Molecular dynamics of heterochromatin protein 1beta, HP1beta, during mouse preimplantation development

To elucidate the molecular dynamics of HP1beta in mouse preimplantation embryos, we examined the localization, dynamics, and mobility of HP1beta in the (pro)nucleus by live cell imaging. Time-lapse observation revealed that the chromatin association of HP1beta is regulated in a cell cycle-dependent manner. HP1beta was localized in the interphase nucleus and was dynamically dissociated from the nucleus during the metaphase stage. The HP1beta assembly and clustered heterochromatin structure were both found in the nuclei of 2-cell and later-stage embryos. Moreover, fluorescent recovery after photobleaching analysis implied that HP1beta is more freely mobile in the pronucleus of the 1-cell embryo than in the 4-cell nucleus. These results suggest that the chromatin configuration may be regulated by the stability and mobility of chromatin-associated proteins including HP1beta during early embryonic stages.     

Changes in expression and localization of GPRC5B and RARalpha in the placenta and yolk sac during middle to late gestation in mice

The mRNA expression of GPRC5B, an orphan G protein-coupled receptor, is induced by retinoic acid (RA). Because RA plays critical roles in embryonic development, reproductive functions, metabolism and homeostasis, GPRC5B is also considered crucial in these physiological events. We investigated the changes in expression of GPRC5B and RA receptor (RAR) alpha mRNAs and immunohistochemical localization of their proteins in the murine placenta and yolk sac at 13.5, 15.5 and 17.5 days post coitus. Stable levels of GPRC5B and RARalpha mRNAs were detected in the placenta and yolk sac. In the placenta, GPRC5B was present in maternal and fetal vascular endothelial cells, stromal cells, fibroblast-like cells and glycogen cells. A strong reaction to RARalpha was detected in maternal and fetal vascular endothelial cells and stromal cells. The levels of GPRC5B and RARalpha proteins in maternal and fetal vascular endothelial cells decreased with gestation. In the yolk sac, GPRC5B and RARalpha proteins were detected in vascular endothelial cells, but their levels did not change during the gestation period. These findings indicate that GPRC5B is involved in RA-dependent morphogenesis/angiogenesis and regulation of extracellular matrix synthesis in the murine placenta and yolk sac.     

Assessment of fertility and reproductive toxicity in adult female mice after long-term exposure to Pueraria mirifica herb

The present study investigated the effects of long-term administration of Pueraria mirifica (PM) at non-toxic doses on the ovarian function and fertility of adult female mice based on evaluation of hematological and biochemical parameters. Female mice were divided into 4 groups (36 mice/group). Groups 1-3 were orally treated with a dose of 0 (PM-0), 10 (PM-10) or 100 mg/kg BW/day PM (PM-100), and group 4 was subcutaneously injected with 200 mug/kg BW/day of synthetic estrogen diethylstilbestrol (DES). The treatment schedule was separated into treatment and post-treatment periods. The duration of each period was 8 weeks. The PM-10 mice exhibited regular estrous cycles, while the PM-100 and DES treatments induced prolonged estrous cycles. Although no changes were observed in the uterus and ovary weights of the mice after the PM-100 and DES treatments, hyperplasia of the uterine endothelium and a decrease in the number of growing ovarian follicles were detected. The changes in the ovarian histologies of the PM-100 and DES mice were related to reductions in the levels of LH and FSH, which subsequently caused a decrease in mating efficiency. Once the PM mice were able to copulate, they were capable of successfully becoming pregnant and mothering offspring. No abnormalities were observed in the external morphologies and reproductive organ weights of the 50-day-old offspring. In conclusion, our results suggest that long-term exposure to 100 mg/kg BW of PM has adverse effects on the mating efficiency and reproduction of adult female mice and that administration of 10 mg/kg BW of PM does not induce any changes in the hypothalamic-pituitary-ovarian-uterine axis.     

Immunological and gene expression responses to a Salmonella infection in the chicken intestine

Besides infection in humans, Salmonella enteritidis can also cause serious illness in young chickens. However, the genetic and immunological parameters important for the disease in chickens are not well characterized. In this study, processes in the chicken intestine in response to a Salmonella infection were investigated in two different chicken lines. One-day-old chickens were orally infected with Salmonella. T-cell subpopulations, phagocytic properties of intestinal mononuclear cells and RNA expression levels of the jejunum were investigated. The two chicken lines differed in the amount of cfu in the liver and growth retardation after the infection. Differences in phagocytic activity of intestinal mononuclear cells were found between control and Salmonella infected chickens. The number of CD4+ T-cells of the intestine decreased after the Salmonella infection in one chicken line, while the number of CD8+ T-cells increased in both chicken lines, but the time post infection of this increase differed between the lines. In one chicken line the expression levels of the genes carboxypeptidase M and similar to ORF2 decreased after the Salmonella infection, which might be related to a decrease in the amount of macrophages. With the microarray, ten genes were found that were regulated in only one of the chicken lines, while we found six genes regulated in response to the infection in both chicken lines. So differences in genetic background of the chickens influence the intestinal host response of the Salmonella infection as observed by phagocytic activity, gene expression and changes in the number of T-cell subpopulations and macrophages.     

Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells

Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F(2alpha) (PGF(2alpha)) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF(2alpha) concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF(2alpha) concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF(2alpha) concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.     

