Oxidative stress and lateral muscle development in Siberian Sturgeon (Acipenser baeri): preliminary observations.

Embryonic development in bony fish is strongly influenced by environmental factors, mainly by temperature and dissolved oxygen, but little is known about their influence on Sturgeons. Since 1998 the international trade of Sturgeons has been regulated under CITES due to depletion of wild stocks. Sturgeon aquaculture as well as larvae production is, therefore, important because it may contribute to the repopulation of the wild stocks (Bronzi et al., 2011; Di Giancamillo et al., 2012).The aim of this study is to monitor oxidative stress status and muscle development during embryonic and precocious larval phases in Siberian Sturgeon (Acipenser baeri), when subjected to different incubating temperatures (13°, 16° and 19°C).Siberian sturgeon eggs were subjected to different incubation temperatures. Data regarding water quality parameters were recorded, as well as mortalities, embryonic period length, and precocious larvae behavior. Sampling was performed in the same developmental stage for each temperature, at five time points: 48 hours post-fertilization, embryo movements, hatching, schooling and yolk-sac full absorption. The observed hatching rates were between 85.5% and 98.8% with significant differences concerning the different experimental temperatures (T19 vs T13 and T19 vs T16, p<0,05). Histological, histochemical and immunohistochemical analyses will be performed to assess ontogeny of the lateral muscle and stress biomarkers (Lushchak, 2011), and gene expression will be analyzed for muscle development (Johnston, 2006). The obtained results will be compared with those concerning teleosts and will possibly contribute to better rearing conditions of sturgeons.This study has been approved by the ethic committee of the Università degli Studi di Milano, with the following authorisation code: OPBA_20_2016.

Effect of different stocking densities on growth, muscle development and fatty acid profile of Acipenser baerii larvae

Sturgeons, as well as paddlefishes, belong to the Acipenseriformes group, which is one of the most primordial 57 orders of the Osteichthyes that comprehends 25 species spread throughout Europe, Asia and North America. The present study aims at investigating muscle growth and development as well as fatty acid profile in Siberian sturgeon free-embryos when subjected to three different rearing densities. Fatty acids, in particular polyunsaturated fatty acids of n-3 series, are generally known as key nutrients in fish larvae.This study was approved by the Ethic Committee of the University of Milan (OPBA_22_2017). Siberian sturgeon larvae were reared at 18°C, at three stocking densities until complete yolk-sac absorption: low (LD, 30 larvae/l), mid (MD, 80 larvae/l) and high (HD, 150 larvae/l). Sampling timepoints were: hatching, schooling and complete yolk-sac absorption stage (YSA). Sacrificed larvae were weighed and histological analyses were performed in order to assess muscle development as described elsewhere ; fatty acid profile was determined by GC-FID analysis as described by Vasconi et al. (2015). Statistical analysis was performed with SAS software (v. 9.3, Cary Inc., NC).At the end of the experiment, LD larvae presented a higher weight than larvae reared at the other two densities (P<0.05). Within the schooling stage (Figure 1), Total Muscle Area was lower for HD larvae (P<0.05); red and white muscle areas in schooling and YSA were higher than at hatching (P<0.05), regardless the density. Concerning fatty acids, no statistical differences were recorded between different rearing densities, while during the development regardless the rearing density, there was a common pattern: linoleic and alfa linolenic acids, significantly decreased their relative content, while others, as arachidonic acid and DHA, significantly increased. Siberian sturgeon larvae reared at LD or MD reveal an anatomically normal muscle development, while in the HD it is possible to observe a slowdown.What the aquaculture industry requires is a set of guidelines that allows the development of a sustainable industry, so that we tried to develop guidelines for stocking density in the very early stage of farming. As a conclusion, it would seem that mid density could be more suitable for this species in this stage of development

Meniscal femoral and tibial surfaces characterization in the swine model.

