Dietary Mannan Oligosaccharides Modulate Gut Inflammatory Response and Improve Duodenal Villi Height in Post-Weaning Piglets Improving Feed Efficiency.

The aim of this study was to evaluate the effects of mannan oligosaccharides (MOS) on gut health and performance in post-weaning piglets. In total, 40 piglets were divided into two experimental groups and fed a basal diet with (TRT) or without (CON) 0.2% mannan oligosaccharides for 35 days. Growth performance was determined weekly and faecal microbial composition on days 0, 14 and 35. On day 36, histometrical evaluations were performed on duodenal, jejunal, ileal, and colon samples. mRNA gene expression of inflammation-related genes was evaluated in samples of ileal Peyer's patches (IPP). MOS administration improved feed efficiency in the last two weeks of the trial (p < 0.05), and a decreased clostridia content was found in faeces at day 14 (p = 0.05). TRT piglets showed increased duodenal villi height (p < 0.05), and reduced mRNA levels of Tumour Necrosis Factor α (p < 0.05) and Toll-Like Receptor 4 (p < 0.01) in IPP. Our results suggest beneficial effects of MOS supplementation on gut morphology and the expression of inflammation-related genes in post-weaning piglets, accompanied by increased feed efficiency.

Development and biological validation of a cyclic stretch culture system for the ex vivo engineering of tendons.

INTRODUCTION: An innovative approach to the treatment of tendon injury or degeneration is given by engineered grafts, made available through the development of bioreactors that generate tendon tissue in vitro, by replicating in vivo conditions. This work aims at the design of a bioreactor capable of applying a stimulation of cyclic strain on cell constructs to promote the production of bioartificial tissue with mechanical and biochemical properties resembling those of the native tissue. METHODS: The system was actuated by an electromagnet and design specifications were imposed as follows. The stimulation protocol provides to scaffolds a 3% preload, a 10% deformation, and a stimulation frequency rate set at 0.5, 1, and 2 Hz, which alternates stimulation/resting phases. Porcine tenocytes were seeded on collagen scaffolds and cultured in static or dynamic conditions for 7 and 14 days. RESULTS: The culture medium temperature did not exceed 37°C during prolonged culture experiments. The applied force oscillates between 1.5 and 4.5 N. The cyclic stimulation of the engineered constructs let both the cells and the scaffold fibers align along the strain direction in response to the mechanical stimulus. CONCLUSION: We designed a pulsatile strain bioreactor for tendon tissue engineering. The in vitro characterization shows a preferential cell alignment at short time points. Prolonged culture time, however, seems to influence negatively on the survival of the cells indicating the need of further optimization concerning the culture conditions and the mechanical stimulation.

Copper sulphate forms in piglet diets: Microbiota, intestinal morphology and enteric nervous system glial cells.

The objective of this study was to evaluate dietary supplementation with different copper sulphate (CuSO4 ) forms on small intestine microanatomy and large intestine microbiota. Ninety weaned piglets were divided into three experimental groups: control diet (CTR), with no added CuSO4 and diets supplemented with 150 ppm of CuSO4 in protected (150P) and unprotected form (150UP). After 18 days of dietary treatment, six piglets per treatment were randomly selected and sacrificed. Duodenum villi length and crypt depths were higher (P < 0.001) in the animals fed 150UP than other groups. Glial fibrillary acidic protein (GFAP), a marker for enteric glial cells, was unaffected by dietary treatments. The total bacteria and Enterobacteriaceae bacteria counts were lower (P < 0.05) in cecum of animals fed 150P in comparison with the other two groups. In the colon the Streptococci spp were lower (P < 0.001) in both CuSO4 supplemented groups than controls. The obtained results revealed a modulation of intestinal structure and microbiota exerted by the studied CuSO4 dietary supplementation. The present data show that dietary supplementation with 150UP in the first period post-weaning may assist in restoring the gut morphology, improving duodenal structure.

Age-related modulation of angiogenesis-regulating factors in the swine meniscus.

