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Mycoplasma pneumoniae, as one of the most common pathogens, usually causes upper respiratory tract infections and pneumonia in humans and animals. It accounts for 10% to 40% of community-acquired pneumonia in children. The alveolar epithelial cells (AECs) are the first barrier against pathogen infections, triggering innate immune responses by recruiting and activating immune cells when pathogens invade into the lung. Alveolar macrophages (AMs) are the most plentiful innate immune cells in the lung, and are the first to initiate immune responses with pathogens invasion. The cross-talk between the alveolar epithelium and macrophages is necessary to maintain physiological homeostasis and to eradicate invaded pathogen by regulating immune responses during Mycoplasma pneumoniae infections. This review summarizes the communications between alveolar macrophages and epithelial cells during Mycoplasma pneumoniae infections, including cytokines-medicated communications, signal transduction by extracellular vesicles, surfactant associated proteins-medicated signal transmission and establishment of intercellular gap junction channels.

Previous studies have shown that melatonin (MT) can ameliorate vitrification-inflicted damage in mouse germinal vesicle (GV) oocytes, however, the key mechanistic basis of this improvement still remains poorly understood. This study was conducted to investigate whether MT can improve in vitro developmental potential of vitrified-warmed GV oocytes through its receptors. The fresh oocytes were randomly divided into four groups: untreated (control group, F), vitrified by open-pulled straw method (vitrification group, V), vitrification group with 100 nmol/L MT supplementation (vitrification + MT group, VM), and with 100 nmol/L MT plus 100 nmol/L luzindole administration (vitrification + MT + luzindole group, VML) or with 50 nmol/L ramelteon addition (vitrification + ramelteon group; VR). After warming, oocytes were cultured in vitro, and MT receptors (MTRs), MAD2 (mitotic arrest deficient 2), Securin and CyclinB1 protein levels and spindle morphology were evaluated. The ratio of oocytes developed to the metaphase I (MI) and metaphase II (MII) stages was also assessed. The results showed that after vitrification-warming, the in vitro maturation rate of GV oocytes was significantly lower compared to the control (F) group. Vitrification also significantly impaired the spindle morphology, decreased the protein level of MTRs and Securin, and decreased MAD2 levels in MI oocytes. However, when MT or ramelteon (MTRs agonist) were added (group wise) to warming and maturation media, the maturation rate of GV oocytes was significantly increased, the normal proportion of the spindle morphology increased, and the expression level of MAD2 increased in their resulting MI oocytes compared to the vitrification group. However, following addition of both MT and ramelteon, the maturation rate of GV oocyte showed no significant difference between VML and vitrification groups. The spindle morphology and MAD2 levels in MI oocytes were comparable to the vitrification group but differed significantly from the VM group. Taken together, finding of the present study shows that MT (100 nmol/L) can ameliorate the in vitro maturation of vitrified-warmed mouse GV oocytes, potentially by improving the spindle morphology, modulating MAD2 protein level and promoting the development of MI stage oocytes through MTRs.

Background This study investigated the effect of melatonin (MT) on cell cycle (G1/S/G2/M) of parthenogenetic zygotes developed from vitrified-warmed mouse metaphase II (MII) oocytes and elucidated the potential mechanism of MT action in the first cleavage of embryos. Results After vitrification and warming, oocytes were parthenogenetically activated (PA) and in vitro cultured (IVC). Then the spindle morphology and chromosome segregation in oocytes, the maternal mRNA levels of genes including Miss, Doc1r, Setd2 and Ythdf2 in activated oocytes, pronuclear formation, the S phase duration in zygotes, mitochondrial function at G1 phase, reactive oxygen species (ROS) level at S phase, DNA damage at G2 phase, early apoptosis in 2-cell embryos, cleavage and blastocyst formation rates were evaluated. The results indicated that the vitrification/warming procedures led to following perturbations 1) spindle abnormalities and chromosome misalignment, alteration of maternal mRNAs and delay in pronucleus formation, 2) decreased mitochondrial membrane potential (MMP) and lower adenosine triphosphate (ATP) levels, increased ROS production and DNA damage, G1/S and S/G2 phase transition delay, and delayed first cleavage, and 3) increased early apoptosis and lower levels of cleavage and blastocyst formation. Our results further revealed that such negative impacts of oocyte cryopreservation could be alleviated by supplementation of warming, recovery, PA and IVC media with 10− 9 mol/L MT before the embryos moved into the 2-cell stage of development. Conclusions MT might promote cell cycle progression via regulation of MMP, ATP, ROS and maternal mRNA levels, potentially increasing the first cleavage of parthenogenetic zygotes developed from vitrified–warmed mouse oocytes and their subsequent development.

Female reproductive (ovarian) aging is characterized by a marked decline in quantity and quality of follicles and oocytes, as well as alterations in the surrounding ovarian stroma. In our previous report, we have shown that dietary selenium (Se) insufficiency and supplementation differentially impacted the reproductive efficiency in aging mice; however, the precise understanding of such modulation is still incomplete. In the present study, we sought to determine the impact of low (mildly low level) and moderately high (medium level) Se diets on expression profile of non-selenoprotein genes in the ovaries of aging mice. For this purpose, the aged mice were divided in two groups and fed either a low Se (Se-L; 0.08 mg Se/kg) diet or a moderately high Se (Se-M; 0.33 mg Se/kg) diet. RNA-seq analysis revealed that a total of 168 genes were differentially expressed between the two groups. From these, 72 and 96 differentially expressed genes (DEGs) were found to be upregulated and downregulated, respectively. Gene Ontology (GO) and pathways enrichment (KEGG) analyses revealed that these DEGs were enriched in several key GO terms and biological pathways including PI3K-Akt signaling pathway, steroid hormone biosynthesis, signaling pathways regulating pluripotency of stem cells, Hippo signaling pathway, ovarian steroidogenesis, and Wnt signaling pathway. Further filtering of RNA-seq data revealed that several DEGs such as Star, Hsd3b6, Scd1, Bmp7, Aqp8, Gas1, Fzd1, and Wwc1 were implicated in key ovarian- and fertility-related functions. In addition, some of the DEGs were related to ER homeostasis and/or proteostasis. These results highlight that dietary low and moderately high (medium level) Se diets, in addition to modulation of selenoproteins, can also have an impact on expression of several non-selenoprotein genes in the ovaries of aging mice. To sum up, these findings add more value to our understanding of Se modulation of ovarian functions and female fertility and will pave a way for the focused mechanistic and functional studies in this domain.

