Apoptosis in the Antral Follicles of Swamp Buffalo and Cattle Ovary: TUNEL and Caspase-3 Histochemistry

The present study was carried out to investigate the pattern of apoptosis in the healthy antral and atretic follicles of Philippine swamp buffaloes (BU) in comparison with Holstein-Friesian (HF) cows. Paraffin sections of healthy follicles and various stages of atretic follicles were stained using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labelling (TUNEL) method to detect DNA fragmentation and cleaved caspase-3 antibody to detect cells committed to undergo apoptosis. Five equidistant areas of a follicle were counted for the presence of TUNEL- and caspase-3-positive cells. Healthy follicles of BU and HF contained no TUNEL-positive cells in the granulosa and theca layer but showed some caspase-3 positivity. The granulosa layer of advanced atretic follicles showed a significantly higher frequency of caspase-3 positivity than the healthy and early atretic follicles in both breeds. The frequency of caspase-3-positive cells of BU was significantly higher than HF in the granulosa layer of healthy, early atretic and advanced atretic follicles. In the theca interna layer, BU and HF showed a significantly lower and higher frequency of TUNEL-positive cells in the late atretic follicles compared with advanced atretic follicle, respectively. However, the frequency of caspase-3-positive cells of both BU and HF in the late atretic follicles was significantly higher than the advanced atretic follicles in the theca interna layer. These results indicate that caspase-3 and DNA fragmentation is involved in the buffalo ovarian apoptotic process.     

Cell Proliferation in the Atretic Follicles of Buffalo and Cattle Ovary

The present study was carried out to describe the proliferative activity of granulosa and theca cells in healthy antral and atretic follicles of Philippine buffaloes (BU) and Holstein-Friesian (HF) cows. Paraffin sections of ovary were immunostained with mouse monoclonal antibody to proliferating cell nuclear antigen (PCNA). Then the follicles were classified into healthy and various stages of atretic follicles. The granulosa layer of healthy follicles had a significantly higher frequency of PCNA-positive cells than the early and advanced atretic follicles in both breeds. In the theca interna, significantly reduced populations of the PCNA-positive cells were found in both breeds as atresia progressed. Moreover, HF had significantly higher PCNA-positive cells in the theca interna of healthy, early atretic and advanced atretic follicles than BU. A reduction of PCNA-positive cells during atresia was also noted in the theca externa in both animals although differences were not significant. The results of the present work suggest that the proliferative activity of granulosa and theca cells decreases in association with follicular atresia in the BU similar to HF. Furthermore, a significantly deficient cell proliferative activity of theca interna was found in BU compared with HF.     

Ergovaline does not alter the severity of ryegrass staggers induced by lolitrem B

AIM: To investigate a possible interaction between lolitrem B and ergovaline by comparing the incidence and severity of ryegrass staggers in sheep grazing ryegrass (Lolium perenne) containing lolitrem B or ryegrass containing both lolitrem B and ergovaline.

METHODS: Ninety lambs, aged approximately 6 months, were grazed on plots of perennial ryegrass infected with either AR98 endophyte (containing lolitrem B), standard endophyte (containing lolitrem B and ergovaline) or no endophyte, for up to 42 days from 2 February 2010. Ten lambs were grazed on three replicate plots per cultivar. Herbage samples were collected for alkaloid analysis and lambs were scored for ryegrass staggers (scores from 0–5) weekly during the study. Any animal which was scored ≥4 was removed from the study.

RESULTS: Concentrations of lolitrem B did not differ between AR98 and standard endophyte-infected pastures during the study period (p=0.26), and ergovaline was present only in standard endophyte pastures. Ryegrass staggers was observed in sheep grazing both the AR98 and standard endophyte plots, with median scores increasing in the third week of the study. Prior to the end of the 42-day grazing period, 22 and 17 animals were removed from the standard endophyte and AR98 plots, respectively, because their staggers scores were ≥4. The cumulative probability of lambs having scores ≥4 did not differ between animals grazing the two pasture types (p=0.41).

CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence for ergovaline increasing the severity of ryegrass staggers induced by lolitrem B. In situations where the severity of ryegrass staggers appears to be greater than that predicted on the basis of concentrations of lolitrem B, the presence of other tremorgenic alkaloids should be investigated.     

