Effects of ecofriendly synthesized calcium nanoparticles with biocompatible Sargassum latifolium algae extract supplementation on egg quality and scanning electron microscopy images of the eggshell of aged laying hens.

In the present study, 200 Brown commercial egg-type layers (60 wk old) were used to study the effects of different levels of ecofriendly synthesis of calcium (Ca) nanoparticles (0.0, 0.50, 1.0, and 1.5 g/kg diet) with biocompatible Sargassum latifolium algae extract (SL-CaNps) on exterior egg quality traits, electronic microscopic view of eggshells, Ca and phosphorus (P) retention, serum Ca and P concentrations, and the histology of the uterus. Hens fed with dietary SL-CaNps powder had higher egg weight and shell weight % values than those of the control group. All SL-CaNps treatment groups had the greatest values of shell weight per unit surface area and shell thickness. Dietary supplementation of SL-CaNps at graded levels up to 1.5 g/kg diet had higher serum Ca and inorganic P levels than that of the control. Laying hens fed with SL-CaNps-added diets had beneficial effects on shell ultrastructure in terms of well-developed palisade and mammillary layers. The numbers of apical cells along the branched tubular gland were greater in SL-CaNps-treated groups than those of control. Conclusively, supplementing SL-CaNps powder up to 1.5 g/kg to the diet of laying hens improved eggshell thickness, shell weight% and shell weight per unit surface and has no adverse effect on their eggshell quality or electronic microscopic view of their eggshell.

The effectiveness of a dietary direct-fed microbial and mannan oligosaccharide on ultrastructural changes of intestinal mucosa of turkey poults infected with Salmonella and Campylobacter.

Salmonella and Campylobacter are considered major public health burdens worldwide, and poultry are known to be one of the main reservoirs for these zoonotic pathogens. This study was conducted to evaluate the effect of a commercial probiotic or direct-fed microbial (DFM) Calsporin (CSP), and prebiotic or mannan oligosaccharide (MOS) (IMW50) on ultrastructural changes and the villous integrity of intestinal mucosa in turkey poults challenged with Salmonella and Campylobacter. A 21-day battery cage study was conducted using 4 dietary treatments including a basal diet (corn and soybean-based) nonsupplemented and uninfected as a negative control (NC); basal diet supplemented with 0.05% DFM (CSP); basal diet supplemented with 0.05% MOS (IMW50); and basal diet supplemented with 0.05% mixture of DFM and MOS at equal proportions. Female large white turkey poults aged 336 days were obtained from a local commercial hatchery and randomly distributed in electrically heated battery cages with 12 treatments of 4 replicates per treatment containing 7 poults per pen. The first 16 pens were not infected with bacteria, poults in pens 17-32 were orally challenged at day 7 with 105 cfu Salmonella Heidelberg, and the poults in pens 33-48 were orally challenged at day 7 with 105 cfu Campylobacter jejuni. Feed and water were provided ad libitum throughout the study. At day 21, ileal tissue samples from 1 bird per cage were collected for intestinal integrity and ultrastructural examination by scanning and electron microscopy. DFM and MOS supplementation was effective in both challenged and nonchallenged (not infected with Salmonella and Campylobacter) birds. Goblet cells and mucus were increased, with the presence of large numbers of segmented filamentous bacteria in DFM- and MOS-supplemented groups compared with birds in control treatments. The number and size of villi were reduced in poults exposed to Salmonella and Campylobacter. Results show that CSP and IMW50 provide protection of ileal mucosal integrity in poults exposed to Salmonella or Campylobacter.

Ultrastructure changes induced by the phloroglucinol derivative agrimol G isolated from Leucosidea sericea in Haemonchus contortus.

Plant extracts used for the treatment of helminth infections in sheep are an alternative to chemical anthelmintic drugs. Previous studies have reported the anthelmintic activity of acetone leaf extracts of Leucosidea sericea. For this study, we evaluate the ultrastructure changes induced by the acetone leaf extract of L. sericea and the component agrimol G (AG) that was isolated for the first time on adult haemonchus parasites. Adult haemonchus parasites harvested from sheep were incubated with the plant extract and AG for 3 h and evaluated by both scanning and transmission electron microscopy in comparison and in combination with albendazole or ivermectin. In all cases the method of evaluation shows ultrastructural changes, with albendazole inducing mitochondrial damage and ivermectin inducing muscle degeneration, both as previously described. Incubation with the plant extract and AG resulted in the formation of numerous non-membrane bound multi-vesicular like bodies and evenly spread disruptions/erosion in the epicuticle. Combining AG with ivermectin or albendazole resulted in an absence of effect of AG. Based on the structural changes induced by AG, together with the absence of an effect in combination with ivermectin and albendazole would suggest a disrupted microtubular network. The latter does however require biochemical confirmation.