Adenocarcinoma metastasis of the intertransversarius cervicis muscle eliciting a right forelimb lameness in a dog

This article describes identification of a metastatic adenocarcinoma to the intertransversarius cervicis muscle using magnetic resonance imaging (MRI) in a dog that presented with chronic lameness of the right forelimb. Magnetic resonance imaging revealed a right sided, ovoid signal abnormality within the intertransversarius cervicis muscle lateral to the sixth cervical (C6) vertebra. The lesion was uniform, hyperintense on T2- and isointense on T1-weighted images to muscle and exhibited uniform contrast enhancement on T1-weighted images. The MRI findings were consistent with a neoplasia. Surgical excision was performed. Histopathological diagnosis was metastatic fibrous adenocarcinoma. The dog recovered rapidly but 6 months post-operatively he was killed because of lung metastases. Necropsy was declined and the primary tumour could not be identified.     

Feline calicivirus

Feline calicivirus (FCV) is an important and highly prevalent pathogen of cats. It belongs to the family Caliciviridae which includes other significant pathogens of man and animals. As an RNA virus, high polymerase error rates convey upon FCV a high genome plasticity, and allow the virus to respond rapidly to environmental selection pressures. This makes the virus very adaptable and has important implications for clinical disease and its control. Being genetically diverse, FCV is associated with a range of clinical syndromes from inapparent infections to relatively mild oral and upper respiratory tract disease with or without acute lameness. More recently, highly virulent forms of the virus have emerged associated with a systemic infection that is frequently fatal. A proportion of FCV infected cats that recover from acute disease, remain persistently infected. In such cats, virus evolution is believed to help the virus to evade the host immune response. Such long-term carriers may only represent a minority of the feline population but are likely to be crucial to the epidemiology of the virus. Vaccination against FCV has been available for many years and has effectively reduced the incidence of clinical disease. However, the vaccines do not prevent infection and vaccinated cats can still become persistently infected. In addition, FCV strain variability means that not all strains are protected against equally. Much progress has been made in understanding the biology and pathogenesis of this important feline virus. Challenges for the future will necessarily focus on how to control the variability of this virus particularly in relation to emerging virulent strains and vaccination.     

Feline herpesvirus

Feline herpesvirus (FHV-1; felid herpesvirus 1 (FeHV-1)) is an alphaherpesvirus of cats closely related to canine herpesvirus-1 and phocine herpesvirus-1. There is only one serotype of the virus and it is relatively homogenous genetically. FeHV-1 is an important cause of acute upper respiratory tract and ocular disease in cats. In addition, its role in more chronic ocular disease and skin lesions is increasingly being recognised. Epidemiologically, FeHV-1 behaves as a typical alphaherpesvirus whereby clinically recovered cats become latently infected carriers which undergo periodic episodes of virus reactivation, particularly after a stress. The primary site of latency is the trigeminal ganglion. Conventional inactivated and modified-live vaccines are available and protect reasonably well against disease but not infection, although viral shedding may be reduced. Genetically engineered vaccines have also been developed, both for FeHV-1 and as vector vaccines for other pathogens, but none is as yet marketed.     

Evaluation of endotoxaemia in the prognosis and treatment of scouring merino lambs

This study looked at measurement of endotoxaemia as a tool in determining prognosis and probable response to treatment in scouring lambs. One hundred eighty-three lambs in the first 15-20 days of life, from eight Merino sheep farms located in the region of La Serena, south-west Spain, were used in this experiment. Scouring and normal/control lambs were selected following a clinical examination, the scouring group was further divided into subgroups, specifically those that did or did not survive 72 h following treatment. At the time of the clinical examination, faecal and blood samples were taken. Faecal culture and commercial faecal antigen tests for detection of enteropathogens in faeces and serum endotoxin measurement using chromogenic lymulus amoebocyte lysate (LAL) were carried out. Scouring lambs received 0.07 mg/kg liveweight halofuginone once a day for 3 days, a single oral dose of 0.20 mg/kg liveweight of spectinomycin and oral rehydration fluid. The pathogens isolated were Cryptosporidium spp. and Escherichia coli. The case fatality rate was 51% in the scouring lambs. Postmortem findings were consistent with enterotoxigenic E. coli infection. The concentration of endotoxin was 0.18 +/- 0.12 ng/ml in the control group, 0.35 +/- 0.17 ng/ml in the surviving lambs and 0.46 +/- 0.14 ng/ml in the non-surviving lambs. Significant differences between groups were found. Case fatality rate of the scouring lambs with endotoxaemia below 0.30 ng/ml was 0%, while it was 100% above 0.50 ng/ml. These results may be utilized as a prognostic indicator in lambs affected by E. coli and Cryptosporidium that will help aid in decision-making as to whether to treat a lamb or not based on its chances of survival.     

Factors affecting the success rate of treatment of recumbent dairy cows suffering from hypocalcaemia

We aimed to investigate the ratio of accompanying diseases in cows suffering from clinical hypocalcaemia and their influence on cure rate. In five veterinary practices in different regions of Germany, all recumbent cows around parturition were included in the study for a period of 1 year. After recording the case history a clinical examination was done and a serum sample was taken to measure the concentrations of calcium and phosphorus magnesium, beta-hydroxybutyrate, total bilirubin, cholesterol, urea, and the activities of ASAT, CK, and GLDH. Only cows with hypocalcaemia entered the statistical analysis. Hypocalcaemia was the major cause of recumbency in cows of the second lactation or elder. Muscle damage was the second frequent diagnose in recumbent cows and the major concomitant disease in hypocalcaemic cows. The overall cure rate was between 89.4% and 94.8%. Calcium and phosphorus concentrations did not have an influence on cure rate. Non-cured cows had higher serum activities of CK (p<0.043) and ASAT (p<0.006). Nevertheless, the activities of CK and ASAT were no good predictors of treatment failure because of their low specificity and the high cure rate of the cows in the five practices.