Staphylococcus aureus infections pose a potential threat to livestock production and public health. A novel strategy is needed to control S. aureus infections due to its adaptive evolution to antibiotics. Autophagy plays a key role in degrading bacteria for innate immune cells. In order to promote S. aureus clearance via Toll-like receptor (TLR)-induced autophagy pathway, the domain fusion TLR2-4 with the extracellular domain of TLR2, specific recognizing S. aureus, and transmembrane and intracellular domains of TLR4 is assembled, then the goat expressing TLR2-4 is generated. TLR2-4 substantially augments the removal of S. aureus within macrophages by elevating autophagy level. Phosphorylated JNK and ERK1/2 promote LC3-puncta in TLR2-4 macrophages during S. aureus-induced autophagy via MyD88 mediated the TAK1 signaling cascade. Meantime, the TRIF-dependent TBK1-TFEB-OPTN signaling is involved in TLR2-4-triggered autophagy after S. aureus challenge. Moreover, the transcript of ATG5 and ATG12 is significantly increased via cAMP-PKA-NF-κB signaling, which facilitates S. aureus-induced autophagy in TLR2-4 macrophages. Overall, the novel receptor TLR2-4 enhances the autophagy-dependent clearance of S. aureus in macrophages via TAK1/TBK1-JNK/ERK, TBK1-TFEB-OPTN, and cAMP-PKA-NF-κB-ATGs signaling pathways, which provide an alternative approach for resistant against S. aureus infection.

Staphylococcus aureus infections pose a potential threat to livestock production and public health. A novel strategy is needed to control S. aureus infection due to its adaptive evolution to antibiotics. Autophagy plays a key role in degrading bacteria for innate immune cells. In order to promote S. aureus clearance via TLR induced autophagy pathway, the domain fusion TLR2-4 with the extracellular domain of TLR2, specific recognizing S. aureus, and transmembrane and intracellular domains of TLR4 is assembled, then the goats expressing TLR2-4 is generated. TLR2-4 substantially augments the removal of S. aureus within macrophages by elevating autophagy level. Phosphorylated JNK/ERK1/2 promote LC3- puncta in TLR2-4 macrophages during S. aureus-induced autophagy via MyD88-mediated the TAK1 signaling cascade. Meantime, the TRIF-dependent TBK1-TFEB-OPTN signaling is involved in TLR2-4-triggered autophagy after S. aureus challenge. Moreover, the transcript of ATG5 and ATG12 is significantly increased via cAMP-PKA-NF-κB signaling, which facilitates S. aureus-induced autophagy in TLR2-4 macrophages. Overall, the novel receptor TLR2-4 enhances the autophagy-dependent clearance of S. aureus in macrophages via TAK1/TBK1-JNK/ERK, TBK1-TFEB-OPTN and cAMP-PKA-NF-κB-ATGs signaling pathways, which provide an alternative approach to resistant against S. aureus infection.

Internal egg and eggshell quality are often deteriorated in aging laying hens, which causes huge economic losses in the poultry industry. Selenium yeast (SY), as an organic food additive, is utilized to enhance laying performance and egg quality. To extend the egg production cycle, effects of selenium yeast supplementation on egg quality, plasma antioxidants and selenium deposition in aged laying hens were evaluated. In this study, five hundred and twenty-five 76-week-old Jing Hong laying hens were fed a selenium-deficient (SD) diet for 6 weeks. After Se depletion, the hens were randomly divided into seven treatments, which included an SD diet, and dietary supplementation of SY and sodium selenite (SS) at 0.15, 0.30, and 0.45 mg/kg to investigate the effect on egg quality, plasma antioxidant capacity, and selenium content in reproductive organs. After 12 weeks of feeding, dietary SY supplementation resulted in higher eggshell strength (SY0.45) (p < 0.05) and lower shell translucence. Moreover, organs Se levels and plasma antioxidant capacity (T-AOC, T-SOD, and GSH-Px activity) were significantly higher with Se supplementation (p < 0.05). Transcriptomic analysis identified some key candidate genes including cell migration inducing hyaluronidase 1 (CEMIP), ovalbumin (OVAL), solute carrier family 6 member 17 (SLC6A17), proopiomelanocortin (POMC), and proenkephalin (PENK), and potential molecular processes (eggshell mineralization, ion transport, and eggshell formation) involved in selenium yeast’s effects on eggshell formation. In conclusion, SY has beneficial functions for eggshell and we recommend the supplementation of 0.45 mg/kg SY to alleviate the decrease in eggshell quality in aged laying hens.

Staphylococcus aureus is an invasive, facultative intracellular pathogen that can colonize niches in various host organisms, making it difficult for the host immune system to completely eliminate. Host autophagy is an intracellular clearance pathway involved in degrading S. aureus. Whereas the accessory gene regulatory system of S. aureus that controls virulence factors could resist the host immune defenses by evading and even utilizing autophagy. This article reviews the interaction between autophagy and S. aureus, providing insights on how to use these mechanisms to improve S. aureus infection control.

The declines in laying performance during the late production period have adverse effects on the length of the production cycle. Improving the nutrition of laying hens is a crucial measure to reverse this declination. This study investigated the effect of selenium yeast (SY) on egg production, ileal gene expression and microbiota, as well as elucidating their associations in aged laying hens. A total of 375 Jinghong laying hens at 76 weeks old were randomly assigned into 5 dietary treatments, which included a selenium-deficient basal diet based on corn-soybean meal, and dietary supplementation of SY at 0.15, 0.30 and 0.45 mg/kg, and sodium selenite at 0.45 mg/kg. The results showed that SY ameliorated the depression in aged laying performance in the 0.30 mg/kg group (

Staphylococcus aureus is an invasive, facultative intracellular pathogen that can colonize niches in various host organisms, making it difficult for the host immune system to completely eliminate. Host autophagy is an intracellular clearance pathway involved in degrading S. aureus. Whereas the accessory gene regulatory system of S. aureus that controls virulence factors could resist the host immune defenses by evading and even utilizing autophagy. This article reviews the interaction between autophagy and S. aureus, providing insights on how to use these mechanisms to improve S. aureus infection control.

Background Egg internal and eggshell quality were deteriorated in aging laying hens. Improving egg and shell quality can prolong the laying cycle. Although, selenium yeast (SY) has been found with the potential to enhance the laying performance and egg quality, the underlying mechanisms have not been investigated. Therefore, we investigated the effect and molecule mechanism of selenium yeast on egg and shell quality in aged laying hens. Methods Three hundred 76-week-old Jing Hong laying hens were divided into four equal treatments and fed with Se-deficient diet (SD), 0.15, 0.30, 0.45 mg/kg selenium yeast diet (SY0.15, SY0.30 and SY0.45). At the end of the trial, we evaluated the plasma and tissue selenium content, plasma antioxidant capacity and egg quality. Transcriptomic analysis was performed to investigate the boosting effect of selenium yeast on eggshell quality. The weighted gene co-expression network analysis (WGCNA) analysis was performed to screen out the key candidate genes related with eggshell quality affected by selenium yeast. Results Dietary selenium yeast supplementation enhanced the eggshell strength (SY0.45) (P P CEMIP), ovalbumin (OVAL), solute carrier family 6 member 17 (SLC6A17), solute carrier family 13 member 5 (SLC13A5), proopiomelanocortin (POMC) and proenkephalin (PENK). Functional enrichment analysis revealed that selenium may alter the biological process including eggshell mineralization, ion transport and laying period to affect eggshell formation. Conclusion Selenium yeast enhanced eggshell strength and reduced the shell translucence by improving antioxidant capacity, selenium status, and regulating the processes of shell mineralization, ion transport and laying period. These findings provide a novel molecule biomarker for affecting eggshell quality regulated by selenium.

