Beneficial Bacteria and Plant Extracts Promote Honey Bee Health and Reduce Nosema ceranae Infection.

The research aims to give new insights on the effect of administering selected bacterial strains, isolated from honey bee gut, and/or a commercial plant extract blend (HiveAlive®) on Nosema ceranae. Analyses were first performed under laboratory conditions such as different infective doses of N. ceranae, the effect of single strains and their mixture and the influence of pollen administration. Daily survival and feed consumption rate were recorded and pathogen development was analysed using qPCR and microscope counts. Biomarkers of immunity and physiological status were also evaluated for the different treatments tested using one bacterial strain, a mixture of all the bacteria and/or a plant extract blend as treatments. The results showed an increase of abaecin transcript levels in the midgut of the honey bees treated with the bacterial mixture and an increased expression of the protein vitellogenin in the haemolymph of honey bees treated with two separate bacterial strains (Bifidobacterium coryneforme and Apilactobacillus kunkeei). A significant effectiveness in reducing N. ceranae was shown by the bacterial mixture and the plant extract blend regardless of the composition of the diet. This bioactivity was seasonally linked. Quantitative PCR and microscope counts showed the reduction of N. ceranae under different experimental conditions. The antiparasitic efficacy of the treatments at field conditions was studied using a semi-field approach which was adapted from research on insecticides for the first time, to analyse antiparasitic activity against N. ceranae. The approach proved to be reliable and effective in validating data obtained in the laboratory. Both the mixture of beneficial bacteria and its association with Hive Alive® are effective in controlling the natural infection of N. ceranae in honey bee colonies.

Vitamin D3 promotes fish oocyte development by directly regulating gonadal steroid hormone synthesis†.

Vitamin D receptors and vitamin D3-metabolizing enzymes have been found to be highly expressed in the ovaries and spermatophores of fish. However, the role of vitamin D3 on fish gonadal development has rarely been reported. In this study, 2-month-old female zebrafish were fed with different concentrations of vitamin D3 diets (0, 700, 1400, and 11 200 IU/kg) to investigate the effects of vitamin D3 on ovarian development. The diet with 0 IU/kg vitamin D3 resulted in elevated interstitial spaces, follicular atresia, and reproductive toxicity in zebrafish ovaries. Supplementation with 700 and 1400 IU/kg of vitamin D3 significantly increased the oocyte maturation rate; upregulated ovarian gonadal steroid hormone synthesis capacity; and elevated plasma estradiol, testosterone, and ovarian vitellogenin levels. Furthermore, the current study identified a vitamin D response element in the cyp19a1a promoter and demonstrated that 1.25(OH)2D3-vitamin D response directly activated cyp19a1a production through activating the vitamin D response element. In conclusion, this study shows that an appropriate concentration of vitamin D3 can promote zebrafish ovarian development and affect vitellogenin synthesis through the vdr/cyp19a1a/er/vtg gene axis.

The effects of thymol, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on viability, the Nosema sp. spore load and the expression of vg and sod-1 genes in infected honey bees.

This study was done to investigate the effects of thymol, fumagillin, oxalic acid (Api-Bioxal) and hops extract (Nose-Go) on Nosema sp. spore load, the expression of vitellogenin (vg) and superoxide-dismutase-1 (sod-1) genes and mortality of bees infected with N. ceranae. Five healthy colonies were assigned as the negative control, and 25 Nosema sp. infected colonies were assigned to five treatment groups including: the positive control: no additive to sirup; fumagillin 26.4 mg/L, thymol 0.1 g/L, Api-Bioxal 0.64 g/L and Nose-Go 5.0 g/L sirup. The reduction in the number of Nosema sp. spores in fumagillin, thymol, Api-Bioxal and Nose-Go compared to the positive control was 54, 25, 30 and 58%, respectively. Nosema sp. infection in all infected groups increased (p < .05) Escherichia coli population compared to the negative control. Nose-Go had a negative effect on lactobacillus population compared to other substances. Nosema sp. infection decreased vg and sod-1 genes expression in all infected groups compared to the negative control. Fumagillin and Nose-Go increased the expression of vg gene, and Nose-Go and thymol increased the expression of sod-1 gene than the positive control. Nose-Go has the potential to treat nosemosis if the necessary lactobacillus population is provided in the gut.

Reprotoxicity induced by acute exposure to aqueous tuber extract of Peruvian Maca (Lepidium meyenii Walp.) in Caenorhabditis elegans.