Menisci are wedge-like structures interposed, in the knee joint, between the femoral and the tibial articular heads (Kohn et al. 1995; Greis et al. 2002). Improving the articular surface, the cellular nutrition and the articular lubrication, they are essential structures for the prevention of gonarthrosis (Proctor et al.1989; Makris et al. 2011). This study is focused upon the relationship between the contact forces at the femoral and tibial surfaces and the corresponding structure of these meniscal surfaces. For this purpose, 20 adult (~9 months old) female pigs (Landrace x Large white, average weight 75–90 kg; n=80 meniscal samples) were obtained from a local slaughterhouse and dissected to isolate the menisci. Swine meniscal samples were evaluated from morphological (Safranin-O, Sirius Red and collagen type I and II) (Di Giancamillo et al. 2014), biochemical (DNA and glycosaminoglycans, or GAGs, contents) and biomechanical (compression and traction tests) points of view at the level of femoral and tibial meniscal surfaces. Results revealed a characterization of the meniscus which is biomechanical-dependent.  The femoral surface, morphologically characterized by the interposition of radial and oblique fibers and biomechanically by the femoral condyles compression, sliding and rolling forces, shows a higher compressive modulus (p<0.05) and a greater amount of cells and GAGs deposition (p<0.01 for each analysis). On the other hand, results from traction test revealed a higher tensile modulus (p<0.05) in the tibial surface, characterized by a circumferential arrangement of the fibers and a poorer GAGs deposition and cellular distribution (p<0.01). Results (summarized in the figure 1) from this work suggest that a biphasic “femoral-to-tibial” scaffold that mimic the different behavior and composition of the two meniscal surfaces could be useful in the light of meniscal replacement.

Stifle anatomic, tomographic and biomechanical features of growing dogs affected by quadriceps contracture.

Quadriceps contracture is a debilitating and uncommon condition, mostly affecting young dogs. Itcan be congenital or acquired ) and is reported to induce muscular hypotrophy/fibrosis, progressive degenerative joint disease, bone hypoplasia and limb hyperextension. The aim of this study was to elucidate anatomic, tomographic and biomechanical features of stifles affected by quadriceps contracture.Seven 2-month-old dead Dobermann Pinschers with unilateral quadriceps contracture were included. Before gross anatomic evaluation, all stifles underwent Computed Tomography before and after intra-articular administration of iodinated contrast medium. Images were acquired in double positioning (stifle extension and flexion) to identify articular cartilage, Ossification Centres’ (OCs) and menisci abnormalities, which were compared between affected and unaffected limbs. In all affected limbs the stifle was back-turned, the distal femur was extra-rotated and the patella was luxated proximo-medially. Severe lack of physiological stifle movements (rolling, gliding, spinning) was observed, so that affected joints could not be flexed. The articular cartilage of the femur was flattened and irregular in thickness, the femoral trochlea was hypoplasic and sloping, the menisci were misshaped. The OC of the distal femur and proximal tibia were misshaped; the tibial plateau was oriented caudodorsally-cranioventrally and significantly smaller (P<0,05). Quadriceps contracture influenced stifle development. The action of quadriceps insertion on the tibia prevented normal development of the plateau, causing wedging and abnormal orientation. Constant compression also induced external rotation of the distal femur (unable to develop distally) and patellar luxation, ending up in genu recurvatum. Static compression was likely responsible for femoral trochlea hypoplasia, articular cartilage and meniscal deformation, due to the lack of physiological stifle movements.Quadriceps contracture induces severe alterations of stifle development in affected puppies. Histology, histochemistry and immunohistochemistry may better define the nature of such bone, cartilage and meniscal alterations

Swine cortical and cancellous bone: histomorphometric and densitometric characterisation

