Quantitative changes in the normal and apoptotic thymocytes of pigs treated with anabolic doses of the beta2 adrenergic agonist clenbuterol

Although the beta2 adrenergic agonists have been seen to have important effects on the mechanisms regulating the development and death of T-cells in the thymus, the side-effects on the immune system of anabolic treatments of these substances have hardly been considered. In order to evaluate the effects exerted by the beta2 adrenergic agonist clenbuterol on the thymocyte population, the thymus of eight pigs treated with anabolic doses of this substance was studied by morphometric methods, regarding apoptotic (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive) and normal (TUNEL-negative) cells. The thymus of another eight pigs fed without clenbuterol served as a control. The clenbuterol treatment had a clear effect on the thymocyte size, decreasing their mean nuclear area. The T-cell apoptosis index was also affected by the clenbuterol, significantly increasing the apoptosis percentage in the treated group with respect to the control. In the light of our results, the clenbuterol induced thymocyte apoptosis throughout the thymus and caused morphometric changes in the thymocyte population, which was in line with the immunosuppressive role attributed to other beta2 adrenergic agonists.     

Influence of the beta 2-adrenergic agonist clenbuterol on insulin-stimulated lipogenesis in mouse adipocytes

Growing mice fed the beta 2-adrenergic agonist clenbuterol (CB; 20ppm) had increased rate of growth and altered composition of gain (greater protein and less fat). Adipocytes prepared from the epididymal fat pads of treated and untreated mice were used to examine the influence of CB on lipid metabolism. Using cells from untreated mice, CB stimulated lipolysis to an equivalent maximum rate as epinephrine (EPI), but CB was far less potent (EC50 (microM); CB = 5, EPI = 0.2). Both CB and EPI inhibited insulin-stimulated lipogenesis over the physiological range of insulin concentrations. This inhibition was expressed as a dose-dependent decrease in tissue sensitivity to insulin and a decrease in maximal lipogenic capacity. Inhibition of maximal rate, but not of insulin sensitivity, could be stimulated by the addition of palmitate without EPI or CB. Adipocytes isolated from CB-treated mice did not differ from controls in sensitivity to insulin or in activity of fatty acid synthetase. Increased lipolysis and reduced lipogenesis as observed in vitro with CB are consistent with reduced fat accretion in CB-treated mice. However, the absence of detectable changes in adipocyte lipogenesis from CB-fed mice leaves open the question of the relevance of altered lipid metabolism to the observed changes in body composition.     

Use of a beta 2-adrenergic stimulant (clenbuterol) for eliminating night-time calving.

To determine whether beta 2-adrenergic stimulant (clenbuterol) would exhibit beneficial effects upon the elimination of night-time calving, 42 Holstein Friesian cows and heifers at the first stage of labour were injected intramuscularly twice with clenbuterol; 300 micrograms at 18:00 and 210 micrograms at 22:00. Other 26 cows and heifers of the same breed at the first stage of labour at 18:00 were not treated and served as controls. All of the treated animals, but one, calved after 5:00 in the next morning, showing a peak of parturition between 5:00 and 10:00. In contrast to this, 42% of the control animals calved at night between 22:00 and 5:00. Thus, the double administrations of clenbuterol were shown to be effective for eliminating night-time calving. The cows treated with clenbuterol tended to show lower incidences of dystocia and retained placenta and a higher first insemination conception rate than the control animals. No adverse effects of the treatment on the viability of newborn calves and milk yields were observed.     

Morphological and quantitative study of the Leydig cells of pigs fed with anabolic doses of clenbuterol

The effects of clenbuterol administered at anabolic doses on the testicular interstitium were studied in 30 pigs allocated to three experimental groups. The diet of two groups was supplemented with clenbuterol (Clb) (1 ppm), but whereas in the Clb+ group the treatment was given until slaughter (treatment period: 3 months), in the Clb- group the clenbuterol was withdrawn 2 weeks before slaughter (treatment period: 2-5 months); in the control group, the pigs were fed without clenbuterol. For histological procedures, a fractional sampling scheme was applied and routine techniques for light and transmission electron microscopy were used. The results of subjective morphology and morphometrics showed slight differences between the treated and the control groups. Conversely, the stereological results identified a prominent hyperplasia of the Leydig cells and ultrastructural analysis of these cells revealed a conspicuous increase in the organelles related to testosterone production, suggesting a functional activation of the interstitial cells in response to the clenbuterol treatment.     