Maca (Lepidium meyenii Walp.) has been used in folk medicine to treat fertility disturbances, a claim that has been evidenced in some studies. However, the clinical trials validating this use have shown paradoxical findings and then maca safety is not well known. This study investigated the effects and mechanisms by which maca affects the reproductive system using an in vivo model, the nematode Caenorhabditis elegans. Tuber maca powder, obtained from local commerce, was used to prepare the aqueous extract. Worms were acutely exposed to maca extracts (40, 120, 240, and 330 µg/µl), and 48 h after treatments, physiological and biochemical assays were conducted. Maca extract caused a significant decrease in total number of eggs and in the number of eggs per worm. These effects were associated to increased lipid peroxidation, reduced triacylglycerol levels, and also impaired vit-2 (vitellogenin) expression, besides increase in the number of apoptotic germline cells. We have found quantifiable levels of alkaloids in this maca extract, which presence could be related to this toxicity. Collectively, our data suggest that maca extract exposure causes reproductive toxicity to worms that could be, at least in part, associated to both an increase in apoptosis of germline cells and also to a decrease in vitellogenin expression, needed for egg yolk production and, consequently, successful reproduction.

Vitellogenin 1 is essential for fish reproduction by transporting DHA-containing phosphatidylcholine from liver to ovary.

Vitellogenins (Vtgs) are essential for female reproduction in oviparous animals, yet the exact roles and mechanisms remain unknown. In the present study, we knocked out vtg1, which is the most abundant Vtg in zebrafish, Danio rerio via the CRISPR/Cas 9 technology. We aimed to identify the roles of Vtg1 and related mechanisms in reproduction and development. We found that, the Vtg1-deficient female zebrafish reduced gonadosomatic index, egg production, yolk granules and mature follicles in ovary compared to the wide type (WT). Moreover, the Vtg1-deficient zebrafish diminished hatching rates, cumulative survival rate, swimming capacity and food intake, but increased malformation rate, and delayed swim bladder development during embryo and early-larval phases. The Vtg1-deficiency in female broodstock inhibited docosahexaenoic acid-enriched phosphatidylcholine (DHA-PC) transportation from liver to ovary, which lowered DHA-PC content in ovary and offspring during larval stage. However, the Vtg1-deficient zebrafish increased gradually the total DHA-PC content via exogeneous food intake, and the differences in swimming capacity and food intake returned to normal as they matured. Furthermore, supplementing Vtg1-deficient zebrafish with dietary PC and DHA partly ameliorated the impaired female reproductive capacity and larval development during early phases. This study indicates that, DHA and PC carried by Vtg1 are crucial for female fecundity, and affect embryo and larval development through maternal-nutrition effects. This is the first study elucidating the nutrient and physiological functions of Vtg1 and the underlying biochemical mechanisms in fish reproduction and development.

Role of adipokinetic hormone in the Colorado potato beetle, Leptinotarsa decemlineata infected with the entomopathogenic nematode Steinernema carpocapsae.

The effects of the entomopathogenic nematode Steinernema carpocapsae on the Colorado potato beetle (CPB) Leptinotarsa decemlineata and the involvement of adipokinetic hormone (AKH) in the responsive reactions were examined in this study. It was observed that nematode application doubled the amount of AKH (Peram-CAH-I and Peram-CAH-II) in the central nervous system of L. decemlineata, indicating mobilization of anti-stress reactions in the body. Furthermore, the external co-application of Peram-CAH-II with the nematode significantly increased beetle mortality (5.6 and 1.8 times, 1 and 2 days after application, respectively). The mechanism underlying this phenomenon was investigated. As the effect on gut characteristics was equivocal, it was assumed that the nematodes profited from the observed mobilization of metabolites from the fat body into the Peram-CAH-II-induced hemolymph. This phenomenon supplied nematodes with a more nutrient-dense substrate on which they propagated. Furthermore, Peram-CAH-II lowered vitellogenin expression in the fat body, particularly in males, thus limiting the anti-pathogen defense capacity of the protein. However, there could be other possible mechanisms underpinning this chain of events. The findings could be theoretically intriguing but could also aid in developing real insect pest control methods in the future.

Green tea powder inclusion promoted hatchability through increased yolk antioxidant activity.

