Suitability of dried olive pulp in slow-growing broilers: performance, meat quality, oxidation products, and intestinal mucosa features.

To assess the effect of dietary dried olive pulp (DOP) on growth performance, meat traits and oxidation, and intestinal mucosa features, a total of 180 male slow-growing broiler chickens (Hubbard) were divided into 3 groups and fed 3 isonitrogenous and isoenergetic diets from 14 d of age until slaughter (49 d). The treatments varied according to 3 DOP levels: a control diet without DOP (DOP0, 0%) and 2 test diets containing 5 and 10% of DOP (DOP5 and DOP10, respectively). Duodenal morphometric indices were measured at the end of the feeding period and included: villus height, crypt depth, villus-to-crypt ratio, and villus surface area. Dietary DOP had no adverse effect on growth performance, dressing percentage, or breast yield of broilers. The breast muscle pH at 24 h was significantly higher in birds fed DOP10 diet compared to those on DOP0 and DOP5 diets. Meat color was also affected by dietary treatments. Feeding DOP did not influence breast meat fatty acid composition, whereas meat from DOP-fed broilers resulted less susceptible to lipid and protein oxidation compared to control diet. Including DOP up to 10% in diet resulted in higher duodenal villus height, crypt depth, and villus height to crypt depth ratio as well as villus surface area. Based on our findings, dietary DOP supported productive traits of slow-growing broilers preserving meat from oxidation and improving intestinal morphometric features. As a result, the current study assessed that olive by-product can be used in broiler ration, resulting in a valuable ingredient as replacement for conventional feeds, which could reduce feeding costs due to the low cost of the olive by-product. Thus, using olive by-products as poultry feed may become economically feasible for producers where the olive oil industries play an important economic role.

Rearing in captivity affects spermatogenesis and sperm quality in greater amberjack, Seriola dumerili (Risso, 1810).

The greater amberjack, (Risso, 1810), is a promising candidate for the diversification of European aquaculture production, but inconsistent reproduction in captivity prevents commercial production. Recent studies showed that greater amberjack confined in sea cages exhibited scarce gonad development and early interruption of gametogenic activity during the reproductive season. The aim of the present study was to improve our understanding of the observed impairment of spermatogenesis. Adult wild and captive-reared males were sampled during 3 different phases of the reproductive cycle: early gametogenesis (EARLY; late April to early May), advanced gametogenesis (ADVANCED; late May to early June), and spawning (SPAWNING; late June to July). Spermatogonial stem cells and proliferating germ cells were identified through the immunohistochemical localization of and proliferating cell nuclear antigen, respectively. Apoptotic germ cells were identified throughout the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling method. Sperm quality of captive-reared fish was evaluated using computer-assisted sperm analysis. Captive-reared males exhibited seminiferous lobules of a smaller diameter, a precocious and progressive decrease of spermatogonial mitosis, and a high level of apoptosis at the beginning of the reproductive season, concomitant with a many-fold higher 17ß-estradiol plasma concentration. The motile spermatozoa percentage of captive greater amberjack was lower than in other teleosts, and a drastic decrease of spermatozoa motility duration, velocity, and ATP content occurred along the reproductive season. An abnormal increase of sperm concentration as well as an increase of dead spermatozoa occurred during the SPAWNING phase, probably because of lack of sperm hydration and ejaculation and consequent sperm ageing. The present study demonstrates the extreme susceptibility of greater amberjack to rearing stress and underscores the need for improvement of the rearing and handling procedures to ameliorate gametogenesis dysfunctions in commercial aquaculture production.

In vitro effect of isotocin on ovarian tunica albuginea contractility of gilthead seabream (Sparus aurata L.) in different reproductive conditions.