An in-depth knowledge of the native meniscus morphology and biomechanics in its different areas is essential to develop an engineered tissue. Meniscus is characterized by a great regional variation in extracellular matrix components and in vascularization. Then, the aim of this work was to characterize the expression of factors involved in angiogenesis in different areas during meniscus maturation in pigs. The menisci were removed from the knee joints of neonatal, young and adult pigs, and they were divided into the inner, intermediate and outer areas. Vascular characterization and meniscal maturation were evaluated by immunohistochemistry and Western blot analysis. In particular, expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and the anti-angiogenic marker Endostatin (ENDO) was analysed, as well as the vascular endothelial cadherin (Ve-CAD). In addition, expression of Collagen II (COLL II) and SOX9 was examined, as markers of the fibro-cartilaginous differentiation. Expression of VEGF and Ve-CAD had a similar pattern in all animals, with a significant increase from the inner to the outer part of the meniscus. Pooling the zones, expression of both proteins was significantly higher in the neonatal meniscus than in young and adult menisci. Conversely, the young meniscus revealed a significantly higher expression of ENDO compared to the neonatal and adult ones. Analysis of tissue maturation markers showed an increase in COLL II and a decrease in SOX9 expression with age. These preliminary data highlight some of the changes that occur in the swine meniscus during growth, in particular the ensemble of regulatory factors involved in angiogenesis.

Neuroprotective effects of low fat-protein diet in the P301L mouse model of tauopathy.

Tauopathies are a class of neurodegenerative diseases associated with the pathological aggregation of tau protein in the human brain. Although numerous studies in mouse models of Alzheimer disease (AD) have shown a correlation among diet, beta-amyloid and AD onset, little is known about the impact of diet on Tau. We investigated whether a low fat-protein diet (LFPD) may improve lifespan, cognitive and locomotor activity in P301L-tg mouse model of tauopathy. Our data indicate that LFPD has a beneficial effect on these parameters. Tg mice fed with standard diet shown a decrease in body weight, food intake and survival rate if compared to wild type animals. In contrast, LFPD counteracted weight loss, increased mortality and ameliorated cognitive and locomotor performances in tg mice. LFPD also reduced the abnormal accumulation of agglomerates of P-Tau (pathological features of tauopathies) and the expression of apoptotic markers (i.e., TUNEL immunopositive neurons) in the prefrontal cerebral cortex and hippocampus of P301L-tg mice. Interestingly, some of these effects are sex-dependent. For instance, tg females, but not males, fed with LFPD had a significant increase of body weight and a reduction of P-Tau agglomerates compared to tg fed with standard diet. These changes correlated with a more pronounced improvement of cognition and locomotor activity in females than in male tg fed with LFPD. Altogether, these results suggest a sex dependent neuroprotective effect of LFPD in P301L-tg mice, suggesting that lifestyle intervention strategies may be clinically relevant for delaying the onset of cognitive impairment and dementia, especially in females.

Sex Impact on Tau-Aggregation and Postsynaptic Protein Levels in the P301L Mouse Model of Tauopathy.

P301L transgenic (tg) mice well mimic features of human tauopathies and provide a good model for investigating the role of tau in neurodegenerative events. We here analyzed the possible interactions among phosphorylation of tau (p-tau), spine injury, neuronal death, and sex in the P301L mouse model of tauopathy. When compared to control mice (ctr), P301L transgenic mice (tg) presented a lower body weight, reduced survival rate, hyperphosphorylated tau, spine injury, and neuronal loss in both cerebral cortex and hippocampus at 15 months of age. Importantly, we found that pathological features were more pronounced in female than male tg mice. Recent reports underline that tau may be localized within both pre- and post-synaptic compartments, suggesting that it may possibly induce or contribute to synaptic dysfunction. Therefore, we focused our attention on tau localization at dendritic spines. We detected high levels of both tau and p-tau in dendritic spine of P301L transgenic mice. In addition, p-tau correlated with a significant reduction of post-synaptic markers, such as GluN2A, GluN2B, GluA1, GluA2, Drebrin, and PSD-95, in P301L mice. The p-tau levels are higher in female than in male mice, and the increased p-tau was consistent with a proportional decrease in the post-synaptic marker levels analyzed. The P301L-tg females showed a more severe synaptopathy compared to males. Future investigations on the postsynaptic role of p-tau will be necessary to understand its toxic effects and provide insights into new therapeutic targets for maintaining spine integrity, highlighting the importance of tau toxicity as well as the impact of sex on tau-pathology.

Effects of different rearing temperatures on muscle development and stress response in the early larval stages of Acipenser baerii.

