Supranuclear Vacuoles Research Articles

Changes in the histology and digestive enzyme activity during digestive system development of silver pomfret (Pampus argenteus)

Silver pomfret (Pampus argenteus) is an economically important marine fish species in the Indian and Western Pacific oceans, it has great aquaculture potential. Research on the ontogeny of the digestive system is critical in improving the larval rearing protocols and weaning process. In this study, an approach that integrates histology and biochemistry was used to illustrate the digestive system ontogeny of silver pomfret from hatching to 50 days post-hatching (dph). At 1 dph, the digestive system was differentiated into the esophageal sac, esophagus, anterior and posterior intestine, liver, pancreas, and gall bladder, and except for pepsin, digestive enzymes were detected. At 4 dph (4.0 ± 0.2 mm in total length, TL), the mouth opened to allow for exogenous feeding, which coincided with the development of papillae and mucous cells in the esophageal sac, indicating their preliminary function of grinding food. At 7 dph (4.5 ± 0.2 mm, TL), the larvae entered an exclusively exogenous feeding stage and numerous supranuclear vacuoles appeared in the posterior intestine. From 1 to 10 dph, alkaline phosphatase increased sharply, suggesting the rapid development of enterocytes and intestine. At 10 dph (4.8 ± 0.6 mm, TL), gastric glands appeared in the stomach, and from 14 to 20 dph (5.2 ± 0.3 to 6.1 ± 0.4 mm, TL), they multiplied greatly. At this time, the other digestive organs, including the esophageal sac, pyloric caeca, intestine, and accessory glands were well developed and tended to mature in structure. From 15 to 20 dph, the activity of pepsin, trypsin, lipase, and α-amylase rapidly increased, suggesting that the digestive system might have become functionally integrated. After reaching enzyme activity peaks, alkaline phosphatase, lipase, and α-amylase decreased significantly while pepsin and trypsin initially decreased and then increased to a second peak at 50 dph (25.0 ± 4.6 mm, TL). Except for the esophageal sac, the ontogeny of the digestive system in silver pomfret larvae appears to be generally similar to that of other marine fish species; it might be feasible to begin weaning this species from 15 to 20 dph, which would reduce the amount of live food as required by current protocols. This study provides a preliminary insight into the digestive system ontogeny of silver pomfret and establishes an initial step towards the rearing and weaning process optimization of this novel species.

The development of the digestive system and the fate of the yolk syncytial layer in postembryogenesis of Stenodus leucichthys nelma (Teleostei).

Stenodus leucichthys nelma is an economically important species for cold-water aquaculture. Unlike other Coregoninae, S. leucichthys nelma is a piscivore. Here, we describe in detail the development of the digestive system and the yolk syncytial layer from hatching to early juvenile stage using histological and histochemical methods to determine their common and specific characteristics and to test the hypothesis that the digestive system of S. leucichthys nelma rapidly acquires adult features. The digestive tract differentiates at hatching and starts to function before the transition to mixed feeding. The mouth and anus are open, mucous cells and taste buds are present in the buccopharyngeal cavity and esophagus, pharyngeal teeth have erupted, the stomach primordium is seen, the intestinal epithelium with mucous cells is folded and the intestinal valve is observed; the epithelial cells of the postvalvular intestine contain supranuclear vacuoles. The liver blood vessels are filled with blood. The cells of exocrine pancreas are loaded with zymogen granules, and at least two islets of Langerhans are present. However, the larvae remain dependent on maternal yolk and lipids for a long time. The adult features of the digestive system develop gradually, the most significant changes take place approximately from 31 to 42 days posthatching. Then, the gastric glands and pyloric caeca buds appear, the U-shaped stomach with glandular and aglandular regions develops, the swim bladder inflates, the number of islets of Langerhans increases, the pancreas becomes scattered, and the yolk syncytial layer undergoes programmed death during the larval-to-juvenile transition. During postembryonic development, the mucous cells of the digestive system contain neutral mucosubstances.

