Autumn grass treated with a hydrolysable tannin extract versus lactic acid bacteria inoculant: Effects on silage fermentation characteristics and nutritional value and on performance of lactating dairy cows.

Hydrolysable tannins (HT) show potential as silage additive for autumn herbage silages, high in (rumen degradable) protein, as they may reduce proteolysis. Additionally, they have abilities to form pH-reversible tannin-protein complexes, non-degradable in the rumen but degradable in the abomasum and intestines of ruminants. Therefore they can improve milk N efficiency and shift N excretions from urine to faeces, possibly mitigating the environmental impact of ruminants. In this study, two small bunker silos were filled with autumn grass. One was treated with 20 g/kg DM HT extract (TAN) (TannoSan-L), the other with 8 mg/kg DM inoculant containing lactic acid bacteria (INO) (Bonsilage Fit G). Secondly, micro-silos (2.75 L) were filled with four treatments; (1) grass without additive (CON) (n = 5); (2) TAN (n = 5); (3) INO (n = 5); and (4) TAN + INO (n = 5). The bunker silos were used in a cross-over feeding experiment with periods of 4 weeks involving 22 lactating Holstein cows (average ± SD: 183 ± 36.3 days in milk, 665 ± 71.0 kg body weight, and 33.8 ± 3.91 kg/day milk yield). The HT dose was insufficient to reduce proteolysis or alter chemical composition and nutritional value in the micro- and bunker silages. Including grass silage added with TAN (3.2 g HT/kg DM) in the diet, did not affect feed intake nor fat and protein corrected milk yield in comparison to feeding the grass silage added with INO in a similar diet. The TAN-fed cows had an increased faecal N excretion and decreased apparent total-tract N and organic matter digestibility, but no improvement in the cows' N utilization could be confirmed in milk and blood urea levels. Overall, feeding an autumn grass silage treated with 20 g/kg chestnut HT extract did not affect the performance of dairy cows in comparison to feeding an autumn grass silage treated with a lactic acid bacteria inoculant.

Suboptimal culture conditions induce more deviations in gene expression in male than female bovine blastocysts.

BACKGROUND: Since the development of in vitro embryo production in cattle, different supplements have been added to culture media to support embryo development, with serum being the most popular. However, the addition of serum during embryo culture can induce high birthweights and low viability in calves (Large Offspring Syndrome). Analysis of global gene expression in bovine embryos produced under different conditions can provide valuable information to optimize culture media for in vitro embryo production. RESULTS: We used RNA sequencing to examine the effect of in vitro embryo production, in either serum-containing or serum-free media, on the global gene expression pattern of individual bovine blastocysts. Compared to in vivo derived embryos, embryos produced in serum-containing medium had five times more differentially expressed genes than embryos produced in serum-free conditions (1109 vs. 207). Importantly, in vitro production in the presence of serum appeared to have a different impact on the embryos according to their sex, with male embryos having three times more genes differentially expressed than their female counterparts (1283 vs. 456). On the contrary, male and female embryos produced in serum-free conditions showed the same number (191 vs. 192) of genes expressed differentially; however, only 44 of those genes were common in both comparisons. The pathways affected by in vitro production differed depending on the type of supplementation. For example, embryos produced in serum-containing conditions had a lower expression of genes related to metabolism while embryos produced in serum-free conditions showed aberrations in genes involved in lipid metabolism. CONCLUSIONS: Serum supplementation had a major impact on the gene expression pattern of embryos, with male embryos being the most affected. The transcriptome of embryos produced in serum-free conditions showed a greater resemblance to that of in vivo derived embryos, although genes involved in lipid metabolism were altered. Male embryos appeared to be most affected by suboptimal in vitro culture, i.e. in the presence of serum.

The need for transparency and good practices in the qPCR literature.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

High oxygen tension increases global methylation in bovine 4-cell embryos and blastocysts but does not affect general retrotransposon expression.

