Testing Hypoxia in Pig Meniscal Culture: Biological Role of the Vascular-Related Factors in the Differentiation and Viability of Neonatal Meniscus.

Menisci play an essential role in shock absorption, joint stability, load resistance and its transmission thanks to their conformation. Adult menisci can be divided in three zones based on the vascularization: an avascular inner zone with no blood supply, a fully vascularized outer zone, and an intermediate zone. This organization, in addition to the incomplete knowledge about meniscal biology, composition, and gene expression, makes meniscal regeneration still one of the major challenges both in orthopedics and in tissue engineering. To overcome this issue, we aimed to investigate the role of hypoxia in the differentiation of the three anatomical areas of newborn piglet menisci (anterior horn (A), central body (C), and posterior horn (P)) and its effects on vascular factors. After sample collection, menisci were divided in A, C, P, and they were cultured in vitro under hypoxic (1% O2) and normoxic (21% O2) conditions at four different experimental time points (T0 = day of explant; T7 = day 7; T10 = day 10; T14 = day 14); samples were then evaluated through immune, histological, and molecular analyses, cell morpho-functional characteristics; with particular focus on matrix composition and expression of vascular factors. It was observed that hypoxia retained the initial phenotype of cells and induced extracellular matrix production resembling a mature tissue. Hypoxia also modulated the expression of angiogenic factors, especially in the early phase of the study. Thus, we observed that hypoxia contributes to the fibro-chondrogenic differentiation with the involvement of angiogenic factors, especially in the posterior horn, which corresponds to the predominant weight-bearing portion.

How different rearing temperatures affect growth and stress status of Siberian sturgeon Acipenser baerii larvae.

Environmental temperature is one of the critical factors affecting fish development. The aim of this study was to examine the impact of three different rearing temperatures (16, 19 and 22°C) throughout the endogenous feeding phase of the Siberian sturgeon Acipenser baerii. This was performed by assessing (a) larval survival and growth; (b) immunofluorescence localization and expression of genes involved in muscle development and growth - myog and Igf1; and (c) stress status through the expression of thermal stress genes - Hsp70, Hsp90α and Hsp90ß - and whole body cortisol. Overall survival rate and larval weight did not differ significantly across temperatures. Larvae subjected to 22°C showed faster absorption of the yolk-sac than larvae subjected to 19 or 16°C. Both at schooling and at the end of the trial, larvae reared at 16°C showed significantly lower levels of cortisol than those reared at 19 or 22°C. IGF-1 immunopositivity was particularly evident in red muscle at schooling stage in all temperatures. The expression of all Hsps as well as the myog and Igf1 genes was statistically higher in larvae reared at 16°C but limited to the schooling stage. Cortisol levels were higher in larvae at 22°C, probably because of the higher metabolism demand rather than a stress response. The observed apparent incongruity between Hsps gene expression and cortisol levels could be due to the lack of a mature system. Further studies are necessary, especially regarding the exogenous feeding phase, in order to better understand if this species is actually sensitive to thermal stress.

Dietary supplementation with a blend composed of carvacrol, tannic acid derived from Castanea sativa and Glycyrrhiza glabra, and glycerides of medium chain fatty acids for weanling piglets raised in commercial farm.

This study aimed to evaluate the dietary administration of a blend composed of carvacrol, tannic acid derived from Castanea sativa mill and Glycyrrhiza glabra, medium chain fatty acids (MCFAs) glycerides for weanling piglets. An in vitro digestion followed by total phenolic content (TPC) and total antioxidant activity (TAC) assessment was performed before the in vivo application. At weaning, a total of 210 piglets were randomly allocated to two experimental treatments (7 replicates/15 piglets for each replicate). Control group (CTR) was fed a standard basal diet while the treated group (T) was fed the basal diet mixed with 1.500 mg/kg of blend. After in vitro digestion, TPC and TAC evidenced peaks at the end of oral and gastric phases in comparison to the intestinal one in line with the high content of phenolic compound (P < 0.05). Treatment conditioned body weight and average daily gain (P < 0.05), fecal score on 6, 7, and 8 d after weaning (P < 0.05). At 35d, the T group showed a decrease in salivary cortisol compared to CTR (P < 0.05). Duodenum and jejunum sections of T piglets revealed higher villi (P < 0.05), deeper crypts (P < 0.01), and increased V/C ratio (P < 0.01). CTR showed a higher expression of duodenal Occludin (P < 0.05). Jejunal E-cadherin and Occludin were more expressed in T jejunum sections (P < 0.05). Twelve differentially abundant genera were identified in T group caecal samples. Potentially harmful Clostridium sensu stricto 13 was reduced by the treatment (P < 0.05). In conclusion, the tested blend positively affected salivary stress markers and the gut health of weaned piglets.

