Transcriptomic analysis of intestinal organoids, derived from pigs divergent in feed efficiency, and their response to Escherichia coli.

BACKGROUND: There is increasing interest in using intestinal organoids to study complex traits like feed efficiency (FE) and host-microbe interactions. The aim of this study was to investigate differences in the molecular phenotype of organoids derived from pigs divergent for FE as well as their responses to challenge with adherent and invasive Escherichia coli (E. coli). RESULTS: Colon and ileum tissue from low and high FE pigs was used to generate 3D organoids and two dimensional (2D) monolayers of organoid cells for E. coli challenge. Genome-wide gene expression was used to investigate molecular differences between pigs that were phenotypically divergent for FE and to study the difference in gene expression after challenge with E. coli. We showed, (1) minor differences in gene expression of colon organoids from pigs with low and high FE phenotypes, (2) that an E. coli challenge results in a strong innate immune gene response in both colon and ileum organoids, (3) that the immune response seems to be less pronounced in the colon organoids of high FE pigs and (4) a slightly stronger immune response was observed in ileum than in colon organoids. CONCLUSIONS: These findings demonstrate the potential for using organoids to gain insights into complex biological mechanisms such as FE.

Transit along the vas deferens results in a high percentage of filiform spermatozoa with compacted chromatin in the rooster (Gallus domesticus).

The present work aimed to evaluate the chromatin compaction of rooster spermatozoa along the male reproductive tract, and to study the vas deferens lining cells, potentially involved in sperm maturation. Chromomycin A3 (CMA3) was used to determine the chromatin compaction of spermatozoa from testis (T), proximal (including epididymis, V1), intermediate (V2) and distal (V3) vas deferens, and ejaculate (E). Six Birchen Leonesa roosters were used. E was obtained in vivo by dorso-ventral massage. V1, V2 and V3 sperm were obtained post mortem (six pairs of vasa deferentia), by flushing. T was obtained by washing the testes, cut in halves. The fixed cells were stained with CMA3 and propidium iodide for flow cytometry assessment. Results showed higher (P P P.

Changes in Blood Metabolites, Intestinal Microbiota Composition and Gene Expression of 95 Weeks Old Laying Hens Differing in Egg Production and Egg Breaking Strength.

Herein, we investigated to what extent molecular phenotypes of the systemic level (blood) and local (intestine) are associated with the performance of laying hens at 95 weeks of age. After the trial had run for 95 weeks, two performance groups were generated, i.e., egg production (PROD) and egg breaking strength (BS). A subset of 21 cages, 116 hens, was measured to indicate the metabolism and disease status. Additionally, a focus group (four cages) was made to perform molecular phenotyping in the intestine. A notifiable observation made during the post-mortem dissection was that approximately 12% of the birds at 95 weeks had developed certain aberrations and/or impairments (denoted as organ morbidity). At the systemic level, we observed five metabolites (γGT, triglycerides, HDL, glucose, and cholesterol) significantly associated to organ morbidity, and only two metabolites (urea and aspartate aminotransferase) to the performance phenotypes. At the local level, when comparing high PROD vs. low PROD, we observed differentially expressed genes involved in cell cycle processes and the extracellular matrix. When comparing high BS vs. low BS differentially, expressed genes were observed mainly involved in immune and cell cycle-related processes. This knowledge is crucial for developing novel strategies of keeping laying hens vital.

New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification.