The present study aims at investigating muscle development and stress response in early stages of Siberian sturgeon when subjected to different rearing temperatures, by analysing growth and development of the muscle and by assessing the stress response of yolk-sac larvae. Siberian sturgeon larvae were reared at 16°C, 19°C and 22°C until the yolk-sac was completely absorbed. Sampling timepoints were: hatching, schooling and complete yolk-sac absorption stage. Histometrical, histochemical and immunohistochemical analyses were performed in order to characterize muscle growth (total muscle area, TMA; slow muscle area, SMA; fast muscle area, FMA), development (anti-proliferating cell nuclear antigen -PCNA or anticaspase) as well as stress conditions by specific stress biomarkers (heat shock protein 70 or 90, HSP70 or HSP90). Larvae subjected to the highest water temperature showed a faster yolk-sac absorption. Histometry revealed that both TMA and FMA were larger in the schooling stage at 19°C while no differences were observed in the SMA at any of the tested rearing temperatures. PCNA quantification revealed a significantly higher number of proliferating cells in the yolk-sac absorption phase at 22°C than at 16°C. HSP90 immunopositivity seems to be particularly evident at 19°C. HPS70 immunopositivity was never observed in the developing lateral muscle.

The taphonomy of blood components in decomposing bone and its relevance to physical anthropology.

OBJECTIVES: The variation and persistence of blood components, in particular red blood cells (RBCs), within bone tissue during the decomposition process, especially at the early stages and in different taphonomic conditions, has never been thoroughly investigated, regardless of the fact that knowing how blood survives or degrades within bone could be of help in solving many anthropological issues, such as trauma analysis and interpretation. MATERIALS AND METHODS: This research investigated the influence of time and taphonomy on the persistence and detectability of blood components in parietal bone fragments (of different post mortem periods and taphonomic conditions) through histological (Hematoxilin and Eosin, HE) and immunohistochemical (Glycophorin A, GYPA) analyses. RESULTS: The immunohistochemical investigation for GYPA showed the presence of RBCs under the form of erythrocyte debris or residues otherwise morphologically unidentifiable using only HE staining. Hence, while well-defined RBCs can be observed only in the first week of decomposition, afterward these structures can be detectable with certainty only by immunohistochemical analysis, which reveals discrete quantities of RBC residues also in dry bone (post mortem interval, or PMI, of 15 years), but not in archaeological samples, in which the greater PMI and the different taphonomic conditions together could be the answer behind such difference. DISCUSSION: This study highlights the usefulness and potential of immunohistochemical detection of GYPA in RBC investigation and gives a realistic idea of the persistence and detectability of erythrocytes in different osteological taphonomic conditions, in contrast to results reported by some authors in literature. Another important result concerns the detection of RBC residues in dry bone, which opens the way to the possible use of RBCs in trauma interpretation.

Repair of osteochondral defects in the minipig model by OPF hydrogel loaded with adipose-derived mesenchymal stem cells.

AIM: Critical knee osteochondral defects in seven adult minipigs were treated with oligo(polyethylene glycol)fumarate (OPF) hydrogel combined with autologous or human adipose-derived stem cells (ASCs), and evaluated after 6 months. METHODS: Four defects were made on the peripheral part of right trochleas (n = 28), and treated with OPF scaffold alone or pre-seeded with ASCs. RESULTS: A better quality cartilage tissue characterized by improved biomechanical properties and higher collagen type II expression was observed in the defects treated by autologous or human ASC-loaded OPF; similarly this approach induced the regeneration of more mature bone with upregulation of collagen type I expression. CONCLUSION: This study provides the evidence that both porcine and human adipose-derived stem cells associated to OPF hydrogel allow improving osteochondral defect regeneration in a minipig model.

Osteochondral repair by a novel interconnecting collagen-hydroxyapatite substitute: a large-animal study.