Bovine Colostrum Supplementation in Rabbit Diet Modulates Gene Expression of Cytokines, Gut-Vascular Barrier, and Red-Ox-Related Molecules in the Gut Wall.

Rabbits, pivotal in the EU as livestock, pets, and experimental animals, face bacterial infection challenges, prompting a quest for alternatives to curb antibiotic resistance. Bovine colostrum (BC), rich in immunoregulatory compounds, antimicrobial peptides, and growth factors, is explored for disease treatment and prevention. This study assesses BC diet supplementation effects on rabbit intestines, examining gene expression. Thirty female New Zealand White rabbits at weaning (35 days) were divided into three experimental groups: control (commercial feed), 2.5% BC, and 5% BC. The diets were administered until slaughtering (81 days). BC-upregulated genes in the jejunum included IL-8, TGF-ß, and CTNN-ß1 at 5% BC, while PLVAP at 2.5% BC. Antioxidant-related genes (SOD1, GSR) were downregulated in the cecum and colon with 2.5% BC. BC 5% promoted IL-8 in the jejunum, fostering inflammation and immune cell migration. It also induced genes regulating inflammatory responses (TGF-ß) and gastrointestinal permeability (CTNN-ß1). BC 5% enhanced antioxidant activity in the cecum and colon, but no significant impact on anti-myxo antibody production was observed. These results suggest that BC has significant effects on the rabbit gastrointestinal tract's inflammatory and antioxidant response, but further research is required to fully understand its histological and physiological impact.

Endocrine-Disrupting Chemicals and Their Effects in Pet Dogs and Cats: An Overview.

Over the past few decades, several pollutants classified as environmental endocrine-disrupting chemicals (EDCs) have become a matter of significant public health concern. Companion animals play a major role in human society, and pet ownership is substantially increasing worldwide. These intimate human-pet relationships imply sharing much of the same environment, thus including exposure to similar levels of EDCs in daily routine. Here, we review the current knowledge on the sources and routes of exposure to EDCs in domestic indoor and outdoor environments and discuss whether endocrine disruption is a health concern in pets. We summarize the phenomenon of endocrine disruption, providing examples of EDCs with a known impact on dog and cat health. Then, we propose an overview of the literature on the adverse effects of EDCs in domestic pets, with a special focus on the health of reproductive and thyroid systems. Finally, we explore the potential role of companion animals as unintentional sentinels of environmental exposure to EDCs and the implications for public health risk assessment in a "shared risk" scenario. Overall, this review supports the need for an integrated approach considering humans, animals, and the environment as a whole for a comprehensive assessment of the impact of EDCs on human and animal health.

How Do Alternative Protein Resources Affect the Intestine Morphology and Microbiota of Atlantic Salmon?

The availability and cost of fishmeal constitute a bottleneck in Atlantic salmon production expansion. Fishmeal is produced from wild fish species and constitutes the major feed ingredient in carnivorous species such as the Atlantic salmon. These natural stocks are at risk of depletion and it is therefore of major importance to find alternative protein sources that meet the nutritional requirements of the Atlantic salmon, without compromising the animals' health. Terrestrial animal by-products have been used in aquaculture feed, but their use is limited by the lack of several essential amino acids and consumer acceptance. In the case of plant ingredients, it is necessary to take into account both their concentration and the extraction methodologies, since, if not dosed correctly, they can cause macro- and microscopic alterations of the structure of the gastrointestinal tract and can also negatively modulate the microbiota composition. These alterations may compromise the digestive functions, growth of the animal, and, ultimately, its well-being. An updated revision of alternative protein sources is provided, with the respective impact on the intestine health in terms of both morphology and microbiota composition. Such information may constitute the premise for the choice and development of Atlantic salmon feeds that guarantee fish health and growth performance without having a significant impact on the surrounding environment, both in terms of depletion of the fish's natural stocks and in terms of pressure on the terrestrial agriculture. The sustainability of aquaculture should be a priority when choosing next-generation ingredients.

Postnatal morpho-functional development of a dog's meniscus.

