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The susceptibility to trace metals and legacy POPs is different between terrestrial and marine mammals. In this study, we established the first cell line from Indo-Pacific finless porpoises and compared the cellular responses of skin fibroblast cells from Pygmy killer whales, Pantropic spotted dolphins, Indo-Pacific finless porpoises, mice, and humans following exposure to copper, methylmercury, cadmium, PCB126, PCB153, and BDE47 to better understand the interspecies sensitivities of mammals to chemical pollutants. We conducted a risk assessment by comparing no-observed effect concentrations (NOEC), lowest-observed effect concentrations (LOEC), and half maximal effective concentrations (EC50) from cell viability assays and previously reported pollutant body burdens in mammals. Based on the in vitro data, Indo-Pacific finless porpoises were more sensitive to copper and methylmercury than other mammals. PCB153 exposure reduced cell viability in all mammals except humans, while PCB126 was more potent, with 13.33 μg/mL exposure reducing cell viability in all mammals. In contrast, BDE47 exposure reduced cell viability only in terrestrial mammals in addition to pantropic spotted dolphin. Based on the in vitro data and the natural context of metal concentrations, both methylmercury and cadmium posed a higher risk to cetaceans than human, while copper posed a lower risk to cetaceans. All three legacy POPs (PCB126, PCB153, and BDE47) posed minor risk to cetaceans for short-term exposure. This study demonstrated that a species-specific in vitro model may provide more accurate information on the potential risk of pollutants to mammals. However, due to the bioamplification of POPs and their potential impact on the endocrine system and immune system of cetaceans, risk assessment with long-term exposure with more in vitro models should be further studied.

Deoxynivalenol (DON) is considered to be a grave threat to humans and animals. Ginsenoside Rb1 (Rb1) has been reported for its antioxidant potential and medicinal properties. However, the shielding effects of Rb1 and the precise molecular mechanisms against DON-induced immunotoxicity in mice have not been reported yet. In the present research, 4-weeks old healthy C57BL/6 mice were randomly assigned into four experimental groups (n = 12), viz., CON, DON 3 mg/kg BW, Rb1 50 mg/kg BW and DON 3 mg/kg + Rb1 50 mg/kg BW (DON + Rb1). Feed intake and body weight gain were monitored during the entire experiment (15 d). Our results demonstrated that Rb1 markedly increased the ADG (30%) and ADFI (25.10%) of mice compared with DON group. Furthermore, Rb1 alleviated the DON-induced immune injury by relieving the splenic histopathological alteration, enhancing the T-lymphocytes subsets (CD4+, CD8+), the levels of cytokines (IL-2, IL-6, IFN-γ, and TNF-α), as well as production of immunoglobulins (IgA, IgM, and IgG). Moreover, Rb1 ameliorated DON-inflicted oxidative stress by reducing the ROS, MDA and H2O2 contents and boosting the antioxidant defense system (T-AOC, T-SOD, CAT, and GSH-Px). Additionally, Rb1 significantly reversed the DON-induced excessive splenic apoptosis via modulating the mitochondria-mediated apoptosis pathway in mice, depicting the decreased percentage of splenocyte apoptotic cells by 26.65%, down-regulated the mRNA abundance of Bax, caspase-3, caspase-9, and protein expression of Bax, cleaved caspase-3, and Cyt-c. Simultaneously, Rb1 markedly rescued both Bcl-2 mRNA and protein expression levels. Taken together, Rb1 mitigates DON-induced immune injury by suppressing the oxidative damage and regulating the mitochondria-mediated apoptosis pathway in mice. Conclusively, our current research provides an insight into the preventive mechanism of Rb1 against DON-induced immune injury in mice and thus, presents a scientific baseline for the therapeutic application of Rb1.