Quantitative modification of the testicular structure in pigs fed with anabolic doses of clenbuterol

Morphological and structural data of the testes of thirty male pigs were recorded in order to evaluate the effects of an anabolic clenbuterol treatment. Pigs aged 6 months were randomly allocated to one of three experimental groups. In two treated groups, the animals were fed with anabolic doses of clenbuterol (1 ppm); in the CLB group (n = 10) clenbuterol was given until they were slaughtered (treatment period = 3 months) whereas in the CLBW group (n = 10) the clenbuterol was withdrawn two weeks before slaughter (treatment period = 2.5 months); clenbuterol was not given to the pigs of the control group (n = 10). Stereological estimations of the tissular volume fraction and tubular volume density were applied to quantify the structural constituents of the testes. The results showed an increased volume fraction of the testicular interstitium especially in the Leydig cell population, as a result of the clenbuterol treatment. The increase in the nuclear volume fraction of the Leydig cells was the more persistent change in the variations recorded in both treated groups with respect to the control. A regression of the seminal epithelium was also recorded in the treated animals. The rest of the structural parameters were closer to the normal figures in the CLBW group, suggesting a recovery of the testicular structure when clenbuterol was withdrawn.     

Fasciola hepatica: a light and electron microscope study of sustentacular tissue and heterophagy in the testis

In order to investigate cytolytic activity in the testis of Fasciola hepatica, flukes belonging to several different triclabendazole (TCBZ)-sensitive and TCBZ-resistant isolates, and wild-type flukes from field infections, were studied by light and electron microscopy with a view to identifying sites of heterophagy and macromolecular hydrolysis. At the periphery of the testis tubules in all the flukes examined, large euchromatic nuclei, each bearing a prominent nucleolus, were seen. These were invested with a thin cytoplasmic layer, extensions of which partially enveloped and probably supported the neighbouring spermatogonia. No lateral cell boundaries were identified in this tissue, possibly indicating syncytial organisation. The tissue, considered to be analogous to Sertoli cells in vertebrate testis, was identified as sustentacular tissue. It displayed cytoplasmic features consistent with protein/glycoprotein synthesis (through a granular endoplasmic reticulum-Golgi mediated mechanism) and intracellular digestion/heterophagy (through a lysosomal system). The intracytoplasmic hydrolytic activity of the sustentacular tissue probably serves to scavenge effete cells and cytoplasmic debris, to recycle useful molecules, to promote spermatozoon maturation and possibly to aid osmoregulation within the tubules. Certain protein-containing macromolecules synthesised in the sustentacular tissue may contribute to the seminiferous fluid, or have pheromonal activity. The presence of numerous mitochondria and abundant smooth endoplasmic reticulum is consistent with facilitation of inward and outward movement of micromolecular nutrients, metabolites including excretory products and water. In the sustentacular tissue of certain flukes with dysfunctional spermiogenesis, there was increased heterophagic and cytolytic scavenging activity. The cytoplasmic residual vacuoles remaining after the release of spermatids were also identified as possible sites for lysosome-mediated intracellular digestion and osmoregulation in the testis tubules of F. hepatica.     

The mucosa of the digestive tract in Micropogonias furnieri: a light and electron microscope approach

The histomorphological aspects as well as the histochemical content and distribution of glycoproteins (GPs) in the mucosa of the digestive tract of the white croaker Micropogonias furnieri were studied. The buccopharyngeal cavity and the esophagous showed a squamous stratified epithelium with mucous cells. The stomach presented three portions: cardias, fundus and pylorus. Tubular glands formed by a single type of gland cell were located along the cardias and fundus. Histochemical tests showed that the buccopharyngeal cavity and the esophagous presented the largest amount of the different types of mucosubstances. Both organs showed abundant secretory mucous cells that synthesize large quantities of neutral, sulphated and sialylated GPs. The surface epithelium in the cardias and fundus synthesized and secreted scarce sialylated and neutral GPs whereas the secretions of the apical surface were abundant. The pylorus secreted large amounts of neutral as well as sulphated and sialylated GPs. Gland cells secreted neutral GPs. The ultrastructural features of the gut cells were quite similar to those of other teleosts. The buccopharyngeal cavity and the esophagous surface epithelial cells, identified by their superficial localization, were characterized by cytoplasmic vesicles of different size. Abundant goblet cells with secretory mucous granules were also present. Gastric glands in the stomach contained just one form of cell with a fine structure similar to cells that secrete pepsinogen.     

Spontaneous Pancreatic beta cell tumor in the rat. A light and electron microscopic study.