This study evaluates the morpho-functional modifications that characterize meniscal development from neonatal to adult dogs. Even if menisci are recognized as essential structures for the knee joint, poor information is available about their morphogenesis, in particular in dog models. Menisci from a group of Dobermann Pinchers aged 0, 10, 30 days, and 4 years (T0, T10, T30, adult, respectively) were analyzed by SEM, histochemistry (Safranin O and Picro Sirius Red Staining analyzed under a polarized light microscope), immunofluorescences (collagen type I and II), biomechanical (compression) and biochemical analyses (glycosaminoglycans, GAGs, and DNA content). SEM analyses revealed that the T0 meniscus is a bulgy structure that during growth tends to flatten, firstly in the inner zone (T10) and then even in the outer zone (T30), until the achievement of the completely smooth adult final shape. These results were further supported by the histochemistry analyses in which the deposition of GAGs started from T30, and the presence of type I birefringent collagen fibers was observed from T0 to T30, while poorly refringent type III collagen fibers were observed in the adult dogs. Double immunofluorescence analyses also evidenced that the neonatal meniscus contains mainly type I collagen fibers, as well as the T10 meniscus, and demonstrated a more evident regionalization and crimping in the T30 and adult meniscus. Young's elastic modulus of the meniscus in T0 and T10 animals was lower than the T30 animals, and this last group was also lower than adult ones (T0-T10 vs T30 vs adult). Biochemical analysis confirmed that cellularity decreases over time from neonatal to adult (p < 0.01). The same decreasing trend was observed in GAGs deposition. These results may suggest that the postnatal development of canine meniscus may be related to the progressive functional locomotory development: after birth, the meniscus acquires its functionality over time, through movement, load, and growth itself.

Intestine Health and Barrier Function in Fattening Rabbits Fed Bovine Colostrum.

The permeability of the immature intestine is higher in newborns than in adults; a damaged gut barrier in young animals increases the susceptibility to digestive and infectious diseases later in life. It is therefore of major importance to avoid impairment of the intestinal barrier, specifically in a delicate phase of development, such as weaning. This study aimed to evaluate the effects of bovine colostrum supplementation on the intestinal barrier, such as the intestinal morphology and proliferation level and tight junctions expression (zonulin) and enteric nervous system (ENS) inflammation status (through the expression of PGP9.5 and GFAP) in fattening rabbits. Rabbits of 35 days of age were randomly divided into three groups (n = 13) based on the dietary administration: commercial feed (control group, CTR) and commercial feed supplemented with 2.5% and 5% bovine colostrum (BC1 and BC2 groups, respectively). Rabbits receiving the BC1 diet showed a tendency to have better duodenum morphology and higher proliferation rates (p < 0.001) than the control group. An evaluation of the zonulin expression showed that it was higher in the BC2 group, suggesting increased permeability, which was partially confirmed by the expression of GFAP. Our results suggest that adding 2.5% BC into the diet could be a good compromise between intestinal morphology and permeability, since rabbits fed the highest inclusion level of BC showed signs of higher intestinal permeability.

Rearing Environment during the Endogenous Feeding Stage of Acipenser baerii.

The aim of this study was to evaluate behaviour, growth, lipid composition, muscle development, and stress status of Siberian sturgeon larvae reared with two types of substrate: Bioballs1 (BB1) and Bioballs2 (BB2), when compared to no substrate (CTR). Sampling points were: hatching (T0), schooling (T1), and yolk-sac full absorption (T2). BB1 larvae were less active and showed no schooling behaviour. At T1 and at T2, BB1 larvae showed a significantly higher weight and total length than larvae reared in either CTR or BB2 (p < 0.05). The lipid content of larvae decreased over time, with little relevant differences between groups. At T2, total muscle area, slow muscle area and fast muscle area were significantly higher in larvae reared in BB1 (p < 0.05). No significant differences in muscle proliferation were found between groups. Real Time PCR was used for evaluating the relative expression of a pool of genes: myod, myog, mrf4, igf2, hsp70, hsp90a, hsp90b, and glut2. The expression of these genes did not seem to be much affected by the type of rearing substrate, except for myog and hsp70 at T1, which was greater in BB2 larvae. Our data suggest that the presence of a substrate during this developmental period seems to have positive effects but further studies would be necessary during the exogenous feeding stage.

Meniscus Matrix Structural and Biomechanical Evaluation: Age-Dependent Properties in a Swine Model.

The analysis of the morphological, structural, biochemical, and mechanical changes of the Extracellular Matrix (ECM), which occur during meniscus development, represents the goal of the present study. Medial fully developed menisci (FD, 9-month-old pigs), partially developed menisci (PD, 1-month-old piglets), and not developed menisci (ND, from stillbirths) were collected. Cellularity and glycosaminoglycans (GAGs) deposition were evaluated by ELISA, while Collagen 1 and aggrecan were investigated by immunohistochemistry and Western blot analyses in order to be compared to the biomechanical properties of traction and compression tensile forces, respectively. Cellularity decreased from ND to FD and GAGs showed the opposite trend (p < 0.01 both). Collagen 1 decreased from ND to FD, as well as the ability to resist to tensile traction forces (p < 0.01), while aggrecan showed the opposite trend, in accordance with the biomechanics: compression test showed that FD meniscus greatly resists to deformation (p < 0.01). This study demonstrated that in swine meniscus, clear morphological and biomechanical changes follow the meniscal maturation and specialization during growth, starting with an immature pattern (ND) to the mature organized meniscus of the FD, and they could be useful to understand the behavior of this structure in the light of its tissue bioengineering.