Dietary supplementation of green tea powder (GTP) changes egg quality of hens, however, whether these changes affect incubation is still unknown. This study was to compare the proteomic difference of incubated eggs from hens with GTP supplemented or not. Huainan partridge chickens (1,080) at 35 wk of age were allocated into 2 groups, one group fed basal diet (CG) and one group fed basal diet plus 1% GTP (EG). After 4 wk feeding, artificially fertilized eggs were collected for yolk cholesterol determination and incubation. During incubation, 6 embryos from each group were randomly selected in each day for yolk protein extraction and quantification. Yolk cholesterol content was significantly lower, while the hatchability was significantly higher in EG than that of the CG group (P < 0.05). Yolk protein concentration at embryonic days (ED) of 0, 2, 6, and 13 showed significant changes and were selected for proteomic analysis by 2-dimensional gel electrophoresis combined with liquid chromatography-tandem mass spectrometry. Fifty-one differentially expressed (DE) protein spots were identified among different incubation stages between CG and EG group which were mainly classified into vitellogenin, immunoglobulin, and ovoinhibitor, and occupied 45.1, 23.5, and 15.7%, respectively, to the total DE proteins. Ovotransferrin, participated in extracellular sequestering of iron ion process, was significantly lower in EG group than that of the CG group (P < 0.05). Ig light chain precursor (Immunoglobulin) exhibited higher expression at ED6 in EG group as compared with that of the CG group, and was participated in immune response related processes. Ovoinhibitor, mainly involved in protease binding activity, showed lower abundance at ED13 in EG group as compared with that of the CG group. Vitellogenin-3, showed lower expression in EG group as compared with that of the CG group, was mainly participated in lipid transportation and localization according to GO enrichment. Chickens fed diet with GTP provided eggs more antioxidant ability that increased hatchability, indicated that GTP could be considered as additive in breeding layer.

Dietary plant pigment on blood-digestive physiology, antioxidant-immune response, and inflammatory gene transcriptional regulation in spotted snakehead (Channa punctata) infected with Pseudomonas aeruginosa.

The current study addressed to investigate the effect of lycopene (LYC) on blood physiology, digestive-antioxidant enzyme activity, specific-nonspecific immune response, and inflammatory gene transcriptional regulation (cytokines, heat shock proteins, vitellogenins) in spotted snakehead (Channa punctata) against Pseudomonas aeruginosa. In unchallenged and challenged fish treated with 200 mg LYC enriched diet the growth performance and digestive-antioxidant enzymes increased after 30 days, whereas with inclusion of 100 or 400 mg LYC in the diets, the increase manifested on or after 45 days. No mortality in fish treated with any LYC diet against P. aeruginosa was revealed. In the unchallenged and challenged fish the phagocytic (PC) activity in head kidney (HK) and spleen were significantly enhanced when fed the control diet or other LYC diets, whereas the respiratory burst (RB) activity and nitric oxide (NO) production significantly increased when fed the 200 mg diet for 45 and 60 days. Similarly, the lysozyme (Lyz) activity in the HK and spleen, and total Ig content in serum were significantly higher in both groups fed the 200 mg LYC diet for 15, 45, and 60 days. Heat shock protein (Hsp 70) was significantly improved in the uninfected group fed the 200 mg LYC diet for 45 and 60 days, but Hsp27 did not significantly change among the experimental groups at any time points. TNF-α and IL-6 mRNA pro-inflammatory cytokine expression significantly increased in both groups fed the 200 mg LYC diet after 45 and 60 days, while the IL-12 mRNA expression was moderate in both groups fed the same diet for 60 days. The IL-10 did not significant mRNA expression between groups at any sampling. The iNOS and NF-κB mRNA expression was pointedly high in both groups fed the 200 mg LYC diet on day 45 and 60. Vitellogenin A (VgA) mRNA was significantly higher in the uninfected fish fed the 100 and 200 mg LYC diets for 45 and 60 days, but VgB did not reveal significant difference between the treatment groups at any time points. The present results suggest that supplementation of LYC at 200 mg significantly modulate the blood physiology, digestive-antioxidant enzymes, specific-nonspecific immune parameters, and cytokines, Hsp, and vitellogenins in spotted snakehead against P. aeruginosa.

Extra-Fortification of Zinc Upsets Vitellogenin Gene Expression and Antioxidant Status in Female of Clarias magur brooders.