Aflatoxin B1 (AFB1) is a serious threat to the poultry industry. Proanthocyanidins (PCs) demonstrates a broad range of biological, pharmacological, therapeutic, and chemoprotective properties. The aim of this study was to investigate the ameliorative effects of PCs against AFB1-induced histopathology, oxidative stress, and apoptosis via the mitochondrial pathway in the bursa of Fabricius (BF) of broilers. One hundred forty-four one-day old Cobb chicks were randomly assigned into four treatment groups of six replicates (6 birds each replicate) for 28 days. Groups were fed on the following four diets, (1) Basal diet without addition of PCs or AFB1 (Control), (2) basal diet supplemented with 1 mg/kg AFB1 from contaminated corn (AFB1), (3) basal diet supplemented with 250 mg/kg PCs (PCs), and (4) basal diet supplemented with 1 mg/kg AFB1 + 250 mg/kg PCs (AFB1+ PCs). The present study results showed that antioxidant enzymes activities of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione S-transferase (GST) in AFB1 treated group were (p &lt, 0.05) decreased, whereas malondialdehyde (MDA) contents were significantly increased in comparison with the control group. Furthermore, we found that dietary PCs treatment ameliorated AFB1-induced oxidative stress in the BF through inhibiting the accumulation of MDA content and enhancing the antioxidant enzymes activities (T-SOD, CAT, GSH-Px, and GST). Similarly, PCs markedly enhanced messenger RNA (mRNA) expression of antioxidant genes (SOD, CAT, GPx1, and GST) in comparison with AFB1 group. Moreover, histological results showed that PCs alleviated AFB1-induced apoptotic cells in the BF of broilers. In addition, both mRNA and protein expression results manifested that mitochondrial-apoptosis-associated genes (Bax, caspase-9, caspase-3, and p53 and cytochrome c) showed up-regulation, while (Bcl-2) showed down-regulation in AFB1 fed group. The supplementation of PCs to AFB1 diet significantly reversed the mRNA and protein expression of these apoptosis-associated genes, as compared to the AFB1 group. Our results demonstrated that PCs ameliorated AFB1-induced oxidative stress by modulating the antioxidant defense system and apoptosis in the BF through mitochondrial pathway in broilers.

Cetaceans accumulate persistent and toxic substances such as polybrominated diphenyl ethers in their tissue. PBDEs are ubiquitous in marine environments, and their exposure to mammals is linked to numerous health effects such as endocrine disruption, neurotoxicity, carcinogenicity, and fetal toxicity. However, the toxicological effects and mechanism of toxicity in cetaceans remains poorly understood. The effects of BDE-47 (0.1–0.5 μg mL−1), BDE-100 (0.1–0.5 μg mL−1), and BDE-209 (0.25–1.0 μg mL−1) exposure on cell viability, oxidative stress, mitochondrial structure, and apoptosis were evaluated using a recently established pantropical spotted dolphin (Stenella attenuata) skin fibroblast cell line (PSD-LWHT) as an in vitro model. However, the production of reactive oxygen species (ROS) increased following exposure to 1.0 μg mL−1 PBDE while superoxide anion, hydroxyl radicals, and inducible nitric oxide increased in a dose-dependent manner. At 0.5–1.0 μg mL−1, PBDEs significantly reduced the mitochondrial membrane potential. In addition, exposure to BDE-47 and -209 significantly affected mitochondrial structure as well as cell signaling and transduction compared to BDE-100. Although PBDE exposure did not affect cell viability, a significant increase in cell apoptosis markers (Bcl2 and caspase-9) was observed. This study demonstrated that BDE-47, -100, and −209 congeners might cause cytotoxic and genotoxic effects as they play a crucial role in the dysregulation of oxidative stress and alteration of mitochondrial and cell membrane structure and activity in the fibroblast cells. Hence, these results suggest that PBDEs might have adverse health effects on cetaceans inhabiting contaminated marine environments.

Spermatogenesis is a complex process producing haploid spermatozoa, and the formation of lipid droplets (LDs) within Sertoli cells is critical to maintaining normal spermatogenesis. However, the utilization of LDs within Sertoli cells is still largely unknown. In the present study, proliferation of spermatogonial cells had begun in May, whereas the meiotic cells occurred predominately in July and majority of spermiogenic cells were observed in the seminiferous tubules in October. However, TEM and Oil Red O staining demonstrated that a larger number of LDs had accumulated within the Sertoli cells in May compared to that in October. There were several LDs attached to the isolation membrane/phagophore, suggesting that the LDs may be a source of endogenous energy for the biogenesis of autophagosomes. The LDs were enclosed within the autophagosomes in May, whereas, autophagosomes and mitochondria were directly attached with large LDs within the Sertoli cells in October. Furthermore, immunohistochemistry results demonstrated the stronger localization of LC3 on the Sertoli cells in May than in October. This study is the first to provide clear evidence of the two different modes of lipophagy for lipid consumption within Sertoli cells, which is a key aspect of Sertoli germ cell communication during spermatogenesis.