Retrotransposons are transposable elements that insert extra copies of themselves throughout the genome via an RNA intermediate using a 'copy and paste' mechanism. They account for more than 44% of the bovine genome and have been reported to be functional, especially during preimplantation embryo development. In the present study, we tested whether high oxygen tension (20% O2) influences global DNA methylation analysed by immunofluorescence staining of developing bovine embryos and whether this has an effect on the expression of some selected retrotransposon families. High oxygen tension significantly increased global DNA methylation in 4-cell embryos and blastocysts. A significant expression difference was observed for ERV1-1-I_BT in female blastocysts, but no significant changes were observed for the other retrotransposon families tested. Therefore, the study indicates that global DNA methylation is not necessarily correlated with retrotransposon expression in bovine preimplantation embryos.

Steroids affect gene expression, ciliary activity, glucose uptake, progesterone receptor expression and immunoreactive steroidogenic protein expression in equine oviduct explants in vitro.

The oviduct undergoes dramatic functional and morphological changes throughout the oestrous cycle of the mare. To unravel the effects of steroids on the morphology, functionality and gene expression of the equine oviduct, an in vitro oviduct explant culture system was stimulated with physiological concentrations of progesterone and 17ß-oestradiol. Four conditions were compared: unsupplemented preovulatory explants, preovulatory explants that were stimulated with postovulatory hormone concentrations, unsupplemented postovulatory explants and postovulatory explants that were stimulated with preovulatory hormone concentrations. The modulating effects of both steroids on oviduct explants were investigated and the following parameters examined: (1) ciliary activity, (2) glucose consumption and lactate production pattern, (3) ultrastructure, (4) mRNA expression of embryotrophic genes, (5) steroidogenic capacities of oviductal explants and (6) progesterone receptor expression. The present paper shows that the equine oviduct is an organ with potential steroidogenic capacities, which is highly responsive to local changes in progesterone and 17ß-oestradiol concentrations at the level of morphology, functionality and gene expression of the oviduct. These data provide a basis to study the importance of endocrine and paracrine signalling during early embryonic development in the horse.

Optimisation of the bovine whole in vitro embryo system as a sentinel for toxicity screening: a cadmium challenge.

Developmental toxicity testing could greatly benefit from the availability of an in vitro alternative model based on the use of animal embryos that have better human-like physiology than the currently-used alternative models. These current models are insufficient, as extrapolation of the results can be challenging. Therefore, an in vitro bovine embryo culture system was used to expose individual morulae to test substances, and to study developmental characteristics up to the blastocyst stage. Cadmium was chosen as the reference toxicant to investigate the sensitivity of the bovine morulae to various concentrations and exposure times. Oocytes from slaughterhouse-obtained bovine ovaries, were maturated, fertilised and cultured up until the morula stage. Morulae were exposed to different cadmium concentrations for 18 or 70 hours, and developmental competence, embryo quality and the expression of cadmium exposure-related genes were evaluated. Cadmium exposure hampered embryonic developmental competence and quality. Compared with the 18-hour exposure, the 70-hour exposure induced a 20-fold higher toxic response with regard to developmental competence and a more 'cadmium-typical' transcript expression. The bovine morula might be a promising tool for toxicity testing as, following exposure, the embryos reacted in a sensitive and 'cadmium-typical' manner to our reference toxicant.

Equine oviduct explant culture: a basic model to decipher embryo-maternal communication.

Equine embryos remain for 6 days in the oviduct and thus there is a need for an in vitro model to study embryo-oviductal interactions in the horse, since this subtle way of communication is very difficult to analyse in vivo. Until now, no equine oviduct explant culture model has been characterised both morphologically and functionally. Therefore, we established a culture system for equine oviduct explants that maintained epithelial morphology during 6 days of culture, as revealed by light microscopy and transmission electron microscopy. We demonstrated the presence of highly differentiated, tall columnar, pseudostratified epithelium with basal nuclei, numerous nucleoli, secretory granules and apical cilia, which is very similar to the in vivo situation. Both epithelium and stromal cells originating from the lamina propria are represented in the explants. Moreover, at least 98% of the cells remained membrane intact and fewer than 2% of the cells were apoptotic after 6 days of culture. Although dark-cell degeneration, which is a hypoxia-related type of cell death, was observed in the centre of the explants, quantitative real-time PCR failed to detect upregulation of the hypoxia-related marker genes HIF1A, VEGFA, uPA, GLUT1 and PAI1. Since the explants remained morphologically and functionally intact and since the system is easy to set up, it appears to be an excellent tool for proteome, transcriptome and miRNome analysis in order to unravel embryo-maternal interactions in the horse.