A novel three-dimensional bicomponent substitute made of collagen type I and hydroxyapatite was tested for the repair of osteochondral lesions in a swine model. This scaffold was assembled by a newly developed method that guarantees the strict integration between the organic and the inorganic parts, mimicking the biological tissue between the chondral and the osseous phase. Thirty-six osteochondral lesions were created in the trochlea of six pigs; in each pig, two lesions were treated with scaffolds seeded with autologous chondrocytes (cell+group), two lesions were treated with unseeded scaffolds (cell- group), and the two remaining lesions were left untreated (untreated group). After 3 months, the animals were sacrificed and the newly formed tissue was analyzed to evaluate the degree of maturation. The International Cartilage Repair Society (ICRS) macroscopic assessment showed significantly higher scores in the cell- and untreated groups when compared with the cell+ group. Histological evaluation showed the presence of repaired tissue, with fibroblast-like and hyaline-like areas in all groups; however, with respect to the other groups, the cell- group showed significantly higher values in the ICRS II histological scores for "cell morphology" and for the "surface/superficial assessment." While the scaffold seeded with autologous chondrocytes promoted the formation of a reparative tissue with high cellularity but low glycosaminoglycans (GAG) production, on the contrary, the reparative tissue observed with the unseeded scaffold presented lower cellularity but higher and uniform GAG distribution. Finally, in the lesions treated with scaffolds, the immunohistochemical analysis showed the presence of collagen type II in the peripheral part of the defect, indicating tissue maturation due to the migration of local cells from the surroundings. This study showed that the novel osteochondral scaffold was easy to handle for surgical implantation and was stable within the site of lesion; at the end of the experimental time, all implants were well integrated with the surrounding tissue and no signs of synovitis were observed. The quality of the reparative tissue seemed to be superior for the lesions treated with the unseeded scaffolds, indicating the promising potential of this novel biomaterial for use in a one-stage procedure for osteochondral repair.

Meniscus maturation in the swine model: changes occurring along with anterior to posterior and medial to lateral aspect during growth.

The meniscus plays important roles in knee function and mechanics and is characterized by a heterogeneous matrix composition. The changes in meniscus vascularization observed during growth suggest that the tissue-specific composition may be the result of a maturation process. This study has the aim to characterize the structural and biochemical variations that occur in the swine meniscus with age. To this purpose, menisci were collected from young and adult pigs and divided into different zones. In study 1, both lateral and medial menisci were divided into the anterior horn, the body and the posterior horn for the evaluation of glycosaminoglycans (GAGs), collagen 1 and 2 content. In study 2, the menisci were sectioned into the inner, the intermediate and the outer zones to determine the variations in the cell phenotype along with the inner-outer direction, through gene expression analysis. According to the results, the swine meniscus is characterized by an increasing enrichment in the cartilaginous component with age, with an increasing deposition in the anterior horn (GAGs and collagen 2; P < 0.01 both); moreover, this cartilaginous matrix strongly increases in the inner avascular and intermediate zone, as a consequence of a specific differentiation of meniscal cells towards a cartilaginous phenotype (collagen 2, P < 0.01). The obtained data add new information on the changes that accompany meniscus maturation, suggesting a specific response of meniscal cells to the regional mechanical stimuli in the knee joint.

Collagen scaffold for cartilage tissue engineering: the benefit of fibrin glue and the proper culture time in an infant cartilage model.

This study (i) developed a scaffold made of collagen I designed for hosting the autologous chondrocytes, (ii) focused on the optimization of chondrocytes seeding by the addition of the fibrin glue, and (iii) investigated the culture time for the ideal scaffold maturation in vitro. In the first part of the study, fresh chondrocytes were isolated from infant swine articular cartilage, and immediately seeded onto the collagen sponges either in medium or in fibrinogen in order to show the contribute of fibrin glue in cell seeding and survival into the scaffold. In the second part of the study, chondrocytes were first expanded in vitro and then resuspended in fibrinogen, seeded in collagen sponges, and cultured for 1, 3, and 5 weeks in order to identify the optimal time for the rescue of cell phenotype and for the scaffold maturation into a tissue with chondral properties. The histological and immunohistochemical data from the first part of the study (study with primary chondrocytes) demonstrated that the presence of fibrin glue ameliorated cell distribution and survival into the chondral composites. The second part of this work (study with dedifferentiated chondrocytes) showed that the prolongation of the culture to 3 weeks promoted a significant restoration of the cell phenotype, resulting in a composite with proper morphological features, biochemical composition, and mechanical integrity. In conclusion, this study developed a collagenic-fibrin glue scaffold that was able to support chondrocyte survival and synthetic activity in a static culture; in particular, this model was able to turn the engineered samples into a tissue with chondral-like properties when cultured in vitro for at least 3 weeks.

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.

Adipose-derived stem cells and rabbit bone regeneration: histomorphometric, immunohistochemical and mechanical characterization.

