Search

Background and Aims: It has been established that the inflammatory component represents a significant contribution in the background of clinically recognizable risk factors of atherosclerosis, for example, hypertension, hyperglycemia, dyslipidemia, obesity. The aim of this study was to evaluate systemic and tissue-specific inflammation in pigs fed with high-fat diet. Methods: 21 pigs were kept for 16 weeks on a standard diet (SD; 3.6% lipids; n = 7) or a high-fat diet (HFD; 22% lipids, 5% cholesterol; n = 14). Seven HFD fed pigs received high-dose atorvastatin (80 mg/die) starting after 8 weeks of hypercholesterolemic diet until the end of the experimental procedure. Results: HFD administration significantly raised the number of circulating leukocytes, leading to a “hypercholesterolemia-associated monocytosis” and promoting the synthesis of several pro-inflammatory molecules. Hypercholesterolemia induced adipocytes hypertrophy and T-lymphocytes infiltration in abdominal white adipose tissue (WAT). HFD-fed pigs displayed liver inflammation, hepatic stellate cells activation with a concomitant increased infiltration of macrophages, T- and B-lymphocytes. Inflammation extended beyond liver and WAT with increased macrophage content in lung parenchyma. Atorvastatin abolished WAT inflammation by reducing adipocyte area and the number of infiltrating T-lymphocytes. In the liver, atorvastatin decreased hepatic stellate cells activation and the inflammatory infiltrate and lowered the amount of macrophages in lung parenchima of HFD-fed pigs. Conclusion: Hypercholesterolemia significantly raised the amount of circulating leukocytes and exacerbated the inflammatory response in WAT, liver and lung. Atorvastatin treatment markedly decreased systemic and tissue-specific inflammatory markers by preventing the development of an inflammatory milieu and the accumulation of infiltrating leukocytes

Intimal hyperplasia (IH) limits the success of many vascular interventions, including bypass grafting endarterectomy and stenting. IH following vascular injury is characterized by vascular smooth muscle cells proliferation and migration, matrix deposition, inflammation. The aim of the study was to investigate, in a mild-hypercholesterolemic pig model of vascular injury: i) the role of Heme Oxygenase-1 (HO-1) in the pathogenesis of IH; ii) the molecular mechanism of prevention and therapy mediated by atorvastatin; iii) the atorvastatin-mediated induction of HO-1 in response to vascular injury. Balloon injury (BI) was used to induce IH in pig carotid arteries. Pigs were divided in 3 groups (n=7 each): standard diet; high-cholesterol diet; high-cholesterol diet and atorvastatin (80 mg/die). Carotid sections were analyzed by morphometric and immunohistochemical analyses. Hemochrome, cholesterolemia, proinflammatory cytokines profiles were also evaluated. Hypercholesterolemia induced a significant increase of total and LDL-cholesterol, exacerbated IH, increased extracellular matrix deposition, inhibited HO-1 expression and increased the local and systemic inflammatory state compared to standard diet. Atorvastatin prevented IH development by enhancing HO-1 expression, reducing the number of T-cell infiltrating the site of vascular injury, and lowering the plasmatic concentration of proinflammatory cytokines. In conclusion: i) a high-cholesterol diet induces a persistent state of vascular inflammation that worsen the vascular response following BI; ii) atorvastatin has a protective role in the pathogenesis and outcome of inflammatory response following vascular injury; iii) atorvastatin prevents IH inducing HO-1 expression in the vascular wall.

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.

Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.