The present experiment was designed to evaluate the effect of graded level of zinc on Vitellogenin gene (Vtg) expression and antioxidant enzymes in threatened catfish, Clarias magur (C. magur). One hundred and eighty female C. magur with an average weight of 145 ± 5 g were allocated in twelve cemented tanks with dimension 4.5 × 2 × 1 m for a period of 60 days. Fish were distributed in four groups with three replicates following the completely randomised design. The first group treated as control (C) fed with basal diet contained normal zinc level, and remaining groups were fed with basal diets having 50, 200 and 300 mg/kg zinc acetate and treated as T1, T2 and T3 respectively. To evaluate the effect of dietary zinc supplementation on Vtg gene expression, three sampling were carried out, I sampling (April, before starting the experimental trail), II sampling (May, after 1 month of feeding trail) and III sampling (June before breeding season). In the present study, a dose-dependent relationship between Vtg gene expression and zinc inclusion in the diet of threatened catfish, C. magur, was reported. Vtg gene expression increased in all groups from I sampling to II sampling but the highest Vtg gene expression was found in T1 group and the lowest in T3 group at II sampling. Vtg gene expression among the treatments differs significantly (P < 0.05) in each sampling. Accumulation of zinc was measured by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) in C. magur and it was reported that the significantly higher (P < 0.05) zinc was accumulated in the liver and ovary of T3 group as compared to other groups. The antioxidant enzyme activities (superoxide dismutase, SOD, catalase and GST) were also measured in different tissues (liver, gill and ovary) to evaluate the effect of extra-supplementation of zinc on the antioxidant status. In T3 group, SOD, catalase and GST activities were significantly higher than those in other groups. In the current study, serum glucose level was also measured and it was found in increasing trend with inclusion of zinc in the diet of C. magur. In the present study, it can be concluded that the zinc exhibits beneficial effect only up to 50 mg/kg. Thus, it is concluded that supplementation of zinc at 200 mg/kg or more disrupts Vtg gene expression and antioxidant status.

Modulation of the PGE2-Mediated Pathway in the Eclosion Blocking Effect of Flumethrin and Terpenoid Subfraction Isolated from Artemesia nilagirica in Rhipicephalus annulatus.

Prostaglandins are a group of important cell-signaling molecules involved in the regulation of ovarian maturation, oocyte development, egg laying and associated behaviors in invertebrates. However, the presence of prostaglandin E2 (PGE2), the key enzymes for PGE2 biosynthesis and its interference by drugs were not investigated previously in the ovary of ticks. The present study was undertaken to assess the modulation of the PGE2-mediated pathway in the eclosion blocking effect of flumethrin and terpenoid subfraction isolated from Artemisia nilagirica in Rhipicephalus annulatus ticks. The acaricidal activities and chemical profiling of the terpenoid subfraction were performed. The localization of the cyclooxygenase1 (COX1) and prostaglandin E synthase (PGES) enzymes and the quantification of PGE2 in the ovaries of the ticks treated with methanol (control), flumethrin and terpenoid subfraction were also undertaken. In addition, the vitellogenin concentration in hemolymph was also assayed. Both flumethrin and the terpenoid subfraction of A. nilagirica elicited a concentration-dependent inhibition of fecundity and blocking of hatching of the eggs. The COX1 could not be detected in the ovaries of treated and control ticks, while there was no significant difference observed in the concentration of vitellogenin (Vg) in them. The presence of PGES in the oocytes of control ticks was confirmed while the immunoreactivities against PGES were absent in the vitellogenic oocytes of ticks treated with flumethrin and terpenoid subfraction. The levels of PGE2 were below the detection limit in the ovaries of the flumethrin-treated ticks, while it was significantly lower in the ovaries of the terpenoid subfraction-treated ticks. Hence, the prostaglandin E synthase and PGE2 were identified as very important mediators for the signaling pathway for ovarian maturation and oviposition in ticks. In addition, the key enzyme for prostaglandin biosynthesis, PGES and the receptors for PGE2 can be exploited as potential drug targets for tick control. The detection of PGES by immunohistochemistry and quantification of PGE2 by LC-MSMS can be employed as valuable tools for screening newer compounds for their eclosion blocking acaricidal effects.

Silencing of a putative alanine aminotransferase (ALT) gene influences free amino acid composition in hemolymph and fecundity of the predatory bug, Cyrtorhinus lividipennis Reuter.