Effect of copper nanoparticles on the mineral content of tissues and droppings, and growth of chickens.

We hypothesised that copper nanoparticles (NanoCu), because of their high physicochemical reactivity and bioavailability, could be used in much smaller quantities than bulk Cu, consequently reducing excretion of Cu into the environment. The objective of the study was to evaluate the effects of various levels of NanoCu on the development and growth of broiler chickens, in order to establish an optimum level of NanoCu dietary supplementation. Broiler chickens were randomly divided into five groups of 10 birds each. The control group received 7.5 mg Cu/kg feed (standard level) as CuSO4, while groups fed with complexes of NanoCu and starch received 25%, 50%, 75% and 100% of the standard level of Cu used in the control group. Chicken growth and excretion of Cu, Fe and Zn were measured during the growth period from d 7 to 42. At d 42, the slaughter characteristics, the content of Cu, Fe and Zn in the breast muscle and liver, and the oxidative status were analysed. The results indicate that using NanoCu can reduce the standard level of Cu from CuSO4 supplementation by 75% without jeopardising animal growth, and at the same time significantly decreasing Cu excretion into the environment.

Evaluation of apoptosis in long-term culture of vitrified mouse whole ovaries.

The purpose of the present study was to investigate the development of follicles and incidence of apoptosis in vitrified neonatal mouse ovaries cultured in vitro in the presence of leukemia inhibitory factor (LIF). The vitrified and non-vitrified ovaries of 1-week-old mouse were cultured in the presence or absence of LIF for 7 days. At the beginning and at the end of culture period in each ovary of all groups of study the mean area and the development of ovarian follicles were analyzed; moreover, the incidence of apoptosis was assessed by transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method, DNA laddering and caspase-3/7 activity technique. The hormonal assay was done on the conditioned media collected during culture period. The proportion of preantral follicles and the levels of hormones increased in all cultured groups and it was significantly higher in LIF treated groups than in their control (P<0.001). The ultrastructural characteristics of cell death, DNA fragmentation and TUNEL positive signals were prominent in vitrified cultured ovaries. The level of caspase-3/7 activity was higher in vitrified cultured ovaries. LIF supplementation during 7 days of culture appeared to significantly preserve cells function and increase the follicular development of both vitrified and non-vitrified ovaries.

The hepatoprotective effect of sea buckthorn (Hippophae rhamnoides) berries on induced aflatoxin B1 poisoning in chickens 1.

The leaves and berries of sea buckthorn (SB; Hippophae rhamnoides; family Elaeagnaceae) are medically claimed as having phytoantioxidant, antiinflammatory, and anticancerous properties in humans. This study evaluated the hepatoprotective activity of oil from SB berries against toxicity induced by aflatoxin B1 (AFB1) in broiler chickens. The toxicity of AFB1 led to lower total serum proteins and specifically reduced albumin (P < 0.001). Serum aspartate aminotransferase increased from 191.14 ± 11.56 to 218.80 ± 13.68 (P < 0.001). When chickens were simultaneously dosed with AFB1 and an extract of SB berries, subsequent histology of the liver showed a significant reduction of necrosis and fatty formation compared with chickens treated with AFB1 alone. Immunohistochemical results indicated that COX2, Bcl-2, and p53 were highly expressed in the liver of AFB1-treated chickens and their expression was significantly reduced by SB oil supplementation. The levels of AFB1 residues in chickens livers were significantly reduced by SB oil from 460.92 ± 6.2 ng/mL in the AFB1 group to 15.59 ± 6.1 ng/mL in the AFB1 and SB oil group. These findings suggest that SB oil has a potent hepatoprotective activity, reducing the concentration of aflatoxins in liver and diminishing their adverse effects.

Enhanced intestinal epithelial barrier health status on European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides.