Background: The declines in both laying performance and egg shell quality during late production period have adverse effects on long production cycle. Improving nutrition of laying hens is a crucial measure to reverse the declination. Selenium (Se) plays important roles in antioxidant defense, redox balance, immune response, and modulation of gut microbiota. However, the mechanism underlying selenium yeast regulating the interaction between transcriptome and gut microbiota to influence laying performance, is still unclear. Here, we use the transcriptome and 16S rRNA analysis to investigate how selenium yeast alters the gene expression and microbiota composition of ileum in aged laying hens.Results: In this study, selenium yeast ameliorated the depression in aged laying performance with a significant increase of laying rate in 0.30 mg/kg group. Furthermore, functional enrichment and STEM analysis were performed using RNA-Seq, which indicated selenium yeast activated metabolic progresses (e.g. Glycerolipid metabolism, Glycerophospholipid metabolism, and fatty acid metabolism), immune response and oxidative stress response. Four hub genes (TXNRD1, DLD, ILK and LZTS2) were involved in intestinal metabolism which was closely associated with Se deposition/status. Additionally, Se increased the abundance of beneficial bacteria including Veillonella, Turicibacter, and Lactobacillus whiledecreasing the abundance of pathogenic bacteria Stenotrophomonas by 16S rRNA-Seq. The Integrated analysis of omics revealed that several microbiotas (Maritalea, Alteromonas, Geobacter, etc.) were positively associated with both Se content and laying rate, and there was a markable correlation between several specific microbiotas (Aliivibrio, Anaerobacillus, Shewanella, etc.) and the immune response pathways (regulation of acute inflammatory response, positive regulation of lymphocyte activation and IFN gamma response). Meanwhile, the “switched on” gene PSCA had a positive relationship with Veillonella, and a negative relationship with the opportunistic pathogens Stenotrophomonas. CCA analysis indicated that both the Se content and the laying rate were highly positively correlated with Anaerobacillus, Alteromonas, Loktanella and the positive regulation of lymphocyte activation, but were negative correlated with Streptococcus, Devosia, Aerococcus, Intestinibacter and fatty acid metabolic progress. Conclusions: Overall, our study provides unprecedented insights showing that selenium yeast supplementation can affect immune response, metabolic processes, and specific microbiota composition to ameliorate the egg production deterioration in aged laying hens.

The Toll-like receptor 4 (TLR4) plays a crucial role in innate inflammatory responses, as it recognizes gram-negative bacteria (or their products) and contributes greatly to host defense against invading pathogens. Though TLR4 overexpressing transgenic sheep, resistant to certain diseases related with gram-negative bacteria, had been bred in our previous research, the effects of overexpression of TLR4 on innate immune response remained unclear. In this study, TLR4 overexpressing ovine macrophages were obtained from peripheral blood, and it was found that the overexpression of TLR4 initially promoted the production of proinflammatory cytokines TNFα and IL-6 by activating TLR4-mediated IRAK4-dependent NF-κB and MAPK (JNK and ERK1/2) signaling following LPS stimulation. However, this effect was later impaired due to increased internalization of TLR4 into endosomal compartment of the macrophages. Then the overexpression of TLR4 triggered TBK1-dependent interferon-regulatory factor-3 (IRF-3) expression, which in turn led to the induction of IFN-β and IFN-inducible genes (i.e.IP10, IRG1 and GARG16). Understandably, an increased IFN-β level facilitated phosphorylation of STAT1 to induce expression of innate antiviral genes Mx1 and ISG15, suggesting that TLR4 overexpressing macrophages were equipped better against viral infection. Correspondingly, the bacterial burden in these macrophages, after infection with live S. Typhimurium, was decreased significantly. In summary, the results indicated that overexpression of TLR4 could enhance innate inflammatory responses, initiate the innate antiviral immunity, and control effectively S. Typhimurium growth in ovine macrophages.

With a broad range of hosts, Salmonella enterica serovar Typhimurium (S. Typhimurium) is the major cause of gastroenteritis in human beings and systemic disease in susceptible mice strains. However, different S. Typhimurium strains differ in regard to virulence and host adaptation. Here, C57BL/6 mice were infected, respectively, with different S. Typhimurium strains SL1344 (calf), CVCC541 (chicken) and CMCC50115 (mutton) to determine their virulence and host immune responses. It was found that mice were less susceptible to infection by S. Typhimurium CVCC541 and CMCC50115 strains, with lower lethality and decreased bacterial burden in liver and spleen. Besides, S. Typhimurium strains CVCC541 and CMCC50115 enhanced host innate immune responses by increased frequencies of macrophages and neutrophils 3 days after infection. But SL1344 strain evaded immune response by inducing apoptosis of macrophages. Moreover, CVCC541 could elicit adaptive immune responses of host 11 days after infection upon examination of the proliferation and activation of CD4+ T cells. In addition, 125 and 138 unique mutant coding genes, respectively, in S. Typhimurium strains CVCC541 and CMCC50115 and 78 shared mutant coding genes were annotated by genomic alignment to SL1344 genome and the signal pathways involving these genes were further analyzed. The acquired results indicate that different original S. Typhimurium strains show differential virulence and may induce diverse immune responses in the same host infected.

Genetic modification provides a means to enhancing disease resistance in animals. Toll-like receptor 4 (TLR4), a member of the TLR family, is critical for the recognition of lipopolysaccharide (LPS)/endotoxin from Gram-negative bacteria by host immune cells, which initiates cell activation and subsequently triggers a proinflammatory response to the invading pathogens. In this study, the first generation of genetically modified (GM) sheep overexpressing TLR4 was produced by microinjection for better disease resistance. Compared with wild-type (WT) rams, the GM rams have similar growth performance, basic semen quality and spermatozoon ultrastructure. The offspring birth rates after cervical artificial insemination were also similar between GM (90.32%) and WT (92.38%) rams. Overall, the presence and expression of the TLR4 transgene in the genome did not appear to interfere with normal semen production, reproductive traits and the ability of transgene transmission to offspring. The expression levels of TLR4, tumor necrosis factor and interferon gamma genes in monocyte/macrophages from GM sheep were significantly higher than that from WT sheep at early stages after LPS stimulation. The GM offspring born from the founder transgenic ram inseminated ewes had similar survival rate with WT offspring (88.89% vs 84.86%) at weaning. The TLR4 transgene showed no deleterious effects on growth performance, reproductive traits and offspring survivability of GM rams. Therefore, the GM sheep overexpressing TLR4 provide a powerful experimental model for analyzing function of TLR4 in vivo during infection and inflammation.

Sheep are an important source of fiber production. Fibroblast growth factor 5 (FGF5) is a dominant inhibitor of length of the anagen phase of the hair cycle. Knockout or silencing of the gene results in a wooly coat in mice, donkeys, dogs, and rabbits. In sheep breeding, wool length is one of the most important wool quality traits. However, traditional breeding cannot accurately and efficiently mediate an advanced genotype into the sheep genome. In this study, we generated 3 knockout sheep via the 1-step clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system. Sequencing analysis confirmed that mutations in the gene existed in all germ lines of 3 founders: besides the intact sequence, 3 kinds of deletions in the gene (including 5, 13, and 33 bp) were detected. The changes in the primary and senior structure of the FGF5 protein due to the 3 deletions in founders suggested that the FGF5 protein was dysfunctional. In addition, the expression level of intact mRNA in heterozygous individuals decreased compared with the wild types ( < 0.01). Functionally, we discovered that wool length in founders was significantly longer than in wild types ( < 0.05). Collectively, the knockout sheep with the longer wool length phenotype will provide an efficient way for fast genetic improvement of sheep breeding and promote the development of wool industry.