In Asian rice systems, Cyrtorhinus lividipennis Reuter is an important predator that preys on rice planthopper eggs and young nymphs, as a primary food source. Alanine aminotransferase (ALT) acts in many physiological and biochemical processes in insects. We cloned the full-length complementary DNA of C. lividipennis ClALT. Expression analysis showed higher expression in the fat body and midgut compared to other tissues. It is expressed in all C. lividipennis developmental stages and at least four organs. Silencing of ClALT by RNA interference significantly decreased the ClALT enzyme activity and ClALT expression compared to dsGFP-treated controls at 2 days after emergence (DAE). Silencing of ClALT influenced free hemolymph amino acid compositions, resulting in a reduction of Aspartic acid (Asp) and Alanine (Ala) proportions, and increased Cysteine (Cys) and Valine (Val) proportions in females at 2 DAE. dsClALT treatments led to decreased soluble total protein concentrations in ovary and fat body, and to lower reduced vitellogenin (Vg) expression, body weight, and the numbers of laid eggs. The double-stranded RNA viruse treatments also led to prolonged preoviposition periods and hindered ovarian development. Western blot analysis indicated that silencing ClALT also led to reduced fat body Vg protein abundance at 2 DAE. These data support our hypothesis that ClALT influences amino acid metabolism and fecundity in C. lividipennis.

Health Effects and Life Stage Sensitivities in Zebrafish Exposed to an Estrogenic Wastewater Treatment Works Effluent.

A wide range of health effects in fish have been reported for exposure to wastewater treatment work (WwTW) effluents including feminized responses in males. Most of these exposure studies, however, have assessed acute health effects and chronic exposure effects are less well established. Using an Estrogen Responsive Element-Green Fluorescent Protein (ERE-GFP)-Casper transgenic zebrafish, we investigated chronic health effects and life stage sensitivities for exposure to an estrogenic WwTW effluent and the synthetic estrogen 17α-ethinylestradiol (EE2). Exposure to the WwTW effluent (at full strength;100%) and to 10 ng/L (nominal) EE2 delayed testis maturation in male fish but accelerated ovary development in females. Exposure to 50% and 100% effluent, and to 10 ng/L EE2, also resulted in skewed sex ratios in favor of females. Differing patterns of green fluorescent protein (GFP) expression, in terms of target tissues and developmental life stages occurred in the ERE-GFP- zebrafish chronically exposed to 100% effluent and reflected the estrogenic content of the effluent. gfp and vitellogenin (vtg) mRNA induction were positively correlated with measured levels of steroidal estrogens in the effluent throughout the study. Our findings illustrate the importance of a fish's developmental stage for estrogen exposure effects and demonstrate the utility of the ERE-GFP zebrafish for integrative health analysis for exposure to estrogenic chemical mixtures.

Pyriproxyfen induced impairment of reproductive endocrine homeostasis and gonadal histopathology in zebrafish (Danio rerio) by altered expression of hypothalamus-pituitary-gonadal (HPG) axis genes.

Pyriproxyfen (PPF), a broad-spectrum insecticide known to cause reproductive and endocrine disruption in invertebrates, while the data is scarce in aquatic vertebrates. The goal of this study is to investigate the impact of PPF on reproductive endocrine system of male and female zebrafish along hypothalamus-pituitary-gonadal (HPG) axis. In brain, PPF caused significant alteration in the transcripts of erα, lhß, and cyp19b genes in male and fshß, lhß, and cyp19b genes in female zebrafish. The downstream genes of steroidogenic pathway like, star, 3ßhsd, 17ßhsd, and cyp19a expression were significantly altered in gonad of both sexes. Subsequent changes in circulatory steroid hormone levels lead to imbalance in hormone homeostasis as revealed from estradiol/testosterone (E2/T) ratio. Further, the vitellogenin transcript level was enhanced in hepatic tissues and their blood plasma content was increased in male (16.21%) and declined in female (21.69%). PPF also induced histopathological changes in gonads such as, reduction of mature spermatocytes in male and vitellogenic oocytes in female zebrafish. The altered E2/T ratio and gonadal histopathology were supported by the altered transcript levels of HPG axis genes. Overall, these findings provide new insights of PPF in zebrafish reproductive system and highlights for further investigations on its potential risks in aquatic environment.

Refinement of an OECD test guideline for evaluating the effects of endocrine disrupting chemicals on aromatase gene expression and reproduction using novel transgenic cyp19a1a-eGFP zebrafish.

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 µg/L). In addition to "classical" endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 µg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 µg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.

Reproductive performance and vitellogenin mRNA transcript abundance in the hepatopancreas of female Litopenaeus vannamei fed diets with different soy lecithin content.