Recombinant adeno-associated viral (AAV) vectors are known to safely and efficiently transduce the retina. Among the various AAV serotypes available, AAV2/5 and 2/8 are the most effective for gene transfer to photoreceptors (PR), which are the most relevant targets for gene therapy of inherited retinal degenerations. However, the search for novel AAV serotypes with improved PR transduction is ongoing. In this work we tested vectors derived from five AAV serotypes isolated from porcine tissues (referred to as porcine AAVs, four of which are newly identified) for their ability to transduce both the murine and the cone-enriched pig retina. Porcine AAV vectors expressing EGFP under the control of the CMV promoter were injected subretinally either in C57BL/6 mice or Large White pigs. The resulting retinal tropism was analyzed one month later on histological sections, while levels of PR transduction were assessed by Western blot. Our results show that all porcine AAV transduce murine and porcine retinal pigment epithelium and PR upon subretinal administration. AAV2/po1 and 2/po5 are the most efficient porcine AAVs for murine PR transduction and exhibit the strongest tropism for pig cone PR. The levels of PR transduction obtained with AAV2/po1 and 2/po5 are similar, albeit not superior, to those obtained with AAV2/5 and AAV2/8, which evinces AAV2/po1 and 2/po5 to be promising vectors for retinal gene therapy.

Gene transfer to both cone and rod photoreceptors (PRs) is essential for gene therapy of inherited retinal degenerations that are due to mutations in genes expressed in both PR types. Vectors based on the adeno-associated virus (AAV) efficiently transduce PRs of different species. However, these are predominantly rods and little is known about the ability of AAV to transducer cones in combination with rods. Here we show that AAV2/8 transduces pig cones to levels that are similar to AAV2/9, and the ONL (mainly rods) to levels that are on average, although not statistically significant, higher than both AAV2/5 and AAV2/9. We additionally found that the ubiquitous Cytomegalovirus (CMV) but not the PR-specific GRK1 promoter transduced efficiently pig cones, presumably because GRK1 is not expressed in pig cones as observed in mice and humans. Indeed, the GRK1 and CMV promoters transduce a similar percentage of murine cones with the CMV reaching the highest expression levels. Consistent with this, the AAV2/8 vectors with either the CMV or the GRK1 promoter restore cone function in a mouse model of Leber congenital amaurosis type 1 (LCA1), supporting the use of AAV2/8 for gene therapy of LCA1 as well as of other retinal diseases requiring gene transfer to both PR types.

Ischemia-reperfusion injury, a clinical problem during cardiac surgery, involves worsened adenosine trisphosphate (ATP) generation and damage to the heart. We studied carbon monoxide ( CO) pretreatment, proven valuable in rodents but not previously tested in large animals, for its effects on pig hearts subjected to cardiopulmonary bypass with cardioplegic arrest. Hearts of CO-treated pigs showed significantly higher ATP and phosphocreatine levels, less interstitial edema, and apoptosis of cardiomyocytes and required fewer defibrillations after bypass. We conclude that treatment with CO improves the energy status, prevents edema formation and apoptosis, and facilitates recovery in a clinically relevant model of cardiopulmonary bypass surgery.

The evaluation of chronic activity of the hypothalamic-pituitary-adrenal (HPA) axis is critical for determining the impact of chronic stressful situations. However, current methods have important limitations. The potential use of hair glucocorticoids as a noninvasive retrospective biomarker of long-term HPA activity is gaining acceptance in humans and wild animals. However, there is no study examining hair corticosterone (HC) in laboratory animals. The present study validates a method for measuring HC in rats and demonstrates that it properly reflects chronic HPA activity. The HC concentration was similar in male and female rats, despite higher total plasma corticosterone levels in females, tentatively suggesting that it reflects free rather than total plasma corticosterone. Exposure of male rats to 2 different chronic stress protocols (chronic immobilization and chronic unpredictable stress) resulted in similarly higher HC levels compared to controls (1.8-fold). HC also increased after a mild chronic stressor (30 min daily restraint). Chronic administration of 2 different doses of a long-acting ACTH preparation dramatically increased HC (3.1- and 21.5-fold, respectively), demonstrating that a ceiling effect in HC accumulation is unlikely under other more natural conditions. Finally, adrenalectomy significantly reduced HC. In conclusion, HC measurement in rats appears appropriate to evaluate integrated chronic changes in circulating corticosterone.