Female reproductive (ovarian) aging is characterized by a marked decline in quantity and quality of follicles and oocytes, as well as alterations in the surrounding ovarian stroma. In our previous report, we have shown that dietary selenium (Se) insufficiency and supplementation differentially impacted the reproductive efficiency in aging mice; however, the precise understanding of such modulation is still incomplete. In the present study, we sought to determine the impact of low (mildly low level) and moderately high (medium level) Se diets on expression profile of non-selenoprotein genes in the ovaries of aging mice. For this purpose, the aged mice were divided in two groups and fed either a low Se (Se-L; 0.08 mg Se/kg) diet or a moderately high Se (Se-M; 0.33 mg Se/kg) diet. RNA-seq analysis revealed that a total of 168 genes were differentially expressed between the two groups. From these, 72 and 96 differentially expressed genes (DEGs) were found to be upregulated and downregulated, respectively. Gene Ontology (GO) and pathways enrichment (KEGG) analyses revealed that these DEGs were enriched in several key GO terms and biological pathways including PI3K-Akt signaling pathway, steroid hormone biosynthesis, signaling pathways regulating pluripotency of stem cells, Hippo signaling pathway, ovarian steroidogenesis, and Wnt signaling pathway. Further filtering of RNA-seq data revealed that several DEGs such as Star, Hsd3b6, Scd1, Bmp7, Aqp8, Gas1, Fzd1, and Wwc1 were implicated in key ovarian- and fertility-related functions. In addition, some of the DEGs were related to ER homeostasis and/or proteostasis. These results highlight that dietary low and moderately high (medium level) Se diets, in addition to modulation of selenoproteins, can also have an impact on expression of several non-selenoprotein genes in the ovaries of aging mice. To sum up, these findings add more value to our understanding of Se modulation of ovarian functions and female fertility and will pave a way for the focused mechanistic and functional studies in this domain.

Salmonella genus represents the most common foodborne pathogens causing morbidity, mortality, and burden of disease in all regions of the world. The introduction of antimicrobial agents and Salmonella-specific phages has been considered as an effective intervention strategy to reduce Salmonella contamination. However, data from the United States, European countries, and low- and middle-income countries indicate that Salmonella cases are still a commonly encountered cause of bacterial foodborne diseases globally. The control programs have not been successful and even led to the emergence of some multidrug-resistant Salmonella strains. It is known that the host immune system is able to effectively prevent microbial invasion and eliminate microorganisms. However, Salmonella has evolved mechanisms of resisting host physical barriers and inhibiting subsequent activation of immune response through their virulence factors. There has been a high interest in understanding how Salmonella interacts with the host. Therefore, in the present review, we characterize the functions of Salmonella virulence genes and particularly focus on the mechanisms of immune escape in light of evidence from the emerging mainstream literature.

In the present report, we determined the impact of dietary selenium (Se) deficiency and supplementation on the expression of two ER-resident selenoproteins i.e., Selenok and Selenom in the ovaries of aging mice. The mRNA expression of Selenok and Selenom (RT-qPCR) was significantly higher in the ovaries of mice fed diets supplemented with inorganic (ISe-S: 0.33 mg Se/kg) and organic (OSe-S: 0.33 mg Se/kg) Se compared to those fed a Se-deficient (Se-D: 0.08 mg Se/kg) diet and both Se-adequate (ISe-A: 0.15 mg Se/kg and OSe-A: 0.15 mg Se/kg) diets. Similarly, the protein signals of SELENOK (immunofluorescence assay) were also significantly higher in the Se-supplemented groups compared to those fed Se-D and Se-adequate (ISe-A and OSe-A) diets. Meanwhile, the rate of in vitro-produced blastocysts developing from MII oocytes was also evaluated and it was revealed that this rate was significantly higher in the Se-supplemented mice compared to those fed a Se-D diet. Altogether, the dietary Se supplementation increased the expression of Selenok (also its protein expression) and Selenom in the ovaries of aging mice, potentially contributing to an improved developmental potential of in vitro-matured M II oocytes.

Selenium (Se) is an important trace mineral having many essential roles at the cellular and organismal levels in animal and human health. The biological effects of Se are mainly carried out by selenoproteins (encoded by 25 genes in humans and 24 in mice). As an essential component of selenoproteins, Se performs structural and enzymic roles; in the latter context it is well known for its catalytic and antioxidative functions. Studies involving different animal models have added great value to our understanding regarding the potential implications of Se and selenoproteins in mammalian fertility and reproduction. In this review, we highlight the implications of selenoproteins in male fertility and reproduction followed by the characteristic biological functions of Se and selenoproteins associated with overall male reproductive function. It is evident from observations of past studies (both animal and human) that Se is essentially required for spermatogenesis and male fertility, presumably because of its vital role in modulation of antioxidant defense mechanisms and other essential biological pathways and redox sensitive transcription factors. However, bearing in mind the evidences from mainstream literature, it is also advisable to perform more studies focusing on the elucidation of additional roles played by the peculiar and canonical selenoproteins i.e., glutathione peroxidase 4 (GPX4) and selenoprotein P (SELENOP) in the male reproductive functions. Nevertheless, search for the elucidation of additional putative mechanisms potentially modulated by other biologically relevant selenoproteins should also be included in the scope of future studies. However, as for the implication of Se in fertility and reproduction in men, though a few clinical trials explore the effects of Se supplementation on male fertility, due to inconsistencies in the recruitment of subjects and heterogeneity of designs, the comparison of such studies is still complicated and less clear. Therefore, further research focused on the roles of Se and selenoproteins is awaited for validating the evidences at hand and outlining any therapeutic schemes intended for improving male fertility. As such, new dimensions could be added to the subject of male fertility and Se supplementation.

This study aimed to investigate the effect of melatonin on the cell cycle of parthenogenetic embryos derived from vitrified mouse metaphase II (MII) oocytes. Fresh oocytes were randomly allocated into three groups: untreated (control), or vitrified by the open-pulled straw method without (Vitrification group) or with melatonin (MT) supplementation (Vitrification + MT group). After warming, oocytes were parthenogenetically activated and cultured in vitro, then the percentage of embryos in the G1/S phase, the levels of reactive oxygen species (ROS) and glutathione (GSH), and the mRNA expression of cell cycle-related genes (P53, P21 and E2F1) in zygotes and their subsequent developmental potential in vitro were evaluated. The results showed that the vitrification/warming procedures significantly decreased the frequency of the S phase, markedly increased ROS and GSH levels and the expression of P53 and P21 genes, and decreased E2F1 expression in zygotes at the G1 stage and their subsequent development into 2-cell and blastocyst stage embryos. However, when 10&minus, 9 mol/L MT was administered for the whole duration of the experiment, the frequency of the S phase in zygotes was significantly increased, while the other indicators were also significantly improved and almost recovered to the normal levels shown in the control. Thus, MT might promote G1-to-S progression via regulation of ROS, GSH and cell cycle-related genes, potentially increasing the parthenogenetic development ability of vitrified&ndash, warmed mouse oocytes.