Supplementation of lactic acid bacteria has positive effects on the mucosal health of Atlantic salmon (Salmo salar) fed soybean meal

We investigated the ability of lactic acid bacteria, when added individually or in combination in feeds, to prevent soybean meal-induced enteritis in Atlantic salmon. A control diet, designed to induce enteritis, was formulated with marine ingredients and 20% soybean meal. Three more diets were produced by coating the control diet with two bacteria, either singly (Lactobacillus plantarum; L. fermentum) or in combination. The fish were fed the abovementioned diets for 38 days. We performed histological assessments and evaluated the expression of selected mucin and antimicrobial peptide genes in the dorsal skin, gills, and distal intestine. Digesta were also collected to study the short chain fatty acids. Feeding bacteria, individually or in combination, altered the short chain fatty acids−acetoacetic acid, lactic acid, succinic acid, propionic acid−and the total fatty acids in the digesta significantly. Of all the determined short chain fatty acids, the concentration of acetoacetic acid was the highest, and the fish fed the combination of the two bacteria had the significantly highest value. Succinic acid was also significantly higher in fish fed the combination compared to the control group and the L. fermentum group. Total fatty acids were significantly higher in fish fed the combination than those fed L. fermentum. Compared to the control and probiotic combination-fed fish, those fed L. plantarum had higher defensin1 expression in the skin. We also observed significantly higher number of gill mucous cells in the fish fed the blend compared to the control group. Lamina propria width was significantly reduced in fish fed the blend. Supra nuclear vacuoles were higher in fish fed the single species or the blend, compared to the control group. Thus, adding the probiotics to a soybean meal diet can elevate the digesta short chain fatty acids and intestine supranuclear vacuoles, and reduce the lamina propria width, which probably indicate prevention of enteritis.

Lesions of the biliary system in Atlantic salmon (Salmo salar L.).

Lesions of the biliary system in Atlantic salmon (Salmo salar L.).

Adequate levels of dietary sulphur amino acids impart improved liver and gut health in juvenile yellowtail kingfish (Seriola lalandi).

The sulphur amino acids methionine (Met) and cysteine (Cys) and their derivative taurine (Tau) are metabolically active molecules with interlinked roles in nutritional requirements. Deficiencies in these nutrients are linked to poor growth and health; however, the impacts of these deficiencies on organ structure and function are largely unknown. This study examined the effects of dietary Met, Cys and Tau fed at different levels on yellowtail kingfish (YTK) liver histology and surface colour, plasma biochemistry and posterior intestine histology. Samples were collected from two dose-response feeding trials that quantified (1) the Tau requirement and sparing effect of Met by feeding YTK diets containing one of seven levels of Tau at one of two levels of Met and (2) the Met requirement and sparing effect of Cys by feeding YTK diets containing one of five levels of Met at one of two levels of Cys. YTK fed inadequate levels of dietary Met, Cys and Tau exhibited thicker bile ducts, less red livers, more intestinal acidic goblet cell mucus and supranuclear vacuoles and less posterior intestinal absorptive surface area. Further, thicker bile ducts correlated with less red livers (a*, R), whereas increased hepatic fat correlated with a liver yellowing (b*). Our results indicate a shift towards histological properties and functions indicative of improved intrahepatic biliary condition, posterior intestinal nutrient absorption and homoeostasis of YTK fed adequate amounts of Met, Cys and Tau. These findings may assist in formulating aquafeed for optimised gastrointestinal and liver functions and maintaining good health in YTK.

Intestinal health in Atlantic salmon post-smolt (Salmo salar) when fed low- and high HUFA diets

It is well established that farmed Atlantic salmon (Salmo salar) need n3-highly unsaturated fatty acids (HUFA) in their diet to thrive and grow. However, the biological functions to the individual HUFAs may differ, implying that future supplementation could require fixed ratios for maximum benefit. The intestinal barrier is essential to fish health, and any disruption of the barrier can have detrimental effects. The current experiment was designed to examine the response of the intestinal hindgut when fed a low HUFA diet with 8.5 g/kg EPA + DHA (4.5% total fatty acid) or two high HUFA diets, with either high DHA (28.7 g/kg and 5.9% total fatty acid) or high EPA (25 g/kg and 14.2% total fatty acid). The diets were fed to Atlantic salmon post-smolt over 10 weeks and thereafter exposed to 3 weeks of chronic stress. After 10 weeks of feeding there were no differences in intestinal permeability and integrity, but intestinal morphology indicated increased intestinal health in the high EPA group. Gene expression also suggest that fish fed the high EPA diet had more regulation of pathways related to protein turnover compared to the high DHA fed fish. There was also indication of lower energy utilization in the low HUFA fed fish than high HUFA. Subjecting fish to 3 weeks of chronic stress led to a reduction in transepithelial resistance, increased ion flux and active l-lysine transport across the intestinal barrier in addition to a decrease in mucosal fold, enterocyte height and supranuclear vacuole density and an increase in thickness of the intestinal muscularis. After stress, the low HUFA group showed signs of inflammation with increased infiltration of MHCII positive cells. Gene expression also showed that low HUFA fed fish had a lower response to chronic stress compared to the high HUFA groups. Comparing fish fed either high DHA or EPA exposed to chronic stress showed few physical effects, but a lower density of supranuclear vacuoles and upregulation in immune-related gene expression indicate inflammation in the high DHA group.

