Effect of Oil Overlay on Inhibition Potential of Roscovitine in Sheep Cumulus‐Oocyte Complexes

Inhibitors of cyclin‐dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus‐oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor‐free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (?) at 38.5°C and 5% CO₂. At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/?) exhibited total expansion while in both Rosco groups (+/?) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/?). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.     

Plasma lipopolysaccharide elevations in cattle associated with early-stage infection by Fasciola hepatica

Fasciolosis is an endemic zoonotic parasitic disease with significant impacts on human health and both animal health and production. Early post-infection impacts on the host remain unclear. The objective of this study was to determine the changes, if any, to levels of endotoxin in cattle plasma in response to early-stage infection with Fasciola hepatica. Thirty-six (36) commercial bred cattle were experimentally infected with approximately 400 viable metacercariae. Plasma lipopolysaccharide (endotoxin) levels were examined on 24 occasions from 0 h before infection to 336 h after infection using the Limulus Amoebocyte Lysate chromogenic end point assay and compared with that of six (6) uninfected control animals. Peak lipopolysaccharide levels in infected animals were reached at 52 h after infection and returned to pre-infection levels at time 144 h after infection. Infected animals had significantly elevated lipopolysaccharide levels between 24 and 120 h after infection when compared to uninfected animals. The mean change in endotoxin units (EU)/mL over time after infection was statistically significant in infected animals. Elevations of lipopolysaccharide occurred in all infected animals suggesting a possible repeatable and titratable endotoxemia conducive to therapeutic agent model development.     

Using a GnRH agonist to obtain an index of testosterone secretory capacity in the cockatiel (Nymphicus hollandicus) and sulphur-crested cockatoo (Cacatua galerita)

Objective   Validation of a stimulation test for determining the steroidogenic capacity of the parrot testis. The major aim was to characterise testosterone secretion after injection of a gonadotropin-releasing hormone agonist (GnRHa), then use the test to investigate seasonal reproduction in the male cockatiel.
Procedure   A synthetic GnRHa (buserelin; 8.0 µg of peptide/kg bodyweight) was injected IM into male cockatiels (n = 7) and sulphur-crested cockatoos (n = 3) and serial blood samples collected at 0, 30, 60, 90 and 120 min after administration. Once validated, the technique was subsequently used to examine seasonal changes (23 months) in the testosterone profile of a captive cockatiel population.
Results   Injection of buserelin resulted in a significant increase in the testosterone concentration of cockatiel plasma, with maximal concentrations occurring at approximately 60 (1.33 ± 0.08 ng/mL) to 90 min (1.22 ± 0.08 ng/mL) after injection. Although no clear pattern of seasonal variation in testosterone secretion was detected in cockatiel plasma, samples taken 60 and 90 min after administration showed a significant increase in all seasons. Injection of buserelin in the sulphur-crested cockatoo also resulted in increased testosterone secretion, with maximal concentrations obtained after 90 min.
Conclusion   Buserelin can be used to obtain a reliable index of the prevailing testosterone capacity of the cockatiel and cockatoo testis. With further studies, this test may be incorporated into clinical assessment of reproductive status.     

Bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep

Objective To study the clinical signs following bluetongue virus serotypes 1 and 3 infection in Poll Dorset sheep.
Design A clinical and pathological study.
Procedure Twenty Poll Dorset sheep were inoculated with bluetongue virus serotypes 1 or 3, each inoculum having a different passage history. The sheep were examined daily and their clinical appearance and rectal temperatures recorded. Heparinised and non-heparinised blood samples were taken at intervals for virological and serological study. Gross pathological findings were recorded for several sheep at necropsy and tissue samples were collected from three sheep for virological studies.
Results All inoculated sheep developed clinical disease. The clinical signs and gross pathological changes varied considerably but were consistent with damage to the vascular endothelial system. There was a decline in the titres of infectious bluetongue virus and of antigen in tissues collected between 7 and 12 days after infection.
Conclusions The severity of disease was related to the speed of onset and duration of pyrexia and not the development or titre of viraemia. Generally, those animals with sensitive mouths, depression, coronitis, recumbency and reluctance to move were the most debilitated. Whole blood was the most reliable source of infectious virus from acutely and chronically infected and convalescent animals. However, tissue samples particularly spleen, collected from dead or killed animals suffering from either peracute or acute forms of disease were most appropriate for the rapid confirmation of a clinical diagnosis.     