Equine oocyte vitrification would benefit the growing in vitro embryo production programs, but further optimization of the protocol is necessary to reach clinical efficiency. Therefore, we aimed to perform a direct comparison of non-permeating and permeating cryoprotective agents (CPAs) during the vitrification and warming of equine immature oocytes. In the first experiment, cumulus oocytes complexes (COCs) were vitrified comparing sucrose, trehalose, and galactose in combination with ethylene glycol (EG) and dimethyl sulfoxide (DMSO). In the second experiment, the COCs were vitrified using three mixtures of permeating CPAs in a 50:50 volume ratio (ethylene glycol-dimethyl sulfoxide (ED), propylene glycol-ethylene glycol (PE), and propylene glycol-dimethyl sulfoxide (PD)) with galactose and warmed in different galactose concentrations (0.3 or 0.5 mol/L). Overall, all the treatments supported blastocyst formation, but the developmental rates were lower for all the vitrified groups in the first (4.3 to 7.6%) and the second (3.5 to 9.4%) experiment compared to the control (26.5 and 34.2%, respectively; p < 0.01). In the first experiment, the maturation was not affected by vitrification. The sucrose exhibited lower cleavage than the control (p = 0.02). Although the galactose tended to have lower maturation than trehalose (p = 0.060) and control (p = 0.069), the highest numerical cleavage and blastocyst rates were obtained with this CPA. In the second experiment, the maturation, cleavage, and blastocyst rates were similar between the treatments. Compared to the control, only the ED reached similar maturation (p = 0.02) and PE similar cleavage (p = 0.1). The galactose concentration during warming did not affect the maturation, cleavage, or blastocyst rates (p > 0.1), but the PE-0.3 exhibited the highest blastocyst rate (15.1%) among the treatments, being the only one comparable to the control (34.2%). As such, PE-galactose provides a valuable option for equine immature oocyte vitrification and should be considered for the future optimization of the protocol.

Environmentally enriched housing conditions affect pig welfare, immune system and gut microbiota in early life.

BACKGROUND: Conventional pig housing and management conditions are associated with gastrointestinal pathophysiology and disease susceptibility in early life. Developing new strategies to reduce both therapeutic and prophylactic antibiotic use is urgent for the sustainable swine production globally. To this end, housing methodology providing effective environmental enrichment could be a promising alternative approach to reduce antibiotic usage, as it has been proven to positively influence pig welfare and immune status and reduce susceptibility to infections. It is, however, poorly understood how this enriched housing affects systemic and local pulmonary immune status and gut microbiota colonization during early life. In the present study, we compared the effects of two housing conditions, i.e., conventional housing: (CH) versus enriched housing (EH), on immune status and gut microbiota from birth until 61 days of age. RESULTS: The expected benefits of enrichment on pig welfare were confirmed as EH pigs showed more positive behaviour, less aggression behaviour during the weaning transition and better human animal relation during the post weaning phase. Regarding the pigs' immune status, EH pigs had higher values of haemoglobin and mean corpuscular volume in haematological profiles and higher percentages of T cells and cytotoxic T cells in peripheral blood. Furthermore, EH pigs showed higher ex vivo secretion of IL1ß and TNF-α after lipopolysaccharide stimulation of whole blood than CH pigs. The structure of the developing faecal microbiota of CH and EH pigs significantly differed as early as day 12 with an increase in the relative abundance of several bacterial groups known to be involved in the production of short chain fatty acids, such as Prevotella_2, Christensenellaceae_R_7_group and Ruminococcus gauvreauii group. Furthermore, the main difference between both housing conditions post weaning was that on day 61, CH pigs had significantly larger inter-individual variation of ileal and colonic microbiota than EH pigs. In addition to housing, other intrinsic factors (e.g., sex) were associated with gut microbiota development and immune competence. CONCLUSIONS: In addition to the known welfare benefits for pigs, environmentally enriched housing also positively drives important aspects of the development of the immune system and the establishment of gut microbiota in early life. Consequently, EH may contribute to increasing productivity of pigs and reducing antibiotic use.

Research Note: Evaluation of two methods for adding cryoprotectant to semen and effects of bovine serum albumin on quality characteristics of cryopreserved rooster spermatozoa.