Aim of this study was to characterize the effects of an ochratoxin A (181 ± 34 ng/g) contaminated diet on growth performances, blood parameters, systemic cytokine levels, cell stress markers and reactivity of immune system of weaned pigs. Growth performance was not affected by OTA consumption even if OTA levels increased in plasma, kidney and liver. OTA diminished the protein content in the serum and increased levels of TNF-alpha and IL-10 in plasma. HO-1 mRNA, indicative for cells stress, was decreased in the kidney but increased in the liver. Additionally, whole blood of the animals of the OTA-group showed a decreased capacity to respond with cytokine expression (mRNA and protein) to ex vivo challenge with LPS. In conclusion our findings indicate that chronic ingestion with OTA-contaminated feed, even at low level, is hazardous for the animal and virtually for human health, pig being an excellent model for human.

Cells derived from porcine aorta tunica media show mesenchymal stromal-like cell properties in in vitro culture. Am J Physiol Cell Physiol 306: C322–C333, 2014. First published December 4, 2013; doi:10.1152/ajpcell.00112.2013.—Several studies have already described the presence of specialized niches of precursor cells in vasculature wall, and it has been shown that these populations share several features with mesenchymal stromal cells (MSCs). Considering the relevance of MSCs in the cardiovascular physiopathology and regenerative medicine, and the usefulness of the pig animal model in this field, we reported a new method for MSC-like cell isolation from pig aorta. Filling the vessel with a collagenase solution for 40 min, all endothelial cells were detached and discarded and then collagenase treatment was repeated for 4 h to digest approximately one-third of the tunica media. The ability of our method to select a population of MSC-like cells from tunica media could be ascribed in part to the elimination of contaminant cells from the intimal layer and in part to the overnight culture in the high antibiotic/antimycotic condition and to the starvation step. Aorticderived cells show an elongated, spindle shape, fibroblast-like morphology, as reported for MSCs, stain positively for CD44, CD56, CD90, and CD105; stain negatively for CD34 and CD45; and express CD73 mRNA. Moreover, these cells show the classical mesenchymal trilineage differentiation potential. Under our in vitro culture conditions, aortic-derived cells share some phenotypical features with pericytes and are able to take part in the formation of network-like structures if cocultured with human umbilical vein endothelial cells. In conclusion, our work reports a simple and highly suitable method for obtaining large numbers of precursor MSC-like cells derived from the porcine aortic wal

Background The swine species represents a perfect model for translational medicine due to its physiological and anatomical resemblance to humans. The development of techniques for spinal catheter insertion in swine is significantly useful but, at the moment, the only technique described requires laminectomy as a surgical approach. New method The proposed techniques represent a transdermal approach for catheter placement in piglets. The study was divided into Phase I (anatomical study on 8 cadavers) and Phase II (in vivo application of the technique in 20 anaesthetised 30-day old piglets). A spinal needle was introduced between the L2 and L3 spinous processes with a ventro-cranial orientation until cerebro-spinal fluid leakage. It was then replaced with a Tuohy needle, used to introduce the catheter into the intrathecal space. Before inserting the catheter, the approximate length from the insertion point to the external projection of the Cisterna Magna was measured using the gradation markings on the device. Results The technique described allowed spinal catheter placement in all piglets. In Phase I, the correct placement was confirmed using fluoroscopy while, in Phase II, cerebrospinal fluid leakage from the needle was relied on. No clinical alterations were detected either during the procedure or during the following days. Comparison with existing method This technique is easy and requires less skilled operators when compared to the other existing method which involves a surgical approach. Moreover, being less invasive, it potentially leads to fewer complications. Conclusions In conclusion, the technique can be performed safely in piglets, and provides an easier and less invasive approach for spinal catheter insertion.

The collection of cerebrospinal fluid is necessary in order to determine its composition. It can then be used to diagnose various diseases. The aim of the study was to develop and optimize a technique for performing safe centesis for the collection of cerebrospinal fluid in piglets and its injection through the cisterna magna. The study was divided into three phases: (1) anatomical study of cadavers, (2) in vivo application of the technique and (3) observation of recovery time. The proposed technique resulted in a safe puncture of the cisterna magna. The authors identified and confirmed the correspondence of the crista occipitalis and the wings of the atlas with the external landmarks on the cadaver by means of direct radiological visualization. The punctures were performed successfully at the first attempt in 11 out of 12 anaesthetized piglets. The technique herein described provides a reproducible safe and easy route for approaching the cisterna magna for cerebrospinal fluid collection, drug administration and gene delivery.