Key Performance Indicators of Common Carp (Cyprinus carpio L.) Wintering in a Pond and RAS under Different Feeding Schemes

Overwintering impacts common carp performance, yet the nature of changes is not known. The aim of the study was to compare the zootechnical and key performance indicators (KPI) of Cyprinus carpio wintering in a pond with no supplementary feeding (MCF), in a Recirculating Aquaculture System (RAS) fed typical (30% of protein and 8% of fat) carp diet (AFC), and in a RAS fed high protein (42%) and fat (12%) diet (ABF). The analysis showed that ABF fish had the highest final body weight and the Fulton’s condition factor, as well as the lowest food conversion rate compared with AFC and MCF fish. Histomorphological assessment revealed that MCF fish had thinner skin layers, a depleted population of mucous cells in skin, an excessive interlamellar mass in the gills, and no supranuclear vacuoles in the intestine compared to fish from RAS. At the molecular level, higher transcript levels of il-1β and il-6 transcripts were found in the gills of MCF than in fish from RAS. The transcript level of the intestinal muc5b was the highest in ABF fish. Relative expression of il-1β and il-6 in gills were presumably the highest due to lamellar fusions in MCF fish. Described KPIs may assist carp production to ensure sustainability and food security in the European Union.

Effects of Yeast Species and Processing on Intestinal Health and Transcriptomic Profiles of Atlantic Salmon (Salmo salar) Fed Soybean Meal-Based Diets in Seawater.

The objective of the current study was to examine the effects of yeasts on intestinal health and transcriptomic profiles from the distal intestine and spleen tissue of Atlantic salmon fed SBM-based diets in seawater. Cyberlindnera jadinii (CJ) and Wickerhamomyces anomalus (WA) yeasts were heat-inactivated with spray-drying (ICJ and IWA) or autolyzed at 50 °C for 16 h (ACJ and AWA), followed by spray-drying. Six diets were formulated, one based on fishmeal (FM), a challenging diet with 30% soybean meal (SBM) and four other diets containing 30% SBM and 10% of each of the four yeast fractions (i.e., ICJ, ACJ, IWA and AWA). The inclusion of CJ yeasts reduced the loss of enterocyte supranuclear vacuolization and reduced the population of CD8α labeled cells present in the lamina propria of fish fed the SBM diet. The CJ yeasts controlled the inflammatory responses of fish fed SBM through up-regulation of pathways related to wound healing and taurine metabolism. The WA yeasts dampened the inflammatory profile of fish fed SBM through down-regulation of pathways related to toll-like receptor signaling, C-lectin receptor, cytokine receptor and signal transduction. This study suggests that the yeast species, Cyberlindnera jadinii and Wickerhamomyces anomalus are novel high-quality protein sources with health-beneficial effects in terms of reducing inflammation associated with feeding plant-based diets to Atlantic salmon.

Mucosal barrier status in Atlantic salmon fed marine or plant-based diets supplemented with probiotics

