the embryotoxic effects of propofol injected into eggs on the cerebellum and spinal cord using histologic methods: an animal study.

Background/aim: This study aims to determine the possible embryotoxic effects of propofol on the cerebellum and spinal cord using fertile chicken eggs. Materials and methods: A total of 430 fertile eggs were divided into 5 groups: control, saline, 2.5 mg.kg-1, 12.5 mg.kg-1, and 37.5 mg.kg-1 propofol. Injections were made immediately before incubation via the air chamber. On the 15th, 18th, and 21st day of incubation, 6 embryos from each group were evaluated. Serial paraffin sections taken from the cerebellum and spinal cord were stained with hematoxylin-eosin, Kluver-Barrera, toluidine blue, and periodic acid-Schiff's reaction. The outer granular layer and total cortex thickness were measured, and the linear density of the Purkinje cells was determined. The ratios of the substantia grisea surface area to the total surface area of the spinal cord were calculated. The transverse and longitudinal diameters of the canalis centralis were also assessed. Results: No structural malformation was observed in any embryos examined macroscopically. No significant difference was observed between the groups in terms of development and histologic organization of the cerebellum and spinal cord. However, on the 15th, 18th, and 21st day, the outer granular layer (p < 0.001 for all days) and the total cortex thickness (p < 0.01, p < 0.001, and p < 0.001, respectively) decreased significantly in different propofol dose groups in varying degrees in the cerebellum. Similarly, in the spinal cord, there were significant changes in the ratios of the substantia grisea surface area to the total surface area (p < 0.01 and p < 0.001, respectively). Conclusion: It was concluded that the in-ovo-administered propofol given immediately before incubation has adverse effects on the developing cerebellum and spinal cord. Therefore, it is important for anesthesiologists always to remain vigilant when treating female patients of childbearing age.

The effect of methionine and folic acid administered in ovo on the blood biochemical parameters of chickens (Gallus gallus domesticus).

Methionine is one of the most frequently supplemented amino acids in raising of poultry. However, an overdose of methionine can cause hyperhomocysteinemia. Folic acid, taking part in the process of homocysteine remethylation, is a factor affecting the reduction of the concentration of this amino acid. The study was carried out in 2 stages. The experiment of step I was to investigate the effect of methionine and/or folic acid administration in ovo in the early stage of embryogenesis (E4), and the experiment of the second stage - in the late stage of embryogenesis (E17) on the following biochemical parameters of chicken blood: glucose concentration in whole blood and concentration of homocysteine and uric acid in plasma of domestic chickens (Gallus gallus domesticus). Our results confirm that methionine supplementation may increase the concentration of uric acid and homocysteine. Moreover, we demonstrated that folic acid administered during embryogenesis decreased homocysteine concentration, also in groups simultaneously supplemented with methionine, especially in the initial stage of postnatal life of the bird.

Developmental toxicity of short-chain chlorinated paraffins on early-stage chicken embryos in a shell-less (ex-ovo) incubation system.

Short-chain chlorinated paraffins (SCCPs) are listed as a category of globally controlled persistent organic pollutants (POPs) by the Stockholm Convention in 2017. However, SCCP toxicity, particularly their developmental toxicity in avian embryos, has not been well studied. In this study, we observed the early development of chicken embryos (Gallus gallus domesticus) by applying a shell-less (ex-ovo) incubation system developed in our previous studies. After exposing embryos at Hamburger Hamilton stage (HHS) 1 to SCCPs (control, 0.1% DMSO; SCCPs-L, 200 ng/g; SCCPs-M, 2000 ng/g; SCCPs-H, 20,000 ng/g), we observed the development of embryos from the 3rd to 9th incubation day. Exposure to SCCPs-M and -H induced a significant reduction in survival, with an LD50 of 3100 ng/g on the 9th incubation day. Significant dose-dependent decreases in body length were observed from days 4-9. We also found that SCCPs-H decreased the blood vessel length and branch number on the 4th incubation day. Additionally, SCCPs-H significantly reduced the heart rate on the 4th and 5th incubation days. These findings suggest that SCCPs may have potential of developmental and cardiovascular toxicity during the early stages of chicken embryos. Quantitative PCR of the mRNA of genes related to embryonic development showed that SLC16A10 (a triiodothyronine transporter) level decreased in the SCCPs-H group, showing a significant positive correlation with the body length of embryos. THRA level, a thyroid hormone receptor, was significantly decreased in the SCCPs-H group, whereas that of DIO3 level, a deiodinase was significantly increased. These results suggest that SCCPs exposure induces developmental delays via the thyroxine signaling pathway. Analysis of thyroid hormones (THs) in blood plasma also indicated a significant reduction in thyroxine (T4) levels in the SCCPs-H group on the 9th incubation day of embryos. In conclusion, SCCPs induce developmental toxicity by disrupting thyroid functions at the early-life stage of chicken embryos.