The study assesses the effects of dietary mannan oligosaccharides (MOS) in European sea bass (Dicentrarchus labrax) posterior intestinal lipid class composition and its possible relation to the potential prostaglandins production and Gut Associated Lymphoid Tissue (GALT) stimulation. Fish were fed 4 g kg(-1) MOS (Bio-Mos(®) Aquagrade, Alltech, Inc., USA) for eight weeks. Fish fed MOS presented higher (P ≤ 0.05) weight gain, total length, and specific and relative growth rates than fish fed the control diet. Stimulated posterior gut of fish fed MOS showed higher (P ≤ 0.05) prostaglandins production than fish fed the control diet. Lipid class analyses of posterior gut revealed a reduction (P ≤ 0.05) in the neutral lipid fraction in fish fed MOS compared to fish fed the control diet, particularly due to a reduction (P ≤ 0.05) in triacylglycerols content. The polar lipid fraction increased (P ≤ 0.05) in fish fed MOS compared to fish fed the control diet, mainly due to an increase (P ≤ 0.05) in phosphatidylethanolamine and phosphatidylcoline contents. Light microscopy of posterior gut revealed increased number or goblet cells as well as higher level of infiltrated eosinophilic granulocytes for fish fed MOS. Transmission electron microscopy qualitative observations revealed a better preserved cytoarchitecture of the intestinal epithelial barrier in the posterior gut of fish fed MOS. Posterior gut of fish fed MOS presented more densely packed non-damaged enterocytes, better preserved tight junctions structure, healthier and more organized microvilli, and a higher presence of infiltrated lymphocytes and granulocytes compared fish fed the control diet. The present study indicates that dietary MOS enhances European sea bass posterior gut epithelial defense by increasing membrane polar lipids content in relation to a stimulation of the eicosanoid cascade and GALT, promoting posterior gut health status.

Changes in intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis in gilthead sea bream (Sparus aurata L.) specimens.

Changes produced in gilthead sea bream (Sparus aurata L.) intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis have been studied. Gilthead sea bream specimens were fed diets containing 0 (control), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)), or B. subtilis + inulin (10(7) cfu g(-1) + 10 g kg(-1)) for four weeks. Curiously, fish fed the experimental diets (inulin, B. subtilis, or B. subtilis + inulin) showed the same morphological alterations when studied by light and electron microscopy, while significant differences in the signs of intestinal damage were detected by the morphometric study. All of the observed alterations were present only in the gut mucosa, and intestinal morphometric study revealed no effect of inulin or B. subtilis on the intestinal absorptive area. Furthermore, experimental diets cause important alterations in the intestinal microbiota by significantly decreasing bacterial diversity, as demonstrated by the specific richness, Shannon, and range-weighted richness indices. The observed alterations demonstrate that fish fed experimental diets had different signs of gut oedema and inflammation that could compromise their body homeostasis, which is mainly maintained by the epithelial lining of the gastrointestinal tract. To our knowledge, this is the first in vivo study regarding the implications of the use of synbiotics (conjunction of probiotics and prebiotics) on fish gut morphology and microbiota.

Interaction between melatonin and follicle-stimulating hormone promotes in vitro development of caprine preantral follicles.

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM(+)) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM(+) alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM(+) alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.

Oxidative status and histological changes in sea bass larvae muscle in response to high dietary content of docosahexaenoic acid DHA.

In previous studies, we observed dystrophic alterations in muscle of 48-day-old sea bass fed imbalanced docosahexaenoic acid (DHA) and vitamin E diets. To understand the whole pathological process associated with oxidative stress, a histological study was performed by feeding 14-day-old sea bass larvae with microdiets containing different ratios of DHA/vitamin E (1/150, 5/150 and 5/300) for a period of 21 days. Larvae fed diet 1/150 showed no lesions in contrast to larvae fed diets 5/150 and 5/300 where the highest incidence of muscle lesions and thiobarbituric acid reactive substances (TBARS) content was observed. Semithin sections revealed focal lesions consisting of degenerated fibres with hypercontracted myofilaments and extensive sarcoplasm vacuolization affecting both red and white muscle. Ultrathin sections of degenerating muscle fibres showed diffuse dilatation of sarcoplasmic reticulum, disorganized myofilaments and autophagic vacuoles containing myelin figures and dense bodies. Additionally, some macrophages were observed among injured fibres as numerous satellite cells. Results from the study agree with those obtained from previous work, proving the pathological potential of free radicals in sea bass larvae musculature. Moreover, high vitamin E inclusion could not completely protect cell membranes from free radicals action.

Pathology of ochratoxin A-induced nephrotoxicity in Japanese quail and its protection by sea buckthorn (Hippophae rhamnoides L.).

