Evaluation of swine fertilisation medium (SFM) efficiency in preserving spermatozoa quality during long-term storage in comparison to four commercial swine extenders

In pig production, artificial insemination is widely carried out and the use of fresh diluted semen is predominant. For this reason, there are increasing interests in developing new extenders and in establishing the optimal storage conditions for diluted spermatozoa. In the last few decades, we utilised a homemade diluent (swine fertilisation medium (SFM)) for spermatozoa manipulation and biotechnological application as the production of transgenic pigs utilising the sperm-mediated gene transfer technique. The purpose of the present study is therefore to analyse the ability of SFM, in comparison to four commercial extenders, in preserving the quality of diluted boar semen stored at 16.58C till 15 days. We utilised some of the main predictive tests as objectively measured motility, acrosome and sperm membrane integrity, high mitochondrial membrane potential and pH. Based on our in vitro study, SFM could be declared as a good long-term extender, able to preserve spermatozoa quality as well as Androhep Enduraguard for up to 6 to 9 days and more

Relative abundance of heat shock proteins and clusterin transcripts in spermatozoa collected form boar routinely utilised in an artificial insemination centre: preliminary results

It is widely accepted that mature sperm contains RNA. The first hypothesis was that sperm RNAs have no functions of their own but are simply residues of spermatogenesis reflecting the events that occurred during their formation in the testes. More recently new discoveries have essentially expanded these views, showing that sperm mRNAs constitute a population of stable full-length transcripts, many of which are selectively retained during spermatogenesis and delivered to oocytes contributing to early embryo development. It is well known that semen quality can be influenced by occasional physical stress, infection, and variation in temperature and the definition of new markers for evaluation of semen could offer knowledge about the fertility potential of a semen sample. The aim of the present study was to evaluate the presence and the relative quantity of transcripts and protein of heat shock protein 70 (HSP70), 90 (HSP90) and clusterin (CLU) in Percoll-selected spermatozoa collected from seven adult boars of proven fertility routinely employed for artificial insemination. Our results showed the presence of HSP70, HSP90 and CLU transcripts with different level of expression: high for HSPs and low for CLU transcripts. The transcript level of both HSPs are similar among selected spermatozoa derived from high quality sperm with the exception of one boar that showed a reduced content of HSP70 and HSP90 mRNA together with a lower semen quality. At protein level, both HSPs were detected with similar amount among all seven boars whilst no band was evidenced for CLU protein

Maternal amoxicillin affects piglets colon microbiota: microbial ecology and metabolomics in a gut model

The first weeks of life represent a crucial stage for microbial colonization of the piglets’ gastrointestinal tract. Newborns’ microbiota is unstable and easily subject to changes under stimuli or insults. Nonetheless, the administration of antibiotics to the sow is still considered as common practice in intensive farming for pathological conditions in the postpartum. Therefore, transfer of antibiotic residues through milk may occurs, affecting the piglets’ colon microbiota. In this study, we aimed to extend the knowledge on antibiotic transfer through milk, employing an in vitro dedicated piglet colon model (MICODE— Multi Unit In vitro Colon Model). The authors’ focus was set on the shifts of the piglets’ microbiota composition microbiom- ics (16S r-DNA MiSeq and qPCR—quantitative polymerase chain reaction) and on the production of microbial metabolites (SPME GC/MS—solid phase micro-extraction gas chromatography/mass spectrometry) in response to milk with different concentrations of amoxicillin. The results showed an effective influence of amoxicillin in piglets’ microbiota and metabolites production; however, without altering the overall biodiversity. The scenario is that of a limitation of pathogens and opportun- istic taxa, e.g., Staphylococcaceae and Enterobacteriaceae, but also a limitation of commensal dominant Lactobacillaceae, a reduction in commensal Ruminococcaceae and a depletion in beneficial Bifidobactericeae. Lastly, an incremental growth of resistant species, such as Enterococcaceae or Clostridiaceae, was observed. To the authors’ knowledge, this study is the first evaluating the impact of antibiotic residues towards the piglets’ colon microbiota in an in vitro model, opening the way to include such approach in a pipeline of experiments where a reduced number of animals for testing is employed

Barrier effect of Esoxx® on esophageal mucosal damage: experimental study on ex-vivo swine model