Pantropical spotted dolphins are apex predators and have a long lifespan, which makes them susceptible to chemical pollutants such as polybrominated diphenyl ethers (PBDEs), which are associated with immunotoxicity in wildlife. However, the effects of PBDEs and their mechanism of immunotoxicity in dolphins is largely unknown. Previously, we established fibroblast cell lines obtained from pantropical spotted dolphins (PSD-LWHT) as an in vitro model for assessing the toxicological implications of chemical pollutants in dolphins. In this study, we used the novel immortalized fibroblast cell line to explore the potential immune stimulation effect of PBDEs via prostaglandins signaling pathways to better understand the immunotoxicity pathway of PBDEs in dolphins. BDE-47, -100, and −209 exposure generally resulted in an increase in inflammatory cytokine expression, PGE2 levels, and COX-2 gene expression but BDE-209 resulted in a suppression in IL-10 production. Both protein and mRNA expression of COX-2 and PTGES increased significantly following exposure to the PBDEs. The results suggested BDE-100 and -209 increased prostaglandin E2 (PGE2) production via increased expression of COX-2 and PTGES expression. Only BDE-100 increased expression level of the prostaglandin E2 receptor EP4 while BDE-47 and BDE-209 decreased its expression. This probably explained why suppressive effect on the expression level of anti-inflammatory cytokines were only found in exposure with BDE-47 and BDE-209 rather than BDE-100. The results showed that PBDEs stimulate innate immune response by triggering PGE2-EPs-cAMP-cytokines signaling.

The presence of polybrominated diphenyl ethers (PBDEs) in the aquatic environment is an issue of major concern which may be a cause of increasing prevalence and severity of diseases in marine mammals. Although, cell culture model development and in vitro investigation approach is a prime need of time to progress immunotoxic research on aquatic mammals. In this study, we stablished fibroblast cell line (pantropic spotted dolphin) to assess the potential effects of PBDEs on cytokines response. Cells were grown in 6 well cell culture plate and complete media (DMEM and Ham's F12 nutrient mixture, fetal bovine serum, antibiotic and essential amino acids) was provided. The primary culture of (PSP-LWH) cells identification was achieved by vimentin (gene and protein) expressions. Karyotyping revealed pantropic spotted dolphin chromosomes 20 pairs with XX. Transfection was achieved by SV40 LT antigen and transfected cells were expended for passages. Stability of cell line was confirmed at various passages intervals using RT-PCR, western blotting and immunofluorescence methods. After confirmation, cell line was exposed to BDE-47 (250 ng/ml), BDE-100 (250 ng/ml) and BDE-209 (1000 ng/ml), with control group (PBS), positive control DMSO (0.1%) and negative control LPS (500 ng/ml) for 24 h. The ELISA results showed significant increase in IL-6 in BDE- 100 and BDE-209 while IL-1β and IL-8 were found higher in BDE-47 and BDE-100. TNFα and IL-10 secretion was noted higher in control and positive control groups. Altogether, these results emphasize importance of transfected (PSP-LWHT) cell line in aquatic research and potential effects of PBDEs on fibroblast provides evident to understand immune modulating effects of PBDEs in marine mammals. The impact of PBDEs on dolphin’s fibroblast cells immune response and altered cytokine response have been presented for the first time.