Laser capture microdissection: should an ultraviolet or infrared laser be used?

Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation, however, are crucial for the success of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user-friendliness to sample quality. The comparison was performed on two types of samples: peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. Not only does the UV system allow faster and more precise sample collection, but also the obtained samples-even single cell samples-can be used for DNA extraction and downstream polymerase chain reaction (PCR) applications. RNA-based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10 cells for reverse transcription quantitative PCR (RT-qPCR) analysis, the low RNA quality should be taken into account when designing the RT-qPCR assays.

Influence of the uterine environment on the development of in vitro-produced equine embryos.

The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not a suitable environment for pre-compact morula stage horse embryos. By contrast, exposure to uterocalin during IVP improved capsule formation, although it did not measurably affect the development or expression of a panel of genes known to differ between in vivo and in vitro embryos. Further studies are required to evaluate whether uterocalin serves purely as a carrier protein or more directly promotes improved capsule development.

Differential microRNA expression analysis in blastocysts by whole mount in situ hybridization and reverse transcription quantitative polymerase chain reaction on laser capture microdissection samples.

There is cumulating evidence that microRNAs (miRNAs) are important regulators of pluripotency and differentiation and, hence, of early lineage segregation in embryo development. To unravel the function of specific miRNAs, it is important not only to analyze miRNA expression in the entire blastocyst but also to determine the site and level of expression in the inner cell mass (ICM) versus trophectoderm (TE). A new strategy has been developed for miRNA expression analysis in ICM and TE using two complementary techniques. By whole mount in situ hybridization (WISH), it was visualized that bta-miR-155 is mainly expressed in the ICM. However, WISH does not provide quantitative data on expression differences between the two cell types. By reverse transcription quantitative polymerase chain reaction (RT-qPCR) on ICM and TE isolates taken from single blastocysts with laser capture microdissection (LCM), it was quantified that bta-miR-155 was 50-fold higher expressed in ICM than in TE. The possibility to quantify both miRNAs and messenger RNAs (mRNAs) in LCM samples offers the opportunity to analyze the expression of both miRNAs and potential targets in one sample. This article shows that a combination of WISH with LCM and subsequent RT-qPCR is a robust strategy to qualitatively and quantitatively analyze differential miRNA expression in discrete cell types of a single blastocyst.

Gene expression profiling of pluripotency and differentiation-related markers in cat oocytes and preimplantation embryos.

During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of early lineage markers can be species specific. The present study also revealed a pool of maternal NANOG mRNA transcripts, the role of which remains to be elucidated. Comparing transcription levels of these genes between in vivo and in vitro blastocysts revealed low levels of OCT4 mRNA in the latter, which may contribute to the reduced developmental competence of embryos under suboptimal conditions.

Nitrogen excretion from beef cattle fed a wide range of diets compiled in an intercontinental dataset: a meta-analysis.

Manure N from cattle contributes to nitrate leaching, nitrous oxide, and ammonia emissions. Measurement of manure N outputs on commercial beef cattle operations is laborious, expensive, and impractical; therefore, models are needed to predict N excreted in urine and feces. Building robust prediction models requires extensive data from animals under different management systems worldwide. Thus, the study objectives were to 1) collate an international dataset of N excretion in feces and urine based on individual observations from beef cattle; 2) determine the suitability of key variables for predicting fecal, urinary, and total manure N excretion; and 3) develop robust and reliable N excretion prediction models based on individual observation from beef cattle consuming various diets. A meta-analysis based on individual beef data from different experiments was carried out from a raw dataset including 1,004 observations from 33 experiments collected from 5 research institutes in Europe (n = 3), North America (n = 1), and South America (n = 1). A sequential approach was taken in developing models of increasing complexity by incrementally adding significant variables that affected fecal, urinary, or total manure N excretion. Nitrogen excretion was predicted by fitting linear mixed models with experiment as a random effect. Simple models including dry matter intake (DMI) were better at predicting fecal N excretion than those using only dietary nutrient composition or body weight (BW). Simple models based on N intake performed better for urinary and total manure N excretion than those based on DMI. A model including DMI and dietary component concentrations led to the most robust prediction of fecal and urinary N excretion, generating root mean square prediction errors as a percentage of the observed mean values of 25.0% for feces and 25.6% for urine. Complex total manure N excretion models based on BW and dietary component concentrations led to the lowest prediction errors of about 14.6%. In conclusion, several models to predict N excretion already exist, but the ones developed in this study are based on individual observations encompassing larger variability than the previous developed models. In addition, models that include information on DMI or N intake are required for accurate prediction of fecal, urinary, and total manure N excretion. In the absence of intake data, equations have poor performance as compared with equations based on intake and dietary component concentrations.