Chicken semen cryopreservation is a tool for programs of genetic diversity management and endangered breeds conservation. Due to physiological features, the fertility rates of cryopreserved poultry sperm are lower than mammal species. Thus, improvement of the semen cryopreservation methods is required. A first study was performed by a 2 × 2 factorial design consisting of 2 methods of adding the cryoprotectant [Direct or Diluted (mixed with extender medium)] and 2 cryoprotectants (glycerol and dimethylacetamide). Then sperm quality indicators were evaluated after freezing. A second study with a 2 × 2 design was conducted to evaluate the effectiveness of bovine serum albumin (BSA) on the optimization of 2 different extenders (Lake and Animal Sciences Group [ASG]). Viability and motility variables were evaluated before and after freezing. There was no significant difference in sperm viability and motility variables between Direct or Diluted methods. Supplementation of extenders with BSA improved most of the sperm motility variables in both extenders before and after freezing. Progressive sperm, non-progressive sperm before freezing, and all post-thaw sperm motility parameters, except amplitude of lateral head displacement and beat-cross frequency, were increased in BSA-supplemented extenders (P < 0.05), and BSA improved sperm viability in ASG extender after thawing (P < 0.05). After thawing, the interaction between extender and BSA (P < 0.05), eliminated the differences between the 2 BSA-supplemented media in curvilinear velocity, straight-line velocity, average path velocity, and amplitude of lateral head displacement which were higher in non-supplemented ASG extender than nonsupplemented Lake medium. In conclusion, the direct or diluted methods of adding glycerol or dimethylacetamide, did not significantly affect the post-thaw sperm characteristics. BSA positively affected most of the post-thaw sperm motility indicators regardless of the type of extender and resulted in significantly higher post-thaw sperm viability in ASG medium.

Organoids: a promising new in vitro platform in livestock and veterinary research.

Organoids are self-organizing, self-renewing three-dimensional cellular structures that resemble organs in structure and function. They can be derived from adult stem cells, embryonic stem cells, or induced pluripotent stem cells. They contain most of the relevant cell types with a topology and cell-to-cell interactions resembling that of the in vivo tissue. The widespread and increasing adoption of organoid-based technologies in human biomedical research is testament to their enormous potential in basic, translational- and applied-research. In a similar fashion there appear to be ample possibilities for research applications of organoids from livestock and companion animals. Furthermore, organoids as in vitro models offer a great possibility to reduce the use of experimental animals. Here, we provide an overview of studies on organoids in livestock and companion animal species, with focus on the methods developed for organoids from a variety of tissues/organs from various animal species and on the applications in veterinary research. Current limitations, and ongoing research to address these limitations, are discussed. Further, we elaborate on a number of fields of research in animal nutrition, host-microbe interactions, animal breeding and genomics, and animal biotechnology, in which organoids may have great potential as an in vitro research tool.

Cryopreservation of Avian Semen.

Cryopreservation protocols for semen exist for bird species used in animal production, fancy and hobby species, and wild bird species. Freezing of bird oocytes or embryos is not possible. Cryopreservation of avian semen is used for preserving (genetic diversity of) endangered species or breeds. Freezing semen can also be used in the breeding industry for maintaining breeding lines, as a cost-effective alternative to holding live birds. Success and efficiency of cryopreservation of bird semen differs among species and breeds or selection lines. This chapter describes important variables of methods for collecting, diluting, cold storage, and freezing and thawing of bird semen, notably the medium composition, cryoprotectant used and its concentration, cooling rate, freezing method, and warming method. Media and methods are described for freezing semen using either glycerol or DMA as cryoprotectant, which both are known in chicken and a number of other bird species to render adequate post-thaw fertility rates.

Cryopreservation of equine oocytes: looking into the crystal ball.

Invitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of invivo-matured oocytes has yielded better results, but is less practical. The presence of the corona radiata seems to partially protect those factors that are necessary for the construction of the normal spindle and for chromosome alignment, but multiple layers of cumulus cells may impair permeation of cryoprotective agents. In addition to the spindle, the oolemma and mitochondria are also particularly sensitive to vitrification damage, which should be minimised in future vitrification procedures. This review presents promising protocols and novel strategies in equine oocyte vitrification, with a focus on blastocyst development and foal production as most reliable outcome parameters.

Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols.

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to -35 °C (40 °C/min), from -35 °C to -65 °C (17 °C/min), and then from -65 °C to -85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to -5 °C (4 °C/min), from -5 °C to -110 °C (25 °C/min), and then from -110 °C to -140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to -10 °C (5 °C/min), and then from -10 °C to -130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.

Plasticity of intestinal gene expression profile signatures reflected by nutritional interventions in piglets.

