Study of potential health effects of electromagnetic fields of telephony and Wi-Fi, using chicken embryo development as animal model.

The objective of this study is to investigate possible biological effects of radiofrequency electromagnetic fields (RF-EMF) as used in modern wireless telecommunication in a well-controlled experimental environment using chicken embryo development as animal model. Chicken eggs were incubated under continuous experimental exposure to GSM (1.8 GHz), DECT (1.88 GHz), UMTS (2.1 GHz), and WLAN (5.6 GHz) radiation, with the appropriate modulation protocol, using a homogeneous field distribution at a field strength of approximately 3 V/m, representing the maximum field level in a normal living environment. Radiation-shielded exposure units/egg incubators were operating in parallel for exposed and control eggs in a climatized homogeneous environment, using 450 eggs per treatment in three successive rounds per treatment. Dosimetry of the exposure (field characteristics and specific absorption rate) were studied. Biological parameters studied included embryo death during incubation, hatching percentage, and various morphological and histological parameters of embryos and chicks and their organs, and gene expression profiles of embryos on day 7 and day 18 of incubation by microarray and qPCR. No conclusive evidence was found for induced embryonic mortality or malformations by exposure to the used EMFs, or for effects on the other measured parameters. Estimated differences between treatment groups were always small and the effect of treatment was not significant. In a statistical model that ignored possible interaction between rounds and exposure units, some of the many pairwise comparisons of exposed versus control had P-values lower than 0.05, but were not significant after correction for multiple testing. Bioelectromagnetics. 38:186-203, 2017. © 2017 Wiley Periodicals, Inc.

Association of Egg Mass and Egg Sex: Gene Expression Analysis from Maternal RNA in the Germinal Disc Region of Layer Hens (Gallus gallus).

Female birds have been shown to manipulate offspring sex ratio. However, mechanisms of sex ratio bias are not well understood. Reduced feed availability and change in body condition can affect the mass of eggs in birds that could lead to a skew in sex ratio. We employed feed restriction in laying chickens (Gallus gallus) to induce a decrease in body condition and egg mass using 45 chicken hens in treatment and control groups. Feed restriction led to an overall decline of egg mass. In the second period of treatment (Days 9-18) with more severe feed restriction and a steeper decline of egg mass, the sex ratio per hen (proportion of male eggs) had a significant negative association with mean egg mass per hen. Based on this association, two groups of hens were selected from feed restriction group, that is, hens producing male bias with low egg mass and hens producing female bias with high egg mass with overall sex ratios of 0.71 and 0.44 respectively. Genomewide transcriptome analysis on the germinal disks of F1 preovulatory follicles collected at the time of occurrence of meiosis-I was performed. We did not find significantly differentially expressed genes in these two groups of hens. However, gene set enrichment analysis showed that a number of cellular processes related to cell cycle progression, mitotic/meiotic apparatus, and chromosomal movement were enriched in female-biased hens or high mean egg mass as compared with male-biased hens or low mean egg mass. The differentially expressed gene sets may be involved in meiotic drive regulating sex ratio in the chicken.

Transcription networks responsible for early regulation of Salmonella-induced inflammation in the jejunum of pigs.

BACKGROUND: The aim of this study was to identify transcription factors/regulators that play a crucial role in steering the (innate) immune response shortly (within a few hours) after the first contact of the intestinal mucosa with an inflammatory mediator, and to test whether the processes regulated by these factors/regulators can be modulated by chemical substances of natural origin. METHODS: We experimentally induced inflammation by perfusion of surgically applied jejunal loops with Salmonella enterica subspecies enterica serovar Typhimurium DT104 in three pigs. Segments of mock and Salmonella treated loops were dissected after 2, 4 and 8 hours of perfusion. IL8 and IL1-beta mRNA expression levels were measured in mucosal scrapings of all segments. Furthermore, intra-animal microarray comparisons (isogenic) between Salmonella and mock treated segments after 8 hours, and inter-animal comparisons between similar Salmonella-treated loops of each pig at 2 and 4 hours, were performed. RESULTS: IL-1beta and IL8 mRNA levels, and intra-animal microarray comparisons at 8 hours between Salmonella and mock treated segments showed that the response-time and type of response to Salmonella was different in all three pigs. This plasticity allowed us to extract a comprehensive set of differentially expressed genes from inter-animal comparisons at 2 and 4 hours. Pathway analysis indicated that many of these genes play a role in induction and/or tempering the inflammatory response in the intestine. Among them a set of transcription factors/regulators known to be involved in regulation of inflammation, but also factors/regulators for which involvement was not expected. Nine out of twenty compounds of natural origin, which according to literature had the potential to modulate the activity of these factors/regulators, were able to stimulate or inhibit a Salmonella-induced mRNA response of inflammatory-reporter genes IL8 and/or nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha in cultured intestinal porcine epithelial cells. CONCLUSIONS: We describe a set of transcription factors/regulators possibly involved in regulation of "very early" immune mechanism which determines the inflammatory status of the intestine later on. In addition, we show that these mechanisms may be modulated by chemical substances of natural origin.