The aim of the present study was to assess the potential barrier effect of Esoxx®, a new nonprescription medication under development for the relief of gastroesophageal reflux symptoms. Esoxx is based on a mixture of hyaluronic acid and chondroitin sulfate in a bioadhesive suspension of Lutrol® F 127 polymer (poloxamer 407) which facilitates the product adhesion on the esophageal mucosa. The mucosal damage was induced by 15 to 90 minutes of perfusion with an acidic solution (HCl, pH 1.47) with or without pepsin (2000 U/mL, acidified to pH 2; Sigma-Aldrich). Mucosal esophageal specimens were histologically evaluated and Evans blue dye solution was used to assess the permeability of the swine mucosa after the chemical injury. The results show that: (1) esophageal mucosal damage is related to the perfusion time and to the presence of pepsin, (2) mucosal damage is associated with an increased permeability, documented by an evident Evans blue staining, (3) perfusion with Esoxx is able to reduce the permeability of the injured mucosa, even after saline washing of the swine esophagus. These preliminary results support further clinical studies of Esoxx in the topical treatment of gastroesophageal reflux symptoms

484. Preclinical Proof of Concept of Transcriptional Silencing and Replacement Strategy for Treatment of Retinitis Pigmentosa Due To RHODOPSIN Mutations

Silencing and replacement strategy is a promising approach to overcome mutational heterogeneity of genetic defects. In autosomal dominant retinitis pigmentosa (adRP) due to rhodopsin gene (RHO) approximately 200 different mutations have been described, posing a challenge for the design of effective therapeutics.We designed a silencing and replacement strategy based on transcriptional silencing through an artificial zinc finger DNA-binding protein lacking effector domains (ZF6DBD), and tested both efficacy and safety in two animal models.In a murine model of adRP, we show that AAV-mediate retinal delivery (AAV2/8-CMV-ZF6-DBD) is associated with selective transcriptional silencing of the human mutated allele resulting in morphological and functional (Electroretinography, ERG a-wave and b-wave responses) rescue. We then tested the effect of transcriptional silencing in the porcine large pre-clinical model. Delivery of a low dose (AAV2/8-CMV-ZF6-DBD, 1×10e10 vector genomes, vg) of the ZF6 transcriptional silencer to the porcine retina resulted in robust transcriptional silencing of the endogenous porcine RHO transcript. Cell sorting of transduced photoreceptors showed an almost complete RHO transcriptional silencing effect (90% RHO transcriptional repression), underscoring the potency of the system. To determine the safety of the zinc-finger silencer we performed extensive RNA-seq analysis on treated and control retinae. The data sets generated demonstrate selective RHO gene transcriptional repression and a remarkably low number of differential expressed genes (DEGs), supporting specificity and thus, safety. The co-administration to the porcine retina of the AAV-ZF6 silencer (AAV2/8-CMV-ZF6-DBD) and the AAV-RHO replacement (5×10e11 vg, AAV2/8-GNAT1-HumanRHO) constructs resulted in a balanced silencing and replacement effect. This data support the use of zinc-finger based RHO transcriptional silencing for the development of a clinical trial for adRP patients

320 transcriptional silencing via synthetic dna binding protein lacking canonical repressor domains as a potent tool to generate therapeutics

Transcription factors (TFs) function by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs). Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain entails complete and gene-specific transcriptional silencing. To silence RHODOPSIN (RHO) gain-of-function mutations, we engineered a synthetic DNA-binding protein lacking canonical repressor domains and targeted to the regulatory region of the RHO gene. AAV-mediate retinal delivery at a low dose (AAV2/8-CMV-ZF6-DBD, 1×10e10 vector genomes, vg) in the porcine retina resulted in selective transcriptional silencing of RHO expression. The rod photoreceptors (the RHO expressing cells) transduced cells when isolated by FACS-sorting showed the remarkable 90% RHO transcriptional repression. To evaluate genome-wide transcriptional specificity, we analyzed the porcine retina transcriptome by RNA sequencing (RNA-Seq). The differentially expressed genes (DEGs) analysis showed that only 19 genes were perturbed. In this study, we describe a system based on a synthetic DNA binding protein enabling targeted transcriptional silencing of the RHO gene by in vivo gene transfer. The high rate of transcriptional silencing occurring in transduced cells supports applications of this regulatory genomic interference with a synthetic trans-acting factor for diseases requiring gene silencing in a large number of affected cells, including for instance a number of neurodegeneration disorders. The result support a novel mode of gene targeted silencing with a DNA-binding protein lacking intrinsic activity

Induced sputum is a reproducible method to assess airway inflammation in asthma.