BACKGROUND: Immediately after birth, the porcine intestine rapidly develops morphologically, functionally, and immunologically. The jejunum, the second part of the small intestine, is of importance for nutrient uptake and immune surveillance. To study the early postnatal development of the jejunum, a meta-analysis was performed on different transcriptomic datasets. These datasets were acquired from different experimental in-house studies or from experiments described in literature of porcine jejunum mucosa. Gene expression was measured under different experimental interventions, such as nutritional intervention, at various time-points (age). RESULTS: The studies included in the meta-analysis provided gene expression data for various time-points (piglet ages) for piglets that had received a treatment versus control piglets. In separate studies, treatments were administered to the sow (i.e. amoxicillin), or nutritional supplementation directly to the piglets with medium chain fatty acids (MCFAs), and oral administration of fructooligosaccharides (FOS) or a high dose of zinc-oxide, respectively. In the meta-analysis, genes were grouped into 16 clusters according to their temporal gene expression profiles for control piglets, i.e. the changes of gene expression level over time. Functional analysis showed that these temporal profile clusters had different dominant processes, such as immune related processes or barrier function. Transcriptomics data of treatment piglets was subsequently superimposed over the control temporal profiles. In this way we could investigate which temporal profile clusters (and which biological processes) were modulated by the treatments. Interestingly, not all 16 temporal profiles were modulated. CONCLUSIONS: We showed that it is possible to re-use (publicly available) transcriptomics data and produce temporal gene expression profiles for control piglets with overexpression of genes representing specific biological processes. Subsequently, by superimposing gene expression data from (nutritional) intervention studies we observed deviations from some of these reference profile(s) and thus the plasticity of the system. By employing this meta-analysis approach we highlighted the importance of birth and weaning and the underlying biological processes.

Seminal plasma amino acid profile in different breeds of chicken: Role of seminal plasma on sperm cryoresistance.

Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat ('good freezer') and Black-Red Andaluza ('bad freezer') showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages.

Semi-quantitative proteomics of mammalian cells upon short-term exposure to non-ionizing electromagnetic fields.

The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture.

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.

Cryopreservation of turkey semen: effect of breeding line and freezing method on post-thaw sperm quality, fertilization, and hatching.

Cryopreservation methods for poultry semen are not reliable for germplasm preservation, especially for turkeys, where fertility rates from frozen/thawed semen are particularly low. The objective was to evaluate cryopreservation methods for effectiveness in promoting cryosurvival and post-thaw function of sperm from five turkey lines: one commercial line and four research (RBC1; E; RBC2; F) lines from Ohio State University (OSU). The model for cryopreservation was set up as a 2×2×2×5 design for cryoprotectant (glycerol or dimethylacetamide (DMA)), cryopreservation medium (Lake or ASG), method of dilution (fixed dilution volume versus fixed sperm concentration) and turkey line, respectively. The final cryoprotectant concentrations were 11% glycerol or 6% DMA. Thawed sperm were evaluated for plasma membrane integrity and quality, motility, acrosome integrity and, after artificial insemination, for egg fertility and hatchability. Commercial turkey hens were used for all fertility trials, regardless of semen source. Turkey sperm frozen with glycerol exhibited higher membrane integrity and membrane quality upon thawing than turkey sperm frozen with DMA although no differences in total motility, and only minimal differences in progressive motility, were detected among the eight cryopreservation treatments. Within line, fertility was affected by cryoprotectant, medium and dilution method, where the overall highest percentages of fertile, viable embryos (Day 7) occurred for the DMA/ASG/fixed sperm concentration method, while high percentages (15.8-31.5%) of fertile, non-viable embryos (Day 1-6) were observed for multiple cryopreservation methods, including two glycerol treatments. From a single insemination, the duration of true and viable fertility in all lines was 10-13 weeks and 9-10 weeks, respectively. The duration of hatchability was 4-6 weeks after insemination for four of the turkey lines. The highest percentage of viable embryos was observed for the commercial line (9.5±2.4%), followed by the E line (5.3±1.3%), F line (3.7±2.0%) and RBC2 line (2.6±0.8%). For the RBC1 line, there was 100% embryonic death by Day 6 of incubation. Overall, better fertility results were obtained with the cryoprotectant DMA, the ASG diluent and fixed sperm concentration. However, the applicability of this method for preserving semen from research populations may be line dependent.