Although melatonin has some of the broadest ranges of actions on the physiology of vertebrates, especially on their reproductive processes, the mechanism by which melatonin regulates animal reproduction is still incompletely understood. This study was designed to determine the effect of oral melatonin on the reproductive performance of female mice. Female ICR mice (7 weeks old) were given melatonin-containing water (3, 30 and 300 &mu, g/mL, melatonin) or water only (control) until 10 weeks of age. Then, some of the mice were successfully mated (confirmed by vaginal plugs), and the number of live births and their weights were recorded. Some mice were used for a histological analysis of the number of follicles in the ovaries. Others were used for oocyte collection after superovulation, and in vitro fertilization (IVF) was performed. The mRNA expression of the apopotosis-related genes (BAX, BCL2) in the IVF embryos were analyzed. After melatonin administration, the mice showed similar serum melatonin levels to that of the control. The number of antral follicles per mm2 unit area in the 30 &mu, g/mL melatonin-treated group (14.60) was significantly higher than that of the control (7.78), which was lower than that of the 3 &mu, g/mL melatonin-treated group (12.29). The litter size was significantly higher in the 3 &mu, g/mL melatonin-treated group (15.5) than in the control (14.3). After IVF, the hatched blastocyst formation rate in the 30 &mu, g/mL melatonin-treated group (85.70%) was significantly higher than that of the control (72.10%), and it was the same for the BCL2/BAX expression ratio. Although oral melatonin did not appear to have an effect on the serum melatonin rhythm in the mouse, melatonin did increase litter size at the 3 &mu, g/mL dose level, and improved the developmental competency of IVF embryos at the 30 &mu, g/mL level.

Salmonella enterica serovar Gallinarum biovar Pullorum ( S. Pullorum ) is responsible for the systemic salmonellosis in different breeds of chickens. Macrophages, as host cells, play a key role in the innate immune response following infection with S. Pullorum . In this study, we first generated macrophages from two breeds of chicken (White Leghorn (WL) and Tibetan Chickens (TC)) peripheral blood monocytes in vitro. Then, we showed that the production of interleukin-1β (IL-1β), macrophage inflammatory protein-1β (MIP-1β) and interleukin-10 (IL-10) in lipopolysaccharide (LPS)-treated macrophages was significantly higher compared with the unstimulated cells in TC. LPS triggered only more expression of IL-10 in WL macrophages. Furthermore, macrophages from TC eliminated intracellular bacteria more efficiently than those from WL after S. Pullorum infection at a multiplicity of infection (MOI) 1. In addition, the variation between individuals and sex had the crucial effect on the immune response to LPS and S. Pullorum invasion.

As key elements and targets for various intracellular pathogens, macrophages play an essential role in host defense. Although ovine peripheral blood monocyte-derived cell lines have been established, their phenotypic characteristics and functional properties remain unknown. We have established several ovine macrophage cell lines from peripheral blood adherent cells that can proliferate spontaneously in long-term culture in vitro. Characteristics of macrophages were shaped by cell morphology, cell adhesion, expression of cell surface markers, phagocytic activity and inflammatory response. Furthermore, the differences of genes expression (such as membrane proteins, pro-inflammatory cytokines and chemokine) were compared between blood macrophages (BMs) and alveolar macrophages (AMs), and between BMs and splenic macrophages (SMs), respectively. The expression of membrane genes (CD11b and CD80), pro-inflammatory cytokines (IL-1β and TNFα) and chemokines (IL-8/CXCL8 and CCL-21) was lower than that in AMs or SMs, but not CD200. Moreover, BMs maintained lower expression level of M1 macrophage related genes (iNOS and IDO), but high expression levels of M2 macrophage related genes (ARG2 and TGFβ1). BMs showed lower phagocytic ability than AMs and SMs. Compared with AMs, BMs showed higher salmonella proliferation rate within cells. Collectively, BMs could suppress inflammatory responses and possessed partly phenotypic characteristics of M2 macrophages.

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long periodin vitrostill remains unexplored. We found that the cESCs showed stem cell-like propertiesin vitrofor a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocytein vitro. Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCsin vitro.

This study was conducted to investigate the effect of vitrification on the dynamics of the global transcriptome in bovine germinal vesicle (GV) oocytes and their in vitro-derived metaphase II (MII) oocytes. The GV oocytes were vitrified using the open-pulled straw method. After warming, GV oocytes and the resulting MII-stage oocytes were cultured in vitro for 2 h and 24 h respectively and were then collected. The fresh GV oocytes and their in vitro-derived MII oocytes were used as controls. Then, each pool (fresh GV, n = 3; vitrified GV, n = 4; fresh MII, n = 1 and MII derived from vitrified GV, n = 2) from the different stages was used for mRNA transcriptome sequencing. The results showed that the in vitro maturation rates of GV oocytes were significantly decreased (32.36% vs 53.14%) after vitrification. Bovine GV oocyte vitrification leads to 12 significantly upregulated and 19 downregulated genes. After culturing in vitro, the vitrification-derived MII oocytes showed 47 significantly upregulated and six downregulated genes when compared with those from fresh GV oocytes. Based on molecular function–gene ontology terms analysis and the Kyoto encyclopaedia of genes (KEGG) pathway database, the differentially expressed genes were associated with the pathways of cell differentiation and mitosis, transcription regulation, regulation of actin cytoskeleton, apoptosis and so on, which potentially result in the lower in vitro development of GV bovine oocytes.

Foot and mouth disease, which is induced by the foot and mouth disease virus (FMDV), takes its toll on the cloven-hoofed domestic animals. The VP1 gene in FMDV genome encodes the viral capsid, a vital element for FMDV replication. Sleeping Beauty (SB) is an active DNA-transposon system responsible for genetic transformation and insertional mutagenesis in vertebrates. In this study, a conserved VP1-shRNA which specifically targets the ovine FMDV-VP1 gene was constructed and combined with SB transposase and transposon. Then, they were microinjected into pronuclear embryos to breed transgenic sheep. Ninety-two lambs were born and the VP1-shRNA was positively integrated into eight of them. The rate of transgenic sheep production in SB transposon system was significantly higher than that in controls (13.04% vs. 3.57% and 7.14%, P VP1 vector had a significant inhibitory effect on the VP1 gene of the FMDV. In conclusion, the VP1-shRNA transgenic sheep were successfully generated by the current new method. The ear fibroblasts from these transgenic sheep possess a great resistance to FMDV. The result indicated that RNAi technology combining the “Sleeping Beauty” transposon system is an efficient method to produce transgenic animals.