Feed ingredients and additives significantly affect the mucosal health of fish. A 3 × 2 factorial experiment was conducted to investigate the mucosal health of Atlantic salmon (Salmo salar) fed three basal feeds (namely, BG1, BG5 and BG2; marine-, plant-, and soybean meal-based feeds, respectively) or the basal feeds with (+) or without (÷) probiotics, Lactobacillus fermentum and Lactobacillus plantarum. Six diets were fed to fish distributed into 12 tanks (approximately 43 fish/tank). The average start weight of the experimental fish was about 122.6 g. After 38-days of feeding, the dorsal skin, gills and distal intestine were obtained for analysing histomorphometry and mucus-related genes. Digesta were also collected to study short chain fatty acids (SCFAs).Fish fed BG2 had significantly higher number of mucous cells/μm2 skin epithelium (SNE) than those fed BG1 and BG5. Addition of probiotics significantly increased SNE in BG5+ and BG2+ group compared to BG1÷ group. Similarly, the area and number of mucous cells/μm2 gill epithelium (GME and GNE) were significantly higher in BG2 group, followed by BG5 and BG1 fish groups. Probiotics significantly increased GME and GNE in all feed types. Concerning intestine, villi height (VH) and enterocyte height (EH) were significantly higher for BG1 group, followed by BG5 and BG2 groups. Compared to fish offered BG2, fish fed BG1 had significantly wider villi (VW) and narrower lamina propria (LPW). The number of mucous cells (NM) and intraepithelial lymphocytes (IEL) in the intestine were significantly higher in BG2 fed fish than those offered BG5 and BG1. The indices VH, VW, EH, and IEL were not affected by probiotics. Although higher NM and IEL were observed in BG2÷ fish compared to those fed the other two diets, probiotics reduced NM and IEL. Fish fed BG2÷ showed symptoms of inflammation, including disappearance of supranuclear vacuoles (SNV). Probiotics improved VH, significantly reduced LPW and aided in the reappearance of SNV in BG2+ fed fish. Some of the gene expression data supported histological findings; notably, levels of muc5ac1 in the skin and defensin3 and cathelcidin1 in the intestine were correlated with histology data. Moreover, the total SCFA concentration was significantly affected by feed ingredients. Only acetoacetic acid was affected by both factors.Our findings suggest that feed ingredients can significantly alter the mucosal protective barrier of the organs. Supplementation of probiotics alleviated the inflammatory responses and activated selected innate immune defence molecules, without affecting growth. The positive effect of the probiotics was similar regardless of the feed ingredients, suggesting that these probiotics can be utilized as immune regulators to evoke favourable responses on the skin, gills and intestine.

P–311 Association between serum sex hormones (progesterone and estradiol) and endometrial morphology during the mid-luteal phase

Abstract Study question Is there a relationship between the serum progesterone and estradiol levels and certain morphological characteristics of human endometrium during the mid-luteal phase? Summary answer Serum progesterone is associated with the stromal edema and the abundance and size of basal vacuoles in the endometrium of women during the mid-luteal phase. What is known already Progesterone and estrogen are essential hormones that are necessary to prepare the endometrium for pregnancy. Their serum concentrations during the mid-luteal phase are important criteria for prediction of successful embryo implantation. In addition, a variety of endometrial morphological markers, such as the presence of pinopodes, subnuclear and supranuclear vacuoles, glandular secretion, and stromal edema have been applied for determination of the window of implantation and endometrial receptivity. However, the relationship between these endometrial morphological characteristics and serum levels of progesterone and estradiol is still scarcely studied. Study design, size, duration This is an observational study of 98 women, 25 to 46 years of age (mean 37 years), who had a blood sample and an endometrial biopsy during the mid-lutheal phase (LH + 7) in a natural cycle. The study was conducted between August 2020 and November 2020. Participants/materials, setting, methods Serum progesterone and estradiol were measured by electrochemiluminescence immunoassay (ECLIA) on the Cobas e411 analyser (Roche Diagnostics, Germany). The following endometrial morphological characteristics were assessed using light microscopy: (1) basal vacuoles (mean size and percentage of vacuolated glandular cells) (2) apical vacuoles (mean size and percentage of vacuolated glandular cells), (3) pinopodes (percentage of luminal epithelium covered in pinopodes), (4) glandular intraluminal secretion (6-level scoring system), (5) stromal edema (6-level scoring system). Main results and the role of chance The serum progesterone levels ranged between 0.39 and 145.3 ng/ml, with a median of 24.36 ng/ml. The serum estradiol levels varied between 26.91 and 842.89 pg/ml with a median of 124.75 pg/ml. The percentage of cells with basal vaculoles ranged from 0 to 90%, with a median of 38.57%, apical vacuoles (0–50%, 16.83%), pinopodes (0–80%, 23.87%), glandular intraluminal secretion (0–80%, 28.57%), and stromal edema (1–6, 1.42). To examine the association between the serum progesterone and estradiol and the studied endometrial morphological characteristics, the Spearman’s Rho Correlation coefficient for non-paramentric data was used. No correlation was found between serum estradiol levels and the studied morphological variables (p > 0.05). In contrast, the serum progesterone concentration showed a significant negative correlation with the percentage of glandular epithelial cells with basal vacuoles (R= - 0.28; p = 0.03), the mean size of the basal vacuoles (R= - 0.24; p = 0.5) and a significant positive correlation with the stromal edema (R = 0.34; p < 0.01). Limitations, reasons for caution The study was limited in sample size. Wider implications of the findings: The results of this study revealed that serum progesterone is more strongly associated with the occurrence of certain endometrial morphological characteristics during the mid-luteal phase than serum estradiol. These findings are valuable for development of new methods for accurate determination of the window of implantation. Trial registration number Not applicable

