In vitro genome editing activity of Cas9 in somatic cells after random and transposon-based genomic Cas9 integration.

Due to its close resemblance, the domesticated pig has proven to be a diverse animal model for biomedical research and genome editing tools have contributed to developing porcine models for several human diseases. By employing the CRISPR-Cas9 system, porcine embryos or somatic cells can be genetically modified to generate the desired genotype. However, somatic cell nuclear transfer (SCNT) of modified somatic cells and embryo manipulation are challenging, especially if the desired genotype is detrimental to the embryo.

Genome-wide association study for frozen-thawed sperm motility in stallions across various horse breeds.

Objective: The semen quality of stallions including sperm motility is an important target of selection as it has a high level of individual variability. However, effects of the molecular architecture of the genome on the mechanisms of sperm formation and their preservation after thawing have been poorly investigated. Here, we conducted a genome-wide association study (GWAS) for the sperm motility of cryopreserved semen in stallions of various breeds.

Single-step genomic evaluation of Russian dairy cattle using internal and external information.

Genomic data are widely used in predicting the breeding values of dairy cattle. The accuracy of genomic prediction depends on the size of the reference population and how related the candidate animals are to it. For populations with limited numbers of progeny-tested bulls, the reference populations must include cows and data from external populations.

Efficiency of Tris-Based Extender Steridyl for Semen Cryopreservation in Stallions.

The fertilizing ability of stallion sperm after freezing is lower than in other species. The search for the optimal extender, combination of extenders, and the freezing protocol is relevant. The aim of this study was to compare lactose-chelate-citrate-yolk (LCCY) extender, usually used in Russia, and Steridyl (Minitube) for freezing sperm of stallions.

Screening of haplotype for cholesterol deficiency genetic defect in the Russian Holstein cattle population.

In our study, we estimated the frequency of haplotype for cholesterol deficiency (HCD) carriers in the Russian Holstein cattle population. We studied 1817 random samples of cows born in 2010-2017 from ten herds and 331 cows and heifers from the other three herds born in 2016-2019, fathers or fathers of mothers of which were HCD carriers. The method of AS-PCR was used for animals genotyping.

Modification and Clinical Application of the Inner Perivitelline Membrane Test in Different Avian Species.

The aim of this study was to adapt an inner perivitelline membrane (IPVM) test as an interspecies penetration assay for avian spermatozoa. The IPVM of different bird species was evaluated to test the penetrating ability of avian spermatozoa in an intra- and interspecies design. Isolation of the IPVM via acid hydrolysis was tested in pre-incubated chicken eggs and in six other avian species.

Comparative gene expression profiling of pig-derived iPSC-like cells: Effects of induced pluripotency on expression of porcine endogenous retrovirus (PERV).

Background: Porcine induced pluripotent stem cells (piPSCs) offer an alternative strategy in xenotransplantation (XTx). As human endogenous retroviruses (HERV), particularly HERV-K, are highly expressed in natural human stem cells, we compared the expression of porcine endogenous retroviruses (PERV) and retrotransposon LINE-1 (L1) open reading frames 1 and 2 (pORF1 and pORF2) in different piPSC-like cell lines with their progenitors (porcine fetal fibroblasts, pFF).

Methods: Cells reprogrammed via Sleeping Beauty-transposed transcription factors were cultured and analyzed on a custom-designed microarray representing the reference pig genome.

Murine pluripotent stem cells with a homozygous knockout of Foxg1 show reduced differentiation towards cortical progenitors in vitro.

Foxg1 is a transcription factor critical for the development of the mammalian telencephalon. Foxg1 controls the proliferation of dorsal telencephalon progenitors and the specification of the ventral telencephalon. Homozygous knockout of Foxg1 in mice leads to severe microcephaly, attributed to premature differentiation of telencephalic progenitors, mainly of cortical progenitors.

Mouse iPSC generated with porcine reprogramming factors as a model for studying the effects of non-silenced heterologous transgenes on pluripotency.