Effect of in-ovo inoculation of betaine on hatchability, serum antioxidant levels, muscle gene expression and intestinal development of broiler chicks.

This study investigated the effects of in-ovo inoculation of betaine on hatchability, hatching weight, and intestinal development, as well as serum and expression levels of some antioxidants in the posthatched chicks. A total of 350 fertile eggs of Hubbard efficiency plus breeder's flock were incubated at normal incubation temperature (37.5°C) and randomly assembled into 3 groups with 4 replicates, and 25 eggs per each. The experimental groups were allocated as noninjected control group (CN), diluent-injected group (CP, 0.1 mL saline), and betaine-injected group (B, 2.5 mg in 0.1 mL saline). The injections were performed in the air cells of the eggs on the 12th day of the embryonic phase. Hatchability percentage, hatching weight, serum-reduced glutathione (GSH), and superoxide dismutase (SOD) were estimated in 7-day-old chicks. Moreover, expression levels of the nuclear factor erythroid 2-related factor 2 (Nrf2) and SOD were determined in the breast skeletal muscles of chicks. Jejunum histo-morphometric analysis was assessed with computerised morphometric measurements. The results revealed that the hatchability percentage was not influenced by in-ovo injection of betaine or vehicle while betaine significantly increased the hatchling's weight of chicks. Moreover, there were a significant increase in SOD and Nrf2 mRNA expression levels. In-ovo injection of betaine significantly induced positive effects on intestinal morphometry by ameliorating the jejunal villus length, the ratio of villus height to villus width, and absorptive surface area.

Mycofabrication of AgONPs derived from Aspergillus terreus FC36AY1 and its potent antimicrobial, antioxidant, and anti-angiogenesis activities.

BACKGROUND: There is an emergency need for the natural therapeutic agents to treat arious life threatening diseases such as cardio- vascular disease, Rheumatoid arthritis and cancer. Among these diseases, cancer is found to be the second life threatening disease; in this view the present study focused to synthesize the silver oxide nanoparticles (AgONPs) from endophytic fungus. METHODS: The endophytic fungus was isolated from a medicinal tree Aegle marmelos (Vilva tree) and the potential strain was screened through antagonistic activity. The endophytic fungus was identified through microscopic (Lactophenol cotton blue staining and spore morphology in culture media) and Internal Transcribed Spacer (ITS) 1, ITS 4 and 18S rRNA amplification. The endophyte was cultured for the synthesis of AgONPs and the synthesized NPs were characterized through UV- Vis, FT- IR, EDX, XRD and SEM. The synthesized AgONPs were determined for antimicrobial, antioxidant and anti- angiogenic activity. RESULTS: About 35 pigmented endophytic fungi were isolated, screened for antagonistic activity against 12 pathogens and antioxidant activity through DPPH radical scavenging assay; among the isolates, FC36AY1 explored the highest activity and the strain FC36AY1 was identified as Aspergillus terreus. The AgONPs were synthesized from the strain FC36AY1 and characterized for its confirmation, functional groups, nanostructures with unit cell dimensions, size and shape, presence of elements through UV-Vis spectrophotometry, FT-IR, XRD, SEM with EDX analysis. The myco-generated AgONPs manifested their antimicrobial and antioxidant properties with maximum activity at minimum concentration. Moreover, the inhibition of angiogenesis by the AgONPs in Hen's Egg Test on the Chorio-Allantoic Membrane analysis were tested on the eggs of Chittagong breed evinced at significant bioactivity least concentration at 0.1 µg/mL. CONCLUSIONS: Thus, the results of this study revealed that the fungal mediated AgONPs can be exploited as potential in biomedical applications.

Modifications in the Intestinal Functionality, Morphology and Microbiome Following Intra-Amniotic Administration (Gallus gallus) of Grape (Vitis vinifera) Stilbenes (Resveratrol and Pterostilbene).