BACKGROUND: In the last few years, several attempts have been made to treat large bone loss, including the use of tissue engineering with osteoinductive scaffolds and cells. This study highlights the role of mesenchymal stem cells from adipose tissue (ASCs; adipose-derived stem cells) in a rabbit bone regeneration model. METHODS: We compared the neoformed bone tissues achieved by treating critical tibial defects with either hydroxyapatite alone (HA, group I) or hydroxyapatite-autologous ASC constructs (ASCs-HA, group II), investigating their histomorphometric, immunohistochemical and biomechanical properties. RESULTS: After eight weeks of follow-up, we observed advanced maturation and a spatial distribution of new bone that was more homogeneous in the inner parts of the pores in group II, not just along the walls (as seen in group I). The new tissue expressed osteogenic markers, and biomechanical tests suggested that the newly formed bone in group II had a higher mineral content than that in group I. Although variability in differentiation was observed among the different cell populations in vitro, no differences in bone healing were observed in vivo; the variability seen in vitro was probably due to local microenvironment effects. CONCLUSIONS: Tibial defects treated with rabbit ASCs-HA showed an improved healing process when compared to the process that occurred when only the scaffold was used. We suggest that implanted ASCs ameliorate the bone reparative process either directly or by recruiting resident progenitor cells.

Burial of piglet carcasses in cement: a study of macroscopic and microscopic alterations on an animal model.

Scarce experimental data exist describing postmortem effects of burial in cement. The scanty literature presents several case reports, but no experimental study. To perform a pilot study, the following experimental system was designed: 4 piglet corpses, who died of natural causes, were encased in concrete. After 1, 2, 3, and 6 months, a block was opened, and autopsy and microscopic analyses were performed. At the first month, initial putrefaction had started, and hindlegs were partly skeletonized. At the second month, both forelegs and hindlegs were partly skeletonized, and the abdomen and back showed advanced putrefaction. At the third month, the samples showed areas of mummification at the abdomen within a general context of initial putrefaction. At the sixth month, the sample showed wide adipocere formation. Histological findings revealed in some analyzed tissues (epithelium, dermis, adipose, and subcutaneous muscular tissues) a well-defined histological pattern even at 3 months after encasement in concrete: this means that microscopic changes may be delayed in concrete and that it may be worth performing histological analyses even in such kind of decomposed material.

Changes in nitrosative stress biomarkers in swine intestine following dietary intervention with verbascoside.

In farm animals, oxidative stress can be involved in several intestinal pathological disorders, and many antioxidant molecules, especially those of plant origin, can counteract free radicals, thus stabilizing the gut environment and enhancing health. The aim of the study was to investigate whether the use of verbascoside (VB), a polyphenol plant compound, in pig feeding could modulate oxidative and/or nitrosative stress in the gut. Eighteen male piglets (Dalland) were assigned to two groups, which were fed with either a control diet (CON) or a diet supplemented with 5 mg/kg of verbascoside (VB) for 166 days. At slaughter, duodenum and jejunum specimens were collected. Immunohistochemistry and Western blot analyses were performed on the samples to evaluate free radical adducts, including acrolein (ACR), 8-hydroxydeoxyguanosine (8-OHdg) and nitrotyrosine (NT). A KRL test was also used to assess the total blood antioxidant activity, and no difference was observed. Immunohistochemistry and Western blot showed that dietary treatment decreased the levels of nitrotyrosine in enteroendocrine cell populations(P<0.05). Characterization of the enteroendocrine cell typology was then performed, and serotonin-immunoreactive cells were revealed to be directly involved in decreasing the nitrosative stress status. This preliminary study demonstrates the important role of dietary VB in decreasing stress biomarkers in swine gut, thus highlighting a possible intervention aimed at building a large prospective for antioxidant dietary supplementation in food animal species.

Is there adaptation of the exocrine pancreas in wild animal? The case of the Roe deer.

BACKGROUND: Physiology of the exocrine pancreas has been well studied in domestic and in laboratory animals as well as in humans. However, it remains quite unknown in wildlife mammals. Roe deer and cattle (including calf) belong to different families but have a common ancestor. This work aimed to evaluate in the Roe deer, the adaptation to diet of the exocrine pancreatic functions and regulations related to animal evolution and domestication. RESULTS: Forty bovine were distributed into 2 groups of animals either fed exclusively with a milk formula (monogastric) or fed a dry feed which allowed for rumen function to develop, they were slaughtered at 150 days of age. The 35 Roe deer were wild animals living in the temperate broadleaf and mixed forests, shot during the hunting season and classified in two groups adult and young. Immediately after death, the pancreas was removed for tissue sample collection and then analyzed. When expressed in relation to body weight, pancreas, pancreatic protein weights and enzyme activities measured were higher in Roe deer than in calf. The 1st original feature is that in Roe deer, the very high content in pancreatic enzymes seems to be related to specific digestive products observed (proline-rich proteins largely secreted in saliva) which bind tannins, reducing their deleterious effects on protein digestion. The high chymotrypsin and elastase II quantities could allow recycling of proline-rich proteins. In contrast, domestication and rearing cattle resulted in simplified diet with well digestible components. The 2nd feature is that in wild animal, both receptor subtypes of the CCK/gastrin family peptides were present in the pancreas as in calf, although CCK-2 receptor subtype was previously identified in higher mammals. CONCLUSIONS: Bovine species could have lost some digestive capabilities (no ingestion of great amounts of tannin-rich plants, capabilities to secrete high amounts of proline-rich proteins) compared with Roe deer species. CCK and gastrin could play an important role in the regulation of pancreatic secretion in Roe deer as in calf. This work, to the best of our knowledge is the first study which compared the Roe deer adaptation to diet with a domesticated animal largely studied.