Treatment of partial seizures and seizure-like activity with felbamate in six dogs

Six dogs with partial seizures or partial seizure-like activity were treated with the antiepileptic drug felbamate between 1993 and 1998. All dogs had a history and results of diagnostic testing suggestive of either primary (idiopathic) or occult secondary epilepsy. Dogs ranged between four months and eight years of age at the onset of seizure activity. The median time period between onset of the first seizure and the start of felbamate therapy was 3.8 months (range 0.75 to 36 months). Median duration of therapy was nine months (range two to 22 months). All dogs experienced a reduction in seizure frequency after felbamate administration. Median total number of seizures post-treatment was two (range 0 to 9). Two dogs had an immediate and prolonged cessation of seizure activity. Steady-state trough serum felbamate concentrations measured at two weeks, and one, 12 and 22 months after the commencement of therapy in four dogs ranged between 13 and 55 mg/litre (median 35 mg/litre). Reversible haematological adverse effects were detected in two dogs, with one dog developing concurrent keratoconjunctivitis sicca. These results suggest that felbamate can be an effective antiepileptic drug without life-threatening complications when used as monotherapy for partial seizures in the dog.     

Immunohistochemical detection of PrP in the medulla oblongata of sheep: the spectrum of staining in normal and scrapie-affected sheep

Sections of the medulla oblongata from the brains of sheep were examined for prion protein (PrP) by immunohistochemistry. On the basis of the morphology and neuroanatomical distribution of the deposits, distinct disease-associated patterns of PrP deposition were identified in scrapie-affected sheep, suggesting at least four distinct phenotypes of scrapie. In addition, clearly defined patterns of PrP deposition, readily distinguished from the disease-associated PrP deposits, were identified in some normal sheep from scrapie-free flocks. In five sheep, believed to be preclinically affected by scrapie, PrP deposition of a disease-specific type but of restricted distribution was identified, demonstrating the sensitivity of the technique for the diagnosis of scrapie. The neuroanatomical distribution of these early PrP deposits suggest that the route of entry of the scrapie agent into the brain is via parasympathetic motor neurons in the vagus nerve which innervate the gastrointestinal tract.     

Refinement of an orthotopic lung cancer model in the nude rat

Over 85% of people with lung cancer eventually succumb to this disease, largely because current chemotherapies are ineffective. The testing and validation of promising new approaches generally rely on achieving responses with cell lines in vitro or in tumor xenografts in nude mice. However, quite often the results seen with these models are not recapitulated in the clinic, thus necessitating the need for better animal models of lung cancer for preclinical testing of new therapies. One promising model is that of orthotopic lung cancer, where xenografts of human lung cancer are established in lungs of immunodeficient rodents. The problems associated with this model include poor rates of engraftment, limited tumor multiplicity, and a heightened risk for surgical trauma. The purpose of our study was to develop an efficient approach to engraftment of orthotopic tumors throughout the lungs of the Rowett nude rat. Initially, we augmented immunosuppression in the rats with whole-body X-irradiation and then used orotracheal cannulas to intratracheally instill human cancer cells from the Calu-6 cell line. This protocol produced a low rate of engraftment and low tumor multiplicity. The hypothesis that slight disruption of the pulmonary epithelium or the surfactant layer would allow better tumor engraftment was tested by coadministration of either pancreatic elastase or ethylenediaminetetraacetic acid (EDTA) along with the cell instillations. Lung tumor engraftment was evaluated 8 weeks after instillation. The inclusion of elastase or EDTA with the Calu-6 cells resulted in an 80-100% engraftment rate, respectively. Coadministration of EDTA resulted in significantly larger and greater numbers of tumors/lung than those in elastase-treated animals. Temporal studies demonstrated that small nodules were scattered throughout the lung parenchyma 5 weeks after instilling Calu-6 cells and EDTA. These nodules grew to coalesce and form large masses that effaced >75% of the parenchyma at 9 weeks postinstillation. The refinements made through our studies have led to the development of an orthotopic lung cancer model that should facilitate the evaluation of novel therapies designed to treat or impede lung cancer development.     