To evaluate the reproducibility of induced sputum analysis, and to estimate the sample size required to obtained reliable results, sputum was induced by hypertonic saline inhalation in 29 asthmatic subjects on two different days. The whole sample method was used for analysis, and inflammatory cells were counted on cytospin slides. Reproducibility, expressed by intra-class correlation coefficients, was good for macrophages (+0.80), neutrophils (+0.85), and eosinophils (+0.87), but not for lymphocytes (+0.15). Detectable differences were 5.5% for macrophages, 0.6% for lymphocytes, 5.2% for neutrophils, and 3.0% for eosinophils. We conclude that analysis of induced sputum is a reproducible method to study airway inflammation in asthma. Sample sizes greater than ours give little improvement in the detectable difference of eosinophil percentages

In Vivo Effects of Einkorn Wheat (Triticum monococcum) Bread on the Intestinal Microbiota, Metabolome, and on the Glycemic and Insulinemic Response in the Pig Model

Einkorn wheat (Triticum monococcum) is characterized by high content of proteins, bioactive compounds, such as polyunsaturated fatty acids, fructans, tocols, carotenoids, alkylresorcinols, and phytosterols, and lower α-, β-amylase and lipoxygenase activities compared to polyploid wheat. These features make einkorn flour a good candidate to provide healthier foods. In the present study, we investigated the effects of einkorn bread (EB) on the intestinal physiology and metabolism of the pig model by characterizing the glycemic and insulinemic response, and the microbiota and metabolome profiles. Sixteen commercial hybrid pigs were enrolled in the study; four pigs were used to characterize postprandial glycemic and insulinemic responses and twelve pigs underwent a 30-day dietary intervention to assess microbiota and metabolome changes after EB or standard wheat bread (WB) consumption. The postprandial insulin rise after an EB meal was characterized by a lower absolute level, and, as also observed for glucose, by a biphasic shape in contrast to that in response to a WB meal. The consumption of EB led to enrichment in short-chain fatty acid producers (e.g., Blautia, Faecalibacterium, and Oscillospira) in the gut microbiota and to higher metabolic diversity with lower content of succinate, probably related to improved absorption and therefore promoting intestinal gluconeogenesis. The observed changes, at both a compositional and metabolic scale, strongly suggest that EB consumption may support a health-promoting configuration of the intestinal ecosystem

The p-ERG spatial acuity in the biomedical pig under physiological conditions

Pigs are becoming an important pre-clinical animal species for translational ophthalmology, due to similarities with humans in anatomical and physiological patterns. Different models of eye disorders have been proposed, and they are good candidates to assess biocompatibility/functionality of retinal prostheses. Electroretinography is a common tool allowing to gain information on retinal function, with several types of electroretinogram (ERG) been implemented including full field (ff-ERG), multifocal (mf-ERG) and pattern (p-ERG). p-ERG represents a valuable tool to monitor Retinal Ganglion Cells (RGCs) activity and can be used to calculate p-ERG spatial acuity. Unfortunately, scarce methodological data are available regarding recording/interpretation of p-ERG and retinal acuity in biomedical pigs yet enhancing knowledge regarding pig vision physiology will allow for more refined and responsible use of such species. Aim of this study was to record p-ERG in juvenile pigs to functionally assess visual acuity. Six female hybrid pigs underwent two p-ERG recording sessions at 16 and 19 weeks of age. Photopic ff-ERG were also recorded; optical coherence tomography (OCT) and histology were used to confirm retinal integrity. ff-ERG signals were repeatable within/across sessions. All p-ERG traces consistently displayed characterizing peaks, and the progressive decrease of amplitude in response to the increment of spatial frequency revealed the reliability of the method. Mean p-ERG spatial acuities were 5.7 +/- 0.14 (16 weeks) and 6.2 +/- 0.15 cpd (19 weeks). Overall, the p-ERG recordings described in the present work seem reliable and repeatable, and may represent an important tool when it comes to vision assessment in pigs

PCV2 infection in vaccinated conventional gilts inseminated with PCV2b-spiked semen

The present trial investigated the effect of PCV2 vaccination on viremia, virus shedding and viral load in maternal tissues and foetuses of conventional gilts inseminated with PCV2b-spiked semen. Twelve gilts were randomly divided into two groups of six animals each (vaccinated infected, VI; non-vaccinated infected, NVI). Estrus synchronization was followed by artificial insemination (AI) with a single PCV2 negative semen dose supplemented with 0.2 mL of a PCV2b suspension containing 104.4 TCID50/50 \u3bcL (total viral dose: 105 TCID50). Vaginal, nasal and faecal swabs, and blood samples were collected weekly from two days before artificial insemination till the end of the experimental period (55 days post AI; DPAI) and tested by real-time PCR (qPCR) for PCV2; sera were tested for anti- PCV2 antibodies. During necropsy foetal and maternal tissues were collected for qPCR and histopathology. In each of the VI and NVI groups three out of the six gilts were pregnant at 29 DPAI. The VI group showed a significantly lower proportion of PCR-positive swabs: 24.6% VI vs 71.3% NVI. PCV2 clearance was demonstrated by qPCR in lymphoid tissue during the trial in the VI group. Only one foetus was PCV2-positive (in the NVI group) and three amniotic fluids of the NVI group. PCV2 was found in a significantly lower proportion of the placenta of foetuses in the VI group (39%) than the NVI group (77%). The PCV2 vaccine seems to play an active role in reducing virus shedding, tissue viral load and foetoplacental infection