Effect of Candida utilis on growth and intestinal health of Atlantic salmon (Salmo salar) parr

In this study, Candida utilis was included as alternative protein source in diets for Atlantic salmon (Salmo salar) parr and the effects on growth performance and distal intestinal (DI) health were assessed. The potential of C. utilis to counteract possible adverse effects of a high soybean meal (SBM) diet was additionally assessed by using graded levels of C. utilis in combination with a 40% SBM diet. Six experimental diets were formulated: Fishmeal control (FM); FM with 20% C. utilis (FM20CU); SBM-based diet containing 40% SBM (SBM); three SBM-based test diets, where wheat gluten and starch were substituted with increasing levels of C. utilis of 5%, 10% and 20% (SBM5CU, SBM10CU and SMB20CU, respectively). A total of 2700 Atlantic salmon parr with an average weight of 4.4 g were distributed into 18 tanks and the diets were fed in triplicates. The experiment lasted 28 days and sampling for histology and gene expression analysis was done at the end of the experiment.The fish grew to an average weight of 14.8 g in 28 days. Fish fed FM20CU obtained a significantly higher specific growth rate (SGR) of 4.59 than the other dietary groups. While fish fed FM and FM20CU displayed normal morphology of the DI, fish fed SBM-based diets showed mild histological changes in the DI that can be related to classical SBM induced enteritis (SBMIE). These changes included reduced height of simple folds and decreased presence of supranuclear vacuoles. The severity of these changes was not altered with increasing levels of C. utilis in the SBM diet. The expression of four of the five selected genes analyzed in the DI were altered by 40% dietary SBM inclusion. Aquaporin 8ab demonstrated the clearest changes among dietary groups, showing down-regulation independent of dietary inclusion of C. utilis. The strong down-regulation of aquaporin has been observed before and may be an indicator for intestinal barrier dysfunction. In conclusion, adding C. utilis to diets for Atlantic salmon parr gave high growth performance without any obvious adverse effects on intestinal health, but were unable to counteract the mild histology changes seen in DI of the SBM fed fish.

Ontogeny of the digestive tract in stinging catfish, Heteropneustes fossilis (Bloch) larvae.

Heteropneustes fossilis (Bloch) is an important candidate species for diversification of freshwater aquaculture in India. However, high mortality rate during larval rearing is the most serious bottleneck in commercial production of this species. A proper understanding of the ontogenic development of digestive system provides the basis to understand the nutritional physiology of larvae and develop appropriate feeding strategies. In the present study, the ontogenical development of the digestive tract in H. fossilis larvae was studied from hatching until 30day post-hatching (dph) at 29°C. At hatching (2.8 ± 0.2mm standard length, SL), the digestive tract was undifferentiated and attached dorsally to the yolk sac. At 1dph (2.9 ± 0.2mm SL), the mouth opened and oral valves were visible. At 2dph (3.0 ± 0.3mm SL), goblet cells were observed in the buccoparyngaeal cavity. At this age, exogenous feeding started and the intestine was differentiated into the anterior and posterior regions, and the rudimentary liver and pancreas were also seen. Small supranuclear vacuoles were observed in the enterocytes of the posterior intestine at 2dph. Zymogen granules were observed in acinar cells of pancreas by 3dph, and islets of Langerhans were visible at 4dph (3.5 ± 0.1mm SL). At the same age, most of the yolk sac reserves were consumed, whereas they were completely exhausted by 5dph (3.9 ± 0.5mm SL). Between 4 and 6dph, the liver elongated in size and started to accumulate lipids in the hepatocytes. Gastric glands were detected at 4dph, and the pyloric sphincter was completely differentiated at 9dph (6.1 ± 0.4mm SL) as an epithelial fold that separated stomach from the anterior intestine. By 13dph (8.6 ± 0.2mm SL), profuse gastric glands were visible inside longitudinal mucosal folds of the stomach. The formation of gastric glands and their development were noticed as the last events in the development of the digestive tract in H. fossilis. This indicated the end of the larval period and the commencement of the juvenile stage. Considering these observations, it is suggested that H. fossilis larvae have a morphologically complete digestive tract by 13dph. The findings of the study on the development of the digestive system in H. fossilis may help in synchronising the larval stage of development and feeding strategies and would be helpful in improving larval rearing techniques for catfish species.

