Glycohistochemical, Immunohistochemical and Ultrastructural Studies of the Bovine Epididymis

In the present work, efferent ductules and epididymal duct from male foetuses as well as from sexually mature bulls were investigated using conventional light and electron microscopical techniques as well as glycohistochemical and immunohistochemical staining techniques. The prenatal development of the bovine epididymis was studied in foetuses ranging from 10 cm CRL (75 pcd) to 90 cm CRL (285 pcd). In foetuses with 10 cm CRL (75 pcd) the main event was the establishment of the urogenital junction between the extratesticular rete testis and mesonephric duct via the growing efferent ductules. At the foetal age of 110 pcd (24 cm CRL), efferent ductules underwent a strong coiling. At the same time the mesonephric duct began to lengthen and coil, forming three distinct regions, namely caput, corpus and cauda epididymidis. The coiling was much more distinct in caput and cauda than in corpus epididymidis. At 130 pcd (30 cm CRL) and upwards efferent ductules were organized in lobules which are then arranged in groups separated from each other by connective tissue septa. A similar organization involved the highly convoluted epididymal duct, particularly in the head and tail regions. In addition to the macroscopical modifications in the morphology of extratesticular excurrent duct system, histological differentiation involved both the tubular epithelium and the peritubular mesenchymal cells. The epithelium of efferent ductules was differentiated into ciliated and nonciliated columnar epithelium. The simple epithelium of the epididymal duct increased in height and developed stereocilia on its apical surface. Distribution of WGA-, PNA- and GSA-I-binding sites on luminal surface of the epithelium of efferent ductules, but not of epididymal duct may indicate earlier differentiation of the former. WGA-binding to the peritubular and interstitial mesenchymal cells, but not to the epididymal epithelium indicated that the mesenchymal structures differentiate before epithelial ones. S-100, FGF-1, FGF-2, ACE, laminin and GT were immunolocalized in the epithelium both of efferent ductules and epididymal duct as early as at 75 pcd (10 cm CRL). Also ?-SMA was immunolocalized in the peritubular mesenchymal cells at 75 pcd (efferent ductules) and at 95 pcd (epididymal duct, CRL 18 cm). The epithelium of the adult bovine efferent ductules is simple columnar including ciliated and nonciliated cells as well as some scattered intraepithelial leucocytes. On the basis of their cytological characteristics, nonciliated cells could be categorized into three sub-types. The epididymal duct of the adult bull is lined with pseudostratified columnar epithelium. It consists mainly of tall, slender, stereocilia-bearing columnar cells and small basal cells. On the basis of several morphometric parameters like epithelial height, luminal diameter and width of peritubular muscle coat the epididymal duct could be subdivided into six segments. Ultrastructural studies revealed a well developed Golgi apparatus, numerous profiles of sparsely granulated endoplasmic reticulum and mitochondria as well as rER in the cytoplasm of principal cells particularly in those of the first three segments. Apical surfaces of principal cells particularly those of the proximal segments were equipped with long stereocilia and their apical cell membrane and cytoplasm displayed a well developed endocytotic apparatus. The narrow basal extensions of principal cells were crowded with numerous pleomorphic mitochondria, lysosomes, heteromorphic electron dense granules and residual bodies. Basal cells were insinuated between the narrow basal extensions of principal cells and the basal lamina. They possessed kidney-shaped, mostly deeply-invaginated nuclei and were characterized by a paucity of organelles. Apical mitochondria-rich cells were frequently found in segments II and III and rarely in segments IV and V. Their hyaloplasm was lighter than that of the neighbouring principal cells and their apical surfaces were provided with short microvilli. Apart from a reasonable number of mitochondria, small