This efficacy trial evaluated the effects of two polyphenolic stilbenes, resveratrol and pterostilbene, mostly found in grapes, on the brush border membrane functionality, morphology and gut microbiome. This study applied the validated Gallus gallus intra-amniotic approach to investigate the effects of stilbene administration versus the controls. Three treatment groups (5% resveratrol; 5% pterostilbene; and synergistic: 4.75% resveratrol and 0.25% pterostilbene) and three controls (18 MΩ H2O; no injection; 5% inulin) were employed. We observed beneficial morphological changes, specifically an increase in the villus length, diameter, depth of crypts and goblet cell diameter in the pterostilbene and synergistic groups, with concomitant increases in the serum iron and zinc concentrations. Further, the alterations in gene expression of the mineral metabolism proteins and pro-inflammatory cytokines indicate a potential improvement in gut health and mineral bioavailability. The cecal microbiota was analyzed using 16S rRNA sequencing. A lower α-diversity was observed in the synergistic group compared with the other treatment groups. However, beneficial compositional and functional alterations in the gut microbiome were detected. Several key microbial metabolic pathways were differentially enriched in the pterostilbene treatment group. These observations demonstrate a significant bacterial-host interaction that contributed to enhancements in intestinal functionality, morphology and physiological status. Our data demonstrate a novel understanding of the nutritional benefits of dietary stilbenes and their effects on intestinal functionality, morphology and gut microbiota in vivo.

In Ovo Early-in-Life Inhalation Exposure to Gas/Aerosol with a Chicken Embryo Model.

To assess the toxicities of gas/aerosol, inhalation exposure model is necessary. Especially important is the inhalation exposure early in life. Traditional inhalation exposure method requires specific instruments and may have to imitate the exposure either days before or after birth. Here, a new inhalation exposure method is introduced, which may be performed without any specific instruments and effectively expose late stage chicken embryos to gas/aerosol very early-in-life by inhalation. This method may facilitate the risk assessment and mechanistic studies regarding the early-in-life effects of gas/aerosol exposure.

Embryotoxic effects of Rovral® for early chicken (Gallus gallus) development.

Rovral® is a fungicide used to control pests that affect various crops and little is known regarding its effects on embryonic development of amniotes. Thus, this study aimed to determine the influence of Rovral® during chicken organogenesis using acute in ovo contamination. Fertilized eggs were inoculated with different concentrations of Rovral® (100, 300, 500 or 750 µl/ml), injected into the egg's air chamber. After 7 days, embryos were examined for possible malformations, staging, weight and mortality. Subsequently, head, trunk, limbs and eyes were measured for morphometry and asymmetry. For blood analysis, eggs were treated with 300 µl/ml Rovral® and glucose, presence of micronuclei and erythrocyte nuclei abnormalities determined. Treatments with Rovral® affected the mortality rate in a concentration-dependent manner. LC50 value was found to be 596 µl/ml which represents 397-fold higher than the recommended concentration for use. Rovral® produced several malformations including hemorrhagic, ocular and cephalic abnormalities. No significant changes were observed in body weight, staging, body measurements, symmetry and glucose levels of live embryos, which indicates this fungicide presents low toxicity under the analyzed conditions. Changes in erythrocyte nuclei were noted; however significant difference was observed only for presence of binucleated erythrocytes. It is important to point out that possibly more significant changes may have occurred at lower concentrations through chronic contamination. Therefore, caution is needed in the use of this fungicide, since it presents teratogenic and mutagenic potential.

A Comprehensive Study Monitoring the Venom Composition and the Effects of the Venom of the Rare Ethiopian Endemic Snake Species Bitis parviocula.

The Ethiopian endemic snake of the species Bitis parviocula, recognized for its colorful patterns, might be more interesting as we look deeper into the venom activity. We assayed the effects of venoms from the most widespread venomous African Bitis arietens and closely related species Bitis parviocula using The Hen's Egg Test-Chorioallantoic membrane test (HET-CAM) and Chicken embryotoxicity screening test (CHEST), acetylcholinesterase (AChE) analysis, cytotoxicity assay performed on cell lines and protein analysis of selected venoms. Our results indicated that B. parviocula venom contains vasoactive compounds that have a direct effect on blood vessels. The AChE analysis showed significant ability inhibiting AChE activity in embryonic tissue. Cytotoxicity observed on A549 ATCC® CCL-185™ cells indicates the possible presence of cytotoxic agents in B. parviocula venom. We proved previously described differences in the composition of venom obtained from B. arietans and B. parviocula by using electrophoresis and total protein concentration. Based on similarities in vasoactive effects observed after administration of venoms onto a chicken chorioallantoic membrane, we suggest that venom from B. arietans and B. parviocula might share certain venom proteins responsible for haemotoxicity. The main active components of B. parviocula venom are unknown. Our results suggest that it might be worth performing proteomic analysis of B. parviocula venom as it might contain medically valuable compounds.