A 30-day nutritional study was conducted for determining the effects of supplementing soy lecithin (SL) in the diet of female Litopenaeus vannamei. Four isonitrogenous and isoenergetic diets were supplemented with graded amounts of SL including 0 (control), 20, 40 and 60 g Kg-1. The brooding specimens fed the 40 g SL/kg diet had the greatest gonadosomatic index (3.4 ±â€¯0.2 %) and the shortest latency period from eyestalk ablation to the first spawning (5 days). In addition, there was a larger content of long chain polyunsaturated fatty acids (LC-PUFA) in the hepatopancreas of brooding specimens fed with the 20 and 40 SL/kg diets compared to the other treatments. Furthermore, brooding specimens fed with the 40 g SL/kg diet had the greatest hemolymph cholesterol and high density lipoprotein concentrations. The abundance of vitellogenin mRNA transcript was greater in the hepatopancreas of brooding specimens fed with the 40 and 60 g SL/kg diets compared with the other groups. Results indicate that supplementing diets with 40 g SL/kg can improve growth and reproductive performance in L. vannamei female brooding specimens probably by enhancing LC-PUFA deposition and enhancing vitellogenin gene expression, as indicated by a greater abundance of mRNA transcript for vitellogenin, in the hepatopancreas.

Genistein improves the reproductive performance and bone status of breeder hens during the late egg-laying period.

Genistein (GEN), a type of soy isoflavones, is similar to estrogen structurally and functionally. The effects of dietary gen on the reproductive performance and bone status of breeder hens were investigated. A total pf 720 laying broiler breeder (LBB) hens were randomly allocated into 3 groups with supplemental dietary GEN doses (0, 40, 400 mg/kg). Each treatment has 8 replicates of 30 birds. The results indicated that supplemental GEN significantly improved the egg production and eggshell strength of LBB hens. Dietary GEN was deposited into the egg yolk, which decreased malonaldehyde in the follicle and egg yolk. The levels of vitellogenin (VTG), progesterone, and follicle-stimulating hormone in the serum of GEN-treated groups were elevated compared with the control group. Furthermore, GEN treatment downregulated the mRNA expression of insulin-like growth factor binding protein in the fallopian tube, whereas 40 mg/kg GEN treatment upregulated estrogen receptor α expression. Both the mRNA expression of VTG-II in the liver and mRNA expression of amphiregulin in the fallopian tube were upregulated after 40 and 400 mg/kg GEN treatment. In the 400 mg/kg GEN-treated group, the levels of calcitonin and alkaline phosphatase in the serum were increased compared with the control group, which was consistent with the increased levels of calcium and phosphorus in the tibia. Supplemental GEN (400 mg/kg) improved the tibia strength of LBB hens, whereas 40 mg/kg GEN had better effects on laying performance. In summary, dietary GEN could improve the egg production and quality, as well as the bone status of LBB hens during the late egg-laying period.

Effect of sub-chronic dietary L-selenomethionine exposure on reproductive performance of Red Swamp Crayfish, (Procambarus clarkii).

The effect of selenium (Se) on the reproductive system has been investigated in both humans and vertebrates, but few studies of female fertility and reproduction in invertebrate have been reported. This study is aimed to investigate the effect of SeMet on growth performance and reproductive system after crayfish were fed with graded levels of dietary SeMet (0, 1.49, 3.29, 10.02, 30.27 or 59.8 µg Se/g dry weight) for 60 days. Crayfish treated with the high levels of SeMet (10.02, 30.27 and 59.76 µg Se/g) exhibited decreasing FW and CL in both male and female. Interestingly, Se accumulation was higher in ovary than in other tissues, suggesting that ovary may serve as a target organ for Se accumulation. We found that dietary Se concentration of 10.02 µg Se/g significantly improved the spawning rate, promoted the synchronized spawning, and up-regulated the expressions of mRNA of cdc2 and vitellogenin, with significantly increased E2 and VTG concentrations in hemolymph of female crayfish. However, a marked decrease of the E2 contents and spawning rate was observed in the groups treated with 30.27 and 59.76 µg Se/g diets. In conclusion, the results of this study indicated that the Se had maximum accumulation in ovary, affecting the reproductive capacity by intervening the expression of cdc2 and vitellogenin in the reproductive system. The LOAEL to induce FW was observed in crayfish fed with 10.02 µg Se/g diet, and its value can cause toxicity within the range of natural concentration, so the addition of Se in the feed should be within 10.02 µg Se/g.

