A Rapid and Reliable Sequential Staining Method for Bovine Muscle

Various different combinations of histochemical stains at differing pH levels on single sections of bovine skeletal muscle were evaluated. The staining sequence that was the most consistent and reliable of those studied included an initial NADH-TR (reduced nicotinamide adenine dinucleotide-tetrazolium reductase) followed by an acid ATPase stain at pH 4.15. This staining combination resulted in easily discernable type I, II A, and II myofibers. Thus, a marked saving of expended time, labor and materials was realized.     

Glycocomposition of the apocrine interdigital gland secretions in the fallow deer (Dama dama)

The secretions of the tubular interdigital glands were investigated by conventional (Periodic-Acid Schiff, Alcian-Blue at different pH, Low Iron Diamine and High Iron Diamine) and lectin (Con-A, UEA-I, LTA, WGA, GSA-II, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA) histochemical methods in adult males and females of different age of fallow deer during the breeding season. Sialidase digestion and deacetylation pre-treatment were also employed in conjunction with lectin histochemistry.The glandular epithelium consisted of a single layer of low columnar cells with typical apical protrusions. No substantial differences of the above histochemical staining in relation to sex and age were observed. Conventional histochemical staining revealed that the interdigital glands secreted neutral glycoproteins whereas acidic glycocomponents did not seem to be present. Lectin histochemical technique allowed us to disclose a great heterogeneity of glycoproteins with N- and O-linked oligosaccharides containing α-d-Man/α-d-Glc, GlcNAc, α-Fuc, terminal β-d-Gal-(1-3)-d-GalNAc, -d-Gal-(1-4)-d-GlcNAc, α-Gal and β-GalNAc residues. β-GalNAc and disaccharide β-d-Gal-(1-3)-d-GalNAc were also found as subterminal to sialyl moieties. The lack of sexual and age-related differences in the glucidic content of the glandular secretions seems to indicate that the glycoderivatives may play only an accessory role in the production of odoriferous signals in fallow deer.     

Comparison of histochemical characteristics in various pork groups categorized by postmortem metabolic rate and pork quality

The purpose of the present study was to investigate the variations in histochemical characteristics of muscle samples segregated according to metabolic rates (MR) and pork quality attributes. A total of 231 crossbred Duroc x (Yorkshire x Landrace) pigs was evaluated. Samples of the LM were taken to evaluate histochemical characteristics, postmortem MR, and meat quality. Samples were classified into fast, normal, and slow MR groups based on muscle pH at 45 min and R-value. Drip loss and lightness (L*) were used to assign samples to 1 of 4 quality classes. Pale, soft, and exudative pork belonging in the fast group had the greatest (P < 0.05) percentage of type IIb fibers, and RSE (reddish-pink, soft, and exudative) pork belonging in the fast group had a similar tendency. Additionally, RFN (reddish-pink, firm, and nonexudative) pork belonging in the normal group showed a lower (P < 0.05) percentage of type IIb fibers than PSE or RSE, regardless of MR, and DFD pork had the lowest (P < 0.05) percentage of type IIb fibers. In general, the fast-glycolyzing PSE pork with the lowest pH at both 45 min and 24 h had greater percentages of type IIb fibers than the fast-glycolyzing RFN pork. There were more fiber-type composition differences between quality classes in pork undergoing a fast rate of metabolism compared with pork undergoing a normal rate of metabolism. It can be concluded that muscle histochemical characteristics are associated with early postmortem MR, the extent of glycolysis, and, thereby, pork quality; however, these effects are limited to the pigs exhibiting a fast glycolytic rate.     

Differences in Fibre Population in Dog Muscles of Different Functional Purpose

Samples were taken from particular sites of the tibialis cranialis (extensor), flexor digitalis medialis (flexor) and pectineus (adductor) muscles of 27 dogs of different breeds--German shepherd, Spanish greyhound, Spanish mastiff and Iberian hound. Samples, which were taken from right and left limbs, were subjected to histochemical analysis using ATPase (pH 9.4, 4.6 and 4.2) and NADH-tr. Fibre population was assessed using an IBAS-2 Kontron image analyser.     