Contractions of ovarian tunica albuginea, the teleostean cystovary wall layer containing smooth muscle fibres, facilitate oocytes and fluids movements within the ovary, oocytes ovulation and spawning. Fish isotocin, the homologue hormone of mammalian oxytocin, plays a significant role in ovulation, oviduct contraction and spawning. In the present study, ovarian wall spontaneous contraction, as well as isotocin in vitro effect on tunica albuginea contractility, was analysed in female seabream in different reproductive conditions: vitellogenesis, regressing (post-spawning) and extensive atresia. Tunica albuginea spontaneous contractility was recorded using ovary wall strips mounted in an organ bath containing modified Ringer's solution. The strips were then exposed to cumulative doses of isotocin (6, 30, 60 µg/ml). Female seabream in regressing condition exhibited the highest level of tunica albuginea spontaneous contraction amplitude compared with the other two groups. Only fish in vitellogenesis state showed a significant increase in contraction amplitude after isotocin administration at the dose of 30 µg/ml. The same group exhibited also a significant isotocin dose-dependent decrease in the contractile frequency. These results confirm the involvement of isotocin in stimulating tunica albuginea contractile activity during the oestrogen-regulated phase of vitellogenesis, whereas the absence of significant effects of isotocin on ovarian contractility in fish at the regressing state might be ascribed to the occurrence of a contractile activity autonomously regulated by the internal pacemaker system. The absence of exposed isotocin receptors could explain the lack of effects of the isotocin administration in seabream showed extensive atresia of the follicular cells.

Liver melanomacrophage centres as indicators of Atlantic bluefin tuna, Thunnus thynnus L. well-being.

Melanomacrophage centres (MMCs), located in different organs of non-mammalian vertebrates, play a role in the destruction, detoxification or recycling of endogenous and exogenous materials. Cytochrome P450 monoxygenase 1A (CYP1A) is involved in xenobiotics biotransformation, and its liver expression is considered as a biomarker for detecting exposure to environmental pollutants. Atlantic bluefin tuna (ABFT), Thunnus thynnus L., liver samples were collected from: wild animals caught in the eastern Atlantic; juveniles reared in the central Adriatic; juveniles reared in the northern Adriatic; adults reared in the western Mediterranean. The samples were processed for basic histology, histochemistry and for CYP1A immunodetection. An unexpected high density of MMCs, containing ferric iron and lipofuscin-ceroids, was detected in the juveniles sampled in the northern Adriatic Sea. These individuals showed also a strong anti-CYP1A immunopositivity in hepatocytes and in the epithelium of bile ducts. This study supports the utility of MMCs as biomarkers of fish 'health status' and gives concern for a potential contaminant accumulation in ABFT.

Productive performance and histological features of intestinal mucosa of broiler chickens fed different dietary protein levels.

To evaluate the effect of decreasing dietary protein on growth performance, carcass traits, and intestinal mucosal morphometry, 180 female Hubbard strain broiler chickens were divided into 3 groups and fed 3 isoenergetic diets ad libitum from 14 d of age until slaughter age (49 d). The treatments varied according to 3 protein levels: high-protein diet (HiP, 22.5% CP, DM basis), medium-protein diet (MedP, 20.5% CP), and low-protein diet (LowP, 18.5%). Diets were obtained by replacing wheat middlings with soybean meal and were formulated to meet or exceed broiler amino acid requirements of the NRC. Morphometric indices of duodenum, jejunum, and ileum were measured at the end of the feeding period and included villus height, crypt depth, villus-to-crypt ratio, and apparent villus surface area. The dietary protein level had a significant effect on final BW of birds, whereas ADG, ADFI, and feed efficiency remained unaffected by dietary treatment. The muscle (breast and drumstick) yields were significantly higher in birds fed the HiP diet compared with those of the MedP and LowP diets. Meat quality traits were not affected by the protein level. The villus surface area of all intestinal segments did not change among groups. Instead, reducing the dietary protein level to 20.5% resulted in a higher villus height and villus height to crypt depth ratio in the duodenum and ileum. On the basis of our findings, even if the high-protein diet promoted meat yield, a medium-protein diet could positively support broiler growth performance, as confirmed by favorable morphometric features of the intestine.

Apoptosis of gut-associated lymphoid tissue in rainbow trout Oncorhynchus mykiss after incubation with Candida albicans and bacterial lipopolysaccharide.

