Potential of exogenous melatonin administration to mitigate heat stress induce pathophysiology of chicken.

Melatonin (MT) is an amine hormone secreted by the body that has antioxidant and anti-inflammatory properties. The aim of this study was to investigate pathophysiological protection of MT in heat-stressed chickens. By modelling heat-stressed chickens and treating them with MT. After 21 days of administration, serum antioxidant enzymes, biochemical indices, inflammatory cytokine and heat-stress indices were detected, along with cardiopulmonary function indices and histological observations in chickens. The results show heat-stress induced a decrease (P < 0.05) in body weight and an increase in body temperature, which was reversed after MT intervention. Treatment with MT inhibited (P < 0.05) the secretion of pro-inflammatory factors interleukin-1ß, interleukin-6, tumor necrosis factor α, serum heat shock protein 70, corticosterone, and elevated (P < 0.05) the levels of biochemical factors total protein, albumin, globulin, and increased (P < 0.05) the activities of antioxidant enzymes superoxide dismutase, glutathione peroxidase and catalase in chicken serum caused by heat stress, and the best effect was observed with the medium dose of MT. The heat-stress caused cardiac atrophy and pulmonary congestion, decreased (P < 0.05) the cardiac function indices creatine kinase isoenzyme, cardiac troponin I, angiotensin receptor I, creatine kinase and lung function indices myeloperoxidase, angiotensin-II, heat shock factor I, and increased (P < 0.05) the lung vascular endothelial growth factor II. Sections of the heart and lungs after administration of MT were observed to be more complete with more normal tissue indices. At the same time, compared with heat stress, heart and lung function indices of grade chickens after MT administration were significantly (P < 0.05)reduced and tended to normal levels, and the best effect was observed in the medium-dose MT. In conclusion, heat stress can cause pathophysiological damage in chickens, and 1 mg/kg/d of exogenous melatonin can attenuate this adverse effect.

MiR-196-5p involvement in selenium deficiency-induced immune damage via targeting of NFκBIA in the chicken trachea.

Dietary selenium (Se) deficiency can induce multifarious immune injury in tissues, accompanied by inflammation and a decreased expression of selenoproteins. The results of previous studies indicated that these issues are associated with Se-mediated microRNAs involved in immune regulation, although the specific mechanisms associated with these interactions have not been reported in the trachea of chickens. To explore the effects of Se deficiency in the trachea of chickens and the role of miR-196-5p, we established correlational models of tracheal injury in chickens. One hundred broilers were divided into four groups, including a control group (C group), a Se deficient group (L group), a lipopolysaccharide (LPS)-induced control group (C + LPS group) and a LPS-induced Se deficient group (L + LPS group). Light microscopy observations indicated that the infiltration of inflammatory cells was the major histopathological change caused by Se deficiency. Furthermore, ultrastructural observation of the tracheal epithelium and ciliary showed typical inflammatory signs owing to Se deficiency. We determined the targeting relationship between miR-196-5p and NFκBIA by bioinformatics analysis. In the case of Se deficiency, the changes were detected as follows: 19 selenoproteins showed different degrees of decrease (p < 0.05). Significant inhibition of both antimicrobial peptides and immunoglobulin production were observed (p < 0.05). IκB-α (NFκBIA) expression degraded with the increasing miR-196-5p (p < 0.05), and the NF-κB pathway was activated. Thereafter, we can see a significant increase in the mRNA levels of inflammatory cytokines-related genes (tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E (PTGE), interleukin (IL)-1ß, IL-6) and protein expression of NF-κB/iNOS pathway-related genes (NF-κB, iNOS, TNF-α, COX-2) (p < 0.05). The release of IL-2, interferon (IFN)-γ inhibited (p < 0.05) and the secretion of IL-4, IL-6 increased, suggesting the imbalance of Th1/Th2 (Th, helper T cell) cytokines. Compared to the control, the mRNA and protein expression levels of the anti-inflammatory system components with antioxidant activity (PPAR-γ/HO-1) were in an inhibitory state (p < 0.05). Antioxidases (SOD, CAT, GSH-Px) activities were suppressed. The activities of the peroxide markers (MDA, H2O2) were enhanced (p < 0.05). In addition, Se deficiency had a positive effect on the pathological changes of inflammation and the exceptional immunity in LPS-treated groups (p < 0.05). The results confirmed the relationship between miR-196-5p and NFκBIA in chickens, revealing that Se deficiency causes respiratory mucosal immune dysfunction via the miR-196-5p-NFκBIA axis, oxidative stress and inflammation. Moreover, Se deficiency exacerbates the inflammatory damage stimulated by LPS. Our work provides a theoretical basis for the prevention of tracheal injury owing to Se deficiency and can be used as a reference for comparative medicine. Furthermore, the targeted regulation of miR-196-5p and NFκBIA may contribute to the protection of the tracheal mucosa in chickens.