The present study was designed to study the protective effect of sea buckthorn (SBT) against renal damage induced by ochratoxin A (OTA) in Japanese quail. Day-old quail chicks were divided into six groups and fed a basal quail chick mash containing 2% SBT leaf powder (group SX), OTA at a dietary level of 3 ppm (group OX), 25 ppm L-beta-phenylalanine (Phe) plus 3 ppm OTA (group OP), 2% dietary level of SBT leaf powder plus 3 ppm OTA (group OS), SBT leaf extract at a level of 10%/L of drinking water plus 3 ppm OTA (group OSS), and a standard toxin-free feed (group CX, control) for 21 days. OTA at 3 ppm level in diet grossly revealed mild to moderate renal swelling in OX birds, and the severity was less in the case of OS, OSS, and OP birds. Microscopically, degenerative, necrotic, and inflammatory changes were observed in OX birds, but the changes were less severe in OS, OSS, and OP birds. Ultrastructural studies revealed remarkable and consistent changes in the proximal convoluted tubules (PCTs), with severe damage of mitochondria and endoplasmic reticulum in OX birds, whereas SBT-treated birds (groups OS, OSS) had mild changes in mitochondria. A moderate to marked increase in number of peroxisomes in the cytoplasm of PCTs was a consistent finding in the Phe- and SBT-treated groups kept on OTA in comparison to the group fed OTA alone. In conclusion, the inclusion of 2% SBT leaf powder in feed and SBT leaf extract in water provided partial protection against OTA-induced nephropathy in Japanese quail.

The disruption of mitochondrial metabolism and ion homeostasis in chicken hearts exposed to manganese.

Exposure to high levels of manganese (Mn) can result in cardiotoxicity in animals. However, little is known about the effect of excess Mn on poultry hearts. The aim of this study was to investigate the effect of dietary Mn on chicken cardiac injuries and the possible mechanisms of this process. In the present study, 400 fifty-day-old Hy-line brown cocks were randomly divided into four groups, and were fed either a commercial diet (containing 100mg/kg Mn) or a Mn-supplemented diet containing 600mg/kg, 900mg/kg, or 1800mg/kg Mn for 30, 60 or 90 days, respectively. Next, we examined several biomarkers of cardiac injury, including biochemical blood serum analyses, electrocardiogram assays, histological analyses, ultra-structural assays and apoptosis assays. To investigate the possible mechanisms of Mn-induced cardiotoxicity, we examined the effect of MnCl(2) on mitochondrial function and metal ion homeostasis. We found that subchronic MnCl(2) exposure induced damage in chicken hearts. Further investigations indicated that possible mechanisms for Mn-induced chicken cardiac injury included the disruption of mitochondrial metabolism and the alteration of ion homeostasis.

Epizootic myocarditis associated with encephalomyocarditis virus in a group of rhesus macaques (Macaca mulatta).

Six cases of fatal myocarditis associated with encephalomyocarditis virus occurred over a 14-month period in a group of outdoor-housed juvenile rhesus macaques. All animals were younger than 3 years of age and died or were euthanized following acute onset of dyspnea or pulmonary effusion (3 of 6) or were found dead without premonitory signs (3 of 6). Gross findings included pulmonary congestion (6 of 6), variable degrees of pleural effusion (4 of 6), multifocal pale tan foci throughout the myocardium (3 of 6), hepatomegaly and hepatic congestion (3 of 6), and pericardial effusion (1 of 6). Histologically, affected myocardium was infiltrated multifocally by lymphoplasmacytic and histiocytic inflammation admixed with necrotic and degenerate myofibers and infrequent mineralization (6 of 6). Pulmonary edema was present in all animals. Encephalomyocarditis virus was confirmed in 6 of 6 hearts by immunohistochemistry, and virus was isolated from one case by polymerase chain reaction. Sequencing of virus isolated from 1 affected animal indicated infection with a novel encephalomyocarditis virus. Encephalomyocarditis virus should be considered as a differential etiology in outbreaks of myocarditis and pulmonary edema in juvenile primates.

Effects of papaya seeds extract on the sperm characteristics of dogs.

The effect of chloroform extracts from seeds of the papaya plant (Carica papaya) on the spermatic characteristics of dogs was evaluated at doses of 50, 100 and 150 mg/kg, Groups 1, 2, and 3, respectively. Structural and ultrastructural changes in sperm cells and testicular parenchyma were also evaluated, and possible side effects were noted. Significant reductions in sperm concentration and motility were observed starting from Days 60 and 75, respectively, in all treated groups (P<0.05), but no azoospermia was noted. A mild osmotic diarrhea occurred in dogs from Group 3 (150 mg/kg), although blood variables were within the normal range of a clinically healthy dog. Arrested spermatogenesis was observed in the seminiferous tubules of all treated groups, and vacuolization and signs of Sertoli cell degeneration were detected in all treated groups, particularly in Group 3 (150 mg/kg). Selective damage to Sertoli cells induced by the extract occurred in all treated groups independently of the extract concentration. Alteration of the epididymal environment may reduce the motility of sperm cells, considering that their structure was normal. Sperm characteristics in treated animals were considered to be similar to those of sub-fertile dogs. However, these effects may be temporary, and dogs may recover normal sperm characteristics when the extract is withdrawn.