Until now a few studies have been carried out on the gut lymphoid system in fish despite its protective role in the host. Here, we have evaluated the effects of Candida albicans (Ca) and lipopolysaccaride (LPS) on the pyloric and terminal segments of gut in the rainbow trout Oncorhynchus mykiss. In particular, data show that both Ca and LPS are able to cause apoptosis of intestinal lymphoid cells as detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) procedure. These findings suggest a further modality of gut response in fish to environmental antigens.

VEGF concentration from plasma-activated platelets rich correlates with microvascular density and grading in canine mast cell tumour spontaneous model.

Canine cutaneous mast cell tumour (CMCT) is a common cutaneous tumour in dog, with a higher incidence than in human. CMCT is classified in three subgroups, well and intermediately differentiated (G1 and G2), corresponding to a benign disease, and poorly differentiated (G3), corresponding to a malignant disease, which metastasize to lymph nodes, liver, spleen and bone marrow. In this study, we have evaluated serum (S), platelet-poor plasma (P-PP), plasma-activated platelet rich (P-APR) and cytosol vascular endothelial growth factor (VEGF) concentrations, microvascular density (MVD) and mast cell density (MCD) in a series of 86 CMCTs and we have correlated these parameters with each other, by means of ELISA detection of VEGF and immunohistochemistry. Results show that VEGF level from cytosol P-APR and MVD were significantly higher in G3 CMCTs as compared to G1 or G2 subgroups. Moreover, a significantly strong correlation among VEGF levels from P-PAR and cytosol, MVD and MCD was found in G3 subgroup. Because VEGF levels from P-APR well correlated with MVD and malignancy grade in CMCT, we suggest that VEGF might be secreted from MCs and it may be a suitable surrogate inter-species angiogenetic markers of tumour progression in CMCT. Finally, CMCT seems to be a useful model to study the role of MCs in tumour angiogenesis and inhibition of MCs degranulation or activation might be a new anti-angiogenic strategy worthy to further investigations.

In vitro elicitation of intestinal immune responses in teleost fish: evidence for a type IV hypersensitivity reaction in rainbow trout.

In fish the gut immune system has been the subject of few investigations until now. Here, we provide novel morphological and immunological data on the gut isolated from rainbow trout Salmo gairdneri. The pyloric (P) and terminal (T) segments of trout gut, when morphologically examined, evidenced lymphocytes and macrophages (MØ) loosely dispersed in the intestinal mucosa and in the lamina propria in the absence of typical Peyer's patches-like structures. Furthermore, incubation of P and T sections with Candida albicans (Ca) and functional analysis of supernatants generated some interesting results. In fact, active supernatants, when compared with controls, exhibited cytokine-like activities attributable to the presence of interferon (IFN)-gamma and migration inhibiting factor (MIF), respectively. In particular, IFN-gamma-like activity gave rise to an enhancement of Ca phagocytosis by MØ, whereas MIF inhibited MØ migration in agarose. Taken together, these in vitro data suggest that the gut-associated lymphoreticular tissue in fish possesses the appropriate armamentarium to mount a type IV hypersensitivity response when challenged by microbial antigens.

Antigenically activated avian erythrocytes release cytokine-like factors: a conserved phylogenetic function discovered in fish.

Fish erythrocytes are endowed with the ability to produce cytokine like factors when stimulated with Candida albicans (Ca). To evaluate whether similar activities are still conserved in bird erythrocytes (BE), a morphological, cytochemical and immunological evaluation was conducted on peripheral cells in chickens (Gallus gallus). BE form rosettes with monocytes (Mo)-macrophages (MØ), and Mo-MØ according to cytochemical analysis to maintain phagocytic functions across the evolution. Finally, Ca-activated BE release in the supernatants cytokine like-factors that enhance Mo-MØ phagocytosis (interferon-gamma-like activity) and inhibit Mo-MØ migration in agarose (migration inhibitory factor activity). In conclusion, bird erythrocytes, as nonimmune cells, are able to participate in the immune response contributing to the host defence.

