Cryopreservation of equine oocytes: looking into the crystal ball

In vitro 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 in vivo-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

The phosphate potential maintained by mitochondria in State 4 is proportional to the proton-motive force

AbstractEvidence is presented for a proportional relationship between the extramitochondrial phosphate potential (ΔGexp) and the proton-motive force (Δ\̃gmH+) across the mitochondrial membrane in rat-liver mitochondria oxidising succinate in State 4, when Δ\̃gmH+ is varied by addition of uncouplers or malonate. This relationship was found when precautions were taken to minimise interference with the determination of ΔGpex and Δ\̃gmH+ by intramitochondrial nucleotides, adenylate kinase activity, the quenching method, and Δ\̃gmH+-dependent changes in matrix volume. A non-proportional ΔGpex/Δ\̃gmH+ relationship was obtained when these precautions were omitted. Our results do not support mosaic protonic coupling, but are not necessarily in conflict with other localised coupling schemes

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

Semi-quantitative proteomics of mammalian cells upon short-term exposure to nonionizing 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

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 (<it>OXT, AVP, POMC, MCHR1</it>), but also genes whose association with estrous behavior is novel and warrants further investigation.</p> <p>Conclusions</p> <p>Several genes were identified whose expression levels in the bovine brain associated with the level of expression of estrous behavior. The genes <it>OXT </it>and <it>AVP </it>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.</p

Gene banking and transplantation of (mammalian) ovarian tissue

The use of cryopreserved gonadal tissue to reconstitute breeds or breeding lines has been demonstrated in birds (Silversides et al., 2013; Liptoi et al., 2013) and mammals (e.g. Huang et al., 2010) as a highly effective, efficient method. For large domestic mammals, such as the pig, proof of principle needs to be demonstrated. We (Egerszegi et al., unpublished) have undertaken a small pilot experiment in which orthotopic homografting of fragments of juvenile pig ovaries into neonate ovariectomized recipient piglets was attempted. This attempt was not successful. The results of the study are briefly presented in this report. This report further presents a literature review to describe current possibilities in various mammalian species and to provide suggestions for potential future improvements of methods in pigs or other mammalian farm animal species

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 pheno-typing 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

Role and state of cryopreservation in local cattle breeds

EU GENRES 870/04 project EURECAvo

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.</p

Meta-analysis of temporal intestinal gene expression data to generate reference profiles: VDI-10

Gene expression is measured because it reflects the status of a certain tissue in the host. Here, wetargeted the jejunum, the second part of the small intestine, because of the importance of the smallintestine for nutrient uptake and immune surveillance. Immediately after birth, the intestine rapidlydevelops morphologically, functionally, and immunologically. We acquired different transcriptomic datasetsfrom literature measuring gene expression at different time-points (age) to perform a metaanalysis.In this meta-analysis, genes were grouped into 16 clusters according to their gene expressionprofiles over time, using data from the control animals of all studies. Within these reference profiles(clusters), we investigated to what extent genes contribute to similar biological processes. From thisfunctional analysis we observed that the different temporal profiles (clusters) had different dominantprocesses, such as immune related processes, or barrier function. Subsequently, we superimposed transcriptomicsdata of piglets of the ‘dietary intervention groups’ of four of the used studies, in which thepiglets themselves, or the sow that farrowed them, had been administered either zinc-oxide, amoxicillin,or medium chain fatty acids). In this way we could investigate which temporal profiles (and which biologicalprocesses) were modulated by the interventions. Interestingly, not all 16 temporal profiles weremodulated. In conclusion, we showed that it is possible to re-use (publicly available) transcriptomicsdata and produce temporal reference gene expression profiles with overexpression of genes representingspecific biological processes. Subsequently, by superimposing gene expression data from (dietary)intervention studies we observed deviations from some of these reference profile(s). The latter suggeststhat it may be possible to modulate intestinal processes in a beneficial way