GIS-based land suitability analysis for the optimal location of integrated multi-trophic aquaponic systems.

Aquaponics has witnessed global proliferation and a notable enhancement in sustainability in recent years. Consequently, it assumes paramount importance to delineate optimal locations for its implementation, in fact, the success of an aquaponic facility also depends on its geographical placement, necessitating consideration of many variables encompassing natural resources, socioeconomic factors, infrastructural availability and environmental constraints, whether natural or artificial. This paper focuses on the definition and test in the Emilia-Romagna region (Italy) of a GIS-based multi-criteria land suitability assessment model aimed at allowing the diffusion and environmental integration of innovative integrated multi-trophic aquaponic systems. The process has been implemented with a Weighted Linear Combination (WLC) model, where decisions and criteria were selected via a participatory mechanism involving experts in various fields. The region has been subdivided into 50 × 50 m grid cells, with each grid cell being associated with a value ranging from 0 to 8. In this context, a rating of 0 means unsuitability, while a rating of 1 denotes minimal suitability, and the highest rating of 8 designates maximal suitability. Notably, a substantial portion of the surveyed territory has been found to be completely unsuitable for the establishment of aquaponic facilities. More than 86.4% of the remaining suitable areas were rated 6, 7, or 8, affirming the overall favourability of the Emilia-Romagna region for aquaponic installations. Finally, the veracity and robustness of the results have been tested through a one-at-a-time sensitivity analysis, that has proven the appropriateness of the proposed model.

X and Y chromosome-bearing spermatozoa are equally able to uptake and internalize exogenous DNA by sperm-mediated gene transfer in swine.

Since proteomic differences between male X/Y chromosome-bearing gametes have recently been described, a question has been raised: could these differences be responsible for different behavior between X and Y chromosome-bearing spermatozoa during the binding and internalization of exogenous DNA in the swine species? In order to investigate this hypothesis, our group studied the process of the uptake and internalization of exogenous DNA in X and Y chromosome-bearing sperm sub-populations. No significant differences were found between sperm types in both the uptake and internalization of exogenous DNA. The quantity of internalized exogenous DNA was significantly lower than that of the uptaken DNA. In conclusion, our results showed that X and Y chromosomes-bearing spermatozoa have the same binding capacity and internalization of DNA, and the proteomic differences between them do not seem to interfere with these complex processes.

In vitro differentiation of porcine aortic vascular precursor cells to endothelial and vascular smooth muscle cells.

Recent findings suggest that progenitor and multipotent mesenchymal stromal cells (MSCs) are associated with vascular niches. Cells displaying mesenchymal properties and differentiating to whole components of a functional blood vessel, including endothelial and smooth muscle cells, can be defined as vascular stem cells (VSCs). Recently, we isolated a population of porcine aortic vascular precursor cells (pAVPCs), which have MSC- and pericyte-like properties. The aim of the present work was to investigate whether pAVPCs possess VSC-like properties and assess their differentiation potential toward endothelial and smooth muscle lineages. pAVPCs, maintained in a specific pericyte growth medium, were cultured in high-glucose DMEM + 10% FBS (long-term medium, LTM) or in human endothelial serum-free medium + 5% FBS and 50 ng/ml of hVEGF (endothelial differentiation medium, EDM). After 21 days of culture in LTM, pAVPCs showed an elongated fibroblast-like morphology, and they seem to organize in cord-like structures. qPCR analysis of smooth muscle markers [α-smooth muscle actin (α-SMA), calponin, and smooth muscle myosin (SMM) heavy chain] showed a significant increment of the transcripts, and immunofluorescence analysis confirmed the presence of α-SMA and SMM proteins. After 21 days of culture in EDM, pAVPCs displayed an endothelial cell-like morphology and revealed the upregulation of the expression of endothelial markers (CD31, vascular endothelial-cadherin, von Willebrand factor, and endothelial nitric oxide synthase) showing the CD31-typical pattern. In conclusion, pAVPCs could be defined as a VSC-like population considering that, if they are maintained in a specific pericyte medium, they express MSC markers, and they have, in addition to the classical mesenchymal trilineage differentiation potential, the capacity to differentiate in vitro toward the smooth muscle and the endothelial cell phenotypes.

Cells derived from porcine aorta tunica media show mesenchymal stromal-like cell properties in in vitro culture.

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. Aortic-derived 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 wall.

Development of a vessel organ culture system: characterisation of the method and implications for the reduction of animal experiments.