First observation of an Hashimoto thyroiditis-like disease in horses from Eastern Europe: histopathological and immunological findings.

Among 622 slaughtered horses from eastern Europe, 156 thyroid glands were selected on the basis of macroscopic alterations (e.g., determination of volume and weight). In the 80% of these thyroids, microscopic alterations consistent with a diagnosis of Hashimoto thyroiditis-like disease were found. In particular, a subverted architecture of the thyroid gland with colloid rarefaction, lymphocytic infiltration, and fibrosis was noted. The confirmation of the histopathological diagnosis of an equine Hashimoto thyroiditis-like disease was provided by the increased serum concentration of thyroglobulin, of antithyroglobulin, and of antithyroid peroxidase autoantibodies. Despite evidence consistent with an Hashimoto thyroiditis-like disease in eastern European horses, the etiopathogenesis of this autoimmune disorder deserves further investigation. In this respect, in some horses histopathological alterations in the pituitary gland may suggest an as-yet-unidentified disorder within the hypothalamus-pituitary adrenal axis associated with Hashimoto thyroiditis.

Modifications of serum and cellular parameters in trotters after a race. Macrophage migration inhibitory activity reduction and serum beta-glucan elevation.

Trotters are exposed to a chronic prolonged stress, such as daily training and frequent races during their active lifespans. There is evidence that trotters undergo very often lethal lung infections after a race, and therefore, is likely that modifications of certain physiologic cellular parameters could account for the increased susceptibility to microbial diseases. Here, we demonstrate that in 7 trotters after a race either serum values (e.g., glycaemia, triglycerides, transaminases, gamma-glutamyltransferase, cholinesterase, amylase, alkaline phosphatase, total proteins, serum albumin, sodium, blood urea nitrogen, lactic dehydrogenase, creatine phosphokinase, and creatinine) or hematological parameters (red blood cell count, hemoglobin, lymphocyte and monocyte count) were increased. At the same time, in the same animals after a race, macrophage migration inhibitory factor activity was depressed, thus indicating an impaired T-lymphocyte response. Finally, increased levels of circulating beta-glucans in some horses, after a race, may suggest a reduced clearance of fungal cell wall components. Taken together, these findings indicate a condition of multiple organ dysfunction, such as the liver, the kidney, the pancreas, and skeletal muscles, as well as a reduced cell-mediated immune response in trotters, after a race.

Do fish thrombocytes play an immunological role? Their cytoenzymatic profiles and function during an accidental piscine candidiasis in aquarium.

Fish (F) thrombocytes (THRs) from healthy trouts were studied in terms of cytoenzyme expression. FTHRs were positive to acid periodic of shiff (PAS) and acid phosphatase (ac. phos.) without tartaric acid (-TA) stainings, as well to alkaline phosphatase. However, when compared with autologous macrophages (M psi's), they were negative to naphthol cloroacetate esterase (AS-D), alpha-naphthyl acetate esterase (Anae), peroxidase (perox) and control ac. phos. with tartaric acid (+TA) stainings, thus indicating a lack of typical lysosomial enzymes. This evidence supports the notion that FTHRs are not true digesting cells. Quite interestingly, trouts and human M psi's were positive for PAS, AS-D, Anae, and perox stainings, thus confirming that cellular cytochemistries are maintained across evolution as their phagocytic functions. Additionally, blood films from trouts, accidentally infected with Candida albicans in aquarium, were morphologically analyzed. Actually, FTHRs interact with erythrocytes, potentiating the formation of rosettes around a central Mpsi. Polymorph nuclear cells and lymphocytes are present in these cellular aggregates, thus suggesting that FTHRs may represent a link between innate and adaptive immunity.

Thymidine phosphorylase profiles in nonmalignant and malignant pancreatic tissue. Potential therapeutic role of capecitabine on tumoral and endothelial cells and tumor-infiltrating macrophages.