The pygmy killer whale (Feresa attenuata) (PKW) is a tropical and subtropical marine mammal commonly found in the Atlantic, Indian and Pacific oceans. Since the PKWs live in offshore protected territories, they are rarely seen onshore. Hence, PKW are one of the most poorly understood oceanic species of odontocetes. The dermal tissue comes primarily from stranding events that occur along the coast of the Shantou, Guangdong, China. The sampled tissues were immediately processed and attached on collagen-coated 6-well tissue culture plate. The complete medium (DMEM and Ham’s F12, fetal bovine serum, antibiotic and essential amino acids) was added to the culture plates. The primary culture (PKW-LWH) cells were verified as fibroblast by vimentin and karyotype analyses, which revealed 42 autosomes and two sex chromosomes X and Y. Following transfection of PKW-LWH cells with a plasmid encoding, the SV40 large T-antigens and the transfected cells were isolated and expanded. Using RT-PCR, western blot, immunofluorescence analysis and SV40 large T-antigen stability was confirmed. The cell proliferation rate of the fibroblast cells, PKW-LWHT was faster than the primary cells PKW-LWH with the doubling time 68.9h and 14.4h, respectively. In this study, we established PKW dermal fibroblast cell line for the first time, providing a unique opportunity for in vitro studies on the effects of environmental pollutants and pathogens that could be determined in PKW and/or Cetaceans.

The present study was designed to evaluate the effects of Saccharomyces boulardii (Sb) and Bacillus subtilis B10 (Bs) on intestinal epithelial Toll like receptors (TLR), and Cytokine expression response to understand the intestinal epithelial innate immune mechanism in broilers. A total of 300 birds (Sanhuang broilers) were allotted into three groups (n = 100) and each divided into five replications (n = 20). Control group (Ctr) birds were fed basal diet, broilers in experimental groups received (1×108cfu/kg feed) Sb and Bs respectively in addition to basal diet for 72 days. The result showed significant increase in mRNA expression level of TLR2, TLR4 and TLR15. Down streaming MyD88, TRAF6, TAB2 and NF-κB mRNA level noted higher, in the jejunum and ileum as compared to control group. Meanwhile, IL-6, TNFα, IL-10, TGF-β expression levels showed high expression in the jejunum of Sb and Bs groups. IL-10 expression level increased in the ileum and IL-6, TNFα, IL-10 and TGF-β expression levels increased in the jejunum of Sb group. Levels of IL-1 β, IL-17, and IL-4, increased merely in Sb group. Ileal cytokines IL-1β, IL-17 and IL-4concentration were noted higher in Sb group, and IL-1β, and IL-4 levels were up-regulated in Bs group. The results indicated that the INF-γ and IL-8 level decreased in Sb and BS groups. Serum IgA and sIgA level increased in both treatment groups. Our findings illustrated that S. boulardii and B. subtilis B10 may have a role to induce mucosal immunity by activating the TLRs and cytokines expressions in broilers.

The recent ban on the use of antibiotics as a feed additive has led to the search for alternative sources of antibiotics in the feed industry. Presently, probiotics are considered as a potential substitute for antibiotic as a live biotherapeutic agent to improve animal health and performance. Accordingly, study was focused on evaluating the effect of Saccharomyces boulardii (Sb) and Bacillus subtilis B10 (Bs) on ultrastructure modulation and mucosal immunity development in broiler chickens. A total of three hundred 1-d-old Sanhuang broilers (a Chinese cross breed) were randomized into 3 groups, each group with 5 replications (n = 20). The control group (Ctr) was fed a basal diet containing an antibiotic (virginiamycin, 20 mg/kg). Meanwhile, broilers in experimental groups received Sb and Bs (1 × 10(8) cfu/kg of feed) in addition to the basal diet for 72 d. The results of the experimental groups revealed a significant improvement in live BW and relative weight of bursa of Fabricius and thymus. Also, intestinal villus height, width, and number of goblet cells increased in the Sb and Bs groups. Meanwhile, modulation in the intestinal ultrastructure and increased mRNA expression levels of occluding, cloudin2, and cloudin3 (P < 0.05) were observed in the Sb and Bs groups. Moreover, IgA-positive cells significantly increased in the jejunum of Sb- and Bs-supplemented groups (P < 0.05). Intestinal cytokines interleukin-6, tumor necrosis factor-α, interleukin-10, transforming growth factor-β, and secretory IgA concentrations were (P < 0.05) improved in the probiotic groups; however, Sb induced inflammatory and antiinflammatory cytokines (P < 0.05) in comparison with the Ctr group. The present findings conclusively revealed that Sb and Bs increased IgA-positive cells in the lumen of the intestinal villus and revealed that Sb and Bs could modulate intestinal ultrastructure through increasing occluding, cloudin2, and cloudin3 mRNA expression levels in broiler intestine.