Salmonella enteric serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen causing public health hazards. Identification of genes related to macrophages resistance to S. Typhimurium and their immune mechanisms can provide a theoretical basis for disease resistance. In this study, sixty significant differentially expressed genes were screened between susceptible and resistant sheep macrophages by transcriptome RNA-seq. Eight significantly enriched GO terms and six canonical pathways were involved by GO and KEGG enrichment analysis. Furthermore, knockdown of HMOX1 and SLPI increased remarkably the clearance of S. typhimurium, but SPP1 had little effect on the clearance of S. Typhimurium within sheep macrophages. Altogether, these results suggest that many genes of macrophages were reprogrammed via S. Typhimurium infection, some of which may facilitate host defence against Salmonella, while others allow Salmonella to escape.

Female reproductive (ovarian) aging is distinctively characterized by a markedly reduced reproductive function due to a remarkable decline in quality and quantity of follicles and oocytes. Selenium (Se) has been implicated in playing many important biological roles in male fertility and reproduction, however, its potential roles in female reproduction, particularly in aging subjects, remain poorly elucidated. Therefore, in the current study we used a murine model of female reproductive aging and elucidated how different Se-levels might affect the reproductive efficiency in aging females. Our results showed that at the end of an 8-week dietary trial, whole-blood Se concentration and blood total antioxidant capacity (TAOC) were significantly reduced in Se-deficient (0.08 mg Se/kg, Se-D) mice, whereas both of these biomarkers were significantly higher in inorganic (0.33 mg/kg, ISe-S) and organic (0.33 mg/kg, OSe-S) Se-supplemented groups. Similarly, compared to the Se-D group, Se supplementation significantly ameliorated the maintenance of follicles and reduced the rate of apoptosis in ovaries. Meanwhile, the rate of in vitro-produced embryos resulting from germinal vesicle (GV) oocytes was also significantly improved in Se-supplemented (ISe-S and OSe-S) groups compared to the Se-D mice, in which none of the embryos developed to the hatched blastocyst stage. RT-qPCR results revealed that mRNA expression of Gpx1, Gpx3, Gpx4, Selenof, p21, and Bcl-2 genes in ovaries of aging mice was differentially modulated by dietary Se levels. A considerably higher mRNA expression of Gpx1, Gpx3, Gpx4, and Selenof was observed in Se-supplemented groups compared to the Se-D group. Similarly, mRNA expression of Bcl-2 and p21 was significantly lower in Se-supplemented groups. Immunohistochemical assay also revealed a significantly higher expression of GPX4 in Se-supplemented mice. Our results reasonably indicate that Se deficiency (or marginal levels) can negatively impact the fertility and reproduction in females, particularly those of an advancing age, and that the Se supplementation (inorganic and organic) can substantiate ovarian function and overall reproductive efficiency in aging females.

In this study, using a laying hen model, we determined the expression of FOXL2 and RSPO1 in different central and peripheral tissue and ovarian follicles at different stages of development. At the same time, mRNA expression of both genes in granulosa and theca cells harvested from follicles at different stages of folliculogenesis was also evaluated. Finally, we assessed the effect of leptin treatment on expression of FOXL2 and RSPO1 in in vitro cultured granulosa cells harvested from 1&ndash, 5 mm to F3&ndash, F1 follicles. Our RT-qPCR results revealed that a comparatively higher expression of FOXL2 and RSPO1 was observed in ovary, hypothalamus, and pituitary. Abundant mRNA expression of FOXL2 was observed in small prehierarchical follicles (1&ndash, 1.9 and 2&ndash, 2.9 mm follicles, p &lt, 0.05), whereas mRNA expression of RSPO1 showed an increasing trend in large hierarchical follicles (F5&ndash, F1), and its abundant expression was observed in post-ovulatory follicles. FOXL2 mRNA expression was stable in granulosa cells harvested from 3&ndash, 5 mm to F4 follicles, and exhibited a significantly higher expression in large hierarchical follicles. Conversely, relatively low mRNA expression of FOXL2 was observed in theca cells. RSPO1 mRNA expression was relatively lower in granulosa cells, however, theca cells exhibited a significantly higher mRNA expression of RSPO1 in F4 to F1 follicles. In the next experiment, we treated the in vitro cultured granulosa cells with different concentrations (1, 10, 100, and 1000 ng/mL) of exogenous leptin. Compared to the control group, a significant increase in the expression of FOXL2 was observed in groups treated with 1, 10, and 100 ng/mL leptin, whereas expression of RSPO1 was increased in all leptin-treated groups. When treated with 100 ng/mL leptin, FOXL2 and RSPO1 expression was upregulated in cultured granulosa cells harvested from both large hierarchical (F3&ndash, F1) and small prehierarchical follicles (1&ndash, 5 mm). Based on these findings and evidence from mainstream literature, we envisage that FOXL2 and RSPO1 genes (in connection with hypothalamic-hypophysis axis) and leptin (via modulation of FOXL2 and RSPO1 expression) might have significant physiological roles, at least in part, in modulating the ovarian mechanisms, such as follicle development, selection, and steroidogenesis in laying hens.

As an important micronutrient, selenium (Se) plays many essential roles in immune response and protection against pathogens in humans and animals, but underlying mechanisms of Se-based control of salmonella growth within macrophages remain poorly elucidated. In this study, using RNA-seq analyses, we demonstrate that Se treatment (at an appropriate concentration) can modulate the global transcriptome of chicken macrophages HD11. The bioinformatic analyses (KEGG pathway analysis) revealed that the differentially expressed genes (DEGs) were mainly enriched in retinol and glutathione metabolism, revealing that Se may be associated with retinol and glutathione metabolism. Meanwhile, Se treatment increased the number of salmonella invading the HD11 cells, but reduced the number of salmonella within HD11 cells, suggesting that enhanced clearance of salmonella within HD11 cells was potentially modulated by Se treatment. Furthermore, RNA-seq analyses also revealed that nine genes including SIVA1, FAS, and HMOX1 were differentially expressed in HD11 cells infected with salmonella following Se treatment, and GO enrichment analysis showed that these DEGs were mainly enriched in an extrinsic apoptotic signaling pathway. In summary, these results indicate that Se treatment may not only affect retinol and glutathione metabolism in macrophages, but could also inhibit salmonella-induced macrophage apoptosis via an extrinsic apoptotic signaling pathway involving SIVA1.

Melatonin as a potent antioxidant exhibits important nutritional and medicinal values. To produce melatonin-enriched milk will benefit the consumers. In this study, a sheep bioreactor which generates melatonin-enriched milk has been successfully developed by the technology that combined CRISPR/Cas9 system and microinjection. The AANAT and ASMT were cloned from pineal gland of Dorper sheep (Ovis aries). The in vitro studies found that AANAT and ASMT were successfully transferred to the mammary epithelial cell lines and significantly increased melatonin production in the culture medium compared to the nontransgenic cell lines. In addition, the Cas9 mRNA, sgRNA, and the linearized vectors pBC1-AANAT and pBC1-ASMT were co-injected into the cytoplasm of pronuclear embryos which were implanted into ewes by oviducts transferring. Thirty-four transgenic sheep were generated with the transgenic positive rate being roughly 35% which were identified by Southern blot and sequencing. Seven carried transgenic AANAT, two carried ASMT, and 25 carried both of AANAT and ASMT genes. RT-PCR and Western blot demonstrated that the lambs expressed these genes in their mammary epithelial cells and these animals produced melatonin-enriched milk. This is the first report to show a functional AANAT and ASMT transgenic animal model which produce significantly high levels of melatonin milk compared to their wild-type counterparts. The advanced technologies used in the study laid a foundation for generating large transgenic livestock, for example, the cows, which can produce high level of melatonin milk.