The drug capecitabine (CAP) is a thymidine Pi-deoxyribosyltransferase (TP) activated oral fluorpyrimidine that generates 5-fluorouracil (5-FU), preferentially, within tumors. Here, in 38 patients with pancreatic cancer we analyzed immunohistochemical TP expression in pancreatic cancer tissue (PCT) and adjacent nonmalignant pancreatic tissue (ANMPT). In addition, a correlation with the main clinical pathological features was made. Furthermore, TP-positive macrophages (MO) isolated from neoplastic tissue were determined. The mean of TP-positive epithelial cells and endothelial cells in terms of microvessel density was significantly higher in PCT than in ANMPT. Because pancreatic cancer is sensitive to 5-FU, TP-activated oral CAP in tumoral and endothelial cells and tumor infiltrating MO could increase the concentration of 5-FU at tumor site, thus resulting in an enhanced antitumor activity.

Lymphoreticular system in fish: erythrocyte-mediated immunomodulation of macrophages contributes to the formation of melanomacrophage centers.

In fish, melanomacrophage centers (MMC) represent the equivalent of germinal centers of birds and mammals. They consist of aggregates of macrophages filled with fragments of cells, mainly erythrocytes, and various pigments such as hemosiderin, lipofuscin, and melanin. The kidney, the spleen, and the liver contain many MMC either moving freely or encapsulated in the context of the lymphoreticular tissue. Here, we have demonstrated for the first time that MMC exist in the kidney, spleen, and liver of the rainbow trout Salmo gairdneri Richardson by using traditional stainings and cytoenzymatic methods: periodic acid-shiff, alpha-naphthyl acetate esterase, and peroxidase. Finally, in the view of the already described peripheral interaction between fish erythrocytes (FE) and macrophages (MO), we postulate that these cellular aggregates (rosettes) travel to distant organs, thus contributing to the formation of MMC in the kidney, spleen, and liver.

Maturation of fish erythrocytes coincides with changes in their morphology, enhanced ability to interact with Candida albicans and release of cytokine-like factors active upon autologous macrophages.

Erythrocytes from the rainbow trout Salmo gairdneri Richardson (Salmo g.R.) were classified into immature and mature populations, respectively, by measuring longitudinal diameters. More elongated fish erythrocytes (FE), classified as mature cells, were those interacting with Candida albicans (CA) in a higher frequency in terms of either binding to the fungus or its intracellular engulfment. At the same time, in the rosetting phenomenon more elongated mature FE surrounded macrophages (Mø) phagocytosing CA. Finally, FE activated by CA released in the supernatants cytokine-like factors able to modulate Mø functions. In particular, these active supernatants were analyzed for their capacity to inhibit Mø migration Macrophage Inhibition Factor (MIF) activity and enhance Mø phagocytosis. Both activities were detected in supernatants from CA stimulated FE but not in control supernatants. MIF activity could play a role in the accumulation of Mø in the context of functional rosettes, while the factor enhancing Mø phagocytosis could promote clearance of CA in a more efficacious way.

A phylogenetic comparison between acute monocytic leukemia cells and monocytes-macrophages in lower vertebrates.

In humans, monocytes and macrophages (Mphi) play a central role in immune regulation, tissue maintenance and pathogen control. In lower vertebrates, a few studies have been conducted on Mphi like cells. In acute monocytic leukemia monocytic cells, as immature cells restrained in one of the phases of their ontogenesis, would offer the opportunity to rebuild an archaic condition helpful to understand the phylogenesis. Therefore, aim of this work was to characterize in the Rainbow trout (Salmo Gairdneri Richardson) Mphi and compare them with acute leukemia monocytic cells. In the trout, Mphi's morphology is similar to that of mammals. In particular, Mphi possess an irregular embryoshaped nucleus occupying 2/3 of the cell, while the peripheral cytoplasmic profile is irregular with extroflexed plasmalemma and pseudopods. A morphological transition towards Mphi is featured by a wavy hyaline classical membrane and an irregular and extroflexed surface. Some aspects of erythrophagocytosis represented a finding of great interest indicating that the hemocatheretic function could take place directly in circulation. This condition, also observed in human acute monocytic leukemia, suggests that the information to the erythrophagocytosis is restrained under physiological conditions. Non-specific esterases, which are positive in human Mphi smear and Mphi from human lymph node tissue, were also positive in the teleost studied but with a dysomogeneous pattern. Consequently non-specific esterase system is phylogenetically conserved. A lack of immune-reactivity with the anti-CD68 monoclonal antibody (MoAb) on smear and trout tissue sections was observed. On the contrary, strong positivity was detected on human lymph node sections. In trout, the presence of Mphi and circulating Mphi like cells exhibiting an erythrocatheretic function in the circulation would indicate a primordial function that has later been replaced by the liver and the spleen.