Fine structural study of a pancreatic neoplasm found at autopsy of a 27-month-old female Long-Evans rat showed that the tumor consisted of, and derived from beta cells of the islets of Langerhans.     

The spleen of Zaedyus pichiy, (Mammalia,Dasypodidae): a light and electron microscopic study

Armadillos are ancient mammals important as models for biomedical, evolutive and ecological studies, because they have adaptive and primitive morpho-physiological characteristics. In this work we study the splenic microarchitecture of the 'Patagonian Piche', Zaedyus pichiy, as an attempt to understand the relationship between the organizational plan of the organ and the particular features of this species. The organ samples were classically processed for light and electron microscopic study. The microanatomy of the organ as well as its different cell types are studied. The spleen is non-sinusoidal, with the typical arrangement for storage functions. White pulp is well defined. Red pulp is a meshwork of circulating, immunocompetent and haemopoietic cells. The general structure of the organ agrees with the semi-fossorial habit and the adaptability of the species. Comparative aspects with other armadillos or other less specialized groups are discussed. Persistence of haemopoietic spleen activity in the adult suggests the existence of specific inductive functions of the stroma. Better knowledge of spleen structure and function in ancient mammals may give important information about their phylogeny.     

Equine synovial tendon sheaths and bursae: a transmission electron microscope study.

A transmission electron microscope study was undertaken to investigate the details of the synovial tendon sheath and bursal lining in horses. The lining cells appeared to be fibroblasts and were buried in a finely granular ground substance. Generally these cells had poor cytoplasmic organelles, sparse short profiles of rough endoplasmic reticulum (RER), few scattered ribosomes and, occasionally, a poorly developed Golgi complex. However, a few lining cells appeared more active, having pronounced RER with dilated cisternane. The surface of the lining cells, particularly those with dilated RER, had filipodia of different shapes, which in some instances appeared to surround a detached matrix. This feature might indicate that these cells are capable of phagocytosis. In young animals, the lining was more or less akin to that of the adult. However, the lining cells were virtually indistinguishable from the fibroblasts in the subconnective tissue.     

Manipulation of growth in pigs through treatment of the neonate with clenbuterol and somatotropin

Neonatal pigs were treated with lipolytic agents to determine whether this would cause a long-term decrease in their ability to deposit fat, with a consequent increase in muscle growth and feed efficiency. Groups of 25 female piglets were given clenbuterol (100 microg/kg BW), porcine somatotropin (pST; 100 microg/kg BW), pST plus clenbuterol, or saline injections from 3 d to 40 d of age. Five piglets from each group were then slaughtered to determine body composition. Clenbuterol and pST both increased ADG up to weaning when given separately (24%, P < 0.05; 20%, P < 0.1 respectively) but did not reduce fat deposition. In contrast, pigs given clenbuterol plus pST showed no increase in ADG and a 41% reduction in carcass fat (P < 0.05). Clenbuterol caused a marked decrease in beta2-adrenoceptor density in porcine adipose tissue (P < 0.001) and skeletal muscle (P < 0.01). This effect was attenuated by concurrent pST treatment, which helps to explain the synergistic effect of these drugs on fat deposition. Once the drugs were withdrawn at 40 d, the anabolic effect of pST gradually disappeared, so that the live weight of pST-treated and control pigs was identical at 168 d. Clenbuterol withdrawal caused the rapid loss of extra weight gained, plus an additional 4 to 5 kg live weight that was never recovered. During the 4-wk finishing period there was an increase in feed intake in pigs that had previously undergone treatment with pST (23%, P < 0.1), with no increase in ADG, and so feed efficiency was impaired (P < 0.05). Pigs that were treated with pST plus clenbuterol showed no marked increase in feed intake during this period. Carcasses from clenbuterol-treated pigs tended to be leaner at 168 d, but there was no long-term effect of pST or the combined treatment on carcass composition. Overall, the treatment of neonatal pigs with repartitioning agents was counter-productive, due to the withdrawal effects of the beta-adrenefgic agonist and the delayed long-term effect of pST on feed intake.     