A reliable method for sexing unincubated bird eggs for studying primary sex ratio.

In birds, offspring sex ratio manipulation by mothers is now well established with potentially important consequences for evolution and animal breeding. In most studies on primary sex ratio of birds, eggs are sexed after incubation by the use of PCR methods targeted to the sex-linked CHD1 genes. Sexing of unincubated eggs would be preferred, but as fertile and infertile blastodiscs cannot be distinguished macroscopically, errors could arise from PCR amplifications of parental DNA associated with the vitelline membrane of infertile eggs. In this study, we stained blastodiscs without the vitelline membrane with Hoechst 33342. This allowed unequivocal distinction between fertile and infertile blastodiscs. Fertile blastodiscs contained thousands of fluorescent nuclei, whereas no nuclei were seen in infertile eggs. In addition, after nucleic acid analysis, fertile blastodiscs yielded much stronger chromosomal DNA and CHD1-targeted PCR bands on agarose gels compared with infertile blastodiscs. These findings indicate that fertile blastodiscs contain much more embryonic DNA than parental DNA, allowing reliable sexing of the fertile eggs. The differences between fertile and infertile blastodiscs in chromosomal DNA and CHD1 PCR banding intensities alone could also be used to distinguish fertile from infertile eggs without using Hoechst staining. We conclude that identifying fertile blastodiscs either by Hoechst staining or by analyzing the yield of chromosomal DNA and CHD1-PCR products, combined with CHD1-targeted PCR amplification, presents an easy and reliable method to sex unincubated eggs.

Gene expression patterns in four brain areas associate with quantitative measure of estrous behavior in dairy cows.

BACKGROUND: The decline noticed in several fertility traits of dairy cattle over the past few decades is of major concern. Understanding of the genomic factors underlying fertility, which could have potential applications to improve fertility, is very limited. Here, we aimed to identify and study those genes that associated with a key fertility trait namely estrous behavior, among genes expressed in four bovine brain areas (hippocampus, amygdala, dorsal hypothalamus and ventral hypothalamus), either at the start of estrous cycle, or at mid cycle, or regardless of the phase of cycle. RESULTS: An average heat score was calculated for each of 28 primiparous cows in which estrous behavior was recorded for at least two consecutive estrous cycles starting from 30 days post-partum. Gene expression was then measured in brain tissue samples collected from these cows, 14 of which were sacrificed at the start of estrus and 14 around mid cycle. For each brain area, gene expression was modeled as a function of the orthogonally transformed average heat score values using a Bayesian hierarchical mixed model. Genes whose expression patterns showed significant linear or quadratic relationships with heat scores were identified. These included genes expected to be related to estrous behavior as they influence states like socio-sexual behavior, anxiety, stress and feeding motivation (OXT, AVP, POMC, MCHR1), but also genes whose association with estrous behavior is novel and warrants further investigation. CONCLUSIONS: Several genes were identified whose expression levels in the bovine brain associated with the level of expression of estrous behavior. The genes OXT and AVP play major roles in regulating estrous behavior in dairy cows. Genes related to neurotransmission and neuronal plasticity are also involved in estrous regulation, with several genes and processes expressed in mid-cycle probably contributing to proper expression of estrous behavior in the next estrus. Studying these genes and the processes they control improves our understanding of the genomic regulation of estrous behavior expression.