Introduction: Swine bone morphology, composition and remodelling are similar to humans’, therefore they are considered good models in bone-related research. They have been used for several studies involving bone growth, bone and cartilage fractures and femoral head osteonecrosis. Nevertheless, the literature about pig normal bone features is incomplete. This work aims to fill the literature gaps on the microarchitecture and Bone Mineral Density (BMD) of swine femoral diaphysis and distal epiphysis and tibial plateau and diaphysis.Materials and methods: Five hind limbs were collected from slaughtered 80-100 kg pigs. Microscopic analysis of cortical and cancellous bone from middle/distal femur and proximal/middle tibia was performed to determine basic histomorphometric parameters at different sites. Dual-energy X-Rays Absorptiometry was also employed to evaluate BMD. ANOVA and correlation between BMD, bone area (BA) and cortical thickness were performed.Results and discussion: Diaphyseal cortical bone was mostly plexiform both in the tibia and the femur; primary/secondary osteons without clear organization were also found. Mean values for bone area, bone perimeter, trabecular width, number and separation and BMD at different anatomical sites were defined. No significant difference was found for these values at different anatomical sites. BMD proved to be positively correlated with cortical thickness (r=0,80; p<0,01). Despite the small sample size, these results seem homogeneous. They could therefore represent reference values for normal bone parameters in pigs. Applied anatomy and regenerative medicine, in fact, demand very precise information about bone micromorphology, composition and density to provide reliable indication in bone substitutes building. Moreover, since the interpretation of bone abnormalities is based on mastering normal bone characteristics, the definition of reference parameters is mandatory to avoid misinterpretation and allow comparative evaluation.Conclusion: The results of this study, although preliminary, may be considered a dependable starting point for the definition of normal bone features in pigs

Meniscus matrix morphological composition: age-dependent evaluation in a swine model

Menisci are fibro-cartilaginous structures interposed between femoral condyle and tibial plateau, which have multiple functions in the stifle joint: act as shock absorbers, bear loaders and allow joint stability, congruity and lubrication (Sweigart et al., 2004; Proffen et al., 2012). It is well known that meniscal injuries lead to osteoarthritis and for these reasons, menisci are considered important target of investigation. Their important role in the knee wellness is only equalled by their deficiency in proper self-repairing.Nowadays, the gold standard technique is not just to remove the damaged meniscus, but to rebuild it or to replace it. For these reasons, studies are necessary to increase the knowledge about these small but essential structures (Streuli, 1999; Deponti et al., 2013). Composition and morphology are basic fundamental information for the development of engineered meniscal substitutes (Di Giancamillo et al., 2014). The analysis of the morphological, structural and biochemical changes, which occur during growth of the normal menisci, represent the goal of the present study. For this purpose, menisci from adult (7-month old), young (1-month old), and neonates (stillbirths) pigs were collected. Cellularity and glycosamiglycans (GAGs) deposition were evaluated by ELISA, while Collagen-1 and Collagen-2 were investigated by immunohistochemistry and Western blot analyses. Cellularity (P<0.01, all comparisons) and Collagen-1 (P<0.05, neonatal-young vs adult) decreased from neonatal to adult stage while GAGs (P<0.01 neonatal vs young-adult) and Collagen-2 (P<0.01 neonatal-young vs adult) showed the opposite trend. Immunohistochemistry revealed similar changes occurring during animal growth thus revealing that cellular phenotype, cellularity and protein expression, as well as fibers aggregation in the matrix, are dissimilar in the three ages analysed categories. These changes reflect the progressive menisci maturation and hyper-specialisation. We observed the correlation between biochemical and phenotype properties of swine menisci follow age-dependent changes during growth: starting with an immature cellular and fiber pattern to the mature organised and differentiated adult menisci.Acknowledgments: This work was funded by the “Finanziamento Piano Sviluppo Ateneo - Linea 2A

Rearing temperature effect on the skeletal muscle fibres of Acipenser baerii yolk-sac larvae