Mouse somatic cells can be reprogrammed to pluripotency by the ectopic expression of four pluripotency transcription factors, Oct4, Sox2, cmyc, and Klf4. Usually, silencing of the exogenous reprogramming factors is considered to be essential for complete reprogramming and differentiation. In the vast majority of studies, murine pluripotency transcription factor sequences have been used for the reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSC).

Evaluation of porcine stem cell competence for somatic cell nuclear transfer and production of cloned animals.

Porcine somatic cell nuclear transfer (SCNT) has been used extensively to create genetically modified pigs, but the efficiency of the methodology is still low. It has been hypothesized that pluripotent or multipotent stem cells might result in increased SCNT efficacy as these cells are closer than somatic cells to the epigenetic state found in the blastomeres and therefore need less reprogramming. Our group has worked with porcine SCNT during the last 20 years and here we describe our experience with SCNT of 3 different stem cell lines.

Systematic in vitro and in vivo characterization of Leukemia-inhibiting factor- and Fibroblast growth factor-derived porcine induced pluripotent stem cells.

Derivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF-derived piPSCs were more successful than their FGF-derived counterparts in the generation of in vitro chimeras and in teratoma formation.

Genetic diversity and genomic signatures of selection among cattle breeds from Siberia, eastern and northern Europe.

Domestication in the near eastern region had a major impact on the gene pool of humpless taurine cattle (Bos taurus). As a result of subsequent natural and artificial selection, hundreds of different breeds have evolved, displaying a broad range of phenotypic traits. Here, 10 Eurasian B.

Long-Term Culture of Porcine Induced Pluripotent Stem-Like Cells Under Feeder-Free Conditions in the Presence of Histone Deacetylase Inhibitors.

The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a complex process that involves significant epigenetic alterations in the reprogrammed cells. Epigenetic modifiers such as histone deacetylase (HDAC) inhibitors have been shown to increase the efficiency of derivation of iPSCs in humans and mice. In this study, we used three HDAC inhibitors, valproic acid, sodium butyrate, and suberoylanilide hydroxamic acid, together with ascorbic acid, for derivation and long-term feeder-free culture of porcine iPS-like cells.

Risk of infection after iatrogenic perforation of the gut wall? Evaluation of preventive strategies in a randomized controlled animal trial.

Background: Interventional endoscopies entail a risk of infection secondary to perforation of the luminal wall. Thereby, bacteria may be introduced into the sterile environment of the peritoneal cavity (PC). Limited data are available regarding the efficacy of prophylactic anti-infective treatments.

Current state of laser synthesis of metal and alloy nanoparticles as ligand-free reference materials for nano-toxicological assays.

Due to the abundance of nanomaterials in medical devices and everyday products, toxicological effects related to nanoparticles released from these materials, e.g., by mechanical wear, are a growing matter of concern.

The small molecule inhibitors PD0325091 and CHIR99021 reduce expression of pluripotency-related genes in putative porcine induced pluripotent stem cells.

Small molecule inhibitors of the mitogen-activated protein kinase kinase (MEK) and glycogen synthesis kinase 3 (Gsk3) have been essential in the establishment and maintenance of embryonic stem cells (ESCs) from rats and from nonpermissive mouse strains. However, conflicting results have been reported regarding their efficacy in the establishment and maintenance of pluripotent stem cells from other species. Here, we investigated the effects of PD0325091 (PD; a MEK inhibitor) and CHIR99021 (CH; a Gsk3β inhibitor) on the reprogramming of porcine fetal fibroblasts to induced pluripotent stem cells (piPSCs).

Passage number of porcine embryonic germ cells affects epigenetic status and blastocyst rate following somatic cell nuclear transfer.

Epigenetic instability of donor cells due to long-term in vitro culture may influence the success rate of subsequent somatic cell nuclear transfer (SCNT). Therefore, the present study was designed (1) to investigate the epigenetic changes after prolonged culture in vitro of porcine embryonic germ (EG) cells, including differences in expression levels of both DNA methylation and demethylation-related genes and catalyses of histone modifications, and (2) to assess the efficiency of SCNT using EG cells from different passages. Results showed that genes either associated with DNA demethylation including DNMTs and TET1 or genes related to histone acetylation including HDACs were highly expressed in EG cells at higher passages when compared to EG cells at lower passages.