Golgi apparatus and sporadic strands of rER, they displayed a paucity of organelles. Intraepithelial macrophages were occasionally encountered in the basal third of the epithelium. They possessed many mitochondria, well developed Golgi apparatus and rER as well as small heterochromatic nuclei. Various profiles of lysosomes and dark residual bodies were found in their cytoplasm. Intraepithelial lymphocytes were characterized by their heterochromatic, round and mostly indented nuclei and narrow peripheral cytoplasmic rim. They were often encountered in immediate proximity to subepithelial capillaries. Fluoresceinisothiocyanate (FITC)-labelled lectins (GSA-I, PNA, ECA, WGA, Con A, LCA, PSA, DBA, HPA, SBA, VVA, LTA and UEA-I) were also used for the study of the regional distribution of saccharide groups in adult bovine epididymal tissues. WGA, Con A, LCA, PSA, DBA and HPA bound distinctly to stereocilia of principal cells in the different segments. However, DBA- and HPA-binding sites were confined to stereocilia in caput region. WGA, LCA, PSA, DBA and HPA possessed distinct binding sites in Golgi zone of principal cells, mostly of the caput epididymidis. Basal cells reacted distinctly with WGA, Con A, LCA, PSA and HPA. Intraepithelial leucocytes displayed moderate binding sites for PNA, WGA, LCA and PSA. The basal membrane reacted moderately only with WGA. Epididymal connective tissue showed weak to moderate binding only with ECA and WGA. GSA-I bound distinctly to vascular endothelium and could be applied as a good marker for bovine endothelium. Sperm cell mass bound WGA and PNA distinctly. No binding sites could be found for VVA, LTA or UEA-I. Immunohistochemical studies used the Avidin-Biotin-peroxidase Complex (ABC) method for localization of S-100, FGF-1, FGF-2, ACE, GT, VEGF, ?-SMA, laminin, connexin 43, CD4, CD8 and CD68 in the epididymis. The epithelium of the efferent ductules showed intense immunoreaction for S-100, FGF-1 and FGF-2 and a moderate immunostaining for ACE and GT. Principal cells of the first three epididymal segments exhibited a distinct immunostaining for S-100. They also showed a distinct immunoreactivity for FGF-1 throughout the different segments. Principal cells in the first, second and sixth segment displayed intense immunostaining for ACE. Immunostaining for GT in Golgi zone of the principal cells was intense (segments II and III), distinct (segments IV and V) and moderate (segments I and VI). Basal cells showed moderate (FGF-1) or intense (FGF-2) immunostaining in different epididymal segments. Intense immunostaining for ACE, laminin and ?-SMA was found respectively in the endothelium, endothelial basal lamina and smooth muscle cells of blood vessels. The basal lamina of the epithelium and the peritubular smooth muscle cells displayed a moderate immunoreactivity for laminin. The peritubular smooth muscle cells manifested an intense immunostaining for ?-SMA. CD4+ T cells and CD68+ macrophages were found within the epithelium and in the interstitium. Mast cells were conventionally stained with Alcian blue and Toluidin blue. They also displayed a distinct immunostaining for VEGF and FGF-2. In conclusion, my study supports the previously proposed 6-segment scheme of bovine epididymis. Moreover, lectin histochemistry and immunohistochemistry were not only helpful tools in emphasising this scheme but also in correlating specific functional activities to certain regions. Lectins- and GT-binding sites as well as ultrastructural characteristics point to high synthetic and secretory activities of principal cells in the first three segments, as indicated by the well developed Golgi apparatus. Ultrastructurally, principal cells of the proximal three epididymal segments displayed a well developed endocytotic apparatus. This was reinforced by intense immunostaining for ACE in this region, which reflects extensive absorptive activities in this region. Existence of mast cells in the epididymal interstitium and T-lympho-cytes and macrophages in the interstitium and within the epithelium may reflect their harmonized co-operation in the induction of immune tolerance in the bovine epididymis