New insights into the obligatory nature of cyclooxygenase-2 and PGE2 during early chick embryogenesis.

Temporal expression patterns and activity of two cyclooxygenase (COX-1 and COX-2) isoforms were analysed during early chick embryogenesis to evaluate their roles in development. COX-2 inhibition with etoricoxib resulted in significant structural anomalies such as anophthalmia (born without one or both eyes), phocomelia (underdeveloped or truncated limbs), and gastroschisis (an opening in the abdominal wall), indicating its significance in embryogenesis. Furthermore, the levels of PGE2, PGD2, PGF2α, and TXB2 were assessed using quantitative LC-MS/MS to identify which effector prostanoid (s) had their synthesis initiated by COX-2. COX-2 inhibition was only shown to reduce the level of PGE2 significantly, and hence it could be inferred that the later could be largely under the regulation of activated COX-2 in chick embryos. The compensatory increase in the activity of COX-1 observed in the etoricoxib-treated group helped to maintain the levels of PGD2, PGF2α, and TXB2. Though the roles of these three prostanoids in embryogenesis need to be further clarified, it appears that their contribution to the observed developmental anomalies is minimal. This study has shown that COX-2 is functionally active during chick embryogenesis, and it plays a central role in the structural configuration of several organs and tissues through its downstream effector molecule PGE2.

In Ovo Inoculation of Vitamin A Modulates Chicken Embryo Immune Functions.

Vitamin A mediates many important biological functions in humans and animals. Presence of vitamin A receptors on immune system cells emphasizes their role in immune functions. To assess the effects of in ovo inoculation of vitamin A on the immune system of chicken embryos, 18 days old embryonated eggs were inoculated with 3 different concentrations of retinoic acid (the active form of vitamin A) at 30, 90, and 270 µmol/egg via the amniotic sac. After 6, 18, and 24 h, the spleen and bursa of the embryos were collected for RNA extraction and real-time polymerase chain reaction. The results were dose dependant. After 24 h, inoculation with 270 µmol/egg downregulated relative expression of interferon IFN-α, IFN-ß, IFN-γ, interleukin (IL)-1ß, IL-2, CXCLi2, IL-12, and IL-13 compared to control in the spleen, indicating an anti-inflammatory effect at this concentration. In comparison, 90 µmol/egg induced greater expressions of the above genes at the same timepoint compared to the 270 µmol. The results of this study indicate that in ovo inoculation of vitamin A can modulate immune functions of the chicken embryo, which might be beneficial for induction of immune responses by in ovo vaccines.

Improvement in the performance and inflammatory reaction of Ross 708 broilers in response to the in ovo injection of 25-hydroxyvitamin D3.

The effects of the in ovo administration of vitamin D3 (D3) and its metabolite, 25-hydroxyvitamin D3 (25OHD3), on the performance, breast meat yield, and inflammatory responses of broilers fed commercial diets were investigated. Live embryonated Ross 708 broiler hatching eggs were randomly assigned to one of the following 5 in ovo injection treatments at 18 d of incubation: 1) noninjected; 2) diluent; diluent containing 3) 2.4-µg D3, 4) 2.4-µg 25OHD3, or 5) 2.4-µg D3 + 2.4-µg 25OHD3. A 50-µL solution volume of each prespecified treatment was injected into each egg using an Inovoject multiegg injector. At hatch, 18 male chicks were randomly assigned to each of 30 floor pens. The BW, BW gain, feed intake, and feed conversion ratio of the birds were determined in each dietary phase. At 14, 28, and 39 d of posthatch age (doa), plasma α-1-acid glycoprotein (AGP) levels in 1 bird in each of 6 replicate pens per treatment were determined at 14 and 39 doa. The pectoralis major and minor weights of those same birds were also determined. The remaining birds were processed at 43 doa, and the weights of their processing parts were determined. At 39 doa, the in ovo injection of 25OHD3 alone decreased plasma AGP concentrations in comparison with the noninjected, diluent, and D3-alone treatment groups. In addition, birds that received 25OHD3 alone had a greater BW at 42 doa than birds in the noninjected, diluent, and D3-alone treatment groups. At 39 and 43 doa, breast meat yield was increased in response to the in ovo injection of 25OHD3 alone in comparison to all other treatments. These results indicate that the in ovo injection of 2.4 µg of 25OHD3 resulted in an improvement in the performance and inflammatory responses of broilers. A reduction in the inflammatory response subsequent to the in ovo injection of 2.4 µg of 25OHD3 may have led to an increase in broiler performance.