Fast growing greater amberjack post-larvae require a high energy-high protein weaning diet

Larvae and early juveniles of fast-growing fish species show tremendous growth potential, leading to higher requirements for protein, high-unsaturated fatty acids, and other nutrients. Several commercial weaning diets with relatively high success have been developed for low/moderate-growing species; however, additional challenges are outlined to meet growth potential and energy requirements of such fast-growing species. The objective of the present study was to evaluate two commercial microdiets for marine fish, one having simultaneously a very high protein and high lipid contents (HIGH), and another (MOD) with a high protein and moderate lipid content, in a growth performance trial with greater amberjack post-larvae from 33 to 78 days after hatching. Moreover, histological preparations of liver, anterior and posterior intestine were assessed for hepatic and intestinal lipid inclusions quantification and gut epithelial brush height measurement. Activities of the digestive enzymes: pepsin, trypsin, chymotrypsin, lipase and amylase were also analyzed. Post-larvae fed HIGH microdiet exhibited higher final weight and lower feed conversion ratio that those fed on MOD microdiet. Liver displayed a higher level of lipid inclusions for the MOD diet than for HIGH diet. Moreover, enterocytes of posterior intestine presented a much higher level of supranuclear vacuoles for the HIGH diet compared to MOD diet. The lower Trypsin/Chymotrypsin ratio observed at the end of the experiment in larvae fed on MOD diet may indicate a deficiency in protein of this diet. Together, these results support that larvae of greater amberjack, and likely other fast-growing marine fish species, require high protein–high lipid microdiets. The use of microdiets developed targeting slower growing marine species may lead to sub-optimal performances in fast-growing larvae.

Ontogenetic development of the digestive tract and ultrastructure of the anterior intestinal epithelia in tiger grouper Epinephelus fuscoguttatus (Forsskål, 1775) larvae

The ontogeny of the digestive tract and ultrastructure of the anterior intestine in Epinephelus fuscoguttatus (Forsskål, 1775) larvae were examined using light microscopy and transmission electron microscopy from hatching to 42 d after hatching (DAH). The first developmental stage started at hatching when the digestive tract was a simple tube. The second stage (2–3 DAH), the endo-exotrophic stage, was the time when the mouth of the tiger grouper larvae developed. The third stage (3–24 DAH) started after the depletion of the yolk-sac (3 DAH). The remarkable changes included the appearance of gastric glands at 9 DAH, eosinophilic supranuclear vacuoles appearing in the posterior intestine at 5 DAH and lipid vacuoles found in the interior intestine at 6 DAH which indicated the beginning of protein and lipid absorption. The last stage (after 24 DAH) started when the gastric glands and pyloric caeca were fully developed. The formation of the gastric glands and pyloric caeca indicated a suitable time for weaning. The ultrastructural features of epithelium cells of the anteria intestine showed large lipid droplets at the beginning of the exotrophic stage. From this time onwards, the lipid droplets became smaller, while the endoplasmic reticulum and Golgi complex were well-developed. Upon metamorphosis, the tiger grouper larvae had eosinophilic granule cells (EGCs) in the intestinal epithelia. This substantially increased immunity capability at this time. This study showed that during a critical period of larval survival, the ontogeny coincided with exogenous feeding, while the ultrastructure showed lipid metabolism, hence highlighting the importance of fatty acid in the development of the larvae.

Histological study of the gastrointestinal tract in longfin yellowtail (Seriola rivoliana) larvae.

This work contributes basic knowledge on larval development of Seriola rivoliana. A histological study describes the development of the digestive tract and accessory glands in S. rivoliana larvae reared under laboratory conditions at 24°C from hatching to 30days post-hatching (DPH). At hatching (2.6±0.12mm), larvae had an undifferentiated digestive tract with a closed straight tube and a large yolk sac with an oil globule. The liver and pancreas were observed at 1 and 2days, and the mouth and anus opened at day 2. Enriched rotifers were visible in their digestive tract. At the beginning of the pre-flexion stage, a mixed nutritional period was observed. At day 3, exogenous feeding began; the digestive tract became differentiated into the buccopharynx, esophagus, an undifferentiated stomach, and the intestines. Zymogen granules were visible in the exocrine pancreas. At day 4, supranuclear vacuoles were present in the posterior intestine, indicating the beginning of intracellular digestion. At day 5, goblet cells were present in the esophagus and became functional at day 7 in the esophagus and intestine. The buccopharynx goblet cells developed at day 15. The presence of gastric glands and differentiation of the stomach in the fundic, cardiac, and pyloric regions during the post-flexion stage occurred at day 20. This was the onset of the juvenile period and the beginning of weaning; however, a long co-feeding phase is recommended. Pyloric caeca were observed at day 30 (13.6±1.6mm). These results provide valuable information on S. rivoliana larvae biology and digestive physiology, which should be useful to improve cultivation techniques and identify ecological features involved in ontogeny.