Muscle Fibre Morphometry in Three Dog Muscles of Different Functional Purpose in Different Breeds

Following a study of fibre population fibre size was determined in three muscles of different functional purpose in four breeds of dog with different locomotory capacities. Samples were taken from specific sites in the tibialis cranialis (extensor), flexor digitalis medialis (flexor) and pectineus (adductor) muscles of 27 dogs of four different breeds—German shepherd, Spanish greyhound, Spanish mastiff and Iberian hound—. Samples were subjected to histochemical analysis using ATPase (pH 9.4) and fibre size was assessed using an IBAS-2 Kontron image analyser.     

The histology and histochemical aspects of gills of the flower fish, <Emphasis Type="Italic">Pseudophoxinus antalyae</Emphasis>

Branchial epithelium of Pseudophoxinus antalyae was lined by both a thick stratified epithelium lining gill arches, gill rakers and primary filaments and a thin epithelium lining the lamellae. Mucous, chloride and rodlet cells, interspersed between pavement cells, were present in the branchial epithelium. With histochemical procedures for the characterization of glycoconjugates, mucous cells showed a strong positive reaction with Periodic acid-Shiff and Alcian Blue at pH 2.5, although with Alcian Blue at pH 0.5 and pH 1.0 the reaction was much weaker. When the combined Alcian Blue (pH 2.5) – Periodic acid-Shiff reaction was performed, most mucous cells were stained purple, whereas by the combined Aldehyde Fuchsin/Alcian Blue (pH 2.5), most cells showed a positive reaction only to Aldehyde Fuchsin. Methylation/ Alcian Blue (pH 2.5) and Methylation/ Saponification/ Alcian Blue (pH 2.5) methods showed the presence of sulphated and carboxylated glycoconjugates in mucous cells. Mucous cells were also detected to stain all metachromatically with Toluidine Blue.     

NADPH-d-positive mast cells in the canine paranal sinus

This study aimed to investigate the enzyme histochemical expression of NADPH-d in mast cells in the wall of the paranal sinus in male and female dogs. NADPH-d-positive cells with weak, medium and strong enzyme histochemical expression were observed in the stroma of the sinus near the blood vessels of the microcirculatory bed and around the apocrine and sebaceous glands. In the same areas, mast cells with similar dimensions and morphology were demonstrated by metachromasia on paraffin and cryostat cross-sections and stained with 0.1% toluidine blue in McIlvane's buffer (pH 3). These findings suggest that the mast cells that are located in the stroma near the blood vessels, the lining epithelium and the glands correspond with the cells with marked NADPH-d activity. The possibility of mast cells having nitric oxide activity could be used in the regulation of mast cells function when treating paranal sinus tumours and inflammations.     

Fibre type proportions of the buccinator muscle in clinically normal adult dogs

In order to provide normal morphological data and reference values for fibre type proportions, against which changes due to facial nerve paralysis or primary facial muscle myopathies can be compared, a histological and histochemical study was carried out on the buccinator muscle of clinically normal adult dogs. Using myosin adenosine triphosphatase under acidic pre-incubation (pH 4.3) conditions, three histochemical fibre types--1, 2A and 2C were recognised. There was no significant difference in mean diameter between left- and right-sided buccinator muscle samples for type 1 versus 2. Mean diameters of fibre types were: type 1, 27.02 +/- 6.61 microns and type 2, 29.05 +/- 7.75 microns. The histographical distribution of fibre type diameters was unimodal. The buccinator muscle had a type 2 fibre predominance with a mean type 1 to type 2 ratio of 32:68. The number of fibres with internal nuclei was less than or equal to 1 per cent, atrophy-hypertrophy factors typically were less than or equal to 200, and variability coefficients approximated 250. Muscle spindles were not observed in any muscle sample.     