The present study evaluated the effects of Saccharomyces boulardii on duodenal digestive enzymes, morphology and cytokine induction response in broiler chicken. A total of 200 birds were allotted into two groups (n = 100) and each group divided into five replications (n = 20). The control group was fed basal diet in addition to antibiotic (virginiamycin 20 mg/kg), and treatment group received (1 × 10

This experiment was conducted to evaluate the effects of dietary inclusion of Bacillus licheniformis on laying performance, egg quality, antioxidant enzyme activities, and intestinal barrier function of laying hens. Hy-Line Variety W-36 hens (n = 540; 28 wk of age) were randomized into 6 groups, each group with 6 replications (n = 15). The control group received the basal diet formulated with maize and soybean meal. The treatment groups received the same basal diets supplemented with 0.01, 0.02, 0.03, 0.06, and 0.09% Bacillus licheniformis powder (2 × 10(10) cfu/g) for an 8-wk trial. The results showed that dietary supplementation with 0.01 and 0.03% B. licheniformis significantly increased egg production and egg mass. However, no significant differences were observed in egg weight, feed consumption, and feed conversion efficiency among the 6 groups. Supplementation with different levels of B. licheniformis was found to be effective in improvement of egg quality by increasing egg shell thickness and strength. Compared with control, d-lactate content, diamine oxidase activity, and adrenocorticotropic hormone level in serum decreased significantly, and the level of estradiol and follicle-stimulating hormone increased significantly in plasma of all the experimental groups. Dietary supplementation with B. licheniformis increased the intestinal villus height and reduced the crypt depth. In conclusion, dietary inclusion of B. licheniformis could improve laying performance and egg quality significantly in a dose-dependent manner by decreasing the stress response, upregulating the growth hormone, and improving intestinal health.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play a critical role to activate immune response. They may be targeted for immunomodulation by microbes, including probiotics. In this study, chicken bone marrow dendrite cells (chi-BMDCs) were stimulated with lipopolysachride (LPS), Saccharomyces boulardii (Sb), Bacillus subtilis B10 (Bs), co-culture of Sb + Bs and phosphate buffer saline (PBS) as a control group (Ctr) at 3, 6, and 12 h intervals. Results revealed that treatment groups modulated the phenotype and biological functions of chi-BMDCs. Scan electron microscopy showed attachment of probiotics on the surface of chi-BMDCs. Additionally transmission electron microscopy (TEM) revealed efficiently engulfing and degradation of probiotics. Gene expression levels of MHC-II, CD40, CD80 and CD86 up-regulated in stimulated groups. Furthermore, toll-like receptors TLR1, TLR2, TLR4, and chicken specific TLR15 expressions were improved and downstream associated factors MyD88, TRAF6, TAB1, and NFκ-B mRNA levels increased in all treatment groups as compared to control. Surprisingly, NFκ-B response was noted significant higher in LPS treatment among all groups. Moreover, IL-1β, IL-17, IL-4, TGF-β, and IL-10 production levels were found higher, and lower concentration of INF-γ and IL-8 were observed in Sb, Bs, and Sb + Bs treatment groups. In contrast, LPS groups showed prominent increase in IL-12, INF-γ, and IL-8 concentration levels as compared to control group. Altogether, these results emphasize a potentially important role of Saccharomyces boulardii and Bacillus subtilis B10 in modulating immunological functions of chi-BMDCs by targeting specific toll like receptors (TLRs) and associated factors. The role of probiotics on chi-BMDCs functionality in a non-mammalian species have been presented for the first time.

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor-alpha, interferon-gamma, interleukin [IL]-1 beta, IL-6, IL-12, IL-10 and macrophage inflammatory protein-2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up-regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.