Hydrogen sulfide-induced oxidative stress leads to excessive mitochondrial fission to activate apoptosis in broiler myocardia.

Hydrogen sulfide (H2S), as an environmental gas pollutant, has harmful effects on many tissues and organs, including myocardium. However, the underlying mechanisms of H2S-induced myocardia toxicity remain poorly understood. The present study was designed to investigate the effect of H2S on myocardia injury in broilers from the perspective of apoptosis. 30 ppm H2S was administered in the broiler chamber for 2, 4 and 6 week, respectively, and the myocardial samples in control groups and H2S groups were collected immediately after euthanized broilers. Transmission electron microscope, test kits, qRT-PCR and western blot were performed. Results showed that H2S exposure decreased the activities of catalase (CAT) and total antioxidant capability (T-AOC), whereas the content of hydrogen peroxide (H2O2) and the activity of inducible nitric oxide synthase (iNOS) enhanced. Besides, we found the excessive expression of mitochondrial fission genes (Drp1 and Mff) by H2S, the dynamic balance of mitochondrial fission and fusion is destroyed. Furthermore, the levels of pro-apoptotic gene (including CytC, Cas3, Cas8, Cas9, TNF-α and Bax) increased after H2S exposure, as well as the expression level of anti-apoptotic gene bcl-2 decreased. At the same time, the activities of ATPase (including Na+-K+-ATPase, Ca2+-ATPase, Mg2+-ATPase and Ca2+-Mg2+-ATPase) weakened under H2S exposure. Therefore, we conclude that H2S induced oxidative stress and then leaded to excessive mitochondrial fission, which involved in apoptosis and damage broiler myocardia.

The Protection of Selenium Against Cadmium-Induced Mitochondrial Damage via the Cytochrome P450 in the Livers of Chicken.

Cadmium (Cd) is a heavy metal in natural environment and has extreme toxicity. Selenium (Se) has protective effect against heavy metal-induced injury or oxidative stress. Cytochrome P450 (CYP450) enzymes are a family of hemoproteins primarily responsible for detoxification functions. In order to investigate whether CYP450 is related to the damage of livers caused by Cd exposure, we chose forty-eight 28-day-old healthy Hailan cocks for four groups: control group, Se group, Cd group, and Se + Cd group. After 90-day treatment, euthanized for experiment. Based on an established subchronic Cd poisoning model in chicken, this experiment was designed to detect mitochondrial structure, malondialdehyde (MDA), glutathione (GSH), DNA and protein crosslink (DPC) and protein carbonyl (PCO) content, the CYP450 and b5 contents, the aminopyrine-N-demethylase (AND), erythromycin N-demethylase (ERND), aniline 4-hydroxylase (AH) and NADPH-cytochrome C reducatase (CR) activities, and mRNA expression level in the livers. The present results indicated that the MDA content, PCO content, and DPC index in Cd group were higher than those observed in other three groups. Most of the mitochondrial structure is incomplete in Cd group. The contents of CYP450 and b5 were decreased in Cd group. The activities of AND, ERND, AH, and CR got reduced after Cd exposure, as observed in CYP450 gene expression. Our results showed that CYP450 system was involved in the entire process of injury and protection. This research provides a comprehensive evaluation of the oxidative stress effects of Cd related to CYP450 in chicken.

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

bFGF signaling-mediated reprogramming of porcine primordial germ cells.

Primordial germ cells (PGCs) have the ability to be reprogrammed into embryonic germ cells (EGCs) in vitro and are an alternative source of embryonic stem cells. Other than for the mouse, the systematic characterization of mammalian PGCs is still lacking, especially the process by which PGCs convert to pluripotency. This hampers the understanding of germ cell development and the derivation of authenticated EGCs from other species. We observed the morphological development of the genital ridge from Bama miniature pigs and found primary sexual differentiation in the E28 porcine embryo, coinciding with Blimp1 nuclear exclusion in PGCs. To explore molecular events involved in porcine PGC reprogramming, transcriptome data of porcine EGCs and fetal fibroblasts (FFs) were assembled and 1169 differentially expressed genes were used for Gene Ontology analysis. These genes were significantly enriched in cell-surface receptor-linked signal transduction, in agreement with the activation of LIF/Stat3 signaling and FGF signaling during the derivation of porcine EG-like cells. Using a growth-factor-defined culture system, we explored the effects of bFGF on the process and found that bFGF not only functioned at the very beginning of PGC dedifferentiation by impeding Blimp1 nuclear expression via a PI3K/AKT-dependent pathway but also maintained the viability of cultured PGCs thereafter. These results provide further insights into the development of germ cells from livestock and the mechanism of porcine PGC reprogramming.