Selenium (Se) is an essential micronutrient that has several important functions in animal and human health. The biological functions of Se are carried out by selenoproteins (encoded by twenty-five genes in human and twenty-four in mice), which are reportedly present in all three domains of life. As a component of selenoproteins, Se has structural and enzymatic functions; in the latter context it is best recognized for its catalytic and antioxidant activities. In this review, we highlight the biological functions of Se and selenoproteins followed by an elaborated review of the relationship between Se and female reproductive function. Data pertaining to Se status and female fertility and reproduction are sparse, with most such studies focusing on the role of Se in pregnancy. Only recently has some light been shed on its potential role in ovarian physiology. The exact underlying molecular and biochemical mechanisms through which Se or selenoproteins modulate female reproduction are largely unknown; their role in human pregnancy and related complications is not yet sufficiently understood. Properly powered, randomized, controlled trials (intervention vs. control) in populations of relatively low Se status will be essential to clarify their role. In the meantime, studies elucidating the potential effect of Se supplementation and selenoproteins (i.e., GPX1, SELENOP, and SELENOS) in ovarian function and overall female reproductive efficiency would be of great value.

Cellular energy metabolism correlates with cell fate, but the metabolic properties of chicken embryonic stem (chES) cells are poorly understood. Using a previously established chES cell model and electron microscopy (EM), we found that undifferentiated chES cells stored glycogen. Additionally, undifferentiated chES cells expressed lower levels of glucose transporter 1 (GLUT1) and phosphofructokinase (PFK) mRNAs but higher levels of hexokinase 1 (HK1) and glycogen synthase (GYS) mRNAs compared with control primary chicken embryonic fibroblast (CEF) cells, suggesting that chES cells direct glucose flux towards the glycogenic pathway. Moreover, we demonstrated that undifferentiated chES cells block gluconeogenic outflow and impede the accumulation of glucose-6-phosphate (G6P) from this pathway, as evidenced by the barely detectable levels of pyruvate carboxylase (PCX) and mitochondrial phosphoenolpyruvate carboxykinase (PCK2) mRNAs. Additionally, cell death occurred in undifferentiated chES cells as shown by Hoechst 33342 and propidium iodide (PI) double staining, but it could be rescued by exogenous G6P. However, we found that differentiated chES cells decreased the glycogen reserve through the use of PAS staining. Moreover, differentiated chES cells expressed higher levels of GLUT1, HK1 and PFK mRNAs, while the level of GYS mRNA remained similar in control CEF cells. These data indicate that undifferentiated chES cells continue to synthesize glycogen from glucose at the expense of G6P, while differentiated chES cells have a decreased glycogen reserve, which suggests that the amount of glycogen is indicative of the chES cell state.

4-Nitroquinoline N-oxide (4-NQO) as an UV-mimetic agent leading to DNA damage is a potent mutagen and carcinogen, and can induce apoptosis in various types of cells. However, the mechanism of apoptosis induced by 4-NQO is still not quite clarified. In this study we found that 4-NQO could not only induce apoptosis in KB cells, but also caused considerable damage to the mitochondrial membrane. Therefore, we inferred that 4-NQO might induce apoptosis through the mitochondrial signaling pathway resulting from DNA damage. Further investigation showed that the apoptosis induced by 4-NQO was p53-dependent. Furthermore, the expression levels of bax and bcl-2, closely related to mitochondrial signaling pathway, were up- and down-regulated, respectively. Meanwhile, the activity of caspase-9 and -3, lying in downstream of mitochondrial, was also enhanced. At the same time, the expression level of p21 also was increased by 4-NQO exposure, leading to the cell cycle arrested in G(1) phase. The results indicated that 4-NQO arrested cell cycle in G(1) phase, thus allowing enough time for DNA repair; on the other hand, if the cellular DNA were not repaired, apoptosis may follow through the p53-dependent mitochondrial signaling pathway, and mechanism of apoptosis induced by 4-NQO is not exactly the same that induced by UV radiation, as the later induces apoptosis through death receptors and mitochondrial signaling pathway.

Foot and mouth disease induced by foot and mouth disease virus (FMDV) is severe threat to cloven-hoofed domestic animals. The gene 3Dpol in FMDV genome encodes the viral RNA polymerase, a vital element for FMDV replication. In this study, a conserved 3D-7414shRNA targeting FMDV-3Dpol gene was designed and injected into pronuclear embryos to produce the transgenic goats. Sixty-one goats were produced, of which, seven goats positively integrated 3D-7414shRNA. Loss of function assay demonstrated that siRNA effectively knockdown 3Dpol gene in skin epithelium cells of transgenic goats. Subsequently, the tongue epithelium cells from transgenic and non-transgenic goats were infected with FMDV O/YS/CHA/05 strain. A significant decrease of virus titres and virus copy number was observed in cells of transgenic goats compared with that of non-transgenic goats, which indicated that 3D-7414siRNA inhibited FMDV replication by interfering FMDV-3Dpol gene. Furthermore, we found that expression of TLR7, RIG-I and TRAF6 was lower in FMDV infected cells from transgenic goats compared to that from non-transgenic goats, which might result from lower virus copy number in transgenic goats’ cells. In conclusion, we successfully produced transgenic goats highly expressing 3D-7414siRNA targeting 3Dpol gene and the tongue epithelium cells from the transgenic goats showed effective resistance to FMDV.

Salmonella enterica serovar Gallinarum biovar Pullorum ( S. Pullorum) is responsible for the systemic salmonellosis in different breeds of chickens. Macrophages, as host cells, play a key role in the innate immune response following infection with S. Pullorum. In this study, we first generated macrophages from two breeds of chicken (White Leghorn (WL) and Tibetan Chickens (TC)) peripheral blood monocytes in vitro. Then, we showed that the production of interleukin-1β (IL-1β), macrophage inflammatory protein-1β (MIP-1β) and interleukin-10 (IL-10) in lipopolysaccharide (LPS)-treated macrophages was significantly higher compared with the unstimulated cells in TC. LPS triggered only more expression of IL-10 in WL macrophages. Furthermore, macrophages from TC eliminated intracellular bacteria more efficiently than those from WL after S. Pullorum infection at a multiplicity of infection (MOI) 1. In addition, the variation between individuals and sex had the crucial effect on the immune response to LPS and S. Pullorum invasion.