Fish immunology. II. Morphologycal and cytochemical characterization and phagocytic activities of head kidney macrophages from rainbow trout (Salmo gairdneri Richardson).

In Salmo gairdneri Richardson trouts, a comparison was made between macrophages (MO) derived from head kidney and peripheral blood monocytes. Morphologically and cytochemically no differences were observed between these two types of mononucleated cells. On the other hand, in parallel studies the ability of trout erythrocytes to engulf Candida albicans (CA) was evaluated and compared to the MO phagocytosis. In erythrocytes, engulfment is preceded by binding to CA and cell membrane invagination, while this was not the case for MO. Finally, MO stimulated with lipopolysaccharides (LPS) did not modify their phagocytic capacities, thus suggesting a lack of LPS receptors or a tolerance to LPS.

Fish immunology. I. Binding and engulfment of Candida albicans by erythrocytes of rainbow trout (Salmo gairdneri Richardson).

The role of fish erythrocytes (FE) as phagocytic cells has poorly been investigated, until now. Here, we have focussed our attention on the interplay between rainbow trout (Salmo gairdneri Richardson) erythrocytes and Candida albicans (CA). At the same time, the intervention of autologous head kidney macrophages (MO) in the CA processing by FE has been studied. Data show that CA particles bind to FE, which, in turn, are able to engulf but not kill them. In the presence of MO, a decrease of FE with bound CA occurs and, in some microscopic images, FE form rosettes with MO. Phagocytosis of CA is higher in rosetting MO than in non-rosetting ones. According to our findings, it appears that FE represent a reservoir of engulfed CA and rosetting is an efficacious phenomenon of presentation of pathogens to MO, where an effective clearance of them can take place.

The rainbow trout (Salmo gairdneri Richardson) granular leukocytes and mast-cells system versus the human one. Cytomorphometrical and cytochemical characters in these two species philogenetically poles apart.

In man, circulating granular leukocytes constitute a cellular system and are able to migrate in the tissues to take part in the immune reactions. This study was to characterize the granular leukocytes and the eventual existence of mast-cells of a low vertebrate, rainbow trout (Salmo gairdneri R.) in order to compare it with man. The blood smears and tissues sections have been tested whit panoptic methods, cytochemistry and toluidine blue. White blood cell count, leukocytes formula and cytomorphometric characterization was performed using an image analyser. Scanning of tissues sections in order to identify mast-cells has been also performed. In this model the granular leukocytes are all neutrophilic like; no eosinophilic, no basophilic no tissue mast-cells, basically existing in allergic and anaphylactic reactions, were found.

Corrosion cast of the vascularization of Mugil Cephalus gills.

The spatial organization of the microcirculation in gills of Mugil Cephalus, was examined by scanning electron microscopic analysis of corrosion cast prepared by intravascular injection of methyl methacrylate. The afferent branchial artery originates from the ventral aorta and gives rise to afferent filamental artery. From the medio-lateral wall of the afferent filamental artery, afferent lamellar arterioles originate which supply one or more lamellae. The lamellar efferent arterioles, which drain the blood coming from the lamellae into the efferent filamental arteries, continue with the efferent branchial arteries and then the dorsal aorta. The techniques used so permitted to evaluate the structure and the interrelationships of the vascular pathways, explaining the regulation and the distribution of the blood flow in the gills.