Histochemical properties of muscle fibres types and enzyme activities in skeletal muscles of Standardbred trotters of different ages

Fibre characteristics and enzyme activities were determined for the gluteus, semitendinosus, vastus lateralis and triceps brachii muscles of 55 Standardbred trotters of different ages. Four fibre types (I, IIA, IIB, IIC) were demonstrated by histochemical staining of myofibrillar adenosine triphosphatase after preincubation at different pH values. Type II fibres predominated in all the muscles and the type IIA/IIB ratio was higher in horses over 5 years than in younger horses, except in the vastus in which the IIA/IIB ratio did not change with age. The vastus had the highest proportion of type IIA fibres and the semitendinosus the highest proportion of type IIB fibres. Histochemical demonstration of NADH dehydrogenase disclosed that almost 100 per cent of the type IIA and many of the type I and IIB fibres were medium-stained; the remaining type I fibres were darkly stained and the type IIB fibres lightly stained. In older horses more fibres were stained for NADH dehydrogenase. The activity of triosephosphate dehydrogenase decreased that that of 3-hydroxy-acyl-coA dehydrogenase and citrate synthase increased in all the muscles except the vastus with increasing age. The greatest increase in oxidative capacity occurred in the gluteus and triceps. Training, rather than age, was regarded as the factor inducing these changes. The results emphasise that histochemical data are only semiquantitative, and there are apparent discrepancies in the intensities of histochemical staining and the biochemical evaluation of various enzymes.     

Globule leukocyte and mast cell in bile ducts of cattle naturally infected with liver flukes

In n previous report dealing with the pathology of bovine fascio-liasis the author described an unknown cell type in the epithelium of bile ducts. The histological and histochemical investigations published in this paper suggest that the cell may be considered a globule leukocyte. Globule leukocytes are rare in uninfected livers but are occurring in abundance in main bile ducts of cattle with spontaneous fascioliasis and also in small perilobular ducts in dicrocoeliasis. Liver fluke infection causes an increase in the population of subepithelial mast cells. Mast cell and globule leukocyte present similarities In their cytochemical properties. However, at low pH toluldinc blue shows a stronger but Alcian blue a weaker affinity for mast cells than for globule leukocytes.     

Complex Carbohydrates Occurring in the Digestive Apparatus of Umbrina Cirrosa (L.) Fry

The glycosoaminoglycans in the digestive apparatus of immature fish have important biological functions and are involved in morphofunctional differentiation. The aim of this study was to investigate the glycoconjugate histochemistry in the different parts of the digestive apparatus (oesophagus, stomach, intestine) of Umbrina cirrosa (L.) fry using classical histochemical reactions (periodic acid-Schiff, Alcian blue pH 2.5, Alcian blue/periodic acid-Schiff, high iron diamine, low iron diamine) in conjunction with glycolytic digestions that degrade different classes of glycosoaminoglycans. No differences were observed in the reactivity to conventional histochemical staining of the oesophagus, stomach or intestine among 27-, 34- or 44-day-old fry. In the oesophagus, the mucopolysaccharides contained chondroitin sulphates B and A and/or C, heparan sulphate and chondroitin. In the stomach, only neutral glycoconjugates were revealed, whereas in the intestine there were only chondroitin sulphates. Some differences in the type and content of glycoconjugates were found in Umbrina cirrosa (L.) fry compared to those of adult subjects, probably related to different dietary habits and to changes in the environmental conditions.     

Histochemical and morphometric studies of the cranial tibial muscle in dogs of different dynamic aptitudes (greyhound, German shepherd and fox terrier)

A histochemical and morphometric study of the cranial tibial muscle of the greyhound, the German shephard and the fox terrier was carried out using hematoxyline-eosin, PAS, Gomori trichrome, SDH, LDH, NADH-tr and ATPase staining (with pH adjustments to both acidic and basic levels), with the goal of comparing the proportion and distribution of type I and type II fibers within the muscle. Significant differences were found between the three breeds of dogs. It is suggested that these differences are related to the locomotor activities of the three breeds.     

Histochemical studies on the zona reticularis of stress-susceptible pigs

During an investigation into the role of the adrenal gland in stress susceptibility of pigs, the large lipid masses in the inner zona reticularis were seen to be particularly related to sex differences, although stress-susceptible castrates had significantly more lipid in this zone than had stress-resistant castrates. Significant histochemical differences were also found in the activity of glucose-6-phosphate dehydrogenase and succinic dehydrogenase between the sexes and between castrates of differing susceptibility to stress. The differences in histochemical activity suggest a different origin for some of the adrenocortical cells in the inner zona reticularis.     