Endostatin in 3D Fibrin Hydrogel Scaffolds Promotes Chondrogenic Differentiation in Swine Neonatal Meniscal Cells.

The success of cell-based approaches for the treatment of cartilage or fibro-cartilaginous tissue defects requires an optimal cell source with chondrogenic differentiation ability that maintains its differentiated properties and stability following implantation. For this purpose, the aim of this study was to evaluate the use of endostatin (COL18A1), an anti-angiogenic factor, which is physiologically involved in cell differentiation during meniscus development. Swine neonatal meniscal cells not yet subjected to mechanical stimuli were extracted, cultured in fibrin hydrogel scaffolds, and treated at two different time points (T1 = 9 days and T2 = 21 days) with different concentrations of COL18A1 (10 ng/mL; 100 ng/mL; 200 ng/mL). At the end of the treatments, the scaffolds were examined through biochemical, molecular, and histochemical analyses. The results showed that the higher concentration of COL18A1 promotes a fibro-chondrogenic phenotype and improves cellularity index (DNA content, p < 0.001) and cell efficiency (GAGs/DNA ratio, p < 0.01) after 21 days. These data are supported by the molecular analysis of collagen type I (COL1A1, a marker of fibrous-like tissue, p < 0.001), collagen type II (COL2A1, a marker of cartilaginous-like tissue, p < 0.001) and SRY-Box Transcription Factor 9 (SOX9, an early marker of chondrogenicity, p < 0.001), as well as by histological analysis (Safranin-O staining), laying the foundations for future studies evaluating the involvement of 3D endostatin hydrogel scaffolds in the differentiation of avascular tissues.

Stages of Gut Development as a Useful Tool to Prevent Gut Alterations in Piglets.

During the prenatal, neonatal, and weaning periods, the porcine gastrointestinal tract undergoes several morpho-functional, changes together with substantial modification of the microbial ecosystem. Modifications of the overall structure of the small intestine also occur, as well as a rapid increase of the volume, mainly in the last period of gestation: intestinal villi, starting from jejunum, appears shortly before the sixth week of gestation, and towards the end of the third month, epithelial cells diversify into enterocytes, goblet cells, endocrine, and Paneth cells. Moreover, in the neonatal period, colostrum induces an increase in intestinal weight, absorptive area, and brush border enzyme activities: intestine doubles its weight and increases the length by 30% within three days of birth. During weaning, intestinal environment modifies drastically due to a replacement of highly digestible sow milk by solid feed: profound changes in histological parameters and enzymatic activity are associated with the weaning period, such as the atrophy of the villi and consequent restorative hypertrophy of the crypts. All these modifications are the result of a delicate and precise balance between the proliferation and the death of the cells that form the intestinal mucosa (i.e., mitosis and apoptosis) and the health conditions of the piglet. An in-depth knowledge of these phenomena and of how they can interfere with the correct intestinal function can represent a valid support to predict strategies to improve gut health in the long-term and to prevent weaning gut alterations; thus, reducing antimicrobial use.

How Different Stocking Densities Affect Growth and Stress Status of Acipenser baerii Early Stage Larvae.

In the present study, a multidisciplinary approach was used in order to evaluate growth, muscle development, and stress status in Siberian sturgeon Acipenser baerii larvae at schooling (T1) and complete yolk sac absorption (T2), reared at three stocking densities (low, medium, and high). Larvae growth, morphological muscle development, and whole-body cortisol levels were assessed. The expression of genes involved in the growth process (igf1 and igf2), in the myogenesis (myog), and in the regulation of cellular stress (glut1, glut2, glut4, and hsp70) was analyzed using a quantitative PCR. Larvae reared at lower densities showed a higher Specific Growth Rate and showed a physiological muscle development. Cortisol levels were low and did not differ significantly, both in different time sampling and across densities, suggesting that either the considered densities are not stressors in this species in the early stages of development or the hypothalamus-pituitary-adrenal (HPA) axis is not yet fully mature. Gene expression of glut1, igf1, and igf2 showed an up-regulation in both developmental stages at all the rearing densities considered, while myog significantly up-regulated at T1 at the highest density. Considering all of the results, it would seem that lower densities should be used in these stages of development, as these showed a higher growth rate, even if it is not economically feasible in commercial hatcheries. Therefore, choosing an intermediate stocking density could be a good compromise between larval performance and economical feasibility.

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.

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.