REGULATION OF TASTE SIGNALING MOLECULES BY HIGH PROTEIN DIET IN THE PIG PYLORUS Vallorani C.*[1], Latorre R.[1], Mazzoni M.[1], Bacci M.L.[1], Monica F.[1], Giancola F.[1], Falconi M.[2], De Giorgio R.[3], Sternini C.[4], Clavenzani P.[1] [1]Department of Veterinary Medical Science, University of Bologna. ~ Bologna, [2]Department of Biomedical and Neuromotorial Sciences, University of Bologna ~ Bologna, [3]Department of Medical and Surgical Sciences, University of Bologna ~ Bologna, [4]CURE/DDRC, Division of Digestive Diseases, Department of Medicine, UCLA, Los Angeles, CA, USA ~ Los Angeles Taste receptors (TRs) and their signaling molecules are widely expressed in extra‐oral sites, including the gastrointestinal (GI) tract mucosa(1). Fasting and refeeding have been shown to modify the expression of α‐ transducin / α‐gustducin in enteroendocrine cells of the pig GI tract(2), however, the effects of individual nutrients on TR‐related molecules remain unknown. The gustatory system is fundamental for detecting dietary nutrients and evoking appropriate functional responses leading to digestion and absorption. Thus, the aim of this study was to test whether a short‐ and long‐term high protein diet (3 and 30 days, respectively) affected the enteroendocrine profile of the TRs signalling molecules α‐transducin / α‐ gustducin expressing cells in the pig gastric mucosa. Twelve hybrid (LW x D) female pigs (weighing 30‐40 Kg) were subdivided in three experimental groups (n= 4 each group): A) fed standard diet (controls, CTR, 14,5% protein); B) fed high protein diet for 3 days (HP‐3, 33% protein); and C) fed high protein diet for 30 days (HP‐30, 33% protein). The protein used to supplement the diet was fish and potatoes protein. Mucosal samples from pylorus were harvested, fixed and processed for single and double labelling immunofluorescence with a mixture of the following primary antisera to Gα‐transducin (Gαtran), Gα‐gustducin (Gαgust) and 5‐hydroxytryptamine (5‐HT). In the pyloric mucosa, the average number of Gαtran immunoreactive (‐IR) cells were 111.5 ± 26.2 (CTR), 148.8 ± 13.2 (HP3), and 218.3 ± 28.8 (HP30) (CTR vs HP‐3 P< 0.04; CTR vs HP‐30 P< 0.002; HP‐3 vs HP‐30 P< 0.005), while Gαgust‐IR cells were 138.8 ± 45.1 8 (CTR), 177.3 ± 14 (HP3), 211.5 ± 29.1 (HP30) (CTR vs HP‐30 P

The present work has the aim to analyze, for the first time, the physiological activity of the pig’s retina using a standardized human electoretinogram protocol approved by the International Society for Clinical Electrophysiology of Vision (ISCEV). The experiment was conducted at the Livestock and Animal Testing unit of the Physiology service of the Department of Veterinary Medical Sciences of the University of Bologna, in accordance with the national and international guidelines on animal experimentation. All procedures were examined by the Ethics Committee of the University of Bologna, authorized in derogate by the Ministry of Health and controlled by the Central Veterinary Service of the University of Bologna during the implementation phase. The subjects are 30 healthy females piglets of 3 months old and weighing 30 kg. The pre medication is performed using Azaperone (2mg/kg of Stresnil) by intramuscular injection behind the ear. Then, the Ketamine (20mg/Kg of Ketavet), Medetomidine (0,04mg/kg of Medetor) and Atropine (0.04 mg/kg of Atropine sulfate) mixture is injected intramuscularly. The subject is left alone until the loss of the righting reflex. Two drops of mydriatic (Tropicamide 1%) per eye are instilled to obtain the necessary paralysis of the pupil. The instrument used for the retinal exam, is a Ganzfeld Stimulator Model GS-MAX for use in humans. The electrical signal data is recorded, amplified and analyzed by the program EREV2000 previously set for ERG sessions running in series. The electrodes are ERG-jet metal conductor model to transfer the bioelectric signal to the amplifier and they are divided in: subcutaneous or inox needle and corneals. Four different light stimulations are sent in order to record the photoreceptors response and to compare the average amplitudes for each protocol with the human and mouse data present in literature. This study confirm that a high cone density and the particular pig’s photoreceptor topography allow this animal to be the better choice for experimental modelling of human cone diseases than mouse. Additionally, this wide data background is a good resource for researchers involved in ophthalmic studies to choose the correct animal model for their experiment and to use the smallest possible number of animals needed to obtain a scientifically valid result.