Fibrin-based model for cartilage regeneration: tissue maturation from in vitro to in vivo.

One of the crucial points for a successful tissue-engineering approach for cartilage repair is represented by the level of in vitro maturation of the engineered tissue before implantation. The purpose of this work was to evaluate the effect of the level of in vitro maturation of engineered cartilaginous samples on the tissue quality after in vivo implantation. Samples were obtained from isolated swine articular chondrocytes embedded in fibrin glue. The cell-fibrin composites were either cultured in vitro or directly implanted in vivo for 1, 5, and 9 weeks. Other experimental samples were precultured for either 1 or 5 weeks in vitro and then implanted in vivo for 4 additional weeks. All the samples were analyzed by histology, immunohistochemistry, biochemistry, and gene expression. The results strongly suggest that the in vivo culture in this model promoted a better tissue maturation than that obtained in the in vitro condition, and that 1 week in vitro preculture resulted in the primary structuring of the engineered composites and their subsequent maturation in vivo, without affecting the cell viability and activity, while a prolonged in vitro preculture caused a cell and matrix degeneration that could not be rescued in vivo.

Distribution of ghrelin-producing cells in the gastrointestinal tract of pigs at different ages.

Ghrelin is involved in many biological processes, ranging from appetite regulation and the release of growth hormone to the regulation of gastrointestinal motility and secretion processes. Ghrelin expression is not homogenously distributed throughout the gastrointestinal tract; expression is species-specific and can also depend on the animal age. This study was performed to investigate ghrelin immunolocalization in the gastrointestinal tract of pigs at different ages: 1 day (birth), 28 days (weaning), 2 months, 4 months, and 7 months (pre-puberty). Tissue samples were collected along the entire gastrointestinal tract and were examined by immunohistochemistry and double-immunofluorescence. Histometry was performed by counting the number of endocrine ghrelin immunopositive cells in the gastrointestinal mucosa. Ghrelin was found to be present along the swine alimentary canal from the stomach to the caecum. In all regions of the alimentary canal of the animals studied, ghrelin-immunoreactive (IR) cells co-localized with chromogranin-A and were therefore identified as endocrine cells. In the gastric fundus, ghrelin-immunoreactivity was partially detected in co-localization with H-K-adenosine triphosphatase and pepsinogen. Ghrelin-IR endocrine cells were abundant in the oxyntic mucosa but less present in the small intestine and rare in the large intestine. The cell density of the ghrelin-IR endocrine cells was lowest in the oxyntic mucosa of 1-day-old pigs. We can conclude that gastric ghrelin expression is not related merely to age but could also potentially be influenced by food intake.

Diatom extraction with HCl from animal tissues: a technical note.

Identification of diatoms from human or animal tissue may be affected by factors concerning techniques of extraction and analysis which cause fragmentation or loss of diatoms. In this brief technical note a novel efficient method of extraction of diatoms from animal tissues is presented. The authors have tested an H(2)O(2) plus HCl and HCl method to detect diatoms in several tissues from pig immersed in water rich in diatoms; they have also compared this method to other methods of extraction including enzymatic ones. The experiment showed that all siliceous frustules of fresh water diatoms are resistant to the treatment proposed and are still recognizable after the digestion. Among the different tests, treatment by 20% HCl (T1) proved to be more simple and time-saving, and seems to be a valuable diagnostic tool for routine forensic procedures in the extraction and detection of diatoms. The positive results obtained in all cases should lead to further studies in order to evaluate the reliability of the proposed methods also from a quantitative point of view.