A quantitative trait locus genome scan for porcine muscle fiber traits reveals overdominance and epistasis

Muscle histochemical characteristics are decisive determinants of meat quality. The relative percentage and diameters of the different muscular fiber types influence crucial aspects of meat such as color, tenderness, and ultimate pH. Despite its relevance, however, the information on muscle fiber genetic architecture is scant, because histochemical muscle characterization is a laborious task. Here we report a complete QTL scan of muscle fiber traits in 160 animals from a F(2) cross between Iberian and Landrace pigs using 139 markers. We identified 20 genome regions distributed along 15 porcine chromosomes (SSC1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and X) with direct and(or) epistatic effects. Epistasis was frequent and some interactions were highly significant. Chromosomes 10 and 11 seemed to behave as hubs; they harbored 2 individual QTL, but also 6 epistatic regions. Numerous individual QTL effects had cryptic alleles, with opposite effects to phenotypic pure breed differences. Many of the QTL identified here coincided with previous reports for these traits in the literature, and there was overlapping with potential candidate genes and previously reported meat quality QTL.     

Histochemical and immunohistochemical characterization of chordoma in ferrets

Chordomas of the tip of the tail in 6 ferrets were examined using histopathological, histochemical and immunohistochemical procedures. Histopathologically, round neoplastic cells containing numerous cytoplasmic vacuoles of varying sizes, categorized as “physaliphorous cells”, were observed in the amorphous eosinophilic or pale basophilic myxoid stroma. Physaliphorous cells were arranged in lobules and in a “chordoid” or “cobblestone” manner. The neoplasms were diagnosed as benign chordoma without local invasion and metastasis. Histochemically, the cytoplasm of small neoplastic cells was positive for periodic acid-Schiff stain and alcian blue (AB) pH 2.5 and pH 1.0 stains, but negative for hyaluronidase digestion-AB pH 2.5 stain. All neoplastic cells were strongly stained with colloidal ion, negative for high iron diamine AB pH 2.5 and toluidine blue pH 2.5 stains, and positive for Mayer’s mucicarmine stain. Immunohistochemistry using antibodies directed against low-molecular-weight cytokeratins (CK18, CK19 and CK20), vimentin and mucin core protein (MUC5AC) revealed that neoplastic cells had both epithelial and mesenchymal elements. The expression of low-molecular-weight cytokeratins suggests that neoplastic cells acquired the properties of glandular epithelial cells and produced epithelial mucus. Furthermore, the expression of cytokeratins, vimentin, S100 protein, brachyury and epithelial membrane antigen indicates that the neoplasms were equivalent to the classic type of human chordoma. Therefore, immunohistochemistry using these antibodies can be useful for the characterization of ferret chordoma.     

Ultrastructure and secretory nature of the seminal glomus in budgerigars (Melopsittacus undulatus)

The seminal glomera from captive budgerigars were dissected and prepared for ultrastructural and histochemical studies of the lining epithelia. The general structure suggested that they are formed by convolutions of the terminal portions of the ductus deferens which appear as a network of tubules. The epithelium lining the tubules was identified as pseudostratified ciliated and non-ciliated columnar epithelium. With the electron microscope it was possible to identify two different cell types in the epithelium: type 1, ciliated cells and type 2, non-ciliated epithelial cells. Light microscopy revealed periodic acid Schiff (PAS) positive material, resistant to diastase digestion in the distal parts of some of the epithelial cells, indicating its glycoprotein nature. Alcian blue/PAS staining showed mixed acidic and neutral glycoproteins. Alcian blue staining at different hydrogen ion concentrations (pH) showed that the acidic glycoprotein was of the weakly sulphated type. Periodic acid thiocarbohydrazide silver proteinase staining, at the ultrastructural level, confirmed the presence of an intracellular glycoprotein.     