Transcriptomic and Epigenetic Preservation of Genetic Sex Identity in Estrogen-feminized Male Chicken Embryonic Gonads.

Whereas in ovo exposure of genetically male (ZZ) chicken embryos to exogenous estrogens temporarily feminizes gonads at the time of hatching, the morphologically ovarian ZZ-gonads (FemZZs for feminized ZZ gonads) are masculinized back to testes within 1 year. To identify the feminization-resistant "memory" of genetic male sex, FemZZs showing varying degrees of feminization were subjected to transcriptomic, DNA methylome, and immunofluorescence analyses. Protein-coding genes were classified based on their relative mRNA expression across normal ZZ-testes, genetically female (ZW) ovaries, and FemZZs. We identified a group of 25 genes that were strongly expressed in both ZZ-testes and FemZZs but dramatically suppressed in ZW-ovaries. Interestingly, 84% (21/25) of these feminization-resistant testicular marker genes, including the DMRT1 master masculinizing gene, were located in chromosome Z. Expression of representative marker genes of germline cells (eg, DAZL or DDX4/VASA) was stronger in FemZZs than normal ZZ-testes or ZW-ovaries. We also identified 231 repetitive sequences (RSs) that were strongly expressed in both ZZ-testes and FemZZs, but these RSs were not enriched in chromosome Z. Although 94% (165/176) of RSs exclusively expressed in ZW-ovaries were located in chromosome W, no feminization-inducible RS was detected in FemZZs. DNA methylome analysis distinguished FemZZs from normal ZZ- and ZW-gonads. Immunofluorescence analysis of FemZZ gonads revealed expression of DMRT1 protein in medullary SOX9+ somatic cells and apparent germline cell populations in both medulla and cortex. Taken together, our study provides evidence that both somatic and germline cell populations in morphologically feminized FemZZs maintain significant transcriptomic and epigenetic memories of genetic sex.

Acute, reproductive, and developmental toxicity of essential oils assessed with alternative in vitro and in vivo systems.

Essential oils (EOs) have attracted increased interest for different applications such as food preservatives, feed additives and ingredients in cosmetics. Due to their reported variable composition of components, they might be acutely toxic to humans and animals in small amounts. Despite the necessity, rigorous toxicity testing in terms of safety evaluation has not been reported so far, especially using alternatives to animal models. Here, we provide a strategy by use of alternative in vitro (cell cultures) and in vivo (Caenorhabditis elegans, hen's egg test) approaches for detailed investigation of the impact of commonly used rosemary, citrus and eucalyptus essential oil on acute, developmental and reproductive toxicity as well as on mucous membrane irritation. In general, all EOs under study exhibited a comparable impact on measured parameters, with a slightly increased toxic potential of rosemary oil. In vitro cell culture results indicated a concentration-dependent decrease of cell viability for all EOs, with mean IC50 values ranging from 0.08 to 0.17% [v/v]. Similar results were obtained for the C. elegans model when using a sensitized bus-5 mutant strain, with a mean LC50 value of 0.42% [v/v]. In wild-type nematodes, approximately tenfold higher LC50 values were detected. C. elegans development and reproduction was already significantly inhibited at concentrations of 0.5% (wild-type) and 0.1% (bus-5) [v/v] of EO, respectively. Gene expression analysis revealed a significant upregulation of xenobiotic and oxidative stress genes such as cyp-14a3, gst-4, gpx-6 and sod-3. Furthermore, all three EOs under study showed an increased short-time mucous membrane irritation potential, already at 0.5% [v/v] of EO. Finally, GC-MS analysis was performed to quantitate the relative concentration of the most prominent EO compounds. In conclusion, our results demonstrate that EOs can exhibit severe toxic properties, already at low concentrations. Therefore, a detailed toxicological assessment is highly recommended for each EO and single intended application.