Effect of corticosterone and hen body mass on primary sex ratio in laying hen (Gallus gallus), using unincubated eggs.

In various studies, chronic elevation of corticosterone levels in female birds under natural or experimental conditions resulted in female biased offspring sex ratios. In chicken, one study with injected corticosterone resulted in a male sex ratio bias. In the current study, we chronically elevated blood plasma corticosterone levels through corticosterone feeding (20 mg/kg feed) for 14 days using 30 chicken hens in each of treatment and control groups and studied the primary offspring sex ratio (here defined as the proportion of male fertile eggs determined in freshly laid eggs, i.e., without egg incubation). Mean plasma corticosterone concentrations were significantly higher in the treatment group but were not associated with sex ratio, laying rate, and fertility rate. Corticosterone treatment by itself did not affect egg sex but affected sex ratio as well as laying rate and fertility rate in interaction with hen body mass. Body mass had a negative association with sex ratio, laying rate, and fertility rate per hen in the corticosterone group, but a positive association with sex ratio in untreated hens. These interactions were already seen when taking the body mass at the beginning of the experiment, indicating intrinsic differences between light and heavy hens with regard to their reaction to corticosterone treatment. The effects on laying rate, fertility rate, and sex ratio suggest that some factor related to body mass act together with corticosterone to modulate ovarian functions. We propose that corticosterone treatment in conjunction with hen body mass can interfere with meiosis, which can lead to meiotic drive and to chromosomal aberrations resulting in postponed ovulation or infertile ova.

Prioritization of candidate genes for cattle reproductive traits, based on protein-protein interactions, gene expression, and text-mining.

Reproduction is of significant economic importance in dairy cattle. Improved understanding of mechanisms that control estrous behavior and other reproduction traits could help in developing strategies to improve and/or monitor these traits. The objective of this study was to predict and rank genes and processes in brain areas and pituitary involved in reproductive traits in cattle using information derived from three different data sources: gene expression, protein-protein interactions, and literature. We identified 59, 89, 53, 23, and 71 genes in bovine amygdala, dorsal hypothalamus, hippocampus, pituitary, and ventral hypothalamus, respectively, potentially involved in processes underlying estrus and estrous behavior. Functional annotation of the candidate genes points to a number of tissue-specific processes of which the "neurotransmitter/ion channel/synapse" process in the amygdala, "steroid hormone receptor activity/ion binding" in the pituitary, "extracellular region" in the ventral hypothalamus, and "positive regulation of transcription/metabolic process" in the dorsal hypothalamus are most prominent. The regulation of the functional processes in the various tissues operate at different biological levels, including transcriptional, posttranscriptional, extracellular, and intercellular signaling levels.

Yolk concentrations of hormones and glucose and egg weight and egg dimensions in unincubated chicken eggs, in relation to egg sex and hen body weight.

Birds can manipulate offspring sex ratio under natural and experimental conditions and maternal hormones have been shown to be involved in this process. Studies also provided evidence for the presence of sex specific concentrations of yolk hormones in avian eggs. These findings led to the suggestion that yolk hormones could influence genetic sex determination in birds. However, in previous studies, yolk hormone concentrations and egg sex were studied in incubated eggs, although incubation of the eggs and embryonic development can alter yolk hormone concentrations and measured sex ratio. This study is the first to determine a wide array of egg components and hen body weight in relation to the sex of the egg in unincubated eggs. Egg parameters studied were yolk concentrations of testosterone, estradiol, androstenedione, progesterone, dihydrotestosterone, and glucose, and egg weight and dimensions. In addition, we studied the associations among all measured parameters. Associations were found between a number of yolk hormones (progesterone associated with testosterone, estradiol and androstenedione; androstenedione with testosterone; dihydrotestosterone with estradiol and androstenedione) as well as between yolk testosterone and egg length and egg weight. There were no significant overall differences between male and female chicken eggs in any of the measured egg parameters. However, there were a few interactions such as the interaction of egg sex with dihydrotestosterone and with hen body weight which predicted estradiol levels and an interaction of estradiol levels with egg width for predicting sex of egg. Their biological relevance need, however, further study.

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.