Laser capture microdissection for gene expression analysis of inner cell mass and trophectoderm from blastocysts.

Isolation of pure inner cell mass (ICM) and trophectoderm (TE) samples from a single blastocyst is necessary to obtain accurate information on the transcriptomes of these cells. Laser capture microdissection (LCM) provides the possibility to isolate small tissue fractions from heterogeneous tissue sections without contamination by the surrounding tissue and without changing the gene expression pattern of the cells. However, the small size of blastocysts hampers tissue processing prior to LCM. This article describes a protocol for the application of LCM to isolate homogeneous ICM and TE cell samples from single bovine blastocysts for downstream gene expression analysis.

In vivo-derived horse blastocysts show transcriptional upregulation of developmentally important genes compared with in vitro-produced horse blastocysts.

In vitro-produced (IVP) equine blastocysts can give rise to successful pregnancies, but their morphology and developmental rate differ from those of in vivo-derived equine blastocysts. The aim of the present study was to evaluate this difference at the genetic level. Suppression subtractive hybridisation (SSH) was used to construct a cDNA library enriched for transcripts preferentially expressed in in vivo-derived equine blastocysts compared with IVP blastocysts. Of the 62 different genes identified in this way, six genes involved in embryonic development (BEX2, FABP3, HSP90AA1, MOBKL3, MCM7 and ODC) were selected to confirm this differential expression by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Using RT-qPCR, five genes were confirmed to be significantly upregulated in in vivo-derived blastocysts (i.e. FABP3, HSP90AA1 (both P<0.05), ODC, MOBKL3 and BEX2 (P<0.005 for all three)), confirming the results of the SSH. There was no significant difference in MCM7 expression between IVP and in vivo-derived blastocysts. In conclusion, five genes that are transcriptionally upregulated in in vivo-derived equine blastocysts compared with IVP blastocysts have been identified. Because of their possible importance in embryonic development, the expression of these genes can be used as a marker to evaluate in vitro embryo production systems in the horse.

Suppression of keratin 18 gene expression in bovine blastocysts by RNA interference.

The expression of the cytoskeleton protein Keratin 18 (KRT18) starts at the onset of bovine blastocyst formation. KRT18 is solely expressed in the trophectoderm and can therefore be used as a marker for trophectodermal differentiation. In the present study, the expression of KRT18 was suppressed by RNA interference to probe its functional importance in bovine blastocyst formation. Microinjection of KRT18 double-stranded RNA into the cytoplasm of zygotes resulted in reduced KRT18 mRNA (76% reduction) and protein expression at the blastocyst stage and a lower developmental competence (41% reduction in the percentage of blastocyst formation) compared with non-injected and phosphate-buffered saline (PBS)-injected controls. KRT18 downregulation was associated with reduced mRNA expression of KRT8, the binding partner of KRT18, but had no effect on the expression of KRT19, CDH1 and DSP, other genes involved in intermediate filament and cytoskeleton formation. The results of the present study demonstrated that KRT18 knockdown in preimplantation embryos results in reduced blastocyst formation, but no further morphological aberrations were observed with regard to the biological function of KRT18. These observations could be due to the function of KRT18 being replaced by that of another gene, the surviving blastocysts expressing the minimum level of KRT18 required for normal blastocyst development or the possibility that further aberrations may occur later in development.

Quantification of fibronectin 1 (FN1) splice variants, including two novel ones, and analysis of integrins as candidate FN1 receptors in bovine preimplantation embryos.