Morphological changes and germ layer formation in the porcine embryos from days 7-13 of development.

Morphogenesis and identification of embryonic differentiation in porcine embryos are crucial issues for developmental biology and laboratory animal science. The current paper presents a study on the asynchronous development of hatched porcine embryos from days 7 to 13 post-insemination. Examination of semi-thin sections of the hypoblast showed that it had characteristics similar to those of the mouse anterior visceral endoderm during embryonic disc formation. Also, a cavity appeared in the epiblast, which was similar to a mouse proamniotic cavity. With the gradual disappearance of Rauber's layer, the cavity opened and contacted the external environment directly, all of which formed the embryonic disc. To confirm the differentiation characteristics, we performed immunohistochemical analyses and showed that GATA6 was detected clearly in parietal endoderm cells during embryonic disc establishment. OCT4 was expressed in the inner cell mass (ICM) and trophoblast of hatched blastocysts and in the epiblast during formation of the embryonic disc. However, OCT4 showed comparatively decreased expression in the posterior embryonic disc, primitive streak and migrating cells. SOX2 was present in the ICM and epiblast. Therefore, both SOX2 and OCT4 can be used as markers of pluripotent cells in the porcine embryonic disc. At the start of gastrulation, staining revealed VIMENTIN in the posterior of the embryonic disc, primitive streak and in migrating cells that underlay the embryonic disc and was also expressed in epiblast cells located in the anterior primitive streak. Together with serial sections of embryos stained by whole mount immunohistochemistry, the mesoderm differentiation pattern was shown as an ingression movement that took place at the posterior of the embryonic disc and with bilateral migration along the embryonic disc borders.

Derivation of putative porcine embryonic germ cells and analysis of their multi-lineage differentiation potential.

Embryonic germ (EG) cells are cultured pluripotent stem cells derived from the primordial germ cells (PGCs) that migrate from the dorsal mesentery of the hindgut to the developing genital ridge. In this study, the morphology of the porcine genital ridge was assessed in embryos harvested on days 22-30 of pregnancy. PGCs from embryos at these stages were cultured to obtain porcine EG cell lines, and EG-like cells were derived from PGCs from embryos harvested on days 24-28 of pregnancy. The EG-like cells expressed Oct4, Sox2, Nanog, SSEA-3, SSEA-4 and alkaline phosphatase (AP). These cells were able to form embryoid bodies (EBs) in suspension culture and differentiate into cells representative of the three germ layers as verified by a-fetoprotein (AFP), α-smooth muscle actin (α-SMA), and Nestin expression. Spontaneous differentiation from the porcine EG-like cells of delayed passage in vitro showed that they could differentiate into epithelial-like cells, mesenchymal-like cells and neuron-like cells. In vitro directed differentiation generated osteocytes, adipocytes and a variety of neural lineage cells, as demonstrated by alizarin red staining, oil red O staining, and immunofluorescence for neuronal class Ⅲ ß-tubulin (Tuj1), glial fibrillary protein (GFAP) and galactosylceramidase (GALC), respectively. These results indicate that porcine EG-like cells have the potential for multi-lineage differentiation and are useful for basic porcine stem cell research.

Inhibitory effects of vitamin E on UVB-induced apoptosis of chicken embryonic fibroblasts.

Apoptosis research has been focused on several model species in the past decades, whereas studies concerned with non-mammalian vertebrate, particularly birds, have rarely been involved. In accord with requirements to expand the biodiversity of apoptotic research, a chicken embryonic fibroblasts model involving UVB (ultraviolet B) as the death stimulus was established through primary explantation and serial passage. Myriads of antioxidants can inhibit UVB-induced apoptosis by virtue of scavenging reactive oxygen species. To improve our understanding of the possible anti-apoptotic effects and mechanisms of Vitamin E against UVB-induced apoptosis in chicken embryonic fibroblasts, cells treated with Vitamin E after UVB irradiation were stained with AO/EB and Fluo-3/AM to visualize chromatin distribution and calcium homoeostasis, respectively. They were also analysed by flow cytometry to detect mitochondrial transmembrane potential, and cell cycle progression and apoptotic rates were recorded. RT-PCR was used to analyse the expression of some apoptosis-related genes. Typical apoptotic events, including cell shrinkage, blebbing and nuclear condensation, occurred after radiation. In the presence of Vitamin E following irradiation, apoptotic cells were reduced. Ca2+ release was temporarily prevented, and cell cycle arrest at S/G2 checkpoint had almost completely reverted to normal. fas decreased, while procaspase-3 remained nearly unchanged with and without Vitamin E, and bcl2/bax ratio was up-regulated, indicating possible anti-apoptotic mechanisms through the mitochondrial pathway. This new investigation of an apoptosis model involving chicken embryonic fibroblasts expands the database of knowledge across a wider spectrum of vertebrate species.