Transforming growth factor type beta (TGF-beta) and adipogenesis in pigs

The present study was performed on s.c. adipose tissue of fetal pigs at 35 to 110 d of gestation to examine the distribution of TGF-beta-positive cells, to localize TGF-beta immunoreactivity at the cellular level using electron microscopy (EM), and to determine the effect of TGF-beta on primary cultures of pig adipose tissue cells. Tissues for EM were fixed and embedded in LR white resin. Sections then were incubated with a polyclonal antibody specific for TGF-beta and TGF-beta was located using 20 nm colloidal gold conjugated second antibody. Tissues were fixed and embedded in paraffin for localization of TGF-beta at the light microscope (LM) level. Tissues were incubated with anti-TGF-beta followed by localization using biotinylated second antibody. Using LM, only a few cells stained positively for TGF-beta within developing blood vessels at 35 d. By 50 d, more TGF-beta-positive cells were associated with forming capillary networks. Between 70 d and 110 d, positively stained adipocytes usually were clustered around blood vessels. Cells surrounding hair follicles stained positive for TGF-beta between 90 to 110 d. Electron microscopy revealed TGF-beta labeling within fat cells. Fibroblasts and endothelial cells did not exhibit TGF-beta immunoreactivity. The addition of TGF-beta to primary cultures of s.c. adipose tissue cells from newborn pigs prevented lipid filling in fat cells. This effect was dose-dependent, with half-maximal inhibition occurring at 3 pM maximum inhibition occurred at 40 pM. These results indicate that TGF-beta may regulate angiogenic activity and lipid filling in s.c. adipose tissue of fetal pigs. Although TGF-beta was present in adipocytes and in cells associated with developing capillary networks, the physiological role of TGF-beta during early adipose tissue development is not known.     

Microscopic anatomy of the oesophagus in the southern white-breasted hedgehog (Erinaceus concolor) a histochemical,stereological, and scanning electron microscope study

The present work was designed to investigate the microscopic structure of the oesophagus in the southern white-breasted hedgehog (Erinaceus concolor) using histochemical staining, Scanning electron microscope (SEM), and stereological procedures. Four adult males were included in our study. Serial sections of the entire length of the oesophagus were stained with aldehyde fuchsin, alcian blue (pH 2.5), Periodic acid Schiff (PAS), and Masson's trichrome. Then, the total volume of the oesophagus, and the total volume of its different layers, were estimated using Cavalieri's principle. The oesophageal epithelium was a non-keratinized stratified squamous type. Muscularis mucosa was present as a thick layer between lamina propria and submucosa and its thickness was increased toward the stomach. Tunica submucosa was a loose connective tissue containing an oesophageal gland with PAS-positive and AB-positive reactions throughout the submucosa and become denser toward the stomach. The tunica muscularis consisted of two distinct striated muscle layers, and its thickness was decreased toward the stomach. On SEM images, the cervical and thoracic oesophagus showed shallow folding, while the abdominal part had deeper folds. The present findings indicate that the histological properties of the oesophagus in southern white-breasted hedgehogs have slight similarities with rodents and considering its epithelium, submucosal glands and tunica muscularis more resemble with dog oesophagus. The obtained results may be quite helpful to improve the current knowledge of the histophysiology of the hedgehog gastrointestinal tract as a member of eulipotyplan mammals and as a pet for biologists and veterinarians.     

A light and electron microscopic study of the leukocytes of channel catfish (Ictalurus punctatus)

A light and electron microscopic study of channel catfish (Ictalurus punctatus) white blood cells was carried out. The white blood cells of channel catfish consist of lymphocytes, neutrophils, thrombocytes and monocytes. A fifth cell type is illustrated and described which could not be characterized. These leukocytes are characterized and compared with their mammalian counterparts.     

Polymorphism at the ovine beta3-adrenergic receptor locus (ADRB3) and its association with lamb mortality

The beta3-adrenergic receptors (ADRB3) are guanine nucleotide-binding protein (G-protein)-coupled receptors predominantly found on the surface of adipocytes. They are major mediators of the lipolytic and thermogenic effects of high catecholamine concentrations, in particular norepinephrine. Recently, variation in the ovine beta3-adrenergic receptor gene (ADRB3) has been associated with lamb survival in Merino sheep. In this study, PCR-single strand conformational polymorphism analysis of part of the ADRB3 intron was used to genotype 13,420 lambs (Borderdale, n = 351; Corriedale, n = 4,260; Coopworth, n = 1,225; Dorset Down, n = 663; Dorset Down x Coopworth, n = 264; Hampshire, n = 230; Merino, n = 4,488; Merino x Coopworth, n = 329; Merino x Polwarth, n = 226; Polwarth, n = 928; Poll Dorset, n = 241; and Suffolk, n = 215) born on 22 farms throughout the South Island of New Zealand. Univariate and multivariate odds ratios calculated for each of the 8 alleles revealed weak to moderate associations of the A and E alleles with cold survival and alleles C and F with cold-related mortality, and a strong association of the D allele with cold-related mortality and total mortality. This variation at the ADRB3 locus can possibly be exploited as a gene marker to increase selection accuracy when breeding for more cold-tolerant lambs.     

