Relationship among growth, steroid production and immunolocalization of transforming growth factor-beta 1 in the normally developing mouse follicles cultured in vitro

This study examined the relationship among growth, steroid production and transforming growth factor-beta 1 (TGF-beta 1) immunolocalization in the mouse follicles cultured in vitro to evaluate the hypothesis that normally developing follicles should express TGF-beta 1 in the granulosa cells around the time of antrum formation. Preantral follicles with 151-175 microns (large category) and 125-150 microns (small category) of initial diameters were used as models for normal and retarded follicles, respectively. Growth rate and timing of antrum formation in both categories were comparable to those of in-vivo grown follicles. At the time of antrum formation, follicular diameters were similar between the two follicle categories; however, antral follicles from the large category showed larger number of granulosa cells, higher estradiol production and proportion of follicles with TGF-beta 1 positive granulosa cells. Two days after antrum formation, there were no differences in the number of granulosa cells and the proportions of follicles with TGF-beta 1 positive granulosa or theca cells between the two categories. Temporal association in large follicles between the increase in estradiol production and proportion of follicles with TGF-beta 1 positive granulosa cells at the time of antrum formation supports our hypothesis. Furthermore, this study demonstrated the usefulness of the follicle culture system in the investigations of follicular physiology.     

Three substrains with different hemagglutination patterns isolated from a Japanese lentogenic strain of Newcastle disease virus

Three substrains were cloned from chick kidney cell monolayers infected with the Ishii strain of lentogenic Newcastle disease virus isolated in Japan. These substrains, which differed in plaque morphology, were distinguished as having no +24R+ and -24R- patterns in the hemagglutination (HA)-elution test.     

Prevalence of dog intestinal parasites and risk perception of zoonotic infection by dog owners in São Paulo State, Brazil

Coprological examination was used to estimate the prevalence of gastrointestinal parasites in stray and domiciled dogs from Botucatu, S?o Paulo State, Brazil. Risk factors for dog infection were assessed in relation to demographic, husbandry and management data. The dog owners completed a questionnaire survey on some aspects of dog parasitism such as parasite species, mechanisms of infection, awareness of zoonotic diseases and history of anthelmintic usage. Parasites were found in the faeces of 138 dogs, with an overall prevalence of 54.3%. Dogs harbouring one parasite were more common (31.4%) than those harbouring two (18.5%), three (3.2%) or four (1.2%). The following parasites and their respective frequencies were detected: Ancylostoma (37.8%), Giardia (16.9%), Toxocara canis (8.7%), Trichuris vulpis (7.1%), Dipylidium caninum (2.4%), Isospora (3.5%), Cryptosporidium (3.1%) and Sarcocystis (2.7%). Stray dogs were found more likely to be poliparasitized (P<0.01) and presented higher prevalence of Ancylostoma, T. canis and Giardia (P<0.01) than domiciled ones. Toxocara canis was detected more frequently in dogs with <6 months of age (P<0.05) and no effect of sex or breed could be observed (P>0.05). Except for Ancylostoma, that showed a significantly higher prevalence in dogs living in a multi-dog household (P<0.01), parasite prevalences were similar in single- and multi-dog household. The answers of dog owners to the questionnaire showed that the majority does not know the species of dog intestinal parasites, the mechanisms of transmission, the risk factors for zoonotic infections, and specific prophylactic measures. The predominance of zoonotic species in dogs in the studied region, associated with the elevated degree of misinformation of the owners, indicates that the risk of zoonotic infection by canine intestinal parasite may be high, even in one of the most developed regions of Brazil.     

Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter,Scortum barcoo (McCulloch & Waite)

In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70–90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus‐specific PCR. The full‐length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV‐like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.     

Immunological effects of glucan and <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> GG,a probiotic bacterium,on Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis> intestine with oral <Emphasis Type="Italic">Aeromonas</Emphasis> challenges

The present study histologically examined the effects of glucan-containing and Lactobacillus rhamnosus GG (LGG)-containing diets on intestinal damages inflicted on Nile tilapia by Aeromonas challenges. Tilapia were fed control, glucan, and LGG diets for 2 weeks and were subsequently challenged with Aeromonas. The intestines were then histologically examined at 1, 7, 14, and 21 days post-infection. Mortality following the challenge was lower for the fish fed the glucan and LGG diets. The intestines of these groups also showed increased inflammatory cell infiltration and reduced intestinal damage from Aeromonas. Moreover, inflammatory cell infiltration occurred more rapidly in the glucan-fed than in the LGG-fed fish following the challenge. Before the challenge, the dominant mucous cell was the acid type in all the tests. After the challenge, the main mucus cell type in the proximal intestine of the glucan-fed fish shifted to AB-PAS double-staining cells, while in the LGG-fed fish, it remained the acid type throughout the test period, and the number of double-staining cells was smaller than in the control fish after the challenge. Thus, the different mucous cell and inflammatory cell responses show that glucan and LGG might have different immunostimulative effects, although they both reduced the intestinal damage following Aeromonas challenges.     

Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein‐4 increases oocyte diameter,but impairs subsequent developmental competence

Bone morphogenetic protein‐4 (BMP‐4) inhibits luteinization of granulosa cells during in vitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than in vivo‐grown oocytes. We herein investigated whether an extended IVG culture with BMP‐4 improves oocyte growth and development to blastocysts after in vitro fertilization. Oocyte‐granulosa cell complexes (OGCs) were cultured for 14 or 16 days with BMP‐4 (10 ng/mL), while a 12 day culture with BMP‐4 served as the in vitro control. OGC viability was maintained for the 16 day culture with BMP‐4 (83.2%), but was significantly lower without BMP‐4 (58.9%) than the control (83.0%). Prolong‐cultured oocytes at 16 days had statistically greater diameter (114.6 μm) than the control (111.7 μm). IVG oocytes with BMP‐4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP‐4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP‐4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes.     