Probiotic Intake Increases the Expression of Vitellogenin Genes in Laying Hens.

A promising approach for slowing down the rate of reproductive aging is the use of probiotic bacteria as a feed additive. In the current study was investigated the influence of the intake of a potential probiotic on the follicle content and expression of vitellogenin genes (vtg1, vtg2, vtg3) in aged hens. RNA was isolated from liver samples collected from 570-day-old laying hens and gene expression levels were measured using RT-PCR. Bacillus subtilis KATMIRA1933 supplementation had a positive effect on the number of formed follicles in hens and also triggered a significant increase in the relative expression levels of vtg1, vtg2, and vtg3. A Bacillus amyloliquefaciens B-1895 enriched diet or a combination of the two strains had a modest effect on both the number of follicles and the expression of vitellogenin genes. Additionally, the study demonstrates that vitellogenin mRNA expression levels can be considered as a biomarker in a convenient approach for analyzing the hen's egg-laying ability.

Advancing the Zebrafish embryo test for endocrine disruptor screening using micro-injection: Ethinyl estradiol as a case study.

Fish (embryo) toxicity test guidelines are mostly based on aquatic exposures. However, in some cases, other exposure routes can be more practical and relevant. Micro-injection into the yolk of fish embryos could offer a particular advantage for administering hydrophobic compounds, such as many endocrine disruptors. Single-dose micro-injection was compared with continuous aquatic exposure in terms of compound accumulation and biological responses. 17α-Ethinyl estradiol (EE2) was used as a model compound. First, the optimal solvent and droplet size were optimized, and needle variation was assessed. Next, biological endpoints were evaluated. The accumulated internal dose of EE2 decreased over time in both exposure scenarios. Estrogen receptor activation was concentration/injected dose dependent, increased daily, and was related to esr2b transcription. Transcription of vitellogenin 1 (vtg1) and brain aromatase (cyp19a1b) was induced in both scenarios, but the cyp19a1b transcription pattern differed between routes. Injection caused an increase in cyp19a1b transcripts from 48 hours post fertilization (hpf) onward, whereas after aquatic exposure the main increase occurred between 96 and 120 hpf. Some malformations only occurred after injection, whereas others were present for both scenarios. We conclude that responses can differ between exposure routes and therefore micro-injection is not a direct substitute for, but can be complementary to aquatic exposure. Nevertheless, vtg1and cyp19a1b transcription and estrogen receptor activation are suitable biomarkers for endocrine disruptor screening in both scenarios. Environ Toxicol Chem 2019;38:533-547. © 2018 SETAC.

Effects of triclosan (TCS) on hormonal balance and genes of hypothalamus-pituitary- gonad axis of juvenile male Yellow River carp (Cyprinus carpio).

Triclosan (TCS) is a broad spectrum antimicrobial agent which has been widely dispersed and determinated in the aquatic environment. However, the effects of TCS on reproductive endocrine in male fish are poorly understood. In this study, male Yellow River carp (Cyprinus carpio) were exposed to 0, 1/5, 1/10 and 1/20 LC50 (96 h LC50 of TCS to carp) TCS under semi-static conditions for 42 d. Vitellogenin (Vtg), 17ß-estradiol (E2), testosterone(T), gonadotropin (GtH), and gonadotropin-releasing hormone (GnRH) levels were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, we also examined the mRNA expressions of aromatase, GtHs-ß, GnRH, estrogen receptor (Er), and androgen receptor (Ar) by quantitative Real-time Polymerase Chain Reaction (qRT-PCR). TCS induced Vtg levels of hepatopancreas, E2 levels of serum, and inhibited Ar and Er mRNA levels, suggesting that the induction of Vtg production by TCS was indirectly caused by non-Er pathways. TCS-induced Vtg levels by interfering with the reproductive axis at plenty of latent loci of male carps: (a) TCS exposure increased the aromatase mRNA expression of hypothalamus and gonad aromatase, consequently increasing serum concentrations of E2 to induce Vtg in hepatopancreas; (b) TCS treatment changed GtH-ß and GnRH mRNA expression and secretion, causing the disturbance of reproductive endocrine; (c) TCS exposure decreased Ar mRNA levels, indicating potential Ar-mediated antiandrogen action. These mechanisms showed that TCS may induce Vtg production in male carp by non-Er-mediated pathways.