Virological and molecular epidemiological investigations into the role of wild birds in the epidemiology of influenza A/H5N1 in central Thailand

A serological and virological surveillance program to investigate the HPAI H5N1 virus in wild bird populations was undertaken from February 2007 to October 2008. The purpose of the survey was to investigate the infection status in free ranging wild birds in Banglane district, Nakhon Pathom province, central Thailand. Samples from wild birds were collected every two months. Choanal and cloacal swabs, serum and tissue samples were collected from 421 birds comprising 44 species. Sero-prevalence of the virus tested by H5N1 serum neutralization test (using a H5N1 virus clade 1; A/chicken/Thailand/vsmu-3-BKK/2004) was 2.1% (8 out of 385 samples; 95% CI 0.7, 3.5). Species that were antibody positive included rock pigeons (Columba livia), Asian pied starling (Gracupica contra), spotted dove (Streptopelia chinensis), oriental magpie robin (Copsychus saularis), blue-tailed bee-eater (Merops philippinus), myna (Acridotheres spp.), and pond heron (Ardeola spp.). Prevalence by H5N1 virus isolation was 0.5% (2 out of 421 samples; 95% CI 0.0, 1.1); the two H5N1 virus-positive samples were from Asian pied starling (Gracupica contra) and white vented myna (Acridotheres grandis). Positive virological samples were collected in June 2007 while all positive serology samples were collected between May and August except for one sample collected in December 2007. No positive samples were collected in 2008. Molecular studies revealed that the wild bird H5N1 viruses were closely related to poultry viruses isolated in other parts of Thailand. However, there was no poultry H5N1 prevalence study performed in the study site during the time of this wild bird survey. Interpretation of source of virus isolates would include spill-over of H5N1 viruses from contaminated sources due to movement of domestic poultry and/or fomites from other areas; or infection of wild birds within the outbreak locations and then translocation by wild bird movement and interaction with wild birds inhabiting distant locations.     

The indirect immunofluorescence assay using cardiac tissue from chickens, quails and ducks for identification of influenza A virus during an outbreak of highly pathogenic avian influenza virus (H5N1): a rapid and simple screening tool for limited resource settings

Here we describe the diagnostic utility of the indirect immunofluorescence assay (IFA) during a recent outbreak of highly pathogenic avian influenza (HPAI) subtype H5N1 virus in southern Thailand and demonstrate the usefulness of the cardiac tissue from infected chickens, quail, and ducks for diagnosis. The most reliable sample for IFA diagnosis of influenza A virus was cardiac tissue (83.0%; 44/53) which when divided by species (chicken, quail and duck cardiac tissues) gave respective positivity rates of 88% (22/25), 88.9% (16/18) and 60.0% (6/10). Cardiac tissue also gave the highest IFA intensity for the three species. We believe that the IFA method has wide applicability in developing countries or remote settings where clinically similar avian diseases with high morbidity and mortality such as Newcastle disease and fowl cholera are common and could be rapidly excluded thereby conserving valuable reference laboratory capacity for true HPAI outbreaks.     

Molecular expression of caprine estrogen receptor gene 1 in reproductive and non‐reproductive tissues

During the last decades, physiological effects of oestrogens have been increasingly explored by scientists and biotechnologists. Estrogens exert a wide range of effects on a large variety of cell types. Oestrogen and its receptors are essential for sexual development and reproduction. Estrogen receptor alpha is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene estrogen receptor gene 1 (ESR1), responsible for better fertility. The ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. Therefore, this is a good startby to study the expression pattern of estrogen receptor 1 gene in high‐fertile (G1) and low‐fertile (G2) bucks of Jamunapari and Barbari breeds identified on the basis of seminal quality traits and fertility trials. RNA was extracted from the tissues by TRIzol method. The identification and expression pattern of caprine ESR1 gene was analysed by real‐time PCR (Roche LC‐480). Our work shows that the relative quantification by RT‐PCR indicates more fold in head of epididymis as compared to spleen of caprine ESR1 gene. Furthermore, the RT‐PCR indicated that fertile bucks of Jamunapari breed have more fold value as compared to Barbari breed in respect of reproductive organ.