Effect of activation treatments of recipient oocytes on subsequent development of bovine nuclear transfer embryos

Effects of recipient oocyte activation methods on the development of nuclear transfer (NT) embryos were investigated. In Exp. 1, cell-cycle phase of serum-starved bovine cumulus cells was examined by flow cytometry. Majority (95.5%) of medium-sized (16-20 microm) cells that made up 56% of total cells was at the G0/G1 phase. NT embryos were constructed by electric fusion with the medium-sized serum-starved cumulus cells and bovine oocytes of 3 different preparations: enucleated oocytes treated with calcium ionophore A 23187 for 5 min and cycloheximide for 5 hr (A 23187/CHX), those treated with ethanol for 7 min and cycloheximide for 2 hr(ethanol/CHX) and those without treatment. In Exp. 2 and 3, developmental competence of NT embryos constructed with A 23187/CHX- and ethanol/CHX-treated oocytes was compared to that of NT embryos constructed with non-treated oocytes, respectively. Further, nuclear behavior in 3 different NT embryos was examined in Exp. 4. Within 1 hr after fusion, majority of the NT embryos constructed with non-treated oocytes showed condensed chromosome. Three hours after fusion, about 50% of NT embryos constructed with non-treated or ethanol/CHX-treated oocytes showed a single pronucleus-like structure. NT embryos constructed with ethanol/CHX-treated oocytes showed similar rates of fusion, cleavage and blastocyst formation to those of the non-treated oocytes. In contrast, NT embryos constructed with A 23187/CHX-treated oocytes did not show any pronucleus-like structure and showed lower cleavage rate and no development to blastocysts. The results indicate that ethanol/CHX-treated oocytes could support development of somatic cell NT embryos to the blastocyst stage at a similar rate to that of non-treated oocytes.     

Neuronal pathway from the liver modulates energy expenditure and systemic insulin sensitivity

Coordinated control of energy metabolism and glucose homeostasis requires communication between organs and tissues. We identified a neuronal pathway that participates in the cross talk between the liver and adipose tissue. By studying a mouse model, we showed that adenovirus-mediated expression of peroxisome proliferator-activated receptor (PPAR)-g2 in the liver induces acute hepatic steatosis while markedly decreasing peripheral adiposity. These changes were accompanied by increased energy expenditure and improved systemic insulin sensitivity. Hepatic vagotomy and selective afferent blockage of the hepatic vagus revealed that the effects on peripheral tissues involve the afferent vagal nerve. Furthermore, an antidiabetic thiazolidinedione, a PPARg agonist, enhanced this pathway. This neuronal pathway from the liver may function to protect against metabolic perturbation induced by excessive energy storage.     

Comparison of field and laboratory microcosm methods on the mass loss ofQuercus serrata andPinus densiflora leaf litter

The decay rates of Japanese Konara Oak (Quercus serrata Murray) and Japanese Red Pine (Pinus densiflora Sieb. et Zucc.) leaf litter were monitored for one year. It aimed to compare the decomposition of leaf litter using microcosms set up in the field (FM) and in the greenhouse (GM), with the litterbag (LB) method as control. Results showed that incubation setting affected the decay rate (k), respiration rates and the changes in the concentrations of nitrogen (N). Thek value ofQuercus in FM was higher than LB, while thek value ofPinus was higher in the LB than in FM. The decay ratesk for both species, however, were significantly lower in GM than FM and LB, clearly suggesting that decay rate was inhibited in the greenhouse. Significant differences in microclimatic variables and soil biological activities (soil respiration) existed between greenhouse and field microcosms, hence, the decay rates were affected. The N concentrations for both litter types increased as decomposition proceeded. Decomposition studies using laboratory microcosm approach alone may lead to erroneous conclusions especially if no appropriate field studies are conducted along with it. Part of this paper was presented at the XXth International Union of Forestry Research Organization (IUFRO) World Congress, August 6–12, 1995, Tampere, Finland.     

Effect of fusion/activation protocol on in vitro development of porcine nuclear transfer embryos constructed with foreign gene-transfected fetal fibroblasts

The effect of fusion/activation protocol on in vitro development of porcine nuclear transfer (NT) embryos constructed with foreign gene-transfected somatic cells were investigated. NT embryos were produced by using enucleated M II oocytes and enhanced green fluorescence protein (EGFP) gene-transfected or non-transfected porcine fetal fibroblasts. One group of NT embryos received a single electrical pulse to induce fusion and activation simultaneously (FAS). The other group was fused 2 hr before activation (FBA) using two kinds of electrical pulses. Electrically activated NT embryos in both groups were treated with cycloheximide (CHX) before culture to assess the development to the blastocyst stage. After 6 days of culture, all morulae and blastocysts derived from EGFP-transfected fibroblasts emitted green fluorescence without mosaicism, and EGFP-gene product was also detected in all morulae and blastocysts examined. NT embryos undergoing FAS showed higher developmental capacity to blastocysts than those undergoing FBA, regardless of the EGFP transfection into the nuclear donor cells. The results also indicated that EGFP-gene transfection into nuclear donor cells has no obvious deleterious effect on the development of NT embryos to blastocysts.