Saturated fatty acids (SFAs) can directly stimulate innate immune responses, thereby exacerbating inflammatory aspects of metabolic syndrome. Dietary SFAs act as ligands of Toll-like receptor 4 (TLR4), triggering associated signaling pathways. In this study, we investigated the role of TLR4 in palm oil SFA-associated inflammatory cytokine gene expression in monocytes/macrophages and adipose tissue using TLR4-overexpressing genetically modified sheep. SFA stimulation resulted in upregulation of interleukin-6 ( IL-6), tumor necrosis factor-α ( TNF-α), interleukin-8 ( IL-8), interferon-γ ( IFN-γ), and interleukin-10 ( IL-10), and TLR4 overexpression enhanced such SFA-induced inflammatory cytokine expression. Moreover, SFAs markedly activated MyD88-dependent signaling, including IL-1 receptor–associated kinase 4 ( IRAK4), TNF receptor–associated factor 6 ( TRAF6), and nuclear factor-κB ( NF-κB). Taken together, our results indicate that TLR4 overexpression enhances the SFA-induced inflammatory response through MyD88-dependent signaling in monocytes/macrophages and adipose tissue.

Genetic modification offers alternative strategies to traditional animal breeding. However, the food safety of genetically modified (GM) animals has attracted increasing levels of concern. In this study, we produced GM sheep overexpressing TLR4, and the transgene-positive offsprings (F1) were confirmed using the polymerase chain reaction (PCR) and Southern blot. The expression of TLR4 was 2.5-fold compared with that of the wild-type (WT) sheep samples. During the 90-day safety study, Sprague-Dawley rats were fed with three different dietary concentrations (3.75%, 7.5%, and 15% wt/wt) of GM sheep meat, WT sheep meat or a commercial diet (CD). Blood samples from the rats were collected and analyzed for hematological and biochemical parameters, and then compared with hematological and biochemical reference ranges. Despite a few significant differences among the three groups in some parameters, all other values remained within the normal reference intervals and thus were not considered to be affected by the treatment. No adverse diet-related differences in body weights or relative organ weights were observed. Furthermore, no differences were observed in the gross necropsy findings or microscopic pathology of the rats whose diets contained the GM sheep meat compared with rats whose diets contained the WT sheep meat. Therefore, the present 90-day rat feeding study suggested that the meat of GM sheep overexpressing TLR4 had no adverse effect on Sprague-Dawley rats in comparison with WT sheep meat. These results provide valuable information regarding the safety assessment of meat derived from GM animals.

The egg white (EW) contains the majority of bioactive components which maintain embryo growth and differentiation. The discovery of new growth promoting factor in egg white will provide vital clue to understand the developmental regulation of early chicken embryo. The egg white heated with different temperatures (63.5, 70 and 95°C) underwent testing on its growth-promoting effect on chicken fibroblast in vitro. The purified c-lysozyme and the expression of related genes in basic fibroblast growth factor (bFGF) pathway were analyzed to ascertain its growth-promoting mechanism. 13 h after egg white treatment, more fibroblast synchronized with serum starvation transited into S phrase from G0/G1 in EW group than in the control group (CM) and reached the phase of peak proliferation at 15 h after treatment. It was found that c-lysozyme had the function of promoting cells growth and was decided by gradient heat inactivation of egg white. The addition of more than 0.25 mg/ml c-lysozyme produced significant increase in the cellular proliferation during 48 to 72 h of culture. At 13 h after c-lysozyme treatment, the bFGF, cyclin D, cyclin A and CDK2 were up-regulated significantly and promoted the transition from G0/G1 into S phrase and the accurate completion of S phrase. C-Lysozyme contains a growth-activating domain to promote the cell proliferation besides its anti-microbe domain.Key words: c-Lysozyme, fibroblast, fibroblast growth factor receptor (FGFR), cell cycle.

The chicken embryo is a classic model used to investigate embryonic development, gene expression, and tissue differentiation, and is also an important research tool in studying the animal functional genomics. The whole blastoderms of fresh unincubated eggs from White Leghorn chickens were collected with a paper ring, mechanically broken into small pieces and cultured in medium. Then the small pieces would develop into blastocyte-like structures (BLS), which could be facilitated by an addition of fetal bovine serum (FBS) to the primary culture and their diameter was nearly doubled from 12 to 24 h. The additional yolk had no positive effect on the development in the first 12 h but encouraged the BLSs attaching and inner cells differentiating instead in 24 h. The inner cells of the BLS showing a high alkaline-phosphatase activity similar to those in mouse embryonic stem (ES) cells and also expressing a large amount of the specific stage embryonic antigen-1 (SSEA-1) on the surface, which was known to be the characteristic of non-differentiated mouse and avian ES cells, could finally differentiate into nerve-like cells, fibroblast cells and so on in the medium. Leukemia inhibitory factor (LIF) facilitated the cells’ proliferation and prevented differentiation in the suspended culture of the BLSs. So we drew the conclusion that the BLS obtained from broken blastoderm can be used to amplify avian ES cells so as to initiate a new method of producing transgenic chickens.

The provirus DNA sequence of porcine endogenous retrovirus (PERV) distributed in the pig genome is the major obstacle that restricts the swine as the organ donors in xenotransplantation, and the copy number of PERV varies greatly among different breeds and individuals. In the experiment, 67 healthy, female Chinese Experimental Mini-Pigs (CEMPs) aged at 3-6 months were selected from the Animal Husbandry Station of China Agricultural University, the copy number of PERV and types of envelope protein gene (env) were then investigated by means of PCR analysis and Southern blotting. It is showed that the distribution of types of envelope protein gene in Landrace and CEMPs makes little difference, but the proportion of individuals carrying two types of envelope protein gene (env-A and env-B, which is denoted as env-AB) is much larger than those which carry only one type of envelope protein gene (env-A or env-B). Meanwhile, two endogenous retrovirus free pigs were found for the first time during our research, and the copy number of others is relatively low, which is about 10 to 20. All the results illuminate the genetic diversity of indigenous pig breeds in China and the potential of CEMPs to serve as organ donors in xenotransplantation.

11 Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement,College of Animal Science and Technology, China Agricultural University, Beijing 100193, China2 State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China3 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China

Long-term in vitro maintenance of embryonic stem cell (ESC) pluripotency enables the pluripotency and differentiation of ESCs in animals to be investigated. The ability to successfully maintain and differentiate chicken embryonic stem cells (cESCs) would provide a useful tool for avian biology research and would be a resource directly applicable to agricultural production. In this study, endogenous chicken pluripotency transcription factors, POUV, Sox-2, Nanog and Lin28 were cloned and expressed as recombinant proteins containing a nine consecutive arginine protein transduction domain (PTD). cESCs were cultured with these recombinant proteins to maintain cESC pluripotency in vitro. Cultured cESCs exhibited typical characteristics of pluripotency, even after six generations of rapid doubling, including positive staining for stage-specific embryonic antigen I, and strong staining for alkaline phosphatase. Expression levels of the pluripotency markers, POUV, Nanog, C-Myc, Sox-2 and Lin28 were the same as in uncultured stage X blastoderm cells, and most significantly, the formation of embryoid bodies (EBs) by 6th generation cESCs confirmed the ability of these cultured cESCs to differentiate into cells of all three embryonic germ layers. Thus, transcription factors could be translocated through the cell membrane into the intracellular space of cESCs by using a PTD of nine consecutive arginines and the pluripotency of cESCs could be maintained in vitro for at least six generations.

Background Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons (breeding and non-breeding season) on superovulation and the imported exogenous gene on growth. Results In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep. Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05). The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group (P