Effect of in ovo application of hydroxyapatite nanoparticles on chicken embryo development, oxidative status and bone characteristics.

Intensive selection in modern lines of fast-growing chickens has caused several skeletal disorders. Therefore, current research is focused on methods to improve the bones of birds. A new potential solution is in ovo technology using nanoparticles with a high specificity for the bone tissue. Thus, the objective of the present study was to evaluate the effect of in ovo application of hydroxyapatite nanoparticles (HA-NP) in different concentrations (50, 100 and 500 µg/ml colloids) on chicken embryo development, with a particular focus on the oxidative status and bone characteristics of the embryo. The results showed that in ovo treatment with HA-NP did not negatively affect hatchability and body weight. However, bone weight was reduced in 500 µg/ml group. The concentrations of calcium, phosphorus and crude ash were not affected. The modulatory effect of HA-NP was observed on the basis of antioxidative markers - superoxide dismutase, total antioxidant status, malondialdehyde in serum and selected tissues. Glutathione concentration in serum suggested higher metabolic stress. Among bone turnover markers, the concentration of osteocalcin was found to be significantly affected by HA-NP injection. Thus, the in ovo application of HA-NP could modify the molecular responses at the stage of embryogenesis but these changes were not reflected in embryo growth and even slowed down bone development. Nevertheless, the question for the follow-up research is whether in ovo administration of HA-NP would affect the antioxidative status and bone turnover resulting in improved bone conditions and body gain in post hatch chickens.

Effects of gonadotropins on testis cell subpopulations of newly hatched chicks treated during embryonic development.

The aim of the present study was to evaluate histological and stereological changes, as well as the variations in the number and size of cells from diverse cell subpopulations in testes of newly hatched chicks treated with follicle stimulating hormone (FSH) and luteinizing hormone (LH) during embryonic development. Stereological results indicated that in FSH-treated chicks total volume of the tubular compartment constitutes most of the testis. In contrast, the total volume of interstitial tissue constitutes most of the testis of LH-treated chicks. Results indicate that the number of germ and Sertoli cells increases as a result of FSH and LH treatment, but in FSH-treated testis, Sertoli cells were the most numerous cell type in seminiferous tubules; whereas germ cells were the most numerous cell type in testis of LH-treated chicks. Results also indicate there was a larger total volume of Leydig cells in the testes of FSH- and LH-treated chicks. The larger volume of Leydig cells in FSH-treated chicks is due to a larger cellular volume of these cells, and not due to the number, which remains constant. In contrast, in testes of LH-treated chicks, there is a larger number and volume of Leydig cells. These results indicate the testes of chick embryos respond to FSH and LH treatment, with there being modifications in the seminiferous tubules and interstitial tissue, but these changes differ markedly, indicating that FSH and LH have differential effects on chick testes.

Codelivery of BV6 and anti-IL6 siRNA by hyaluronate-conjugated PEG-chitosan-lactate nanoparticles inhibits tumor progression.

AIMS: Increased expression of inhibitor of apoptosis (IAP) genes has been associated with progressive cancer and chemoresistance. Accordingly, blockade of IAPs by BV6 has resulted in ameliorative outcomes. Interleukin (IL)-6 is another important mediator involved in the growth and survival of tumor cells. Therefore, we hypothesized that simultaneous inhibition of IAPs and IL-6 could be a new promising anti-tumor treatment strategy. MATERIALS AND METHODS: In this study, we generated and characterized hyaluronate-PEG-Chitosan-Lactate (H-PCL) nanoparticles (NPs) to simultaneously deliver IL6-specific siRNA and BV6 to 4T1 (breast cancer) and CT26 (colon cancer) cells, and investigate the anti-tumor properties of this combination therapy both in vitro and in vivo. KEY FINDINGS: H-PCL NPs exhibited good physicochemical properties leading to efficient transfection of cancer cells and suppression of target molecules. Moreover, combination therapy synergistically increased apoptosis, as well as decreased cell migration, proliferation, colony formation, and angiogenesis in both 4T1 and CT26 cell lines and suppressed cancer progression in tumor-bearing mice that was associated with enhanced survival time. SIGNIFICANCE: These findings imply the effectiveness of cancer combination therapy by using H-PCL NPs loaded with anti-IL-6 siRNA and BV6.