Fetal adenocarcinoma of the lung: a clinicopathologic analysis of six cases

To investigate the clinicopathologic features, diagnosis and differential diagnosis, and genetic alterations of fetal adenocarcinoma of lung (FACL). Six cases of FACL were collected from Fujian Provincial Hospital, the clinicopathologic features and immunophenotype were retrospectively evaluated, PAS/PASD staining and genetic mutation analysis of epidermal growth factor receptor (EGFR) were performed. There were three male and three female patients, aged 40 to 75 years (median 59 years). Tumor size ranged from 2.5-6.0 cm (mean 3.8 cm). Histologically, the tumors showed classic glandular structure similar to fetal lung tubules. The neoplastic glands were lined by non-ciliated pseudostratified columnar epithelium with clear or granular cytoplasm. Some cells showed subnuclear or supranuclear vacuoles. Squamoid morules were also noted. Immunohistochemically, the tumor cells were positive for TTF1, Napsin A, CKL, and focally positive for CKH (2/6), synaptophysin (1/6), chromogranin A (1/6), CD56 (2/6), and EGFR (4/6). PAS/PASD staining was positive in the tumor cells. Five cases showed EGFR mutation, one case was of wild type. All six patients underwent surgical resection, and four had chemotherapy. All patients had no evidence of recurrence or metastasis during 1-40 months follow-up period. FACL is a rare tumor with low malignant potential and has distinct morphologic feature. Clinically and pathologically, it needs to be differentiated from alveolar adenocarcinoma, adenosquamous carcinoma, pneumoblastoma, and metastatic endometrioid adenocarcinoma. The primary treatment for FACL is complete surgical excision and chemotherapy, with good outcome.

Response of European seabass (Dicentrarchus labrax) to canola oil diets: effect on growth performance, fish health and liver and intestine histomorphology

This study was undertaken to assess the effects of feeding European seabass (Dicentrarchus labrax) canola oil-added diets on growth, health status and liver and intestine histomorphology. Seabass (56.18 ± 0.16 g initial body weight) were fed one of three fish meal-based diets with ~48 % crude protein and ~16.0 % lipid, combining fish oil (FO) and canola oil (CO) for 12 weeks. The diets contained: zero (control, CTRL), 45 (CO50) or 63 (CO70) g CO kg−1 assigned in triplicates to three dietary groups. The results indicated that neither dietary oil type (FO or CO) nor CO level adversely affected (P > 0.05) the growth, feed utilization or major blood constituents’ composition as an indicator of the overall health status of fish. Despite the CO diets influence on head kidney macrophage activity being unappreciable (P > 0.05), there was a reducing trend with an increase in CO level incorporation. The CO70 diet induced a minor fat infiltration in hepatocytes and leucocytes infiltration, hyperplasia of the basal nuclei and supranuclear vacuolization of the enterocytes of the distal intestine. The present observations suggest that it is possible to incorporate up to 63 g CO Kg−1 in the feed for European seabass juveniles without major negative effects on growth, health status or liver and intestinal histomorphology.

Ontogeny of the digestive tract of Centropomus parallelus larvae.

Development of the digestive tract and accessory glands of larvae of the fat snook Centropomus parallelus was examined under light microscopy, from hatching to 60 day post-hatching (dph). At hatching, the digestive tract is straight and composed by a cubic cell layer. The exogenous feeding starts at 3 dph, concomitantly with the mouth opening and subdivision of the rudimentary stomach and esophagus. At 4 dph, the intestine has three sectins, and vacuoles are observed in the posterior section, indicating the beginning of protein digestion and absorption. The pharyngeal teeth appear at 9 dph, and goblet cells appear at 13 dph in the esophagus. Gastric glands appear at 30 dph, marking the beginning of weaning. The disappearance of supranuclear vacuoles in the posterior intestine occurs at 35 dph, suggesting efficiency of extracellular digestion. This study shows that C. parallelus larvae is able to start weaning 15 days earlier than reported in earlier studies, increasing the success of larviculture.