Background: Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions. Methodology/Principal Findings: To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3′UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy. Conclusions: We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters. © 2011 Karali et al.

Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy of retinal diseases caused by defects in the retinal pigment epithelium (RPE). In contrast, evidence of the efficacy of AAV-mediated gene transfer to retinal photoreceptors, the major site of inherited retinal diseases, is less robust. In addition, although AAV-mediated RPE transduction appears efficient, independently of the serotype used and species treated, AAV-mediated photoreceptor gene transfer has not been systematically investigated thus so far in large animal models, which also may allow identifying relevant species-specific differences in AAV-mediated retinal transduction. In the present study, we used the porcine retina, which has a high cone/rod ratio. This feature allows to properly evaluate both cone and rod photoreceptors transduction and compare the transduction characteristics of AAV2/5 and 2/8, the two most efficient AAV vector serotypes for photoreceptor targeting. Here we show that AAV2/5 and 2/8 transduces both RPE and photoreceptors. AAV2/8 infects and transduces photoreceptor more efficiently than AAV2/5, similarly to what we have observed in the murine retina. The use of the photoreceptor-specific rhodopsin promoter restricts transgene expression to porcine rods and cones, and results in photoreceptor transduction levels similar to those obtained with the ubiquitous promoters tested. Finally, immunological, toxicological and biodistribution studies support the safety of AAV subretinal administration to the large porcine retina. The data presented here on AAV-mediated transduction of the cone-enriched porcine retina may affect the development of gene-based therapies for rare and common severe photoreceptor diseases. © 2011 Macmillan Publishers Limited All rights reserved. Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy of retinal diseases caused by defects in the retinal pigment epithelium (RPE). In contrast, evidence of the efficacy of AAV-mediated gene transfer to retinal photoreceptors, the major site of inherited retinal diseases, is less robust. In addition, although AAV-mediated RPE transduction appears efficient, independently of the serotype used and species treated, AAV-mediated photoreceptor gene transfer has not been systematically investigated thus so far in large animal models, which also may allow identifying relevant species-specific differences in AAV-mediated retinal transduction. In the present study, we used the porcine retina, which has a high cone/rod ratio. This feature allows to properly evaluate both cone and rod photoreceptors transduction and compare the transduction characteristics of AAV2/5 and 2/8, the two most efficient AAV vector serotypes for photoreceptor targeting. Here we show that AAV2/5 and 2/8 transduces both RPE and photoreceptors. AAV2/8 infects and transduces photoreceptor more efficiently than AAV2/5, similarly to what we have observed in the murine retina. The use of the photoreceptor-specific rhodopsin promoter restricts transgene expression to porcine rods and cones, and results in photoreceptor transduction levels similar to those obtained with the ubiquitous promoters tested. Finally, immunological, toxicological and biodistribution studies support the safety of AAV subretinal administration to the large porcine retina. The data presented here on AAV-mediated transduction of the cone-enriched porcine retina may affect the development of gene-based therapies for rare and common severe photoreceptor diseases. Gene Therapy (2011) 18, 637-645; doi:10.1038/gt.2011.3; published online 17 March 2011