Siberian sturgeon farming is important because it provides an alternative source of caviar and meat, but also for the conservation of the endangered natural stocks. Farmed fish is continuously subjected to stress factors, of which, water temperature is considered a major one (Schram et al., 2006). It has been demonstrated that physiological stress may have serious negative consequences on growth (Wendelaar Bonga, 1997) and that fish larvae appear less tolerant than adults to temperature variations (Stefanovich et al., 2016). The present study aims at investigating the stress response and development in precocious stages of siberian sturgeon when subjected to different rearing temperatures, by analysing ontogeny, growth and stress response of yolk-sac larvae. This study was approved by the Ethic Committee of the University of Milan (OPBA_20_2016). Fertilized Siberian sturgeon eggs were reared at 16°C, 19°C and 22°C until complete yolk-sac absorption. Sampling timepoints were: hatching, schooling and complete yolk-sac absorption stage. Water parameters and larvae development data were registered. Histological, histochemical and immunohistochemical analyses were performed in order to assess ontogeny and stress biomarkers and whole body cortisol was measured by a specific microtitre radioimmunoassay (RIA). Statistical analysis was performed with SAS software (v. 9.3, Cary Inc., NC). Larvae subjected to the highest water temperature showed a faster yolk-sac absorption and larvae body weight significantly increased from hatching onwards. Structural normal development considering the three different temperatures investigated from hatching until the end of the trial was observed. Significant differences were found between temperatures regarding body weight and cortisol levels (P<0.01). A qualitative stronger expression of stress markers was noticed in larvae subjected to the lower temperature. Even if this study indicates that lower rearing temperatures would appear more suitable for Siberian sturgeon rearing, further studies would be necessary to evaluate the temperature effect on a mid-long term basis

Derivation of canine hepatocyte in vitro models to study Branched-Chain Amino Acid effects on liver functions.

Branched chain amino acids (BCAA), have been shown to affect human gene expression, proteinmetabolism, apoptosis, and regeneration of hepatocytes. Furthermore, they have been demonstratedto inhibit proliferation of liver cancer cells in vitro, and to be essential for lymphocyte proliferation.In veterinary medicine, the use of BCAAs as integration of a normal dietary plan, is likely to be a validchoice for the same benefit found in human clinical nutrition, although this aspect is still debated.Indeed, long-term oral supplementation with BCAAs in the prevention of liver fibrosis and injury in thedog's liver is still unclear. Aim of the present study will be to determine how BCAAs preserve liverfunctions in vitro. To this purpose we have selected and set up three different in vitro models: hepaticdog cells and canine hepatocellular carcinoma cells plated in 2D monolayer and hepatic dog cellscultured onto 3D scaffolds, obtained from decellularized rabbit liver. All cells adhered and proliferatedonce plated. Cells grown in monolayer quickly entered G0 end arrested growth, ELISA test confirmedtheir ability to produce albumin. Cells grown on scaffold vigorously replicated and showed theircapability to recellularize ECM rabbit liver. These results, although preliminary, demonstrate that theculture conditions used well preserved the original phenotype and function and further support thepossibility to use in vitro models to successfully study BCAA efficacy in dog

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection

Isolated slaughterhouse liver as model for normothermic perfusion after warm and cold ischemia: single case report

AbstractLiver transplantation is an ultimate procedure in patients suffering end-stage liver diseases. In these last years the donation after cardiac death (DCD) has increased the pool of potential liver donors. Different studies and procedures are involved in the prevention of the main ischemic problems during the reconditioning and resuscitation of the marginal livers. Normothermic extracorporeal liver perfusion (NELP) avoids prolonged cold storage damage that is the main cause of steatosis and biliary tract ischemia in transplanted patiens. Different porcine models have been studied and developed to understand the ischemia mechanism and to select the better technique for NELP.We conducted our study using a DCD pig liver model collected from slaughterhouse. Using extracorporeal membrane oxygenation, 2000 ml of total fluid containing autologous blood, lidocaine, heparin, antibiotics, glucose 10 % solution and flunixin, the NELP was achieved. The liver was perfused over 7 hours after 48 hours of cold storage (4C°), using Eurocollins solution. During the liver withdrawal in the slaughterhouse 20 minutes were waited to simulate the warm ischemia (WI) time. Histological samples, swab for bacterial grow, blood sample, temperature and pulse oximetry saturation were collected to assess the liver viability and function. These analyses revealed stable metabolism throughout perfusion identifying a cycles 2 hours length, coinciding with recovery of oxygen uptake rates to fresh liver, as described in literature.In summary the preliminary established model of isolated hemoperfused slatherhouse liver reveals the important role of the relation between cold storage and normothermic perfusion. Moreover this preliminary study justifies further investigation of the optimization of the treatment protocols and perfusion media