BACKGROUND: Fibronectin 1 (FN1), a glycoprotein component of the extracellular matrix, exerts different functions during reproductive processes such as fertilisation, gastrulation and implantation. FN1 expression has been described to increase significantly from the morula towards the early blastocyst stage, suggesting that FN1 may also be involved in early blastocyst formation. By alternative splicing at 3 defined regions, different FN1 isoforms are generated, each with a unique biological function. The analysis of the alternative FN1 splicing on the one hand and the search for candidate FN1 receptors on the other hand during early bovine embryo development may reveal more about its function during bovine preimplantation embryo development. RESULTS: RT-qPCR quantification of the FN1 splice isoforms in oocytes, embryos, cumulus cells and adult tissue samples revealed a large variation in overall FN1 expression and in splice variant expression. Moreover, two new FN1 transcript variants were identified, the first one expressed in bovine preimplantation embryos and the second one expressed in cumulus cells. In the search for candidate receptors for the new embryo specific FN1 isoform, RNA expression analysis identified 5 alpha integrin subunits (ITGA2B, ITGA3, ITGA5, ITGA8, ITGAV) and 2 beta integrin subunits (ITGB1 and ITGB3) with a similar or overlapping RNA expression pattern as compared to FN1. But double immunofluorescent stainings could not confirm complete co-localisation between FN1 and one out of 3 selected integrins alpha subunits (ITGA3, ITGA5, ITGAV). CONCLUSION: The existence of a new FN1 transcript variant, specifically expressed in morulae and blastocysts strengthens the idea that FN1 is involved in the process of compaction and blastocyst formation. Analysis of the integrin expression could not identify the binding partner for the embryo specific FN1 transcript variant making further steps necessary for the identification of the FN1 receptor and the downstream effects of FN1-receptor binding.

Differential gene expression in the honeybee head after a bacterial challenge.

Bidirectional interactions between the immune and nervous systems are well established in vertebrates. Insects show similar neuro-immune-behavioral interactions to those seen in vertebrates. Using quantitative real-time PCR, we present evidence that gene expression in the honeybee head is influenced by activation of the immune system 8h after a bacterial challenge with Escherichia coli. Seven genes were selected for quantitative analysis in order to cover both typical functions of the head such as exocrine secretion (mrjp3 and mrjp4) and olfactory processes (obp17) as well as more general processes such as structural functions (mlc2 and paramyosin), stress response (ERp60) and energy housekeeping (enolase). In this way, we show at the molecular level that the immune system functions as a sensory organ in insects -- as it does in vertebrates -- which signals to the head that a bacterial infection is present, and leads to regulation of expression of several genes in the head by a yet unidentified mechanism.

Supplementing goat kids with coconut medium chain fatty acids in early life influences growth and rumen papillae development until 4 months after supplementation but effects on in vitro methane emissions and the rumen microbiota are transient.

The aim of this study was to investigate the methane (CH4) reducing potential of a combination of prenatal and/or postnatal treatment with coconut oil medium chain fatty acids (CO MCFA) in goat kids. The hypothesis is that influencing rumen function during early life has more chances for success than in the adult life, related to the resilience of the mature rumen microbiota. Forty-eight pregnant does were split into two experimental groups: treated does (D+) received 40 g/d of CO MCFA in a test compound feed, while control does (D-) received a control compound feed, during the last 3 wk of gestation. Twin kids from 10 does of each group were split up into a treated (K+) and nontreated (K-) group, resulting in four experimental groups: D+K+, D+K-, D-K+, and D-K-. The K+ kids received 1.8 mL/d of CO MCFA from birth until 2-wk postweaning (11 wk). Irrespective of treatment, the experimental rearing conditions resulted in absence of rumen protozoa at all sampling times, assessed by quantitative PCR (qPCR). In vitro incubations with rumen fluid at 4 wk old showed 82% lower CH4 production of inoculum from D+K+ kids compared to D-K- kids (P = 0.01). However, this was accompanied by lower total volatile fatty acids (tVFA) production (P = 0.006) and higher hydrogen accumulation (P = 0.008). QPCR targeting the mcrA and rrs genes confirmed a lower abundance of total methanogens (P < 0.02) and total eubacteria (P = 0.02) in D+K+ kids at 4 wk old. Methanogenic activity, as assessed by mcrA expression by RT-qPCR, was also lower in these kids. However, activity did not always reflect methanogen abundance. At 11 and 28 wk old, prenatal and postnatal effects on in vitro fermentation and rumen microbiota disappeared. Nevertheless, lower milk replacer intake in the first 4 wk resulted in reduced BW in K+ kids, persisting until 28 wk of age. Additionally, differences assigned to postnatal treatment were found in papillae density, width, and length in different areas of the rumen, recorded at 28 wk old. CONCLUSION: prenatal and postnatal supplementation with CO MCFA reduced in vitro CH4 emissions until 4 wk old by depressing methanogen abundance and activity but at the expense of rumen fermentation and eubacterial abundance. Unfortunately, daily gain of K+ kids was suppressed. Some rumen papillae characteristics differed at 28 wk old due to postnatal treatment which ended at 11 wk old, indicating rumen papillary development can be affected by the early-life nutritional circumstances.