Muscular dystrophy in female dogs

The most common form of muscular dystrophy in dogs and humans is caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome, and, therefore, disease-causing mutations in dystrophin occur most often in males. Therefore, females with dystrophin deficiency or other forms of muscular dystrophy may be undiagnosed or misdiagnosed. Immunohistochemistry was used to analyze dystrophin and a number of other muscle proteins associated with muscular dystrophy in humans, including sarcoglycans and laminin alpha2, in muscle biopsy specimens from 5 female dogs with pathologic changes consistent with muscular dystrophy. The female dogs were presented with a variety of clinical signs including generalized weakness, muscle wasting, tremors, exercise intolerance, gait abnormalities, and limb deformity. Serum creatine kinase activity was variably high. One dog had no detectable dystrophin in the muscle; another was mosaic, with some fibers normal and others partly dystrophin-deficient. A 3rd dog had normal dystrophin but no detectable laminin alpha2. Two dogs could not be classified. This study demonstrates the occurrence of dystrophin- and laminin alpha2-associated muscular dystrophy and the difficulty in clinical diagnosis of these disorders in female dogs.     

Pharmacokinetics and toxicity of bromide following high-dose oral potassium bromide administration in healthy Beagles

The pharmacokinetics of a multidose regimen of potassium bromide (KBr) administration in normal dogs was examined. KBr was administered at 30 mg/kg p.o. q 12 h for a period of 115 days. Serum, urine, and cerebrospinal fluid (CSF) bromide (BR) concentrations were measured at the onset of dosing, during the accumulation phase, at steady-state, and after a subsequent dose adjustment. Median elimination half-life and steady-state serum concentration were 15.2 days and 245 mg/dL, respectively. Apparent total body clearance was 16.4 mL/day/kg and volume of distribution was 0.40 L/kg. The CSF:serum BR ratio at steady-state was 0.77. Dogs showed no neurologic deficits during maintenance dosing but significant latency shifts in waves I and V of the brainstem auditory evoked response were evident. Following a subsequent dose adjustment, serum BR concentrations of approximately 400 mg/dL were associated with caudal paresis in two dogs. Estimated half-life during the accumulation phase was shorter than elimination half-lives reported in other studies and was likely related to dietary chloride content. The range of steady-state concentrations achieved suggests individual differences in clearance and bioavailability between dogs. The described protocol reliably produced serum BR concentrations that are required by many epileptic patients for satisfactory seizure control.     

Cryopreservation of Piau‐Breed Wild Boar Sperm: Assessment of Cooling Curves and Centrifugation Regimes

This study aimed to assess the effects of different cooling curves and centrifugation regimes used in cryopreservation protocols on the post‐thaw viability of Piau‐breed wild boar (Sus scrofa) sperm using in vitro assessment tests. Two centrifugations (800  g for 10 min and 2400  g for 3 min) and two cooling curves (conventional cooling using nitrogen vapour – freezing 1 and automated cooling using a programmed freezing machine – freezing 2) were tested. Therefore, the treatments were divided into M3 – centrifugation at 2400  g for 3 min and freezing 2; M10 – centrifugation at 800  g for 10 min and freezing 2; R3 – centrifugation at 2400  g for 3 min and freezing 1; and R10 – centrifugation at 800  g for 10 min and freezing 1. No significant differences (p > 0.05) between treatments occurred post‐thawing regarding the total sperm motility means recorded. The mean values of the different treatments were not different from each other regarding the supravital staining (SV), hypo‐osmotic test (HO), sperm–egg binding assay or sperm morphology. This study showed that both the cooling curve and the centrifugation regime affected the quality of post‐thaw sperm, and centrifugation for shorter times and cooling curves using automated cooling are the most suitable for minimizing sperm injury.     

Dynamics of plasma free amino acids in rainbow trout (Oncorhynchus mykiss under variety of dietary conditions

Plasma amino acid profiles in the systemic circulation of rainbow trout acclimated to diets containing different protein sources, with and without supplementation with free amino acids, were monitored for up to 120 hours after consumption of the diets. Plasma concentrations of lysine, methionine, and isoleucine increased more rapidly after feeding a diet supplemented with these amino acids in free form and, depending upon the increase in plasma concentration, remained at concentrations above those in fish fed the basal diet for at least 24h after feeding. Dietary supplementation with isoleucine increased plasma concentrations of leucine and valine as well as of isoleucine. Maximum plasma concentrations for most amino acids were attained between 12 and 24h postprandial. Dietary inclusion of gelatin caused more rapid elevations in plasma glycine and serine than did free glycine. Feeding at three hour intervals resulted in stable plasma concentrations of individual amino acids in contrast to the fluctuations occurring when fish were fed once daily. Frequent feeding favoured a higher proportion of protein to lipid in body composition.