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Stress inevitably occurs during any fish handling and manipulation in culturing, research, or clinical examination situations that require capture and removal of fish from water. Different stress factors can affect the changes in the relative numbers and function of cells of the fish immune system. Catching, transportation and over-crowding caused stress-induced changes in the total number of leukocytes and thrombocytes, as well as changes in the leukocyte formula in Prussian carp (Carassius gibelio, Bloch, 1782) presented here. Cytochemical characterization of leukocyte cell types was performed by applying Myeloperoxidase (MPO), Periodic Acid Schiff (PAS) and Sudan Black B (SBB) staining of Prussian carp blood smears. Cytochemical characterization is a rapid and efficient method for white leukocyte differentiation and insight in their functional status. Comparison and analysis in Prussian carp hematological parameters from fish with and without exposure to stressful conditions such as capture, manipulation, transport and holding, revealed significant differences between stressed and non-stressed fish. Significant reduction in the total number of thrombocytes and lymphocytes and the increase in total neutrophil count were observed in stressed animals. However, differences in total leukocyte number and the number of monocytes were not observed. Deviations from the estimated reference intervals for Prussian carp hematological parameters clearly indicated the presence/absence of a stress reaction and to some extent its intensity. Estimated reference intervals and characterization of morphological and cytochemical appearance of blood cells form a solid basis for further research of the cellular immune function and hematology changes in Prussian carp.

Graphical abstract Highlights: • Granulocytes were more phagocytic than hyalinocytes, independently of the challenge. • Microbiological challenge increased acid phosphatase activity in hyalinocytes. • Humoral response differed depending on the nature of the microbiological challenge. • Hemocyte damage and gill antioxidant response increased only under E. coli challenge. Abstract Immune cell characterization, immunological response and the associated gill oxidative balance were studied in the Patagonian freshwater mussel, Diplodon chilensis, using two microbiological immunostimulant models: Saccharomyces cerevisiae and Escherichia coli. Mussels were collected out of the breeding season in Paimún Lake and acclimated in the laboratory. Two exposure experiments were performed during two consecutive weeks: (1) mussels challenged with 500 yeast cells mL−1; and (2) mussels challenged with 1000 bacteria cells mL−1. Microorganisms were added in the water every two days, alternating with 6000 lyophilized cells of the green algae Scenedesmus vacuolatus mL−1. A control group, fed with S. vacuolatus , was set for each treatment. Morphological cell characterization was carried out in adherent hemocytes of D. chilensis hemolymph under control conditions. The most important cell type observed were the hyalinocytes (representing ca. 98% of the circulating cells), agranular cells with non-central polymorphic nucleus surrounded by cytoplasm; granulocytes (cells with cytoplasmic granules and non-central rounded nucleus) represented ca. 2%. Another two cell types were occasionally detected, binucleated hyalinocytes and hemoblast-like cells but were not considered for the analyses. Both adherent hyalinocytes and granulocytes exhibit phagocytic activity towards Congo red stained yeast, which was two-fold higher in granulocytes than in hyalinocytes, regardless of the applied challenge. Total hemocyte counts were diminished in mussels challenged with S. cerevisiae or E. coli. Hydrolytic and defense cellular enzyme activities were analyzed only for hyalinocytes. Both, S. cerevisiae and E. coli increased acid phosphatase activity. E. coli challenge diminished hemocyte lysosomal membrane stability and increased humoral phenoloxidase activity, while S. cerevisiae challenge did not affect any of these variables. Mussels challenged with E. coli showed increased gill antioxidant response without oxidative damage, while those challenged with S. cerevisiae showed no change in these variables. [ABSTRACT FROM AUTHOR]

Abstract: Studies of innate immunity in fish species of special concern are essential for better understanding of their health status during hatchery rearing conditions. The cytochemical and morphological characterizations of blood granulocytes have been used to provide information about phylogenetic differences and determine the potential use of neutrophil function assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian granulocytes have been used to characterize granulocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), alkaline phosphatase (AP), Periodic Acid Schiff (PAS) and Diff-quick stain; examined using bright field and differential interference contrast microscopy. Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of the specific chromogen. Presence of lymphocytes, monocytes, platelets/thrombocytes and granulocytes was determined in all fish species. Arctic grayling, June sucker, and cutthroat trout had MPO positive granulocytes, while shovelnose sturgeon heterophils had positive reaction for leukocyte AP, but not MPO. Presence of MPO indicated potential to measure oxidative burst and degranulation of neutrophil primary granules in Arctic grayling, cutthroat trout and June sucker. Absence of MPO in shovelnose sturgeon suggested use of different enzyme marker (AP) in degranulation assay for this species. Standardization of cytochemical techniques allowed for rapid screening of leukocyte types, reducing the number of fish, time and effort to select adequate neutrophil function assays to be used in studies of health status in species of special concern. [Copyright &y& Elsevier]

The fine structure, nature and fate of the components of the nucleolus were studied in young (steps 1, 2), intermediate (steps 3, 4, 5) and mature spermatids (steps 6, 7, 8) of man and monkey, by use of several cytochemical techniques (alcoholic PTA; sodium tungstate; EDTA; HAPTA; nuclease-gold complexes; NOR silver staining). As controls, comparative ultrastructural and cytochemical observations of the nucleolus in spermatids and Sertoli cells were made in the same sections of seminiferous tubules. In the young spermatids of the two species studied, the nucleolar masses exhibited identical features. Segregation of the nucleolar components took place in the nuclei of step 1 spermatids. No typical fibrillar center was observed. In spermatids at steps 1 and 2, the nucleolar masses appeared to be made up of two fibrillar components of equal density, one spherule-shaped, the other forming cords, both surrounded by clusters of 15-20 nm-diameter granules. Alcoholic PTA and sodium tungstate yielded a selective positive contrast of the two fibrillar components whereas EDTA and RNase-gold reacted with the peripheral granular material. Treatment with RNase-gold and DNase-gold complexes resulted in preferential labeling at the periphery of the fibrillar components. After NOR silver staining, numerous small silver grains were localized over the fibrillar cords, suggesting the persistence of specific acidic non-histone proteins. On the contrary, the spherule was never stained. In intermediate spermatids, when the nucleolar components were dissociated, scattered clusters of granules stained by EDTA and HAPTA remained in the entire nucleoplasm. Nucleolar disintegration was accompanied by dispersion of argyrophilic material. In mature spermatids granular material revealed by PTA and silver staining methods was found in the nuclear pockets bounded by the redundant nuclear envelope. [ABSTRACT FROM AUTHOR]