Endothelin (ET)-1 is a potent vasoconstrictor peptide involved in the derangement of respiratory mechanics during endotoxic shock.We measured the kinetics of pulmonary mRNA expression of the key components of the ET system [i.e., ET-1, ET-converting enzyme (ECE), and ETA and ETB receptors] by quantitative real-time reverse transcriptase-polymerase chain reaction in a swine model of endotoxic shock (0, 1, 2, 3, and 4 h of continuous LPS infusion at 40 μg/kg/hour; sham group, 4 hour saline infusion).A significant increase inmRNAexpression levels was observed for ET-1 in LPS-treated piglets; the increase began as early as 1 hour. In contrast, no significant variations were observed for the ECE, ETA, or ETB genes. Small gene expression differences observed with respect to our previous results suggest a possible effect of the anesthesia or surgical protocol on ET system regulation.

Treponema denticola is an oral spirochete that is associated with periodontal disease and detected occasionally in extraoral lesions associated with systemic disorders such as cardiovascular diseases. The effect of specific bacterial products from oral treponemes on endothelium is poorly investigated. This study analyzed the ability of components of the outer membrane of T. denticola (OMT) to induce apoptosis and heat shock proteins (HO-1 and Hsp70) in porcine aortic endothelial cells (pAECs), compared with results obtained with classical pro-inflammatory lipopolysaccharide (LPS) treatment. Cellular apoptosis was detected when pAECs were treated with either OMT or LPS, suggesting that OMT can damage endothelium integrity by reducing endothelial cell vitality. Stimulation with OMT, similarly to LPS response, increased HO-1 and Hsp-70 protein expression in a time-dependent manner, correlating with a rise in HO-1 and Hsp-70 mRNA. Collectively, these results support the hypothesis that T. denticola alters endothelial cell function. Moreover, our in vitro experiments represent a preliminary investigation to further in vivo study using a pig model to elucidate how T. denticola leaves the initial endodontic site and participates in the development of several systemic diseases.

Therapeutic Riding (TR) is a global therapeutic method in which the horse becomes an active component of therapy; nevertheless TR is recognized as potential stressor. The aim was the identification of abnormal behaviours in TR horses. Firstly we conducted an investigation by means of questionnaires to TR centres. Then we made direct ethological observations on 4 TR horses. Those approaches highlighted a late identification of stress-related behaviours; frequent onset of physical-behavioural problems (due to features of TR). Therefore further ethological studies were performed on 3 experimental groups (n=4): TR (A), destined to sport activity (B) and semi-wild breeding (C) horses. A data sheet of behavioural assessment was utilised. Group A showed a prevalence of reactive type anomalies (tendency toward hypo- with sporadic demonstrations of hyper-reactivity), in group B motor/oral-ingestive anomalies resulted more frequent. Group C didn’t show abnormal behaviours. Our results confirm previous observations and seem to reflect that specific stressors, conflicting with horse’s motivations and adaptive skills, may determine disturbs related to a state of constraint or anxiety.

In the last twenty years many acknowledgements were conceived about Sperm Mediated Gene Transfer (1), but it is whether to define differential construct uptake on sperm membrane, in order to operate efficiently with different exogenous DNAs. The aim of this study was to develop a simple and reliable method for differential uptake quantification during the co-incubation phase between exogenous DNA and spermatozoa. We recurred to construct specific-fluorophores (Cyanine 3, 3.5 and 5) and spectrophotometric readings at every time point. Experimental data were then validated through Real Time PCR: both the assays showed clearly that there are no preferences in construct uptake by sperm membrane, and moreover, that most of the exogenous DNA (>70% for each construct) was seized from the solution by the spermatozoa.

In the last ten years it has been described the possibility to produce “one-step” multi-transgenic pigs through the application of Sperm Mediated Gene Transfer. The aim of this study was to obtain primary lines of swine mesenchimal cells from newborn piglets produced through SMGT technique and to perform exogenous DNA detection on the isolated cells. The mesenchimal origin was established with immunocitochemistry. Cell lines resulted to be proliferant and transgenic, disclosing further applications for the production of transgenic pigs.