In the field of cardiovascular research, the pig is considered to be an excellent animal model of human diseases. It is well-known that primary cultures of endothelial cells (ECs) are a powerful tool for the study of vascular physiology and pathology, and, according to the principles of the Three Rs, their use results in a substantial reduction in the numbers of experimental animals required. However, a limitation of EC culture is that the cells are not in their physiological context. Here, we describe and characterise a method for the culture of porcine vessels that overcomes the limitation of EC cultures, with the advantage of reducing the number of animals used for research purposes. The organ cultures were set-up by using an aortic cylinder obtained from the arteries of control pigs sacrificed for other experimental purposes. In order to characterise the method, vascular endothelial growth factor (VEGF) secretion, matrix metalloproteinase (MMP) activation and the vessel's structural features were evaluated during organ culture. These analyses confirm that the culture of aortic cylinder lumen, in a medium specific for ECs, results in a stable system in terms of VEGF and MMP secretion. The ECs do not undergo cell division during the organ culture, which is also the case in vivo, if no stimulation occurs. Overall, we show that this novel system closely resembles the in vivo context. Importantly, porcine aortas can be collected from either veterinary surgeries or slaughterhouses, without having to sacrifice animals specifically for the purposes of this type of research.

Expression of fluorescent reporter protein in equine embryos produced through intracytoplasmic sperm injection mediated gene transfer (ICSI-MGT).

Sperm mediated gene transfer (SMGT) has been reported to be a powerful tool for producing transgenic livestock with applications in biomedicine and agriculture. To date, two studies have reported the production of transgenic equine embryo with, however, low efficiency in blastocyst production and transgene expression. The aim of the present study was to develop a method which allowed the efficient production of transgene-expressing embryos of the equine species through SMGT. To overcome problems due to in vitro fertilization (IVF) in horses, the ICSI procedure was associated with SMGT. The uptake of exogenous DNA in equine spermatozoa was assessed using a spectrophotometric approach and its internalisation using real time PCR and confocal laser scanning microscopy (CLSM). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of eGFP and then for the transmission of the transgene. Our results suggested that the maximal uptake of exogenous DNA in equine spermatozoa occurs from 30 to 60min of co-incubation. Furthermore, real time PCR analysis suggested that the internalisation of exogenous DNA in the highest quality spermatozoa was slightly greater than in those having the poorest quality parameters. Confocal laser scanning microscopy analysis confirmed that exogenous DNA is internalised by membrane intact spermatozoa. In this study, 22 embryos were produced, 8 of which reached the 8-cell stage or greater. Our data confirmed the transmission of the transgene in 86.3% of the cleaved embryos and the expression of the transgene in 25% of the embryos. These data allowed us to affirm that this method is highly efficient in producing equine embryos which are able to express high levels of the exogenous protein.

Procalcitonin gene expression after LPS stimulation in the porcine animal model.

Procalcitonin (PCT), recognised as a marker of sepsis, was investigated in a porcine model of endotoxic shock. The results showed that continuous IV infusion (1-4 h) of LPS (40 µg/kg) in pigs was able to induce a generalised increase of PCT expression in lung, heart, kidney and liver. The increase in PCT was significant only in kidney and was accompanied by an increase in IL-6 gene expression. In vitro results demonstrated that peripheral blood mononuclear cells (PBMCs), as well as endothelial cells, were potentially capable of contributing to in vivo extrathyroidal PCT production. These findings support previous data from pigs concerning the occurrence of widespread activation of PCT extrathyroidal gene expression during endotoxic shock in pigs. Nevertheless, the levels of PCT detected were very low, suggesting the need for additional studies to validate the pig as a reliable animal model for investigating the role of PCT in sepsis.

Validation of a rat model of cardiopulmonary bypass with a new miniaturized hollow fiber oxygenator.

Cardiopulmonary bypass (CPB) is an essential component of cardiac surgery, with still unknown device/patient interactions. To evaluate the response of CPB to hemodynamic, biochemical, inflammatory, as well as thermo-pharmacodynamic interactions, a novel miniaturized oxygenator with controlled and standardized specifications has been developed together with an improved surgical central cannulation technique. A hollow-fiber small priming volume (6.3 ml) oxygenator was manufactured according to specifications resulting from engineering, heart surgery and perfusion expertise (Dideco-Sorin Group, Italy) with the following characteristics: Gas Exchange Surface--450 cm2, and Heat Exchange Surface--16 cm2. The oxygenator was tested in vitro and in vivo in five anesthetized, ventilated, open-chest rats using a miniaturized roller pump. Pressures were monitored in the animal before and after the oxygenator. Central venous cannulation through the superior vena cava and aortic cannulation through the carotid artery were used. In vitro: blood oxygenation increased 10-fold (from room air to 100% O2) and PCO2 removal was 2.5-fold. In vivo: CPB was performed without blood prime for 90 minutes (no ventilation) maintaining stable hemodynamics. A maximal blood flow rate of 124 ml/min/kg was obtained. Arterio-venous PO2 gradients were 10-fold (O2 100%) with only small variations when changing blood flow rates. This new, standardized and miniaturized hollow fiber oxygenator, new cannulation technique and CPB circuit achieved optimal gas transfer with small asanguinous priming volumes. This study opens new potentials for various CPB-related study protocols in the small animal.