Histological and immunohistochemical studies on the epididymal duct in the dromedary camel (Camelus dromedarius)

This study was conducted to underscore the spatial distribution of some biologically active proteins within the epididymal duct in the dromedary camel. Paraffin-embedded sections from different regions of epididymis were stained by conventional histological techniques and by immunohistochemistry. A battery of primary antibodies against six proteins (S100, alpha smooth muscle actin [α-SMA], connexin-43 [Cx43], galactosyltransferase [GalTase], angiotensin converting enzyme [ACE], and vascular endothelial growth factor [VEGF]) were used. The epididymal epithelium consisted of five cell populations: principal, basal, apical, dark, and halo cells. The histochemical findings indicated the absence of binding sites for VEGF and Cx43. The principal cells (PCs) showed variable immunoreactivity (IR) for ACE, S100, and GalTase throughout the whole length of the duct. The apical surfaces of most PCs (at the caput) and some PCs (at the corpus) exhibited intense ACE-IR, whereas those at the cauda displayed alternating negative and strong immunostaining. Similarly, moderate S100-IR was found in cytoplasm and nuclei of all PCs at the caput, few PCs at the corpus, and several PCs alternating with negative PCs at the cauda. In contrast, only some PCs showed weak to strong GalTase-IR in different regions. Apart from negative to weak positive S100-IR, basal cells failed to show IR for all other proteins. Apical cells displayed strong IR for ACE, S100, and GalTase with some regional differences. The peritubular and vascular smooth muscle cells revealed strong α-SMA-IR in all regions. In conclusion, the spatial distribution of different proteins in camel epididymis showed similarities and differences to other mammalian species. The region-specific topographic distribution of different proteins and cell types might indicate that the caput and cauda are metabolically more active than that of the corpus

Pharmacokinetics and bioavailability of ceftiofur following intravenous and intramuscular administrations in broiler chickens

The study focussed on pharmacokinetics and bioavailability of ceftiofur (CFT) after receiving a single dose (2 mg/kg BW) through either intravenous (IV) or intramuscular (IM) injection. Eight broiler chickens, were used in a crossover design with a washout period of two weeks to analyze the behaviour of CFT. Ceftiofur concentrations in the plasma were determined by HPLC with UV detector. The pharmacokinetics of CFT was analyzed using non- compartmental analysis. Following IV injection, CFT elimination half-life (t1/2β) was 2.43 h, volume of distribution at steady state (Vdss) was 0.63 L/kg, and total body clearance (Cl) was 0.24 L/h/kg. Following a single intramuscular (IM) injection of CFT at the same dose, the drug was quickly absorbed into the bloodstream with an absorption half- life (t1/2ab) of 0.31 h. The maximum concentration of the drug in the plasma (Cmax) was 2.85 μg/mL and reached at a time (Tmax) of 0.57 h after injection and the bioavailability (F) of CFT was 96.25%. The results of the study revealed that CFT was absorbed rapidly and showed high bioavailability when administered by IM route. This suggests that CFT has a promising disposition in chickens, and its use could help determine the best dosage regimens for effective eradication of various infections in chickens

Cell Hierarchy and Lineage Commitment in the Bovine Mammary Gland

The bovine mammary gland is a favorable organ for studying mammary cell hierarchy due to its robust milk-production capabilities that reflect the adaptation of its cell populations to extensive expansion and differentiation. It also shares basic characteristics with the human breast, and identification of its cell composition may broaden our understanding of the diversity in cell hierarchy among mammals. Here, Lin− epithelial cells were sorted according to expression of CD24 and CD49f into four populations: CD24medCD49fpos (putative stem cells, puStm), CD24negCD49fpos (Basal), CD24highCD49fneg (putative progenitors, puPgt) and CD24medCD49fneg (luminal, Lum). These populations maintained differential gene expression of lineage markers and markers of stem cells and luminal progenitors. Of note was the high expression of Stat5a in the puPgt cells, and of Notch1, Delta1, Jagged1 and Hey1 in the puStm and Basal populations. Cultured puStm and Basal cells formed lineage-restricted basal or luminal clones and after re-sorting, colonies that preserved a duct-like alignment of epithelial layers. In contrast, puPgt and Lum cells generated only luminal clones and unorganized colonies. Under non-adherent culture conditions, the puPgt and puStm populations generated significantly more floating colonies. The increase in cell number during culture provides a measure of propagation potential, which was highest for the puStm cells. Taken together, these analyses position puStm cells at the top of the cell hierarchy and denote the presence of both bi-potent and luminally restricted progenitors. In addition, a population of differentiated luminal cells was marked. Finally, combining ALDH activity with cell-surface marker analyses defined a small subpopulation that is potentially stem cell- enriched