Mesenteric lymph node cells from neonates present a prominent IL-12 response to CpG oligodeoxynucleotide via an IL-15 feedback loop of amplification.

At birth, the immune system is still in development making neonates more susceptible to infections. The recognition of microbial ligands is a key step in the initiation of immune responses. It can be mimicked to stimulate the immune system by the use of synthetic ligands recognising pattern recognition receptors. In human and mouse, it has been found that neonatal cytokine responses to toll-like receptor (TLR) ligands differ in many ways from those of adults but the relevant studies have been limited to cord blood and spleen cells. In this study, we compared the responses in neonate and adult sheep to CpG oligodeoxynucleotides (ODN), a TLR9 ligand, in both a mucosal and a systemic organ. We observed that in response to CpG-ODN more IL-12 was produced by neonatal than adult sheep cells from mesenteric lymph nodes (MLN) and spleen. This higher IL-12 response was limited to the first 20 days after birth for MLN cells but persisted for a longer period for spleen cells. The major IL-12-producing cells were identified as CD14+CD11b+. These cells were poor producers of IL-12 in response to direct stimulation with CpG-ODN and required the cooperation of other MLN cells. The difference in response to CpG-ODN between neonates and adults can be attributed to both a higher proportion of CD14+CD11b+ cells in neonate lambs and their higher capacity to produce IL-15. The IL-15 increases IL-12 production by an amplifying feedback loop involving CD40.

Anti-Giardia activity of Syzygium aromaticum essential oil and eugenol: effects on growth, viability, adherence and ultrastructure.

The present work evaluates the anti-Giardia activity of Syzygium aromaticum and its major compound eugenol. The effects were evaluated on parasite growth, adherence, viability and ultrastructure. S. aromaticum essential oil (IC(50)=134 µg/ml) and eugenol (IC(50)=101 µg/ml) inhibited the growth of G. lamblia. The essential oil inhibited trophozoites adherence since the first hour of incubation and was able to kill almost 50% of the parasites population in a time dependent manner. The eugenol inhibited G. lamblia trophozoites adherence since the third hour and not induce cell lyses. The main morphological alterations were modifications on the cell shape, presence of precipitates in the cytoplasm, autophagic vesicles, internalization of flagella and ventral disc, membrane blebs, and intracellular and nuclear clearing. Taken together, our findings lead us to propose that eugenol was responsible for the anti-giardial activity of the S. aromaticum essential oil and both have potential for use as therapeutic agents against giardiasis.

Characterisation of biofilm formation by a Streptococcus suis meningitis isolate.

Biofilm formation by a strain of Streptococcus suis serotype 2 isolated from a case of meningitis in pigs was characterised. Using a polystyrene microtitre plate assay, S. suis 95-8242 produced a dense biofilm when glucose, fructose or sucrose was used as the carbohydrate source, whereas no biofilm formed in the presence of lactose. Polysaccharide production by the biofilm-forming strain was demonstrated by the Congo red agar assay. Transmission electron microscopy revealed that bacterial cells were surrounded by a thick layer of polycationic ferritin-labelled material. S. suis 95-8242 was more resistant to both penicillin G and ampicillin in biofilms than in planktonic cultures on the basis of minimal inhibitory and minimal bactericidal concentrations.

Cerebellar degeneration in cattle grazing Solanum bonariense ("Naranjillo") in Western Uruguay.

Cattle in western Uruguay that were eating Solanum bonariense developed periodic episodes of ataxia, hypermetria, hyperesthesia, head and thoracic limb extension, opisthotonus, nystagmus, and falling to the side or backward. Similar clinical signs were experimentally reproduced in cattle by administration of S. bonariense via rumen cannula at a dose of 1,024 g/kg body mass. No significant gross lesions were observed in field cases or experimentally induced cases. Spontaneous and induced histologic lesions were similar and included vacuolation, degeneration, and loss of Purkinje cells. Axonal spheroids, microcavitations, and other changes of wallerian-type degeneration in cerebellar white matter were also observed. Ultrastructural changes included increased number of electron-dense residual storage bodies in membrane-bound vesicles in affected Purkinje cells, and similar vesicles and mitochondria in axonal spheroids. No histologic lesions were detected in the other examined tissues. The Purkinje-cell swelling and vacuolation with subsequent cerebellar degeneration are suggestive of Purkinje-cell specific toxin that produces abnormal lysosome function and cell specific axonal transport. This is the first report of S. bonariense toxicity.