"Somatic-type" malignancies arising from testicular germ cell tumors: a clinicopathologic study of 124 cases with emphasis on glandular tumors supporting frequent yolk sac tumor origin.

Somatic-type malignancies (SMs) in patients with testicular germ cell tumors (GCT) are rare and mostly attributed to "transformation" of teratoma, although yolk sac tumor (YST) origin has also been proposed. We studied 124 cases of "SM" of testicular GCT origin from 106 patients to evaluate their morphology, immunohistochemical features (especially the utility of SALL4), and relationship to YST. Primitive neuroectodermal and nephroblastomatous tumors were excluded because of prior studies. Patients ranged in age from 15 to 68 years (mean, 33 y). The tumors ranged from 0.7 to 30 cm (mean, 7.6 cm) and involved the retroperitoneum (64%), abdomen/pelvis (10%), lung (10%), mediastinum (6%), supraclavicular region/neck (4%), testis (4%), and thigh (1%). Most initial diagnoses were sarcomas (n=68) or carcinomas (n=51). On review and immunohistochemical analysis, 7 of 45 adenocarcinomas were reclassified as glandular YSTs (GYST) on the basis of glypican-3 (GPC3) and/or α-fetoprotein positivity and scant/absent reactivity for EMA and CK7. These occasionally (29%) had subnuclear and sometimes supranuclear vacuoles (endometrioid-like), whereas adenocarcinomas were more frequently mucinous (17%) or enteric-type (11%) than endometrioid-like (9%). Both expressed CDX2 frequently (83% and 63%, respectively). MUC protein 2, 4, 5, and 6 expression was more common in adenocarcinomas (7% to 36%) than in GYSTs (0% to 20%) but was infrequent. Both were often positive for SALL4, BerEP4, and MOC31; all were negative for TTF-1. On follow-up (GYST: range, 23 to 169 mo; mean, 81mo; adenocarcinoma: range, 1 to 170 mo; mean, 55 mo), 50% and 33% of patients with GYST and adenocarcinoma, respectively, died of disease. We reclassified 26 of 76 sarcomatoid tumors as sarcomatoid YSTs (SYST) on the basis of positive reactivity for both AE1/3 and GPC3. These tumors often had spindled and epithelioid cells in a fibromyxoid stroma. SYSTs were often (60%) SALL4 positive, whereas sarcomas were all negative. On follow-up (SYST: range, 1 to 259 mo; mean, 62 mo; sarcoma: range, 1 to 327 mo; mean, 70 mo), 50% and 29% of patients with SYST and sarcoma, respectively, died of disease, with most mortality occurring in those with high-grade tumors. We conclude that, on the basis of a panel of immunoreactivities, a significant number of "SMs" in testicular GCT patients are more accurately classified as either GYSTs or SYSTs. Ambiguous glandular tumors should be evaluated for GPC3, α-fetoprotein, CK7, and EMA reactivity and sarcomatoid ones for GPC3, AE1/3, and SALL4 reactivity.

Ontogeny of the digestive tract in mud loachMisgurnus anguillicaudatuslarvae

This study on histological and mucous histochemistry characteristics of the digestive system of loach (Misgurnus anguillicaudatus) was carried out from hatching (0 day after hatching, DAH) until 45 DAH. The peculiar development of both digestion processes and air-breathing functions of the intestine was revealed. At 3 DAH, both the mouth and anus opened along with the first feed. At 4 DAH, lipid vacuoles appeared in the anterior part of the intestine and at 5 DAH the acidophilic supranuclear vacuoles appeared in the posterior part of the intestine. Mucous cells occurred in the buccopharynx and oesophagus after mouth opening and grew both in number and size as larvae grew. At 15 DAH, blood capillaries were found in the posterior part of the intestine. At 20 DAH, as a valve appeared in the intestine, the whole intestine could be divided into anterior, mid and posterior parts. With large numbers of blood capillaries and a very thin wall, a gas–blood barrier formed in the posterior intestine, which indicated that the dual roles of intestine were formed. These results suggested that the air-breathing function of the digestive tract formed from 15 to 20 DAH, which is a critical period for loach larvae.