Identification and expression analysis of genes associated with bovine blastocyst formation.

BACKGROUND: Normal preimplantation embryo development encompasses a series of events including first cleavage division, activation of the embryonic genome, compaction and blastocyst formation. First lineage differentiation starts at the blastocyst stage with the formation of the trophectoderm and the inner cell mass. The main objective of this study was the detection, identification and expression analysis of genes associated with blastocyst formation in order to help us better understand this process. This information could lead to improvements of in vitro embryo production procedures. RESULTS: A subtractive cDNA library was constructed enriched for transcripts preferentially expressed at the blastocyst stage compared to the 2-cell and 8-cell stage. Sequence information was obtained for 65 randomly selected clones. The RNA expression levels of 12 candidate genes were determined throughout 3 stages of preimplantation embryo development (2-cell, 8-cell and blastocyst) and compared with the RNA expression levels of in vivo "golden standard" embryos using real-time PCR. The RNA expression profiles of 9 (75%) transcripts (KRT18, FN1, MYL6, ATP1B3, FTH1, HINT1, SLC25A5, ATP6V0B, RPL10) were in agreement with the subtractive cDNA cloning approach, whereas for the remaining 3 (25%) (ACTN1, COPE, EEF1A1) the RNA expression level was equal or even higher at the earlier developmental stages compared to the blastocyst stage. Moreover, significant differences in RNA expression levels were observed between in vitro and in vivo produced embryos. By immunofluorescent labelling, the protein expression of KRT18, FN1 and MYL6 was determined throughout bovine preimplantation embryo development and showed the same pattern as the RNA expression analyses. CONCLUSION: By subtractive cDNA cloning, candidate genes involved in blastocyst formation were identified. For several candidate genes, important differences in gene expression were observed between in vivo and in vitro produced embryos, reflecting the influence of the in vitro culture system on the embryonic gene expression. Both RNA and protein expression analysis demonstrated that KRT18, FN1 and MYL6 are differentially expressed during preimplantation embryo development and those genes can be considered as markers for bovine blastocyst formation.

Exploring the methanogen and bacterial communities of rumen environments: solid adherent, fluid and epimural.

The rumen microbiome occupies a central role in animal health and productivity. A better understanding of the rumen ecosystem is essential to increase productivity or decrease methane production. Samples were collected from the three main rumen environments: the solid-adherent fraction, the liquid fraction and the epithelium. For the liquid and solid fraction, two alternative sample processing protocols were compared, resulting in a total of five sample types: crude solids (S), the eluted solid-adherent fraction (Ad), free-living species in the crude rumen liquid (CRL), strained liquid samples (Lq) and epimural scrapings (Ep). The bacterial and methanogen communities of these sample types were analysed using 16S metabarcoding and qPCR. The results indicate that the liquid and solid-adherent environments are distinguished mainly by the differential abundance of specific taxonomic groups. Cellulolytic bacteria that pioneer biofilm formation, together with secondary colonisers are prevalent in solid-adherent samples, while dominant species in the fluid samples are primarily identified as consumers of soluble nutrients. Also, methanogen species are found to have a preference for either a solid-adherent or free-living occurrence. The epimural environment is characterised by a different microbial profile. Ten bacterial families and two methanogen genera are almost exclusively found in this environment.