Early transcriptional response in the jejunum of germ-free piglets after oral infection with virulent rotavirus.

Germ-free piglets were orally infected with virulent rotavirus to collect jejunal mucosal scrapings at 12 and 18 hours post infection (two piglets per time point). IFN-gamma mRNA expression was stimulated in the mucosa of all four infected piglets, indicating that they all responded to the rotavirus infection. RNA pools prepared from two infected piglets were used to compare whole mucosal gene expression at 12 and 18 hpi to expression in uninfected germ-free piglets (n=3) using a porcine intestinal cDNA microarray. Microarray analysis identified 13 down-regulated and 17 up-regulated genes. Northern blot analysis of a selected group of genes confirmed the data of the microarray. Genes were functionally clustered in interferon-regulated genes, proliferation/differentiation genes, apoptosis genes, cytoskeleton genes, signal transduction genes, and enterocyte digestive, absorptive, and transport genes. Down-regulation of the transport gene cluster reflected in part the loss of rotavirus-infected enterocytes from the villous tips. Data mining suggested that several genes were regulated in lower- or mid-villus immature enterocytes and goblet cells, probably to support repair of the damaged epithelial cell layer at the villous tips. Furthermore, up-regulation was observed for IFN-gamma induced guanylate binding protein 2, a protein that effectively inhibited VSV and EMCV replication in vitro (Arch Virol 150:1213-1220, 2005). This protein may play a role in the small intestine's innate defense against enteric viruses like rotavirus.

The early transcriptional response of pig small intestinal mucosa to invasion by Salmonella enterica serovar typhimurium DT104.

Salmonella enterica serovar typhimurium (S. typhimurium) species are a leading cause of human invasive gastroenteritis. There is increasing in vitro evidence about Salmonella interaction with isolated cells or cell lines (macrophages, and enterocytes) on the molecular level, however, very little is known about in vivo interactions during actual invasion. We investigated the early interaction of S. typhimurium with intact small intestinal mucosa, in a pig model. Intestinal segments were infected with or without S. typhimurium DT104, and perfused. Whole mucosal gene expression was analyzed by cDNA array on 0, 2, 4, and 8h post-infection. Invasion resulted in the upregulation of only eight transcripts in jejunal mucosa, among those the proinflammatory IL-8 (at 4h only), and the antiinflammatory STAT3 (at 4 and 8h). The limited number of differentially expressed genes found here in vivo compared to in vitro is most likely due to the presence of multiple, heterogenous cell interactions in intact mucosa. Furthermore, it is concluded that S. typhimurium evades strong host responses by downregulating the local inflammatory response.

Transcriptome expression profiles in prenatal pigs in relation to myogenesis.

Myogenesis, the formation of muscle fibers, is a complex process. Pigs have been selected for efficient muscle growth for the past decades making them interesting to study myogenesis. We studied expression profiles of genes known to affect myogenesis, muscle structural proteins, and energy metabolism in prenatal pigs from 14 to 91 days of gestation. Primary and secondary muscle fiber formation takes place during days 30-60 and 54-90 of gestation, respectively. Differential expression and expression levels of the genes were studied using microarray technology. Gene activation and repression profiles were studied counting the number of spots with detectable signal. The number of spots for muscle tissue structural protein genes showing upregulated expression increased constantly from day 14 until day 91 of gestation indicating continued activation of genes during this period. The mRNA expression level of the genes showed a peak around day 35 of gestation. The expression levels of genes affecting myogenic differentiation (stimulating and inhibiting) showed a peak at day 35 of gestation. The number of spots for differentiation-stimulating genes showing differential expression reaches a first peak around day 35 of gestation and a nadir at day 49 of gestation while the number of spots for differentiation-inhibiting genes reaches a nadir at day 35 of gestation. Myogenic differentiation seems less a matter of the expression level of genes affecting differentiation, but depends on the balance between the number of significantly activated genes for stimulating and inhibiting differentiation. Genes stimulating myoblast proliferation showed a small peak expression prior to day 35 of gestation indicating myoblast proliferation before differentiation. The number of spots and the expression levels of genes for glycolysis and ATP-metabolism are at a nadir around days 35 and 49-63 of gestation suggesting that the energy metabolism is low during fusion of myoblasts into multinucleated muscle fibers.