The choice of expression vector promoter is an important factor in the reprogramming of porcine fibroblasts into induced pluripotent cells.

Porcine induced pluripotent stem cells (iPSCs) are an important animal model for development of regenerative therapies in human medicine. To date, the majority of the porcine cell lines with iPSC characteristics have been generated with the use of viral vectors harboring human or mouse reprogramming factors. Here, we report on the use of Sleeping Beauty transposon vectors based on the porcine transcription factor sequences to reprogram porcine fetal fibroblasts into iPSC-like cells.

Ultrastructure of rabbit embryos exposed to hyperthermia and anti-Hsp 70.

The aim of the study was to determine the effect of short-term hyperthermia and Hsp70 blockage on ultrastructural changes in cell organelles and nucleoli of rabbit preimplantation embryos. The embryos were cultured either at 37.5°C (control, C) or 41.

Galanin immunoreactivity increased in chicken supraoptic neurons after activation of the vasotocin system at oviposition.

The avian arginine vasotocin (AVT) synthesized in the hypothalamic magnocellular neurons and released from the posterior pituitary is known to be involved in the regulation of uterine contractions for oviposition in chickens. However, regulation of AVT synthesis and release within the magnocellular hypothalamus has not been elucidated. Galanin, the oviposition inducing factor in the oviduct of the hen, has been demonstrated to have sexually dimorphic stimulatory action in oxytocin- and vasopressin neurons in the mammalian hypothalamus.

Effect of recombinant bovine somatotropin (rbST) on cytoplasmic maturation of bovine oocytes and their developmental competence in vitro.

The objectives of this study were to evaluate the effects of recombinant bovine somatotropin (rbST) on the nuclear and cytoplasmic maturation of bovine oocytes and their further developmental competence to blastocysts in vitro. We analyzed the mitochondrial activity and concentration of intracellular stored calcium ([Ca(2+)](is)) in matured oocytes and the morphology and chromatin status of produced embryos after in vitro fertilization. Cumulus-oocyte complexes were incubated in TCM 199 containing 10% fetal calf serum (control medium 1: CM 1) or 10% estrus cow serum (control medium 2: CM 2).

Behavioural asymmetry is involved in regulation of autonomic processes: Left side presentation of food improves reproduction and lactation in cows.

It is known that the right and left brain hemispheres differ in their ability to regulate autonomic processes in the organism. Direct unilateral stimulation of the brain provokes side-dependent endocrine, immune and other visceral reactions. Since brain hemispheres are mainly involved in the regulation of muscles and sensory organs on the contra lateral side of the body the activation of behavioural asymmetry stimulates the contra lateral half of the brain.

[Structural changes of the nucleolar organizing regions in rhabdomyosarcoma RA-23 tumor cells induced by okadaic acid].

It has been recently shown that okadaic acid (OA), an inhibitor of dephosphorilation, is capable of inducing changes in the nucleolar organizer regions (AgNORs) of some mammalian cells. Our work was focused on studying the structural changes in AgNORs of tumour cells of rat rhabdomyosarcoma RA-23 by their exposure to 100 nM OA. A standard silver staining procedure of interphase AgNORs in tumour cells was used.

Prolactin in follicular fluid and intracellular store calcium in follicular cells are related to morphological signs of ovarian follicle atresia in cows: work in progress.

It is known that prolactin (PRL) is the third pituitary hormone serving gonadotropic function in mammals. However, its role in the regulation of ovarian folliculogenesis and, in particular, its relationship to follicular atresia as well as the mechanism of its influence on follicular cells are poorly understood. We investigated PRL levels in follicular fluids (FFs) and intracellular store calcium ([Ca2+]is) in cell walls of bovine ovarian follicles with diameters of 10 to 20 mm and their relationship to follicular atresia.