The novel polymorphism of the beta 3-adrenergic receptor gene and its distribution in domestic pigs and wild boars in Asia

The beta 3‐adrenergic receptor (ADRB3) is a G protein‐coupled receptor that is involved in regulating energy homeostasis. We have studied DNA sequences of porcine ADRB3 to find candidate genetic polymorphisms for economically important growth and performance traits in pigs. Five novel haplotypes derived from the three In/dels and 44 SNPs were identified among domestic pigs and wild boars. Three of them encode non‐synonymous amino acid sequences by five missense polymorphisms and a frameshift by a thymine insertion. The amino acid polymorphic sites were distributed as follows: one substitution was in extracellular loop 1, three substitutions were in intracellular loop3 and one substitution and the deletion of two amino acids were at the carboxyl‐terminal. There was no polymorphism in the transmembrane domains. In addition, we surveyed the allelic frequency of the thymine insertion that cause frameshift in South‐east Asian local pigs, including some commercial breeds and wild boars. This thymine insertion was distributed widely in the domestic pigs and wild boars. The frequencies of this allele were relatively low in Western improved breeds, while they were very common in Asian breeds and wild boars in Asia. This result indicates that this insertion originally occurred in ancient Asian wild boars and then circulated among Asian domestic pigs. This allele also spread over Western breeds, probably through the introgression of Asian pigs into European stocks during the 18th and 19th centuries.     

Development of the areola in the early placenta of the one-humped camel (Camelus dromedarius): a light, scanning and transmission electron microscopical study

This study aimed to elucidate development of the areola in the early dromedary placenta in comparison with that of the pig and mare. Placental tissues from 25 pregnant camels were obtained from Cairo abattoir and prepared for light, scanning and transmission electron microscopy by routine methods. Vascular casts were made by injection of 4 : 1 liquid plastic mixture of mercox and methylmethacrylate. Areolar formation was first observed at 4.5 cm curved-crown-rump CVR length, while by 5-9 cm CVR length, the endometrial surface was uneven and studded with numerous uterine gland openings, where corresponding foetal areolae were barely detectable and the foetal areolar cells were of variable appearance and covered with long microvilli. At 10-13 cm CVR length the uterine gland openings developed irregular folds and the maternal areolar cells showed numerous apical blebs. At 14-29 cm CVR length the foetal areolae showed a great increase in height at the expense of their width. At 30-34 cm (CVR) length the maternal areolae appeared discoid and sharply demarcated from the surrounding inter-areolar tissues and the foetal areolae were rounded to irregular in shape with well-developed areolar rims. The vascular casts showed a widely meshed capillary network on the maternal areola, connecting with the pre- and post-capillary vessels, whereas the foetal side showed a relatively dense capillary meshwork. These studies indicate that the areola in the placenta of the one-humped camel is of the regular type like in the pig, and is poorly vascularized.     

Beta 3-adrenergic agonist up-regulates uncoupling proteins 2 and 3 in skeletal muscle of the mouse

Chronic stimulation of the beta3-adrenergic receptor (AR) in obese animals resulted in a reduced adiposity associated with an increased expression of thermogenic uncoupling protein (UCP)1 in adipose tissues. In this study, the mRNA expression of newly cloned UCP isoforms (UCP2 and UCP3) were examined in obese yellow KK and C57BL control mice. UCP2 mRNA was found in all tissues examined, with higher levels in adipose tissues and skeletal muscle of the obese mice. UCP3 mRNA was expressed in skeletal muscle, heart and brown adipose tissue similarly in the two mouse strains. Daily injection of a selective beta3-adrenergic agonist, CL316,243 (0.1 mg/kg), for 10 days resulted in a marked reduction of white fat pad weight and 1.8-4.8-fold increase in the mRNA levels of UCP2 and UCP3 in skeletal muscle of obese mice. No noticeable change in the UCP2 and 3 mRNA levels was found in brown and white adipose tissues. It was also found that CL316,243 injection produced a marked and sustained elevation of the plasma free fatty acid level. These results, together with our previous findings of the fatty acid-induced UCP expression in a myocyte cell line in vitro, suggest that the beta3-AR agonist-induced UCP expression in skeletal muscle may be mediated through the elevated plasma free fatty acids. It was also suggested that anti-obesity effect of beta3-AR agonists is attributable to increased thermogenesis not only by UCP1 but also by UCP2 and UCP3.