Helal A. Immunohistochemical studies on the bovine lactating mammary gland (Bos taurus

a b s t r a c t The study aimed to evaluate the validity of immunohistochemistry in the differential labeling of the diverse components of the lactating mammary gland. Paraffin-embedded sections of lactating bovine mammary glands were stained by conventional and histochemical techniques. Primary antibodies against S100, alpha smooth muscle actin (␣-SMA), connexin-43 (Cx43), cytokeratin-14 (Ck14), galactosyltransferase (GalTase), angiotensin converting enzyme (ACE) and vascular endothelial growth factor (VEGF) were applied on paraffin sections. Strong cytoplasmic and nuclear S100 immunoreactivity was mainly expressed by alveolar epithelium and to a lesser variable extent by ductal epithelium. The Golgi zone of the epithelial cells expressed strong GalTase immunostaining. Myoepithelial cells displayed a strong immunostaining for ␣-SMA, Cx43 and Ck14, but not for S100. Vascular endothelium showed a moderate (for VEGF) to strong (for ACE) immunostaining. The presence of VEGF-immunoreactive mast cells within the interstitium may reflect their functional significance in angiogenesis, vascular permeability and migration of mononuclear leukocytes, suggesting their regulatory role in the secretory and immunological functions of the mammary glands

The antioxidant responses of gills, intestines and livers and blood immunity of common carp (Cyprinus carpio) exposed to salinity and temperature stressors

Aquaculture activity is affected by various environmental factors, including water salinity and high temperatures. The present study investigated the impact of using varying water salinity (0, 5, 10, 15 and 20 ppt) on the growth behavior, immune responses and antioxidative responses of common carp. Fish were raised under optimal conditions except for water salinity for 8 weeks; fish were then subjected to high-temperature stress (32 °C) for 48 h. The results indicated a reduced final weight (FBW), weight gain (WG), specific growth rate (SGR), condition factor (CF), feed intake and feed efficiency ratio (FER) in common carp reared in 15 and 20 ppt (p \u3c 0.05). The lowest FBW, WG, SGR, CF, feed intake and FER values were observed in fish reared in 20 ppt water salinity (p \u3c 0.05). In gills, the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were markedly decreased, but malondialdehyde (MDA) levels increased in fish challenged with 15 and 20 ppt before they were subjected to heat stress (p \u3c 0.05). After heat stress, the SOD, CAT and GPx were decreased, and the MDA increased in fish reared in varying salinity levels (p \u3c 0.05). Before heat stress, the intestinal SOD, CAT and GPx markers were decreased by 15 and 20 ppt, while the MDA level was increased by 15 and 20 ppt (p \u3c 0.05). Generally, heat stress lowered the SOD, CAT and GPx activity in the intestines and liver tissues but increased MDA levels in common carp stressed by varying salinity levels (p \u3c 0.05). The most decreased lysozyme activity, SOD, CAT and GPx and increased MDA levels were observed in common carp exposed to 20 ppt before and after heat stress (p \u3c 0.05). After heat stress, fish exposed to 15 and 20 ppt had lower NBT than the remaining groups, and fish exposed to 20 ppt had the lowest values (p \u3c 0.05). Overall, the heat stress markedly suppressed the antioxidant and immune responses of common carp reared in hypersalinity conditions