Mucus cells in the digestive tract in prenatal platyfish larvae (Poeciliidae, Teleostei)

The appearance and histochemical properties of mucus cells in various parts of digestive tract in prenatal larvae of platyfish (Xiphophorus maculatus), a stomachless teleost, are described and compared. Such cells occurred regularly throughout the entire epithelial cell layer in mouth, pharynx, oesophagus and intestine in 6 mm long larvae i.e. approximately 1 week before delivery, but were very scarce in 4 mm long larvae. The mucin in these cells was strongly stained by alcian blue at pH 2.5 and weakly coloured by this dye at pH 1.0. It displayed a clean blue colour after high iron diamine followed by alcian blue (pH 2.5), except in some very few cells which displayed a purple-brown colour after this treatment. The mucus cells displayed various colours between purple-blue and magenta when the alcian blue (pH 2.5) was followed by periodic acid-Schiff (PAS) solution; the magenta-coloured cells were mainly confined to oesophagus. Together these findings suggest that the mucin in the digestive tract of prenatal platyfish consists mainly of non-sulphated, carboxylated glycoconjugates. The observations from the present lectin histochemistry suggest that the amount of galactosamine and glucosamine in this mucin varies much from cell to cell. Also, the various colours after PAS suggest a significant cellular heterogeneity in the mucus cell population. This may reflect different stages in development and maturation or a true and permanent cellular heterogeneity, suggesting that this mucus is able to play a number of important roles even a week before the larvae start to feed and digest.     

Morphological Studies on the Harderian Gland in the Ostrich (Struthio camelus domesticus) on the Embryonic and Post‐natal Period

The present investigation was performed on 50 ostriches from 28th day of incubation until the 7th month of life. The morphological (morphometric, histological, histometric and histochemical) studies were conducted. Tissue sections were stained with haematoxylin–eosin, methyl green‐pyronin Y, periodic acid–Schiff, alcian blue pH 2.5, aldehyde fuchsin and Hale's dialyzed iron studies. The Harderian gland becomes macroscopically visible on the 28th day of incubation. It is situated in the ventronasal angle of the orbit near inter‐orbital septum, between medial rectus muscle, pyramidal and ventral oblique muscles. The Harderian gland of ostrich is a tubulo‐acinar gland. The acini were composed of tall conical cells which formed a small lumen and were surrounded by myoepithelial cells. These cells had a granular basophilic, vacuolated cytoplasm. Each of the lobes has a system of complex branching ducts – tertiary, secondary and primary. In the III of research group (3rd week of life), the presence of few plasma cells was demonstrated, which were located within acini and tertiary and secondary ducts, whereas the biggest concentration of plasma cells was observed in group IV of research tissue (4th month of life). The dark cells were observed first time in main ducts 72 h after hatching of nestlings (group II). The morphometric and histometric studies showed that the most intensive growth of Harderian gland occurred between the third week and the seventh month of birds' life. The histochemical study indicated the presence of neutral and acidic mucins, glycoproteins and carboxylated acid mucopolysaccharides.     

Skeletal muscle fibre characteristics in young and old bulls and metabolic response after a bullfight

Fibre type composition, activities of enzymes such as citrate synthase (CS), 3-hydroxyacyl coenzyme A dehydrogenase (HAD), lactate dehydrogenase (LDH), as well as glycogen, lactate and pH levels were analysed in muscle biopsies (m. gluteus medius) obtained after bullfighting from 10 young and 10 old bulls. No changes were seen in fibre type composition between groups, but the older bulls had higher HAD and LDH activities. Low glycogen concentrations and low pH values were found in both groups, but the lactate concentration after bullfighting was higher in the older group of bulls. The histochemical stain for glycogen revealed that type IIB fibres in both young and old bulls contained more glycogen than seen in type IIA and type I fibres. These results show that young and old bulls have similar muscle fibre type composition, but the metabolic capacity differs, with a higher glycolytic capacity and lactate production in older bulls. Furthermore, it seems that the physical and emotional stress in connection with a bullfight causes a marked depletion of glycogen, especially of type I and IIA fibres.